Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, Suppl. 1, pp. S1-S35 © Pleiades Publishing, Ltd., 2025.

Russian Text © The Author(s), 2025, published in Uspekhi Biologicheskoi Khimii, 2025, Vol. 65, pp. 3-54.

REVIEW

The Big, Mysterious World of Plant 14-3-3 Proteins

Ilya A. Sedlov

1,2

and Nikolai N. Sluchanko

1,a

Bach Institute of Biochemistry, Federal Research Center of Biotechnology, Russian Academy of Sciences,

119071 Moscow, Russia

Department of Biochemistry, Faculty of Biology, Lomonosov Moscow State University,

119991 Moscow, Russia

e-mail: nikolai.sluchanko@mail.ru

Received August 28, 2024

Revised September 6, 2024

Accepted September 20, 2024

Abstract— 14-3-3 is a family of small regulatory proteins found exclusively in eukaryotic organisms. They

selectively bind to phosphorylated molecules of partner proteins and regulate their functions. 14-3-3 pro-

teins were first characterized in the mammalian brain approximately 60 years ago and then found in plants,

30 years later. The multifunctionality of 14-3-3 proteins is exemplified by their involvement in coordination

of protein kinase cascades in animal brain and regulation of flowering, growth, metabolism, and immunity in

plants. Despite extensive studies of this diverse and complex world of plant 14-3-3 proteins, our understanding

of functions of these enigmatic molecules is fragmentary and unsystematic. The results of studies are often

contradictory and many questions remain unanswered, including biochemical properties of 14-3-3 isoforms,

structure of protein–protein complexes, and direct mechanisms by which 14-3-3 proteins influence the func-

tions of their partners in plants. Although many plant genes coding for 14-3-3 proteins have been identified,

the isoforms for in vivo and in vitro studies are often selected at random. This rather limited approach is

partly due to an exceptionally large number and variety of 14-3-3 homologs in plants and erroneous a  priori

assumptions on the equivalence of certain isoforms. The accumulated results provide an extensive but rather

fragmentary picture, which poses serious challenges for making global generalizations. This review is aimed to

demonstrate the diversity and scope of studies of the functions of plant 14-3-3 proteins, as well as to identify

areas that require further systematic investigation and close scientific attention.

DOI: 10.1134/S0006297924603319

Keywords: 14-3-3 protein, plant biochemistry, phosphorylation, protein–protein interactions, enzyme regulation,

signaling pathways

* To whom correspondence should be addressed.

INTRODUCTION

14-3-3 proteins are small acidic dimeric proteins

found only in eukaryotes. They lack enzymatic activ-

ity, but regulate functions of other proteins via bind-

ing to specific phosphorylated sites in partner proteins

and altering their functional properties.

Mammalian 14-3-3 proteins were first described

about 60 years ago; among them, human 14-3-3 pro-

teins have been studied in most detail. The decades

of research have led to the understanding that 14-3-3

proteins are involved in the regulation of many funda-

mental cellular processes, such as intracellular signal-

ing, gene expression, cell cycle control, and cell death.

14-3-3 proteins also play an important role in human

and animal diseases, e.g., viral infections and cancer.

Plant 14-3-3 proteins were discovered 30 years lat-

er than their mammalian homologs. Because plants are

sessile organisms, they must regulate many biochemi-

cal processes in order to adapt to constantly changing

environmental conditions, so regulatory molecules are

of particular importance to them. This is why 14-3-3

regulatory proteins are essential in crucial processes

of plant life, such as response to phytohormones, flow-

ering, growth, mineral nutrition, and plant immunity.

Despite the importance of plant 14-3-3 proteins,

there are still many unexplored issues, or “blank

spots”. Thus, the structure and biochemical properties

SEDLOV, SLUCHANKOS2

BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

of plant 14-3-3 proteins have been studied much less

compared to their mammalian counterparts. An insuf-

ficient information on the structure of complexes of

plant 14-3-3 proteins with their interaction partners

explains the lack of understanding of mechanisms

by which 14-3-3 proteins affect the functions of their

partners. In most cases, there is no information on

the details of 14-3-3 interaction with their partners

and effects this binding produces on their activity of

the latter. Many 14-3-3 genes have been identified, but

the isoforms for in vivo and in vitro studies are chosen

almost at random, and the obtained results provide a

very fragmented picture that precludes global gener-

alizations.

This review aims, on one hand, to demonstrate

the diversity and broad scope of studies of the func-

tions of plant 14-3-3 proteins, and, on another hand, to

highlight the fragmentary nature of current concepts

and lack of consistency in the research of plant 14-3-3

proteins.

DIVERSITY AND CLASSIFICATION

OF 14-3-3 PROTEINS

Proteins of the 14-3-3 family are common among

eukaryotes. They were first isolated in the 1960s from

the mammalian brain, where they account for a sig-

nificant fraction (up to 1% [1]) of all proteins. The

name comes from the purification procedure, as these

proteins were found in fraction 14 during chromatog-

raphy on diethylaminoethyl cellulose and produced

spot 3.3 during electrophoresis in a starch gel [2].

Further studies have led to the discovery of seven ho-

mologous 14-3-3 proteins in mammals that have been

designated with the first letters of the Greek alphabet:

α, β, γ, δ, ε, ζ, and η. However, the α and δ isoforms

were later discovered to be the phosphorylated ver-

sions of β and ζ, respectively. We should mention that

defining identified 14-3-3 proteins as “isoforms” was

incorrect, as each 14-3-3 “isoform” is encoded by a

separate gene. Nevertheless, since the term “isoform”

has been commonly accepted for describing 14-3-3

proteins, we will use it in our review. Two specifically

expressed isoforms, σ and τ (or θ), have been discov-

ered later [3]. 14-3-3 proteins have been rediscovered

several times by independent research groups and

in different organisms, which resulted in the emer-

gence of alternative names, for example, Leonardo in

Drosophila fruit fly, BMH1 and BMH2 in Saccharomy-

ces cerevisiae yeast, GRF in Arabidopsis, etc. [3]. Plant

14-3-3 proteins were discovered almost three decades

after their mammalian homologs. In 1992, several pa-

pers on plant 14-3-3 proteins were published at the

same time [4-7]. Perhaps, no other group of organ-

isms has so many alternative names for 14-3-3 pro-

teins as plants, for example, fusicoccin-binding protein

(FCBP) [8], general regulatory factor (GRF) [9], G-box

factor 14-3-3 (GF14) [5, 7], nitrate reductase inhibitor

protein (NIP) [10], tomato fourteen-three-three (TFT)

[11, 12], and rare cold-inducible (RCI) [13]. All these

names belong to members of the same protein family.

They refer to important biological functions of these

proteins and are associated with the history of 14-3-3

protein research. Currently, the names GRF and GF14

are used to classify 14-3-3 proteins from different

plant species. Also thirteen 14-3-3 isoforms from the

model plant Arabidopsis thaliana are named with

Greek letters, by analogy with mammalian proteins,

but starting from the end of the alphabet: omega(ω),

psi  (ψ), chi  (χ), phi  (φ), upsilon  (υ), pi  (π), omicron  (ο),

nu  (ν), mu  (μ), lambda  (λ), kappa  (κ), iota  (ι), and ep-

silon  (ε). The orthologs of A. thaliana isoforms from

other plants can also be designated with Greek letters,

which creates the third system for the naming and

classification of plant 14-3-3 isoforms. Table 1 shows

the correspondence between the three nomenclatures

of 14-3-3 proteins from A. thaliana.

Compared to mammalian species, which have

seven 14-3-3 isoforms, the number of isoforms encod-

ed by plant genomes can vary significantly. For ex-

ample, there are 5 isoforms in strawberry (Fragaria

vesca)  [14], 8 in rice (Oryza sativa)  [15] and cocoa

tree (Theobroma cacao) [16], 13 in tomato (Solanum

lycopersicum)  [11], 18 in soybean (Glycine max) [17],

Table  1. Nomenclatures for 14-3-3 isoforms from

A. thaliana

UniProt

Greek

letters

GRF

nomenclature

GF14

nomenclature

P42643 chi χ GRF1 GF14χ

Q01525 omega ω GRF2 GF14ω

P42644 psi ψ GRF3 GF14ψ

P46077 phi φ GRF4 GF14φ

P42645 upsilon υ GRF5 GF14υ

P48349 lambda λ GRF6 GF14λ

Q96300 nu ν GRF7 GF14ν

P48348 kappa κ GRF8 GF14κ

Q96299 mu μ GRF9 GF14μ

P48347 epsilon ε GRF10 GF14ε

Q9S9Z8 omicron ο GRF11 GF14ο

Q9C5W6 iota ι GRF12 GF14ι

F4IA59 pi π GRF13 GF14π

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BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

26 in tea tree (Camellia sinensis) [16], and 36 in apple

tree (Malus domestica) [18].

The existence of numerous orthologs and par-

alogs, as well as the variability of the total number

of 14-3-3 isoforms in different plant species, are due

to several whole-genome and segmental duplications

that had occurred in plants in the course of evolu-

tion [18-22]. It is believed that an ancient duplica-

tion has led to the emergence of two separate large

14-3-3 phylogenetic lineages called epsilon group and

non-epsilon group. Numerous phylogenetic studies

have confirmed this divergence for 14-3-3 isoforms

from different plant species [11, 14, 16, 18, 19, 22].

There is no established opinion on the time of the

phylogenetic divergence, but it might have occurred

before the emergence of green plants [14]. This de-

marcation of isoforms is fundamental. It can be

traced in all studied species of seed plants (Sperma-

tophyta), as each species in this clade has both ep-

silon and non-epsilon isoforms  [14,  19]. The epsilon

and non-epsilon isoforms differ in the gene structure.

In A.  thaliana, genes coding for the epsilon isoforms

contain 6 to 7 exons separated by 4 to 6 introns, while

genes for the non-epsilon isoforms contain 4 exons

and 3 to 4 introns [23, 24]. Despite the fact that clas-

sification of 14-3-3 paralogs into epsilon and non-ep-

silon groups is commonly recognized, the authors of

some recent studies on the evolution of 14-3-3 proteins

do not divide 14-3-3 isoforms into these two large

groups [24, 25].

In the course of evolution, large phylogenetic

groups have diverged into smaller subgroups. Based

on the analysis of gene sequences from three species

of monocots and nine species of dicots, Ren et al. [18]

identified 11 subgroups (subfamilies) of 14-3-3 genes,

four of which were in the epsilon group and sev-

en – in the non-epsilon group [18]. Some subgroups

in the study were represented exclusively by 14-3-3

genes from the monocots. In another study, which ex-

amined 14-3-3 isoforms from 12 plant species, 4 sub-

groups were identified in the non-epsilon group, and

no subgroups were found in the epsilon group [19].

A very extensive phylogenetic study of the 14-3-3 fam-

ily analyzed isoforms from 46 species of angiosperms,

Fig. 1. Phylogeny and structure of plant 14-3-3 proteins. a) Phylogenetic tree of thirteen 14-3-3 isoforms from A.  thaliana,

constructed with MEGA11 using the maximum likelihood method and rooted using human ζ isoform. Evolutionary dis-

tance scale (0.1) is shown below. b)Structure of 14-3-3 λ dimer from A. thaliana (PDB ID: 8QT5 [26]). The left monomer is

colored according to the ConSurf residue conservation score [27] (default settings), with residues conserved across 14-3-3

species shown in magenta and non-conserved residues shown in cyan, with the corresponding color scale below. AG is

the amphipathic groove (the binding site for the partner protein phosphorylated sequence). The right monomer is colored

green, with the N- and C-termini marked; the alpha-helices are indicated with Roman numerals. c)Amino acid sequences of

phosphorylated motifs in 14-3-3 partner proteins. Phosphorylated residues are shown in red. Positions of residues relative

to the phosphorylated residue (position 0) are shown in gray.

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BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

Table 2. Distribution of plant 14-3-3 isoforms in phylogenetic groups and subgroups (according to Mikhaylova et al. [14]).

Systematic groups

(according to APG IV)

Family Plant species Epsilon

group

Non-epsilon

group

Epsilon

isoforms

Non-

epsilon

isoforms

Total

number

of isoforms

μιεωψκ

Basal angiosperms Amborellales Amborellaceae Amborella trichopoda – 1 1 – 1 1 2 2 10 (6)

Monocots

Zingiberales Musaceae

Musa acuminata

(banana)

611–11 8 2 10

Poales

Bromeliaceae

Ananas comosus

(pineapple)

2– 331 2 7 9

Panicum virgatum

(millet)

– – 3 1 9 - 3 10 13

Sorghum bicolor

(sorghum)

––114- 1 5 6

Hordeum vulgare

(barley)

––115- 1 6 7

Triticum aestivum

(wheat)

– – 1 2 10 - 1 12 15 (2)

Dicots Superasterids

Ranunculales Ranunculaceae

Aquilegia coerulea

(aquilegia)

121212 4 5 9

Caryophyllales Amaranthaceae

Amaranthus

hypochondriacus

(amaranthus)

33323811

Lamiales Phrymaceae

Mimulus guttatus

(yellow monkeyflower)

121322 4 7 11

Solanales Solanaceae

Solanum tuberosum

(potato)

211422 4 8 12

Solanum lycopersicum

(tomato)

221422 5 8 13

Nicotiana tabacum

(tobacco)

112522 4 9 13

Apiales Apiaceae

Daucus carota

(carrot)

1–– 413 1 8 9

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Table 2 (cont.)

Systematic groups

(according to APG IV)

Family Plant species Epsilon

group

Non-epsilon

group

Epsilon

isoforms

Non-

epsilon

isoforms

Total

number

of isoforms

μιεωψκ

Dicots Superrosids

Brassicales

Caricaceae Carica papaya (papaya) 2 1 1 1 – 2 4 3 7

Brassicaceae

Brassica rapa (turnip) 1 3 3 7 4 5 7 16 23

Arabidopsis thaliana 113332 5 8 13

Arabidopsis lyrata 111332 3 8 11

Capsella rubella

(pink shepherd’s purse)

111232 3 7 10

Sapindales Rutaceae Citrus sinensis (orange) 2 1 1 2 1 2 4 5 9

Myrtales Myrtaceae

Eucalyptus grandis

(eucalyptus)

111111 3 3 6

Malvales Malvaceae

Theobroma cacao (cacao) 1 1 1 2 1 1 3 4 7

Gossypium raimondii (cotton) 1 1 2 8 3 2 4 13 17

Cucurbitales Cucurbitaceae

Cucumis sativus

(cucumber)

122212 5 5 10

Fabales Fabaceae

Glycine max (soy) 6 3 - 4 3 2 9 9 18

Trifolium pratense (clover) 4 1 - 1 - 1 5 2 7

Rosales Rosaceae

Prunus persica (peach) 2 1 1 2 - 2 4 4 8

Fragaria vesca (strawberry) 1 - - 1 1 2 1 4 5

Malpighiales

Salicaceae

Populus trichocarpa

(poplar)

322232 7 7 14

Salix purpurea (willow) 2 2 2 2 1 2 6 5 11

Euphorbiaceae

Ricinus communis (castor

bean plant)

111 1 -1 3 2 5

Linaceae Linum usitatissimum (flax) 4 3 1 3 2 4 8 9 17

* APG IV (Angiosperm Phylogeny Group) is a current classification system in botany [28].

** Number of non-classified isoforms is shown in parentheses.

SEDLOV, SLUCHANKOS6

BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

including 13 monocots, 32 eudicots, and one represen-

tative of basal angiosperms [14]. The epsilon group

was divided into three subgroups (iota, mu, and ep-

silon), and also three subgroups were distinguished

within the non-epsilon group (omega, psi, and kappa).

This classification was based on the phylogenetic re-

lationships of A.  thaliana isoforms, and the subgroups

were named according to the Greek letter designations

of the most characteristic isoforms. The distribution

of 14-3-3 isoforms in A.  thaliana was the following:

5genes were in the epsilon group [one (ι) in the iota,

one (μ) in the mu, and three (ο, π, ε) in the epsilon

subgroups] and 8 genes were in the non-epsilon group

[three (χ, φ, ω) in the omega, three (υ, ν, ψ) in the

psi, and two (κ, λ) in the kappa subgroups] (Fig. 1a).

As can be noticed from Fig. 1a, each subgroup con-

tained at least one isoform, making A.  thaliana a con-

venient model for a systematic study of 14-3-3 family

representatives. The classification based on the 14-3-3

isoforms of A.  thaliana is complete and the most rep-

resentative, and we will be using it further in this

review.

Numbers of isoforms in subgroups, as well as

the representation of subgroups themselves in plants

may vary significantly. For example, legumes (Faba-

ceae) lack the epsilon subgroup, while the iota and

epsilon subgroups are absent in carrot (Daucus caro-

ta) (Table2). Representatives of the class Monocotyle-

doneae stand out in terms of the isoform distribution

among subgroups. All analyzed representatives of this

class, with the exception of banana (Musa acuminata),

lacked isoforms of the iota subgroup. Plants of the

Poaceae family (grasses) also lack the mu and kappa

subgroups, but the number of isoforms in the psi sub-

group is greatly increased, e.g., up to 10 in wheat (Trit-

icum aestivum) (Table 2). In cereals, the non-epsilon

group contains only ω isoforms. Such distribution has

led to the fact that the psi subgroup has the largest

number of isoforms among the analyzed plants, while

omega is the most widespread subgroup [14].

The high variability in the number and phyloge-

netic affiliation of 14-3-3 paralogs among plant species,

together with the evolution of these proteins through

multiple duplications, have created grounds to assume

that the functions of 14-3-3 isoforms are redundant

and can overlap. Thus, experiments in yeast S.  cerevi-

siae have revealed a high degree of functional similar-

ity between 14-3-3 proteins. S.  cerevisiae contain only

two 14-3-3 genes: BMH1 and BMH2 [29-31]. Knocking

out either of them altered the phenotypes of the re-

sulting mutants only slightly compared to the wild-

type cells, while the double knockout of both genes

was lethal  [29,  31]. However, introduction of plant

14-3-3 gene to the double knockout mutants rescued

a viable phenotype, which indicated interchangeabili-

ty of 14-3-3 proteins even from systematically distant

groups [32]. However, evolutionary analysis of various

angiosperm species revealed the effect of purifying

selection on 14-3-3 proteins, which may be associated

with an acquisition of a specific function or functions

by them [18, 24, 33-35].

Phenotypic analysis of mutant plants deficient by

particular 14-3-3 isoforms can shed light on specific

functions of these proteins in vivo or their redun-

dancy; however, such studies are often unsystemat-

ic and incomplete. Even for the model organism as

A. thaliana, there is no description of phenotypes of

mutants deficient by each of all 13 14-3-3 isoforms,

which hinders elucidation of their in vivo functions.

The studies are often focused on a particular feature

of mutant plants, while neglecting the other traits. In

their extensive analysis of mutations in plant 14-3-3

isoforms, van Kleeff et al. [36] produced single, dou-

ble, triple, and quadruple knockout mutants for 14-3-3

isoforms from the non-epsilon group (λ, κ, ν, υ, φ,

and χ) in A. thaliana. The authors found that the

length of the main root in single and even double mu-

tants did not differ from that in the wild-type plants;

in six triple and three quadruple knockouts, the main

root was shorter than in the wild-type plants  [36].

When three 14-3-3 isoforms from the epsilon group

(ε, μ, and ο) were knocked down simultaneously, the

mutant plants demonstrated serious growth impair-

ments and reduced length of the root and the hypo-

cotyl [37]. Only the knockout of the μ isoform (but

not the ν isoform) resulted in the reduced root length

in A.  thaliana plants grown at constant illumination;

however, both μ- and ν-deficient mutants had shorter

roots when grown under red light [38]. The knockout

of the μ isoform in A.  thaliana reduced the length of

lateral roots, while the overexpression of this isoform

increased it [39]. The transition to flowering under

the long-day conditions was delayed in single μ or ν

knockout mutants [40]. Overexpression of the GF14c

isoform from rice (O.  sativa) also delayed flowering,

while its knockout caused an earlier transition to

flowering compared to the wild-type plants [41]. Over-

expression of the Me14-3-3VII isoform from cassava

(Manihot esculenta) in A.  thaliana increased the con-

tent of starch and sugar in the leaves [42]. However,

overexpression of another cassava isoform, Me14-3-3II,

decreased the amount of starch in the mutant plants

[43]. The antisense-mediated knockdown of 14-3-3 ε

and μ resulted in a 2 to 4-fold increase in the amount

of accumulated starch in the mutant plants compared

to the wild-type controls [44]. Overexpression of the

SiGRF1 isoform from the fox millet (Setaria italica) in

A.  thaliana caused an earlier transition to flowering

under the salt stress conditions [25]. Overexpression

of the MdGRF13 isoform from apple tree (M.  domesti-

ca) in A.  thaliana increased plant tolerance to drought

and salt stress [18]. Overexpression of another apple

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BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

isoform (MdGRF6), on the contrary, led to the increase

in the plant sensitivity to exogenous salt, while the

knockdown of this isoform induced plant tolerance to

salt stress [45].

Apparently, mutations in 14-3-3 proteins mostly

affect plant growth (especially root growth), timing of

transition to flowering, tolerance to salt stress, and

starch accumulation. The phenotype of 14-3-3 mutant

plants is determined by the influence of these proteins

on specific physiological and biochemical reactions,

which will be discussed below.

BIOCHEMICAL CHARACTERISTICS

OF PLANT 14-3-3 PROTEINS

Structure of 14-3-3 proteins. Members of the

14-3-3 family are acidic proteins (pI~  4-5) with a mo-

lecular weight of ~30  kDa. In cells, they typically exist

as homo- or heterodimers consisting of the same or

different subunits, respectively. The monomers bind

to each other in an antiparallel fashion through their

N-terminal regions, with the formation of a W-like

structure that has the central symmetry and two

phosphopeptide-binding (amphipathic) grooves, one in

each monomer (Fig.1b). Both plant and animal 14-3-3

proteins consist of 9 α-helices, each located antipar-

allel to a previous one. Helices 1-4 of the monomers

(N-terminal regions) form a surface necessary for di-

merization, while helices 3, 5, 7, and 9 participate in

the formation of the ligand-binding groove. The most

conserved residues are located within the groove,

while the outer surface of the molecule is less con-

served [46, 47] (Fig. 1b).

The C-terminal region of plant 14-3-3 proteins

contains a number of important functional elements,

including a conserved nuclear export signal (NES) in-

herent in mammalian, plant, and fungal 14-3-3 pro-

teins [48, 49]. The role of NES in the functioning of

14-3-3 proteins was shown in Schizosaccharomyces

pombe yeast. Mutation in the NES sequence of Rad24

(14-3-3 family protein) abolished the nuclear export

of phosphorylated Cdc25 which normally signals DNA

damage [50]. Similarly, 14-3-3 was found to regulate

the signaling associated with DNA damage through

COP1 (constitutive photomorphogenic  1) protein, a

ubiquitin ligase of p53 protein. In the case of DNA

damage, COP1 undergoes phosphorylation, interacts

with 14-3-3  σ, and is exported from the nucleus [51].

Mutations in the NES of 14-3-3  σ prevented the export

of COP1 induced by DNA damage [51]. It is believed

that the NES sequence is essential for the regulation of

transcription factors by 14-3-3 proteins through their

retention in the cytoplasm (cytoplasmic sequestering)

and blockade of their entry to the nucleus. Thus, the

binding of 14-3-3  ε to the mitosis-regulating protein

Cdc25 from the clawed frog (Xenopus) prevented

Cdc25 from entering the nucleus and resulted in its

cytoplasmic localization [52].

The C-terminus of 14-3-3 is disordered, non-con-

served, and has different length and amino acid se-

quence in different 14-3-3 isoforms and homologs

from different organisms [53]. Based on the secondary

structure prediction by bioinformatics methods and

data of circular dichroism analysis of the C-terminal

peptide of 14-3-3  ω from A.  thaliana, Shen et al. [54]

suggested that the C-terminal region of this protein

might contain a tenth α-helix. The question on the ex-

istence of this additional C-terminal α-helix remains

unresolved, since the tenth α-helix is absent from any

resolved spatial structure of plant 14-3-3.

It was suggested that the C-terminus of mamma-

lian 14-3-3 isoforms has an autoinhibitory function.

Thus, 14-3-3  ζ lacking the C-terminus exhibited a high-

er affinity for its protein partners Raf-1 and Bad [55].

Using the FRET method, Silhan etal. [56] demonstrat-

ed that the C-terminus is located in the amphipathic

groove and is displaced from it upon phosphopeptide

binding [56]. The autoinhibitory function of the C-ter-

minus in plant 14-3-3 proteins has been proposed

in several studies. Shen et al. [54] showed that the

14-3-3  ω isoform truncated at the C-terminus had a

higher inhibitory activity towards its partner protein

nitrate reductase (NR), thus indirectly indicating the

autoinhibitory role of the C-terminus [54]. However,

Athwal etal. [57] found that the C-terminal truncation

of 14-3-3  ω, on the opposite, significantly reduced the

inhibitory capacity of this protein towards NR  [57].

The autoinhibitory role of the C-terminus was also

observed in the study of the plasma membrane

-ATPase (AHA1), a well-known partner protein of

14-3-3. Thus, 14-3-3  ω and ε truncated at the C-termi-

nus activated AHA1 more efficiently than the wild-

type isoforms [58]. Deletion of the C-terminus from

the T14-3c isoform of tobacco (Nicotiana tabacum)

increased the protein affinity to sucrose phosphate

synthase (SPS) [59].

Plant 14-3-3 proteins can bind divalent Ca

and

ions in a region close to the C-terminus. The

binding of calcium and magnesium ions to 14-3-3

and their effect on the protein structure have been

confirmed by various experimental approaches. The

tryptic cleavage patterns of 14-3-3  ω in the presence

and absence of Ca

were different, suggesting confor-

mational rearrangements induced by the binding of

calcium ions [60]. Incubation with radioactive isotope

showed that unlike bovine serum albumin, mem-

brane-immobilized 14-3-3 ω was able to bind Ca

ions

[60]. Equilibrium dialysis experiments demonstrated

that 14-3-3 ω bound calcium at a ratio of one Ca

ion

per one 14-3-3 monomer [60]. The ability of 14-3-3  ω

to bind multiply charged cations was shown using

SEDLOV, SLUCHANKOS8

BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

terbium (Tb

)-induced fluorescence, as the appear-

ance of characteristic fluorescence peaks indicated

binding [61]. The binding of Ca

to the 14-3-3

κ and λ isoforms was demonstrated by thermopho-

resis [62]. It was suggested that the ion-binding site

of 14-3-3 is located in the loop between the helices 8

and 9 [57,  60]. Using manual comparison of sequenc-

es of A. thaliana 14-3-3  ω and calmodulin, Lu et al.

[60] found a similarity between individual amino acid

residues in 14-3-3  ω with the residues in the EF-hand

domain of calmodulin [60]. However, automated bio-

informatics search for A.  thaliana proteins containing

EF-hands performed by Day et al. revealed no EF-hand

domains in 14-3-3 proteins [63]. Using the surface plas-

mon resonance method, it was shown that increasing

concentrations of Ca

and Mg

promoted the binding

of 10 tested 14-3-3 isoforms from A. thaliana (χ, κ, λ,

ν, ω, φ, ψ, υ, ε, and μ) with synthetic phosphopeptides

from the partner proteins nitrate reductase (NR2) and

plasma membrane H

-ATPase (AHA2), which indirectly

indicates that the binding of these divalent ions by

14-3-3 can strengthen its interaction with the partner

proteins [64]. It was also noted that the presence of

and Mg

in the reaction buffer increased the in-

hibitory activity of 14-3-3  ω against NR [57] and its

ability to bind phosphopeptides [61]. On the contrary,

ions decreased the inhibitory function of 14-3-3  λ

and κ toward the partner protein SOS2 kinase [62].

It should be emphasized that at the moment, there

is no direct experimental evidence of divalent cation

binding in the region between the helices 8 and 9 ob-

tained based on the spatial structure of 14-3-3. Also,

since 14-3-3 proteins are acidic, nonspecific binding of

cations cannot be excluded. Therefore, the questions

about specific binding of Ca

and Mg

, as well as

the identity and location of the cation-binding site in

14-3-3 remain unresolved and controversial.

The properties of 14-3-3 dimers have been studied

in most detail for mammalian 14-3-3 isoforms. Thus,

the dissociation constant (Kd) of human 14-3-3  ζ dimer

measured by several methods was found to be ~5  nM

[65], indicating very strong binding. Human 14-3-3 iso-

forms have also been studied for the monomer pref-

erences upon dimerization. It was found that 14-3-3  ε

tends to form heterodimers with other isoforms [66,

67], while 14-3-3  σ preferentially homodimerizes [68,

69]. A possible explanation lies in the number and

representation of certain amino acid residues and

chemical contacts in the region of dimerization. The

14-3-3 σ monomers are linked in the homodimer by

three pairs of salt bridges (three symmetrical contacts:

Arg19–Glu91, Asp21–Lys87, Lys9–Glu83) and several

additional contacts, which provides a high stability of

the homodimer. According to the hypothesis of Yang

et  al.  [67], 14-3-3  ε monomers form only one pair

of salt bridges in the homodimer (two symmetrical

contacts of Arg19 of one monomer and Glu92 of the

other monomer), while heterodimerization with other

14-3-3 isoforms involves formation of an additional

salt bridge in 14-3-3  ε [e.g., Glu92(ε)–Arg18(ζ) upon in-

teraction with 14-3-3 ζ], which makes 14-3-3 ε heterod-

imerization more preferable [67, 69]. Interestingly, in

some cases, 14-3-3 heterodimers can bind to partner

proteins, as established from the spatial structure of

the human PEAK3 pseudokinase complex with the

14-3-3  ε/β heterodimer [70]. Heterodimers containing

human 14-3-3  ε can efficiently form in vitro under na-

tive conditions via exchange of different homodimer

subunits [71, 72], but the functions of such heterodi-

mers in vivo remain a subject of debate.

Plant 14-3-3 are likely capable of heterodimeriza-

tion as well. 14-3-3 isoforms from A. thaliana were

found to form χ  +  φ/ω/ψ, ω  +  φ/υ/ψ, φ  +  υ/ψ, and ψ  +  υ

heterodimers [73]; the ω isoform was shown to het-

erodimerize with κ and λ isoforms [74]. However, in

the experiments on subunit heterodimerization, the

mixed 14-3-3 isoforms were first denatured in the

presence of guanidine chloride [73] or deoxycholate

[74] and then subjected to renaturation, followed by

analysis of the homo- and heterodimers formed. These

experimental conditions are very far from the native

ones and only indirectly suggested the possibility of

formation of certain dimers in plant cells. Yet, there

is evidence that 14-3-3 heterodimerization can occur

in vivo, although the isoforms involved in this process

remain unknown [73,  75,  76]. It is important to note

that heterodimerization of plant 14-3-3 isoforms has

been studied only for proteins from the non-epsilon

phylogenetic group, while the data on heterodimeriza-

tion of epsilon group isoforms are lacking. At the same

time, an interesting information was obtained by us-

ing the yeast two-hybrid method to study pairwise

dimerization of six 14-3-3 isoforms from the cotton

plant (Gossypium hirsutum), three of which were from

the epsilon group and three – from the non-epsilon

group. The studied isoforms formed only certain het-

erodimers (Gh14-3-3L and Gh14-3-3e, Gh14-3-3L and

Gh14-3-3g, Gh14-3-3a and Gh14-3-3e, Gh14-3-3a and

Gh14-3-3g, Gh14-3-3g and Gh14-3-3h), but not homod-

imers [77]. Verification of these results by direct bio-

chemical methods will be of great interest.

14-3-3 proteins can be phosphorylated, and this

modification can influence dimer dissociation. Phos-

phorylation of Ser58 in the dimer interface of human

14-3-3  ζ resulted in dimer destabilization and dissoci-

ation [78]. In plant 14-3-3 proteins, residues homolo-

gous to mammalian Ser58 can also be phosphorylated

in vivo. The phosphomimetic modification of homol-

ogous Ser62 residue in 14-3-3  ω facilitated dimer

dissociation [79,  80]; this effect was even more pro-

nounced upon introduction of two phosphomimetic

substitutions simultaneously (at Ser62 and Ser67) [79].

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BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

Fig.  2. Interaction of 14-3-3 proteins with phosphopeptides. a) Binding of phosphopeptide from BZR1 transcription factor

(motif II) in the amphipathic groove of 14-3-3 ω from A. thaliana (PDB ID: 8QTF [26]). Amino acid residues forming polar

contacts (black dashed line) with the phosphate group and peptide backbone are shown. b) Interaction of 14-3-3 with the

BZR1 phosphopeptide in the absence of fusicoccin (motifII; PDBID: 8QTF [26]); orange, phosphopeptide; red, phosphoser-

ine residue. c) Interaction of 14-3-3 with the PMA2a phosphopeptide and fusicoccin (motif III; PDB ID: 1O9F [88]); blue,

phosphopeptide; red, phosphothreonine residue; pink, fusicoccin molecule. Images were created with the PyMol program.

The phosphomimetic substitution of Ser62 in 14-3-3  ω

disrupted protein heterodimerization preferences and

decreased 14-3-3 affinity for its ligands (peptide difo-

pein and N-terminal fragment of NR) [74]. Interest-

ingly, phosphorylation of 14-3-3  χ at Ser72 (analog of

Ser67 in 14-3-3 ω) decreased its inhibitory activity to-

ward NR [81]. Phosphorylation of residues homologous

to Ser58 in plant 14-3-3 proteins was found to yield

dissociation of calcium-dependent protein kinase  3

(CPK3) from 14-3-3 and the kinase degradation [82].

Therefore, phosphorylation of 14-3-3 proteins at the

dimer interface can significantly alter the biological

functions of these proteins by shifting the dimer/mono-

mer equilibrium. Plant 14-3-3 proteins contain other

phosphorylation sites [46, 83], but the consequences

of their modification and its effect on the structure

and functions of 14-3-3 proteins have been studied

insufficiently.

Principles of 14-3-3 binding to partner proteins.

The binding partners of 14-3-3 proteins are typical-

ly phosphorylated at residues located in structurally

disordered regions [84]. Based on numerous experi-

mental data accumulated through the years of studies,

it was found that 14-3-3 proteins bind phosphoserine

(pS) or phosphothreonine (pT) residues in a specif-

ic amino acid context (consensus motif), and only

in very rare cases interact with nonphosphorylated

partners [85]. There are three types of 14-3-3-bind-

ing phosphorylated motifs: type I – RXXp(S/T)X(P/G),

type II – RX(Y/F)Xp(S/T)X(P/G), and type III (C-termi-

nal)– p(S/T)X

0-2

–COOH, where p(S/T) is phosphoserine/

phosphothreonine and X is any amino acid residue

[48, 86] (Fig.1c). There are data indicating that motifI

in 14-3-3 partner proteins in plants can be slightly dif-

ferent. According to Johnson etal. [87], who conducted

bioinformatics analysis of 14-3-3 recognition sequences

in partner proteins, in many cases, plant motif I is

extended at the N-terminus: LX(R/K)SX(pS/pT)XP [87].

The authors associated this feature with the fact that

many light-dependent processes in plants are regulat-

ed by specific protein kinases that require the pres-

ence of a leucine residue in the beginning of motif I.

However, the number of analyzed 14-3-3 recognition

sites in plants was much smaller than for mammali-

an proteins (14 plant sequences vs. 201 mammalian

sequences) [87], so that the validity of extrapolating

these findings to all partners of plant 14-3-3 proteins

is questionable.

The phosphopeptide binds along its entire length

in the amphipathic groove via polar contacts and hy-

drophobic interactions. The main residues of 14-3-3

that form polar contacts with the phosphopeptide

SEDLOV, SLUCHANKOS10

BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

backbone and phosphate group are Lys53, Arg60,

Arg64, Arg133, Tyr134, Asn179, Asn230, and Trp234,

which are highly conserved in plant and animal 14-3-3

proteins [88] (Fig. 2a; residue numbering for Arabi-

dopsis 14-3-3 ω).

14-3-3 isoforms have different affinity for the

partner protein phosphopeptides. Assessment of all 7

human 14-3-3 isoforms for their affinities to phospho-

peptides derived from CTFR  [89], USP8  [90], LRRK2

[91], etc., has shown that γ and η had the highest af-

finities, while σ and ε had the lowest affinities, and

the remaining isoforms had intermediate affinities

for the tested phosphopeptides. The affinity of 14-3-3

isoforms for the phosphopeptides from RSK1, HSPB6,

and papillomavirus E6 protein decreased in the fol-

lowing order: γ>η>ζ>τ>β>ε>σ. The same hierarchy

in the 14-3-3 isoform affinity was observed for large

and diverse samples of phosphopeptides from various

partner proteins [92]. It is quite likely that plant 14-3-3

proteins display a similar hierarchy of affinities for

phosphorylated peptides.

Using the surface plasmon resonance method,

it was demonstrated that 9 (out of 13 known) 14-3-3

isoforms from A. thaliana exhibited different affinities

for the phosphorylated C-terminal peptide of the plas-

ma membrane proton ATPase [47] that decreased in

the following order: φ>χ>ν>ψ>υ>ε>ω>κ>λ. However,

these data contradict the results obtained in the study

on the effect of A. thaliana 14-3-3 isoforms (7 out 13)

on the activity of the same plasma membrane ATPase

[58], which decreased in the order: χ>ω>κ>λ>μ>ε>ο.

The differences in the activity of several 14-3-3 iso-

forms from A. thaliana toward spinach leaf NR was

demonstrated in [93]; in this case, the effect decreased

in the following order: ω>φ>χ>ν [93]. According to

Lambeck et al. [94], who studied the influence of

14-3-3 isoforms from A. thaliana on the NR activity,

the inhibitory effect decreased in the following order:

ω>λ>κ>ν>ψ>χ≫ε (the effect of 14-3-3 ε was so insig-

nificant that the authors were unable to determine

the half-inhibition constant) [94]. In a recent study,

7 isoforms of A. thaliana were examined (μ, ε, and ο

from the epsilon group; λ, ν, ψ, and φ from the non-ep-

silon group) for their binding with the phosphopeptide

from the FD protein (transcription factor and compo-

nent of the florigen activation complex that triggers

flowering). According to the data obtained, the affinity

decreased as follows: ψ>μ>λ>ν>φ>ο>ε [95]. Based on

the above, the data on the affinity of 14-3-3 proteins

to the phosphopeptides of partner proteins are scarce

and contradictory. No correlation was found between

the affinity of a 14-3-3 isoform to the partner proteins

and isoform attribution to the epsilon or non-epsilon

group. Apparently, more systematic studies using all

rather than a few randomly selected isoforms are

required to make confident statements about the ex-

istence and nature of the hierarchy of the binding

affinity of plant 14-3-3 isoforms. It should be noted

that even though such hierarchy has been proven for

7 relatively conserved human 14-3-3 isoforms [92], its

nature remains unclear, especially given that all 7 iso-

forms have the same structure of the phosphorylated

residue-binding pocket in the amphipathic groove. This

suggests that the binding strength may be influenced

by currently unknown allosteric or long-range effects.

In addition to interactions at the primary binding

site located in the amphipathic groove, the partner

proteins can form contacts with other 14-3-3 regions,

as it was observed for the florigen activating com-

plex Hd3a [96] (vide infra). Since the outer regions of

14-3-3 dimers are the least conserved, their interac-

tions may be specific to particular isoforms.

MAIN FUNCTIONS

OF PLANT 14-3-3 PROTEINS

General principles of functioning of 14-3-3

proteins. Similar to their animal and fungal ortho-

logs, plant 14-3-3 proteins lack enzymatic activity and

function by directly interacting with phosphorylated

partner proteins. As a rule, this interaction alters the

activity of the partner protein, thus affecting its bi-

ological function. The mechanisms leading to such

changes can be divided into the following categories.

Enzyme inhibition. In some cases, the binding

of 14-3-3 protein can lead to the enzyme inhibition.

Aclassic example is NR, whose binding to 14-3-3 pre-

sumably causes conformational changes leading to

the suppression of its enzymatic activity [97] (Fig.3a).

Interaction of 14-3-3 with SPS also results in the en-

zyme inhibition [98]. Unfortunately, the lack of struc-

tural data makes it difficult to decipher the mecha-

nism behind the inhibitory effect of 14-3-3.

Enzyme activation. The binding of 14-3-3 to an en-

zyme can also lead to the increase in its catalytic activ-

ity. Thus, the binding of 14-3-3 to the plant H

-ATPase

PMA2 results in the removal of the autoinhibitory se-

quence from the catalytic site and enzyme activation

[58]. The spatial structure of the 14-3-3 complex with a

fragment of the plasma membrane ATPase was one of

the first resolved 3D structures. It was found that the

binding of three 14-3-3 dimers induced the formation

of ATPase hexamers  [99] (Fig.  3b). The interaction of

14-3-3 with the cytosolic enzyme glutamine synthase

(GS1) also promoted enzyme activation [100].

Changes in protein location. The binding of 14-3-3

to a partner protein can alter the intracellular loca-

tion of the latter. Thus, the interaction between phos-

phorylated transcription factors BZR1 (BRASSINAZOLE

RESISTANT  1) and BES1 (BRI1 EMS SUPPRESSOR  1)

involved in the intracellular signaling mediated by

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BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

Fig.  3. Regulation of functional activity of partner proteins by plant 14-3-3 proteins. a)  NR inhibition by 14-3-3 binding.

b)  Activation of plasma membrane H

-ATPase PMA2 by 14-3-3 binding. c)  Changes in the intracellular location (cytoplas-

mic retention) of the transcription factor BZR1 mediated by 14-3-3 proteins. d)  Import of the precursor of Rubisco small

subunit (preSSU) into chloroplasts with the assistance of 14-3-3 and HSP70. e)  Phosphorylation and activation of protein

kinase MAPKKK5 by protein kinase PBL19 in complex with 14-3-3. f) Inhibition of degradation of the transcription factor

WRI after binding to 14-3-3.

brassinosteroids (plant hormones) prevents the entry

of these factors to the nucleus and further regulation

of gene expression [101, 102] (Fig. 3c). Other tran-

scription factors, such as RSG (Repression of Shoot

Growth)  [103] and PIF7 (Phytochrome-Interacting

Factor  7)  [104], also appear to be regulated through

cytoplasmic retention.

Protein transport into chloroplasts and mitochon-

dria. Participation of 14-3-3 in protein transport into

chloroplasts has been suggested already in the early

studies. It was shown that a complex of 14-3-3 with

HSP70 promoted the import of Rubisco small subunit

precursor (preSSU) to chloroplasts [105] (Fig.3d). Also,

14-3-3 was found to interact with the leader sequence

of Arabidopsis photosystem I N-subunit (PSI-N) [106].

According to [107], 14-3-3 promoted the import of at

least 11 chloroplast proteins into chloroplasts. 14-3-3

proteins also participate in protein transport into mi-

tochondria. It is known that the signal sequences of

many proteins imported into mitochondria are phos-

phorylated [108]. In this case, the role of 14-3-3 might

be negative: the binding of MORF3 (Multiple Organel-

lar RNA editing Factor) phosphorylated at the signal

sequence with a complex of 14-3-3 and cytosolic HSP70

slowed down the import of MORF3 into mitochondria

[109]. The details of mechanisms by which 14-3-3 af-

fects the transport of proteins into double-membrane

organelles remain unknown.

Assembly of active protein complexes. 14-3-3 pro-

teins participate in the assembly of functional com-

plexes, e.g., the transcriptional complex with VP1

(Viviparus1) and EmBP1 (Em promoter binding pro-

tein) that activates expression of the Em gene [110].

Formation of complexes with 14-3-3 isoforms can lead

to the protein activation, as was demonstrated for the

activation of MAPKKK5 by a complex of PBL19 kinase

with 14-3-3  λ [111] (Fig. 3e). 14-3-3  κ and λ interact

with the transcription factor PIF3 (Phytochrome-

Interacting Factor  3) and photoactivated phytochrome

phyB and stabilize the complex of these two proteins

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BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

Fig.  4. Mechanisms by which animal 14-3-3 proteins regulate the functions of partner proteins (no similar activities have

been identified for plant 14-3-3 proteins). a)  Protection of PI4KIIIβ from dephosphorylation by 14-3-3  τ binding. b)  Disaggre-

gation of apocytochromec by the chaperone-like activity of 14-3-3  ζ. c)Liquid–liquid phase separation (LLPS) after 14-3-3  ζ

binding to the wild-type tau protein.

in thenucleus, which ultimately results in PIF3 degra-

dation and initiation of photomorphogenesis process-

es [112].

Effect on protein half-life. 14-3-3 proteins can mod-

ulate stability of partner proteins by influencing their

susceptibility to intracellular degradation. For exam-

ple, the binding of 14-3-3 to the key enzyme of eth-

ylene biosynthesis 1-aminocyclopropane-1-carboxyl-

ate synthase (ACS) increases stability of this enzyme,

while 14-3-3 binding to the components of E3 ubiqui-

tin ligase ETO1/EOL, on the contrary, promotes deg-

radation of these proteins [113]. Associated with the

transcription factor WRI1, 14-3-3 prevents its degra-

dation by shielding the binding site for E3 ubiquitin

ligase [114] (Fig. 3f).

Suppression of dephosphorylation. The binding

of 14-3-3 can prevent dephosphorylation of partner

proteins by shielding the phosphate group from phos-

phatases, as demonstrated in studies on human 14-3-3

isoforms. For example, the interaction of 14-3-3 τ with

phosphatidylinositol 4-kinase IIIβ (PI4KIIIβ) increased

the content of phosphorylated active PI4KIIIβ [115]

(Fig.  4a). The binding of 14-3-3 prevented dephos-

phorylation and degradation of the transcription fac-

tor FoxO3 involved in the control of cell proliferation

and apoptosis  [116]. No inhibitors of dephosphoryla-

tion have been found among plant 14-3-3 proteins yet;

however, given their structural and functional similar-

ity to mammalian 14-3-3 proteins, this function can be

expected in plant proteins as well.

Chaperone-like activity. Mammalian 14-3-3 pro-

teins exhibit chaperone-like activity, i.e., prevent ag-

gregation of substrate proteins in an ATP-independent

manner. Mammalian 14-3-3  ζ was found to prevent

aggregation of insulin, alcohol dehydrogenase, and

phosphorylase kinase in  vitro  [117]; 14-3-3 ζ from

Drosophila prevented aggregation of citrate synthase,

as well as prevented aggregation and even promoted

disaggregation of apocytochrome  c aggregates in vivo

[118] (Fig. 4b). Since this activity does not depend on

phosphorylation of partner proteins, it can be consid-

ered as a “moonlighting” function of 14-3-3 [53]. There

are reasons to believe that plant 14-3-3 proteins also

possess chaperone-like activity, especially considering

a potentially greater susceptibility of plant cells to en-

vironmental changes (e.g., in ambient temperature).

Regulation of liquid-liquid phase separation (LLPS).

Recent studies have demonstrated that mammalian

14-3-3 proteins can modulate LLPS by interacting

with partner proteins. Thus, it was shown that 14-3-3  ζ

binds to unmodified tau protein, promoting formation

of droplets of the tau protein phase during LLPS and

stabilizing them [119] (Fig.4c). It cannot be ruled out

that plant 14-3-3 proteins perform a similar function

in LLPS in plants.

14-3-3 proteins as regulators of plant metabo-

lism. In plants, 14-3-3 proteins regulate primary me-

tabolism by interacting with and controlling the ac-

tivity of key enzymes of nitrogen, carbohydrate, and

sulfur metabolism.

Regulation of nitrogen metabolism. The most stud-

ied protein partner of 14-3-3 in plants is NR, an enzyme

involved in nitrogen metabolism. It is a cytoplasmic

protein that catalyzes the first step of nitrogen assim-

ilation in plants, namely, NADH-dependent conversion

of nitrate to nitrite (Fig.5). Nitrite is reduced in chlo-

roplasts to ammonium ion, which is then incorporat-

ed into amino acids [120, 121]. NR is a large protein;

the molecular weight of its dimer is ~200 kDa [122].

The N-terminal domain of NR contains molybdenum

as a cofactor and is followed by the cofactor-free di-

merization domain, central domain with the heme b5,

and C-terminal domain that contains bound FAD and

has the NADH-binding site [122] (Fig.  5). Because of

its important biological role and dependence on reduc-

ing equivalents (which are actively supplied during the

daytime), NR is tightly regulated by many factors, in-

cluding 14-3-3 proteins [121]. The enzyme is phosphor-

ylated at a serine residue located approximately in

the middle of its polypeptide chain (Ser543 in spinach

NR). The sequence containing this residue corresponds

to the 14-3-3 recognition motif I [61]. Indeed, this mo-

tif is recognized by 14-3-3 proteins [61], leading to NR

inhibition [93, 94, 123]. The binding of 14-3-3 blocks

WORLD OF PLANT 14-3-3 PROTEINS S13

BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

Fig.  5. Regulation of nitrate reductase (NR) activity by 14-3-3 proteins. Illumination induces formation of reducing and

energy equivalents via photosynthesis. Active NR reduces nitrate ions to nitrites in the cytoplasm. During catalysis, elec-

trons are transferred from NADH to the FAD-containing domain (FAD) and then to the heme  b5-containing domain (b5)

and molybdenum-containing domain (Mo), where nitrate is reduced to nitrite. Nitrite is reduced to ammonium, which is

then incorporated into organic compounds (e.g., amino acids). In the dark, the amount of reducing equivalents decreases

and nitrate reduction stops, so NR should be inhibited. A decrease in the ATP/ADP ratio leads to the SnRK1 activation and

phosphorylation of NR at the 14-3-3 recognition motif located between the Mo and b5 domains (at Ser543 in spinach NR).

The binding of 14-3-3 to this site causes conformational changes in the enzyme, resulting in the spatial separation of the

Mo and b5 domains, which hinders electron transfer between them and leads to NR inactivation.

electron transfer from the heme to the molybdenum

cofactor [97]. Apparently, the binding of 14-3-3 in the

region between the two cofactors causes conforma-

tional rearrangements that move the cofactors away

from each other and make them to adopt a confor-

mation that hinders electron transport [97] (Fig. 5).

Chi et al. [124] provided a more detailed explanation

of this proposed mechanism [124]. They showed that

in NR from A.  thaliana, 14-3-3 binds not only to motif  I

in the proximity of phosphorylated serine, but also to

the conserved acidic N-terminal motif and that com-

plete inhibition of NR requires simultaneous 14-3-3

binding to both sites [124]. Interestingly, the binding

to the acidic N-terminal motif probably occurs outside

the phosphopeptide-binding groove of 14-3-3 [124].

The key serine residue in the 14-3-3-recognizing site

of NR is phosphorylated by the protein kinase SnRK1

(SNF1-related kinase  1) [125]. This enzyme is known to

suppress anabolic and to activate catabolic processes

upon the deficit of energy equivalents in cells [126,

127]. Thus, phosphorylation of NR by SnRK1 under the

energy deficit conditions (in the dark) promotes the

binding of 14-3-3 and conformational rearrangements

leading to the enzyme inhibition. Despite an overall el-

egance of the proposed mechanism, its details remain

poorly understood due to the lack of information on

the structure of the NR complex with 14-3-3 protein.

Cytosolic GS1, a downstream enzyme in the nitro-

gen assimilation pathway, is also regulated by 14-3-3

proteins. GS1 converts glutamic acid to glutamine, re-

sulting in the ammonium incorporation into organic

compounds (Fig. 6a). Finnemann et al. [100] showed

that the binding of 14-3-3 to phosphorylated GS1 in-

creased its catalytic activity in aging rapeseed (Bras-

sica napus) leaves [100]. The phosphorylated residue

in GS1, as well as kinase and phosphatase involved in

the enzyme regulation, remain unknown. Activation

of GS1 in aging leaves is necessary for the nitrogen re-

moval from tissues in the form of organic compounds

[100]. It was also shown that 14-3-3 interacts with

phosphorylated GS1 from Chlamydomonas reinhardtii

[128]. In this case, GS1 is presumably phosphorylat-

ed by the Ca

/calmodulin-dependent protein kinase,

however, neither phosphorylation, nor the binding

of 14-3-3 had any effect on the GS1 activity [128].

Glutamine synthetase GS2 is a GS isoform located in

chloroplasts. Western blotting of native GS2 oligomers

isolated from tomato chloroplasts demonstrated that

SEDLOV, SLUCHANKOS14

BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

a b

Fig. 6. Hypothetical scheme of plant metabolism regulation by 14-3-3 proteins. The scheme is based on the most reliable

data (in the opinion of the authors of this review) on the effects of 14-3-3 on the functional activity of partner proteins.

a) Regulation of nitrogen metabolism. b) Regulation of carbohydrate metabolism. Designations: Glc, glucose; Fru, fructose;

UDP-Glc, UDP-glucose; Fru-6-P, fructose 6-phosphate; Sucrose-1-P, sucrose-1-phosphate; GA-3-P, glyceraldehyde 3-phosphate;

3-PGA, 3-phosphoglycerate; 1,3-PGA, 1,3-bisphosphoglycerate; NR, nitrate reductase; GS1, glutamine synthetase 1; SPS, su-

crose-phosphate synthase; CINV1, alkaline invertase 1; GAPN, non-phosphorylating glyceraldehyde 3-phosphate dehydro-

genase; GAPC1, cytosolic glyceraldehyde 3-phosphate dehydrogenase 1.

enzymatically active GS2 octamer contained 14-3-3

[129]. It was suggested that dissociation of 14-3-3 from

the complex results in the loss of GS2 activity [129].

This is an unusual finding, since 14-3-3 typically lo-

calizes to the cytosol or the nucleus. The question of

14-3-3 presence in chloroplasts requires further inves-

tigation. A detailed study on the effect of glutamine

synthetase phosphorylation at various residues on its

interaction with 14-3-3 and enzymatic activity in vitro

will provide better understanding of the regulation of

this important enzyme.

Regulation of carbohydrate metabolism. 14-3-3 pro-

teins regulate carbohydrate metabolism at various lev-

els. Several interactome studies have identified dozens

of carbohydrate metabolism enzymes interacting with

14-3-3 proteins [130-133].

SPS catalyzes conversion of fructose-6-phosphate

and UDP-glucose into sucrose-1-phosphate, an imme-

diate precursor of sucrose (Fig.6b). SPS from spinach

(Spinacia oleracea) is regulated by reversible phos-

phorylation at Ser158 that depends on the time of day

and illumination [134]. In the dark, when there is no

photosynthesis and the flow of metabolites decreas-

es, SPS is phosphorylated to inhibit its activity. Expo-

sure to light induces enzyme dephosphorylation and

its activation [134, 135]. It was shown that SPS, like

NR, is phosphorylated by SnRK1 (metabolic regulator

that inhibits anabolic reactions) at Ser158 [125]. Phos-

phorylated SPS binds to 14-3-3 [98, 136]. Using surface

plasmon resonance, Toroser et al. [98] showed that

14-3-3 binds spinach SPS at Ser229 residue. However,

further analysis of 14-3-3 affinity to phosphopeptides

demonstrated that none of the 7 examined A. thaliana

14-3-3 isoforms bound A.  thaliana SPS phosphopeptide

homologous to the Ser229-containing motif from the

spinach SPS; 14-3-3 instead interacted with A. thaliana

SPS phosphopeptide homologous to the Ser158-con-

taining fragment of the spinach enzyme [58]. The stud-

ies using site-directed mutagenesis and yeast two-hy-

brid assay failed to confirm the binding of 14-3-3 to

tobacco SPS at the phosphorylated site homologous to

Ser229 in spinach SPS [59]. Moreover, Ser229 is less

conserved among SPS enzymes from different spe-

cies than Ser158 [137]. These data cast doubts on the

identity of 14-3-3-binding site characterized by Toroser

et al. [98]. It was also shown that 14-3-3 binding

WORLD OF PLANT 14-3-3 PROTEINS S15

BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

prevents SPS degradation in vitro [138]. The data on

the effect of 14-3-3 on the SPS activity are controver-

sial. According to Toroser et al., 14-3-3 binding inhib-

ited SPS [98]. However, Moorhead et al. [136] showed

that the phosphopeptide from Raf-1 (mammalian

partner protein of 14-3-3) exhibited a weak inhibito-

ry effect toward SPS, which may indicate activation of

SPS by 14-3-3 proteins [136]. Unfortunately, conflicting

data and lack of information on the structure of 14-3-3

complexes with SPS raise many questions that require

further research.

Alkaline/neutral invertase CINV1 catalyzes the

breakdown of sucrose to glucose and fructose (Fig.6b).

Using a yeast two-hybrid system, it was demonstrated

that CINV1 from A. thaliana binds to 14-3-3 isoforms ε,

κ, λ, υ, φ, χ, ψ, and ω, but not to μ [139]. The binding

is mediated by Ser547, which can be phosphorylat-

ed by calcium-dependent protein kinases [139], and

results in CINV1 activation [139]. Unfortunately, the

information on the structure of CINV1 complexes with

14-3-3 is lacking.

Another carbohydrate metabolism enzyme inter-

acting with 14-3-3 proteins is non-phosphorylating

glyceraldehyde 3-phosphate dehydrogenase (GAPN)

from wheat [140]. This enzyme catalyzes irreversible

oxidation of glyceraldehyde 3-phosphate into 3-phos-

phoglycerate with the reduction of NADP

(Fig. 6b).

Itis believed that GAPN is especially important in pro-

viding non-photosynthetic tissues with NADPH [140,

141]. The studies on the effect of phosphopeptides on

the activity of GAPN complex with 14-3-3 revealed

that the interaction between these two proteins caus-

es enzyme inhibition and a 3-fold decrease in the

maximum rate of the GAPN-catalyzed reaction [140].

However, both the 14-3-3 recognition site and the ki-

nase that phosphorylates it remain unknown.

14-3-3 proteins also interact with the cytosolic

glyceraldehyde 3-phosphate dehydrogenase (GAPC1)

from A.  thaliana [138] (Fig. 6b). Most likely, the res-

idue involved in the 14-3-3 binding is Ser126 in the

KVVIpSEP sequence, which is similar to the type  II

recognition motif [58, 142], but the effect of this in-

teraction remains unknown.

Plant 14-3-3 proteins can bind to and influence

the functions of proteins regulating carbohydrate

metabolism. Thus, 14-3-3 has been shown to bind

A.  thaliana trehalose phosphate synthase TPS5 [143],

which catalyzes formation of trehalose 6-phosphate,

a signaling molecule regulating carbohydrate metabo-

lism. The binding is mediated by Ser22 and Thr49 res-

idues located close to the protein N-terminus. In both

cases, the recognition motifs for 14-3-3 are similar to

type motifs, but differ from the canonical ones by

theabsence of proline residue at position  +2  [58,  143].

In  vitro, these residues are phosphorylated by SnRK1

[143]. Another important regulatory enzyme of car-

bohydrate metabolism, 6-phosphofructo-2-kinase/fruc-

tose-2,6-bisphosphatase (F2KP), also interacts with

14-3-3. F2KP synthesizes fructose-2,6-bisphosphate, a

signaling metabolite which is believed to direct sug-

ar metabolism toward the synthesis of either sucrose

or starch [144]. Using pull-down assay, Kulma et al.

[145] showed that A. thaliana F2KP specifically bound

14-3-3 in cell extracts, presumably through Ser220 and

Ser303 residues [145]. However, no significant effect

of 14-3-3 on the F2KP activity has been found in vitro,

which might be due to poorly designed experimental

conditions [145].

Many 14-3-3 interaction partners involved in

glycolysis and other carbohydrate metabolism re-

actions have been identified in interactome studies.

These include phosphoenolpyruvate carboxylase, glu-

cose-6-phosphate dehydrogenase [133], phosphoglycer-

ate isomerase, pyruvate kinase, transketolase, sucrose

phosphate synthase [130], triosephosphate isomerase,

enolase, and fructose-bisphosphate aldolase [132].

Unfortunately, this information has not yet been con-

firmed by direct studies of the interaction of these

enzymes with 14-3-3 proteins, which could be an in-

teresting area of future research.

Regulation of sulfur metabolism. Shin et al. [146]

identified a number of enzymes of the sulfur assimi-

lation pathway as 14-3-3 partners [146]. Among them,

two enzymes of cysteine synthesis, o-acetylserine (thiol)

lyase (OASTL) and serine acetyltransferase (SAT), have

been investigated in most detail, and their interaction

with 14-3-3 χ was confirmed by coimmunoprecipita-

tion [146]. However, the details of 14-3-3 interaction

with sulfur metabolism proteins remain enigmatic.

Therefore, there is a large body of evidence indi-

cating that 14-3-3 proteins are involved in the regu-

lation of key enzymes of nitrogen, carbohydrate, and

sulfur metabolism in plants. However, a clear under-

standing of the global role of 14-3-3 in these important

processes is lacking. Moreover, structural and func-

tional details of 14-3-3 interaction with metabolic en-

zymes remain unknown, and reported experimental

data are often contradictory.

Plant 14-3-3 proteins as regulators of mem-

brane transport. 14-3-3 are soluble cytosolic proteins;

however, they can regulate the activity of membrane

transporters and enzymes by binding to them from

the cytosolic side. In a number of studies, plant 14-3-3

proteins have been found in the proximity of the plas-

ma membrane [37, 76, 81, 147].

Regultion of trnsport cross the plsm mem-

brne. One of the most well-characterized protein-pro-

tein interactions of plant 14-3-3 proteins is binding

to the plasma membrane H

-ATPase (PMA), or AHA

(Arbidopsis H

-ATPase/autoinhibited H

-ATPase). PMA

belongs to the family of P-type cation-transporting

ATPases and transfers protons from the cell to the

SEDLOV, SLUCHANKOS16

BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

extracellular medium with the expenditure of energy.

It is responsible for the formation of the transmem-

brane proton gradient used as an energy source for

the secondary active transport in plant cells [148].

The C-terminus of PMA contains the autoinhibitory

sequence [149]. PMA binds 14-3-3 at the phosphor-

ylated C-terminal motif YpTV-COOH (type III motif).

The residue that undergoes phosphorylation in PMA2

from Nicotiana plumbaginifolia is Thr948 [150, 151].

This C-terminal motif is a part of the autoinhibitory

domain [152]. The binding of 14-3-3 to PMA abolish-

es autoinhibition and activates the enzyme [58, 151]

(Fig.3b). Activated PMA oligomerizes with the forma-

tion of the active complex containing six PMA subunits

and three 14-3-3 molecules, as was shown by a com-

bination of X-ray crystallography and cryo-electron

microscopy [99] (Fig.3b; Fig. 7a). The activity of PMA

provides a decrease in the apoplast pH and mainte-

nance of the transmembrane proton gradient, which is

necessary for normal plant growth and development,

e.g., elongation of hypocotyl and roots [153], activity of

stomatal cells [99, 154], and pollen tube growth [155].

The binding between 14-3-3 and PMA is a target of

the pathogenic ascomycete fungus Phomopsis amygda-

li (Fusicoccum amygdali). This fungus synthesizes and

secretes the toxin fusicoccin, which greatly increases

the affinity of 14-3-3 for the typeIII motif in PMA by

occupying a free space in the amphipathic groove of

the 14-3-3 complex with the short C-terminal phospho-

peptide [88] (Fig. 2, b and c). Fusicoccin itself binds

rather weakly to 14-3-3 (dissociation constant Kd,

~66 μM), and simultaneous presence of the phospho-

peptide is important for increasing its binding strength

[88, 156]. The mechanism of action of fusicoccin was

elucidated after resolving the spatial structure of its

complex with 14-3-3 and PMA2 phosphopeptide. This

discovery has initiated an entire new direction in the

studies of 14-3-3 proteins. Currently, the structures of

plant 14-3-3 proteins most represented in the Protein

Data Bank are those of N.  tabacum 14-3-3 complexes

with fragments of N.  plumbaginifolia PMA2 (7 out of

19 structures: 3 complexes with the phosphopeptide

[88, 157] and 4 complexes with a longer C-terminal

fragment of PMA2 [99,  158,  159]) (Table  3). PMA acti-

vation leads to membrane hyperpolarization, opening

of stomata, and uncontrolled transpiration [160]. The

inability to regulate water exchange leads to a patho-

logical phenotype with yellowing and drying leaves

[160]. Notably, one of the first names of plant 14-3-3

was fusicoccin-binding protein (FCBP) due to the dis-

covery of 14-3-3 in the fusicoccin-containing mem-

brane-bound protein complex with PMA [8, 161-163].

Later, it has become clear that fusicoccin stabilizes

not only the binding between 14-3-3 and PMA, but

also other interactions of 14-3-3 with plant and animal

partner proteins through the typeIII motifs [164, 165].

Structural studies have identified a number of small

molecules that can stabilize the interaction of 14-3-3

and PMA2, including cotylenin A [157], pyrrolinone

derivative pyrrolidone 1 [158], dipeptide epibesta-

tin [158], and pyrazole derivative [159]. Analysis of

the obtained spatial structures of protein complexes

with these compounds revealed that the sites of their

binding to 14-3-3 were similar to that of fusicoccin

(Table 3).

Despite generally recognized function of fusicoc-

cin as a PMA activator, there is a growing body of

evidence that this small molecule has other physiolog-

ical effects, such as regulation of potassium ion cur-

rents, enzymes, phytohormones, and protein kinase

Fig.  7. Spatial structures of 14-3-3 complexes with partner

proteins. a) Complex of 14-3-3 isoform C from N. tabacum

(green) with two C-terminal fragments of PMA2 from

N. plumbaginifolia (blue and light blue) and fusicoccin

(red) (PDB ID: 2O98 [99]). b) Complex of 14-3-3 GF14C iso-

form from O.  sativa (green) with florigen Hd3a (orange)

and phosphopeptide from OsFD1 transcription factor (pink,

phosphoserine residue is shown in red) (PDBID: 3AXY) [96].

Images were created with PyMol.

WORLD OF PLANT 14-3-3 PROTEINS S17

BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

Table 3. Spatial structures* of complexes formed by plant 14-3-3 proteins (in reverse chronological order)

PDB ID 14-3-3 protein Partner protein or ligand

Recognition

motif type

Year

released

References

8QTC A. thaliana 14-3-3 ω

phosphopeptide from A. thaliana

transcription factor BZR1

2023 [26]

8QTF A. thaliana 14-3-3 ω

phosphopeptide from A. thaliana

transcription factor BZR1

8QT5 A. thaliana 14-3-3 λ

phosphopeptide from A. thaliana

transcription factor BZR1

8QTT A. thaliana 14-3-3 ω

phosphopeptide from A. thaliana BRI1

kinase inhibitor

–

8HEW S. tuberosum St14f phosphopeptide from S. tuberosum StFDL1 I 2023 [182]

7XBQ S. tuberosum St14f – – 2022 [183]

5NWI N. tabacum 14-3-3 C

C-terminal peptide from A. thaliana KAT1

potassium channel

III 2017 [165]5NWJ N. tabacum 14-3-3 C

C-terminal peptide from A. thaliana KAT1

potassium channel

5NWK N. tabacum 14-3-3 C

C-terminal peptide from A. thaliana KAT1

potassium channel and fusicoccin

4DX0 N. tabacum 14-3-3 E

C-terminal peptide from N. plumbaginifolia

ATPase PMA2 and pyrazole derivative

III 2012 [159]

3AXY O. sativa GF14-C

O. sativa florigen Hd3a, peptide from

O. sativa transcription factor OsFD1

I 2011 [96]

3M51 N. tabacum 14-3-3 C

C-terminal peptide from N. plumbaginifolia

ATPase PMA2 and pyrrolidone1

III 2010 [158]

3M50 N. tabacum 14-3-3 C

C-terminal peptide from N. plumbaginifolia

ATPase PMA2 and epibestatin

3E6Y

N. tabacum 14-3-3 C

C-terminal peptide from N. plumbaginifolia

ATPase PMA2 and cotylenin A

III 2009 [157]

2O98 N. tabacum 14-3-3 C

C-terminal peptide from N. plumbaginifolia

ATPase PMA2 and fusicoccin

III 2007 [99]

1O9E N. tabacum 14-3-3 C fusicoccin -

2003 [88]

1O9C N. tabacum 14-3-3 C – -

1O9D N. tabacum 14-3-3 C

C-terminal peptide from N. plumbaginifolia

ATPase PMA2

III

1O9F N. tabacum 14-3-3 C

C-terminal peptide from N. plumbaginifolia

ATPase PMA2 and fusicoccin

III

Note. * As of July 2024.

signaling cascades. It was suggested that plants have

an endogenous molecule that has a similar function

as fusicoccin and triggers a coordinated physiological

response involving a number of proteins, i.e., acts as

a phytohormone [166]. Hence, the physiological activ-

ity of fusicoccin remains unclear. It is possible that

new research directions will elucidate it and help to

describe it in detail.

SEDLOV, SLUCHANKOS18

BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

Fig. 8. The effect of 14-3-3 on the functions of membrane proteins. The binding of 14-3-3 to the cytosolic parts of plasma

membrane integral proteins regulates their activity: the binding of 14-3-3 to PMA2 leads to the enzyme activation; the

interaction with potassium channels KAT1 and GORK promotes ion currents through the channels; the binding to the

aquaporin PIP2;1 maintains its open state. In the vacuole membrane, 14-3-3 interaction with the potassium channel TPK1

decreases the ion current through the channel; the binding of 14-3-3 to SV channels also slows down the cation current

through them; the putative interaction of 14-3-3 with V-ATPase leads to the enzyme activation.

Another function of 14-3-3 proteins is the regu-

lation of potassium ion transport across the plasma

membrane. It has been shown that 14-3-3 enhances

ion currents through the voltage-dependent potassium

channel KAT1 (K

channel in A.  thaliana) that conducts

potassium into the cell [167, 168] (Fig. 8). Sapona-

ro et al. [165] showed that 14-3-3 interacts with KAT1

through the C-terminal type  III motif YFSpSN-COOH

(phosphorylated residue in A. thaliana is Ser676)

[165]. The authors obtained the spatial structures

of N. tabacum 14-3-3 complex with the phosphopep-

tide from KAT1 (type III motif) in the presence and

absence of fusicoccin [165]. The data suggested that

14-3-3 influences the opening of stomata via two

parallel mechanisms: by acting via PMA ATPase to

generate the proton gradient and hyperpolarize the

membrane in order to promote ion current into the

cells and by affecting KAT1 channels to directly in-

crease K

entry into the cells [165]. Both processes ul-

timately lead to the entry of water into stomatal guard

cells and their opening. The role of 14-3-3 proteins in

the regulation of the functioning of stomatal guard

cells is discussed in detail in the review by Cotelle

et al. [147].

Van Kleeff et al. investigated interactions between

14-3-3 and GORK (Guard Cell Outward Rectifier K

)

channels from A. thaliana [169] (Fig. 8). These chan-

nels remove potassium ions from cells, thus partici-

pating in the stomata closure and maintenance of K

homeostasis in root cells [170]. Using pull-down assay,

the authors showed that GORK channels interact with

14-3-3  λ,  ν, and χ isoforms. However, they failed to

confirm a direct binding of 14-3-3 with GORK channels

in the yeast two-hybrid assay. It was found that 14-3-3

interacts with the calcium-dependent kinase CPK21

and activates its, while CPK21, in turn, phosphorylates

GORK channels at Thr344, Ser518, and Ser649 [169].

The studies of mutant plants deficient by CPK21, 14-3-3

χ, and 14-3-3 φ have shown that these proteins are

required to maintain normal potassium ion currents

through the GORK channels [169].

14-3-3 proteins interact with aquaporins and reg-

ulate their functions. It was found that plant aquapo-

rins from the PIP (Plasma membrane Intrinsic Protein)

WORLD OF PLANT 14-3-3 PROTEINS S19

BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

group can bind 14-3-3 proteins [171, 172]. Aquaporins

facilitate water transport across the membrane. Thus,

PIP2;1 from A. thaliana regulates circadian fluid fluxes

in leaves [173]. Using 14-3-3 knockout mutants, it was

demonstrated that 14-3-3 is necessary for maintaining

normal functions of PIP2;1 [172]. It is possible that

PIP2;1 interacts with 14-3-3 through the Ser280 and

Ser283 residues located closer to the C-terminus [172].

It is believed that phosphorylation of these sites has

a regulatory function [173]. These studies again lack

structural information that could shed light on the

mechanism of aquaporin regulation by phosphoryla-

tion and 14-3-3 protein binding.

Regulation of transport across the vacuolar mem-

brane. 14-3-3 proteins also regulate transport through

the vacuolar membrane (tonoplast). In particular,

14-3-3 regulates potassium ion transport to the vacu-

ole lumen through the channels of the TPK/KCO (tan-

dem-pore K

channels/K

-like channels) family (Fig.8).

The functions of these channels in plants are poorly

understood. They contain the 14-3-3 recognition motif

I at the N-terminus at the cytosolic side of the mem-

brane (phosphorylated residue in A. thaliana TPK1

is Ser42) and the EF-hand domain at the C-terminus

[174]. The interaction between 14-3-3 λ and A. thaliana

TPK1 was shown using the pull-down assay and the

surface plasmon resonance method [175]. The binding

of three 14-3-3 isoforms (14-3-3 A, B, and C) from bar-

ley (Hordeum vulgare) and HvKCO1 channel (a homo-

log of A. thaliana TPK1) was demonstrated using sur-

face plasmon resonance [176]. The binding of 14-3-3

to TPK1 activates this channel [175]. Interesting data

were obtained when studying TPK1 from H. vulgare.

Two of the three isoforms, 14-3-3 B and C, inhibited

this channel, while 14-3-3 A had no effect on the ion

current [176]. Latz et al. showed that the serine resi-

due in the 14-3-3 recognition motif in A. thaliana TPK1

is phosphorylated by the calcium-dependent protein

kinase CPK3 [177]. The activity TPK1 is important for

maintaining ion homeostasis during plant response to

salt stress [177].

Slow cation (SV) channels are also regulated by

14-3-3 proteins (Fig. 8). These channels conduct Ca

, and Na

cations when activated by the increased

intracellular Ca

concentrations [170]. 14-3-3 proteins

slow the current through these channels [175, 178],

although the details of interaction between 14-3-3 and

SV channels remain unknown.

Another protein interacting with 14-3-3 is V-ATPase

[179] (Fig. 8). V-ATPase performs a number of import-

ant functions in the vacuole membrane. By transfer-

ring protons across the membrane, it forms the elec-

trochemical potential, controls turgor pressure, and

stabilizes pH of the cytoplasm [180, 181]. Presumably,

14-3-3 interacts with the A subunit of V-ATPase [179],

but the exact recognition site of 14-3-3 remains un-

known. The amount of 14-3-3 proteins associated with

V-ATPase increases upon illumination with blue light,

as does the V-ATPase activity, but the direct effect of

14-3-3 on the enzyme activity has not been charac-

terized [179].

From the above examples, it is evident that plant

14-3-3 proteins regulated the functions of many mem-

brane proteins, including proton-transporting ATPases,

ion channels, and aquaporins. Many of these 14-3-3

partner proteins are involved in controlling the activi-

ty of stomatal guard cells. Therefore, the opening/clos-

ing of stomata is regulated by 14-3-3 proteins not only

during receiving and transmitting blue light signal via

phototropins, but also directly through the regulation

of ion currents.

14-3-3 proteins as regulators of signaling cas-

cades. Having a sedentary lifestyle, plants cannot

escape from stress factors and have to adjust bio-

chemical characteristics of their cell to environmen-

tal conditions. The effects of various external factors

have led to the formation of a branched signaling net-

work designed to fine-tune the process of biochem-

ical adaptation. By binding phosphorylated residues

in proteins, 14-3-3 proteins can participate in protein

kinase/protein phosphatase signaling cascades or to

add another level of regulation.

Signaling pathways involved in biotic stress re-

sponse. The mitogen-activated protein kinase (MAP)

cascade is one of the best studied in plants. It is a lin-

ear cascade of sequentially phosphorylating kinases:

MAP kinase kinase kinase (MAPKKK) phosphorylates

MAP kinase kinase (MAPKK), which phosphorylates

terminal MAP kinase (MAPK) and activates it [184].

The activation of this signaling pathway can lead to

cell division and differentiation, cell death, and re-

sponse to abiotic and biotic stresses [184]. Oh et al.

[185] found that MAPKKKα from tomato (S. lycoper-

sicum) interacts with the 14-3-3 isoform TFT7 in the

phosphopeptide-binding groove of 14-3-3 [185]. A pos-

sible phosphorylated residue in MAPKKKα is Ser535,

whose amino acid environment corresponds to the

14-3-3 recognition motifII [185]. The presence of 14-3-3

promotes the stability of MAPKKKα protein in cells

and increases the amount of proteins phosphorylated

by this kinase [185]. Overexpression of TFT7 promotes

the pathogen-induced programmed cell death induced

by MAPKKKα [185]. In another study, Oh et  al. [75]

showed that TFT7 binds S. lycopersicum MAPKK iso-

form SlMKK2 (MAPKKKα target) [75]. The putative

recognition site for 14-3-3 is a sequence that includes

phosphorylated Thr33 and corresponds to the type II

motif [75]. In this case, however, the authors did not

find a direct effect of TFT7 on the activity and stability

of SlMKK2 [75]. It was suggested that 14-3-3 has an

adaptor function, since its dimer contains two recog-

nition sites for phosphorylated motifs, one of which

SEDLOV, SLUCHANKOS20

BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

can bind SlMKK2, and the other binds MAPKKKα (as

was shown previously), leading to the convergence of

the two kinases and acceleration of signal transduction

[75]. New data to the concept of MAP kinase cascade

regulation by 14-3-3 were added by a recent study by

Dong etal. [111], who demonstrated that 14-3-3 κ and

λ interact with MAPKKK5 and PBL19 from A. thaliana.

PBL19 is a protein kinase of the RLCK family that

phosphorylates the C-terminus of MAPKKK5 and acti-

vates it, but the access of PBL19 to the phosphoryla-

tion site is limited by the autoinhibitory N-terminus of

MAPKKK5. By binding to the C-terminus of MAPKKK5,

14-3-3 λ eliminates this inhibition and allows PBL19 to

phosphorylate and activate MAPKKK5 [111] (Fig. 3e).

Hence, 14-3-3 λ functions as a scaffold that facilitates

MAPKKK5 activation [111], leading to the initiation of

plant immune response against pathogens [111]. How-

ever, as in many other cases, there is no complete un-

derstanding of the MAPK cascade regulation by 14-3-3

proteins due to the lack of structural information on

the relevant complexes.

The 14-3-3 involvement in the regulation of MAPK

cascade explains an important role of 14-3-3 proteins

in the plant immune response to pathogens. It was

found that some effector proteins of pathogenic pro-

karyotes, eukaryotes and viruses can directly interact

with 14-3-3 proteins of host plants. Thus, the coat pro-

tein (CP) of the beet black scorch virus (BBSV, a  (+)RNA

virus) interacts with 14-3-3a of Nicotiana benthamiana

[186]. It was proposed that the recruitment of 14-3-3

by CP leads to a decrease in the activity of another

14-3-3 partner protein, MAPKKKα, which prevents ini-

tiation of cell death and promotes virus replication

[186]. A similar effect was shown for the 14-3-3 part-

ner protein XopQ from the phytopathogenic bacterium

Xanthomonas euvesicatoria. Recruitment of 14-3-3 by

XopQ resulted in the suppression of the MAPKKKα-me-

diated cell death, presumably due to the disruption of

14-3-3 interactions with proteins, including those with

MAPKKKα [187]. Bacteria also secrete other 14-3-3-

interacting effector proteins. For example, Xanthomo-

nas bacteria produce XopX  [188] and XopN1  [189],

while Pseudomonas syringae secretes HopM1  [190]

and HopQ1 [191]. The binding of 14-3-3 with these

effector proteins suppresses plant immune response

to pathogens and facilitates infection. The pathogenic

oomycete Phytophthora palmivora, which causes rot

diseases of many tropical crops, secretes the FIRE

protein into the plant cell. FIRE interacts with 14-3-3

proteins of the host plant via the type I recognition

motif, leading to successful infection; however, the

molecular mechanism of this process remains unclear

[192]. As can be assumed from the presented data,

recruitment of cellular 14-3-3 proteins by binding to

the effector protein is widely used by plant pathogens

to suppress plant immune response, which character-

izes 14-3-3 as an important element in the regulation

of plant immunity [193]. However, both the details of

such interactions and molecular mechanisms of 14-3-3

functioning in plant immune response have not been

clarified so far.

Signaling pathways involved in response to abiot-

ic stress. 14-3-3 proteins participate in the SOS (Salt

Overly Sensitive) signaling cascade, which regulates

plant response to salt stress. The main participants

of the SOS cascade are the serine/threonine protein

kinases SOS2 and PKS5 (SOS2-Like Protein Kinase 5),

exchanger of the plasma membrane SOS1, and

calcium-binding protein SOS3 [194]. Under normal

conditions, the activities of SOS2 and SOS1 are low. In

the case of salt stress, the concentration of Ca

in cells

increases, which induces SOS3 interaction with SOS2,

SOS2 activation, and phosphorylation of SOS1. Acti-

vated SOS1 pumps sodium ions out of the cell, thus

controlling salt homeostasis [195]. The role of 14-3-3

proteins in the SOS cascade was demonstrated by Yang

etal. [62], who showed that under normal conditions,

PKS5 phosphorylates SOS2 at Ser294, leading to its

interaction with 14-3-3 λ and decrease in the SOS2

activity [62]. However, the amino acid environment

of Ser294 (DGIEGS

294

YVAENV) does not correspond to

the canonical 14-3-3 recognition motifs. Using thermo-

phoresis, the authors showed that 14-3-3  κ and λ are

able to bind Ca

[62]. When the concentration of Ca

increases during the salt stress, 14-3-3 dissociates from

SOS2, resulting in the increase in its kinase activity,

SOS1 activation, and pumping of sodium ions out of

cells [62]. Interestingly, the authors also showed that

14-3-3  λ interacts with PKS5 and inhibits it [62].

Phototropin signaling pathways. Another import-

ant role of 14-3-3 proteins in plants is mediated by

their interaction with the phototropins PHOT1 and

PHOT2. Phototropins are membrane-associated blue-

light receptor protein kinases that initiate intracel-

lular signaling cascade regulating the opening of the

stomata, phototropism, and chloroplast movements

[196,  197]. Phototropins consist of two LOV domains

located at the N-terminus and responsible for captur-

ing light quanta, and the C-terminal kinase domain

that ensures signal transmission [196, 198]. During

photoreceptor activation, a quantum of blue light is

directly captured by flavin mononucleotide (FMN; ab-

sorption maximum, 447  nm) within the LOV domain.

FMN covalently attaches to a cysteine residue, caus-

ing conformational changes leading to phototropin

autophosphorylation and initiation of the signaling

cascade  [196, 198]. Sullivan etal. [199] analyzed inter-

action of PHOT1 and PHOT2 from A. thaliana with six

14-3-3 isoforms, four of which belonged to the non-ep-

silon group (κ, λ, υ, φ), and two were from the epsilon

group (ε, ο). Using far-western blotting, it was demon-

strated that PHOT1 specifically bound the non-epsilon

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BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

isoforms κ, λ, and φ, while the epsilon isoforms (ε, ο)

did not interact with the protein on the membrane

[199]. The authors showed that residues responsible

for the interaction with 14-3-3 (Ser350, Ser376, and

Ser410) were located between the two LOV domains

[199]. At the same time, PHOT2 did not interact with

14-3-3 λ and κ [199]. In a later study, Tseng et al. used

far-western blotting and yeast two-hybrid assay to

demonstrate that PHOT2 interacts with 14-3-3  λ [200].

According to the yeast two-hybrid assay and function-

al studies in mutant plants, the residue responsible for

the interaction with 14-3-3 was Ser747 located in the

kinase domain [200]. The authors demonstrated that

in A. thaliana plants, the effect of PHOT2 on the sto-

mata opening was impaired by mutations in 14-3-3 λ,

but not in 14-3-3  κ [200], indicating a narrow isoform

specificity of 14-3-3 effects. In the absence of stringent

controls, this causes some surprise, since the λ and κ

isoforms are phylogenetically close and have 93% ami-

no acid sequence identity [200]. The results of studies

by Sullivan et al. [199] and Tseng et al. [200] on the

interaction of 14-3-3 and PHOT2 contradict each other

and suggest different 14-3-3 binding sites for such sim-

ilar proteins as PHOT1 and PHOT2. A detailed study

of the interaction of phototropins with 14-3-3 proteins

using structural biology methods could clarify these

issues and explain conflicting observations.

Phytohormone-induced signaling cascades. The sig-

naling cascade of the gaseous phytohormone ethylene

is also regulated by 14-3-3 proteins. In A. thaliana

plants, 14-3-3 proteins interact with ACS, the key

enzyme of ethylene biosynthesis. Yoon  et  al.  [113]

demonstrated that ACS coimmunoprecipitated with

14-3-3  ω, as well as confirmed this interaction in vivo

for four tested 14-3-3 isoforms (ι, ο, κ, φ) using bimo-

lecular fluorescence complementation method. Over-

expression of 14-3-3  ω in cells increased the stability

of ACS, while the treatment of plants with the R18

peptide (which inhibits the binding of 14-3-3 to other

peptides by blocking the amphipathic groove) accel-

erated a decrease in the total ACS content, likely due

to protein degradation. The authors also showed that

14-3-3 ω interacts with ETO1-like (EOL) proteins, com-

ponents of the E3 ubiquitin ligase, which target some

ACS isoforms for degradation  [113,  201]. In a later

study, Catalá etal. [202] also demonstrated the binding

of 14-3-3  ψ (RARE COLD INDUCIBLE 1A, RCI1A) with

ACS using coimmunoprecipitation and bimolecular

fluorescence complementation assays [202]. Howev-

er, the stability of the ACS protein in the presence of

14-3-3  ψ in cells did not increase (as in the case of

14-3-3  ω), but rather decreased. The authors explained

this discrepancy by the specificity of the 14-3-3 ω and

14-3-3  ψ interactions with their partner proteins [202].

In our opinion, these undoubtedly interesting data re-

quire systematization and new studies, in particular,

with the use of structural biology methods, in order

to explain the observed discrepancies.

Plant 14-3-3 proteins as regulators of gene ex-

pression. One of the first names of plant 14-3-3 pro-

tein– G-box-associated factor (GF14)– was due to the

discovery of this protein in a complex with the G-box

element in DNA [7], which suggests participation of

14-3-3 proteins in the regulation of gene transcription.

In A. thaliana, 14-3-3 was found to regulate tran-

scription factors BZR1 and BES1, which are compo-

nents of the brassinosteroid (plant hormone) signaling

pathway. In the absence of brassinosteroids, BZR1 and

BES1 are phosphorylated by the GSK3 family protein

kinase BIN2 at Ser173 [102] and Ser171 [101], respec-

tively. The amino acid environments of the phosphor-

ylated residues represent typeII motifs. Phosphoryla-

tion results in the binding of 14-3-3 (λ and ω isoforms

for BZR1 [26, 102] and λ isoform for BES1 [101]), re-

sulting in the retention of the partner proteins in the

cytoplasm and prevention of their entry to the nucle-

us (Fig. 3c). In the presence of brassinosteroids, the

signaling cascade from the receptor complex of BRI1

(Brassinosteroid Insensitive 1) and BAK1 (BRI1-Asso-

ciated Kinase 1) leads to the inhibition of protein ki-

nase BIN2 by the BSU1 phosphatase [203]. This causes

dephosphorylation of BZR1 and BES1, dissociation of

their complexes with 14-3-3, and import of BZR1 and

BES1 into the nucleus, where they initiate transcrip-

tional programs required for the response to brassi-

nosteroids [101, 102]. Hence, 14-3-3 is involved in the

cytoplasmic sequestration of transcription factors,

possibly due to the presence of the nuclear export

sequence (NES) in the C-terminal region of 14-3-3, as

has been shown for human and yeast 14-3-3 partner

proteins [50, 52].

14-3-3 proteins can be directly involved in the

assembly of transcriptional complexes. It was shown

in O. sativa suspension culture and Zea  mays embry-

os, that 14-3-3 is a component of the protein complex

that binds the DNA sequence of the Em1a regulatory

element in the Em gene promoter, whose transcription

is activated by signaling initiated by the phytohor-

mone abscisic acid [110]. The complex also includes

transcription regulators Viviparous1 (VP1) and EmBP1

(b-ZIP family factor) [110]. The association of 14-3-3

with the AtEm1 gene promoter was also shown in

A. thaliana embryonic cell culture [204].

An important role of 14-3-3 proteins is regula-

tion of flowering at the stage of florigen-activating

protein complex formation. The GF14c isoform from

O. sativa interacts in vitro and in vivo with the so-

called florigen, or Hd3a (heading date  3a) protein,

whose homolog in A. thaliana is encoded by the FT

(FLOWERING LOCUST) gene [41,  96]. Florigen is syn-

thesized in the leaves and transported to the apical

meristem of shoots, where it enters the cell cytoplasm

SEDLOV, SLUCHANKOS22

BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

and interacts with GF14c. The resulting complex en-

ters the nucleus and binds to the transcription fac-

tor OsFD1 (FD is OsFD1 homolog in A. thaliana) [96].

The florigen-activating complex (FAC) formed by the

GF14c dimer, OsFD1 dimer, and two Hd3a proteins,

activates transcription of OsMADS15 (its homolog in

A. thaliana is called APETALA1, or AP1), thus trigger-

ing the flowering [96]. The spatial structure of a FAC

fragment was solved by X-ray crystallography; it is

the only structure of plant 14-3-3 complex with a full-

length partner protein in the PDB (PDBID: 3AXY; 164

amino acid residues out of 179 resolved in Hd3a struc-

ture) (Fig.7b) [96]. OsFD1 is phosphorylated at Ser192

(type I motif) and bound in the groove of 14-3-3 (as

follows from the structure of 14-3-3 complex with a

OsFD1 phosphopeptide) [96]. In A. thaliana, FD tran-

scription factor is phosphorylated at Thr282 (residue

homologous to Ser192 in OsFD1) by calcium-depen-

dent protein kinases, such as CPK6 and CPK33 [205].

14-3-3 proteins can indirectly regulate gene ex-

pression by affecting the stability of transcription fac-

tors. This regulatory mechanism has been shown for

the transcription factor WRINKLED1 (WRI1), a master

regulator of triacylglyceride biosynthesis [114]. WRI1

likely interacts with 14-3-3 at the site that was also

predicted to bind E3 ubiquitin ligase [114]. Hence,

14-3-3 can protect WRI1 from proteasomal degrada-

tion and promote expression of WRI1-regulated genes

of triglyceride biosynthesis [114] (Fig. 3f).

Guo et al. showed that 14-3-3 (HbGF14a isoform)

from the rubber tree (Hevea brasiliensis) regulates the

activity of the transcription factor HbRZFP1 (H. brasil-

iensis RING zinc finger protein) [206]. HbRZFP1 binds

to the promoter of the gene coding for Hevea rub-

ber transferase (HRT2), an enzyme that adds isoprene

units to the growing rubber polymer, and inhibits its

expression. HbGF14a has been shown to interact with

HbRZFP1 in vivo and in vitro in bimolecular fluores-

cence complementation, yeast two-hybrid, and pull-

down assays [206]. A regulatory mechanism has been

proposed in which the binding of HbGF14a to the

transcription factor HbRZFP1 disrupts HbRZFP1 in-

teraction with the HRT2 gene promoter, resulting in

upregulated expression of this transferase [206]. Sev-

eral studies have also investigated the role of 14-3-3

proteins in rubber biosynthesis. HbGF14c (another

14-3-3 isoform from H.brasiliensis) has been shown to

interact with the small rubber particle protein (SRPP),

which is present in rubber particles and involved in

rubber biosynthesis [207, 208]. However, the details

of this interaction and the corresponding regulatory

mechanism remain unknown.

As can be seen from the above, 14-3-3 proteins

participate in the regulation of many transcription

factors and DNA–protein complexes. However, the ef-

fects and the mechanisms of this regulation are often

poorly understood, while the details of protein–pro-

tein interactions still remain enigmatic. Using mod-

ern methods of structural biology and studying the

processes at the protein level might help in resolving

these issues.

CONCLUSION. “BLANK SPOTS”

IN THE STUDIES OF PLANT 14-3-3 PROTEINS

As can be seen from the data presented in this

review, plant 14-3-3 proteins are involved in the reg-

ulation of many important biochemical and physiolog-

ical processes. The fact of 14-3-3 family involvement

in these processes is beyond any doubt. Hundreds of

research papers and dozens of reviews have been

devoted to the functions of 14-3-3 proteins in plants.

However, despite more than thirty years of research,

many issues about the structure and functions of plant

14-3-3 proteins remain unexplored. The majority of

these fundamental problems arise due to the lack of

systematic approach and, as a consequence, a frag-

mentary nature of the obtained data. 14-3-3 proteins

are a multigene family, and plant species often have

several 14-3-3 isoforms, so to obtain a complete pic-

ture, it is necessary to study either all representatives

of the family or a representative set of these isoforms.

In the first case, the task is extremely labor-intensive

even for plant species with a relatively small number

of isoforms. In the second case, it is unclear which

criteria should be used to select 14-3-3 isoforms for

analysis. Therefore, in most studies, isoforms are cho-

sen at random, which complicates data interpretation

and makes global generalizations impossible. Below,

we present some of the most interesting unresolved

issues – “blank spots” – in the field of plant 14-3-3

research.

Are biochemical properties of plant 14-3-3 ho-

modimers similar or different? One of the funda-

mental questions is related to the biochemical char-

acteristics of 14-3-3 proteins. These proteins are quite

conserved; the pairwise identity of amino acid se-

quences of 14-3-3 isoforms from A. thaliana is 60% on

average and reaches 80-90% in some cases. However,

the question whether their biochemical characteristics

are the same has not been properly addressed. When

we started to investigate this problem, we were sur-

prised to find that 14-3-3 isoforms from the epsilon

and non-epsilon subgroups in A. thaliana differ sig-

nificantly in the stability of formed homodimers, hy-

drophobicity of protein surface, thermal and proteo-

lytic stability, and other properties [209]. Thus, unlike

non-epsilon isoforms, isoforms from the epsilon group

are prone to dissociation into monomers, have a lower

half-transition temperature (the greatest difference in

the half-transition temperature between the epsilon

WORLD OF PLANT 14-3-3 PROTEINS S23

BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

and non-epsilon isoforms was over 20°C), and are

more susceptible to limited proteolysis [209]. Even the

homodimers of plant 14-3-3 proteins can significantly

differ from each other. The search for such distinctive

features and biochemical characteristics is of funda-

mental importance, since they can strongly impact

the half-lifetime (stability) and biological functions

of 14-3-3 isoforms in plants. Also, understanding spe-

cific properties of isoforms from different subgroups

can help in phylogenetic analysis and projecting the

properties inherent in a subgroup or in individual

isoforms in some plants onto other less-studied iso-

forms (and in less-studied plants). In the absence of

such information, the growing body of data cannot be

properly systematized and understood.

Which plant 14-3-3 isoforms form heterodi-

mers? Is there a preference for particular isoforms

during heterodimer formation? Heterodimerization

of plant 14-3-3 proteins, both in vitro and in vivo, has

been demonstrated in several studies. It was found

that 13 isoforms of A. thaliana 14-3-3 proteins can

theoretically form 78 different heterodimers, but only

11 of them have been reported to generate heterod-

imers, all of these isoforms belonging to the non-ep-

silon phylogenetic group. Moreover, there is a lack of

information on the ability of plant 14-3-3 proteins to

heterodimerize in their native form, without the use

of denaturation/renaturation. It remains unknown

whether plant 14-3-3 isoforms from the epsilon sub-

group heterodimerize with each other, and whether

epsilon and non-epsilon isoforms can form heterod-

imers. The biological function of heterodimerization

and advantages it provides (if any) remain obscure.

Is there a hierarchy of affinity of plant 14-3-3

proteins for phosphorylated peptides? What is the

reason for the differences in the affinity of 14-3-3 iso-

forms? It has been shown that regardless of the type

of the partner protein, the hierarchy in the affinity

of animal 14-3-3 isoforms remains more or less the

same [92]. It is unknown whether this is true for plant

14-3-3 proteins. Several studies have shown that 14-3-3

isoforms from A. thaliana bind phosphopeptides (or

partner proteins) with different affinity. A systematic

approach is needed to figure out the patterns in the

affinity of plant 14-3-3 proteins, since none of the

studies have examined the full set of isoforms even

for such model plant as A. thaliana. The maximum

number of isoforms analyzed in one study was 9 (out

of 13). The reason for the difference in the affinity

between the 14-3-3 proteins still has to be elucidated.

The residues exposed in the ligand-binding groove of

14-3-3 and responsible for the binding of phosphopep-

tides are fully conserved, so it remains obscure what

provides the differences in the binding affinity from

the structural point of view. This question is relevant

for both plant and mammalian 14-3-3 proteins.

How widespread and universal is the regula-

tion of plant 14-3-3 proteins by phosphorylation?

What are the functional effects of phosphoryla-

tion? 14-3-3 proteins can undergo phosphorylation,

which regulates their binding to the partner proteins

and the 14-3-3 dimer structure [74, 82]. Large-scale

phosphoproteomic studies have identified dozens of

residues capable of being phosphorylated in plant

14-3-3 proteins [83, 210]. However, only two sites locat-

ed at the 14-3-3 dimer interface have been functionally

characterized. It also remains unclear to what extent

the regulation by phosphorylation is conserved and

universal across plant 14-3-3 isoforms.

Are the functions of plant 14-3-3 isoforms spe-

cific or redundant? This question has already been

raised in several reviews on plant 14-3-3 proteins [46,

211, 212]. Does each 14-3-3 isoform bind specific part-

ner proteins or can all 14-3-3 isoforms interact with

all formally suitable partner proteins and compensate

for each other’s functions? A clear answer to this ques-

tion is currently lacking. For example, 14-3-3  κ and λ

isoforms from A. thaliana have appeared as a result

of a relatively recent duplication of a single gene [14].

Their amino acid sequences are as high as 93% identi-

cal, which is the highest identity of the primary struc-

tures of 14-3-3 isoforms in A. thaliana. For such closely

related isoforms, one would expect a strong overlap

of functions and the least degree of subfunctionaliza-

tion. This is consistent with some experimental data

characterizing the functions of these isoforms as re-

dundant. Thus, single mutations in either 14-3-3  λ and

κ had no effect on the flagellin-induced expression

of marker genes [111]. Both single mutants, as well

as the double mutants by 14-3-3 λ and κ, did not dif-

fer from the wild-type plants in the root length [36].

However, other studies have demonstrated distinct

properties and effects of these highly related isoforms:

14-3-3  λ (but not κ) bound RPW8.2 protein in immu-

noprecipitation experiments [213]; 14-3-3  λ (but not  κ)

was shown to play a role in the PHOT2-mediated

opening of stomata in vivo [200]; both 14-3-3  λ and

κ interacted with the PHOT1, but 14-3-3  λ bound the

protein several-fold more strongly than 14-3-3 κ [199];

the affinity of 14-3-3  λ for various targets was consis-

tently higher than that of 14-3-3  κ  [58]. Thus, even

very similar 14-3-3 isoforms can have both specific

and overlapping functions. Apparently, there is no

general rule about redundancy or specificity of plant

14-3-3 proteins, and the extent of manifestation of the

functional effects of particular isoforms depends on

specific protein–protein interactions.

Lack of information on plant 14-3-3 partner

proteins. Another serious problem is the lack of in-

formation on the interactions of 14-3-3 isoforms with

partner proteins, in particular, on the 14-3-3 binding

site, functional consequences of such interactions,

SEDLOV, SLUCHANKOS24

BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

and spatial structures of complexes formed. Solving a

spatial structure naturally helps to identify the mech-

anism of regulation of partner proteins by 14-3-3 iso-

forms. At present, only two structures of plant 14-3-3

complexes with a partner protein (florigen Hd3a from

O. sativa) or a large fragment (C-terminal fragment of

the plasma membrane H

-ATPase PMA2 from N.plum-

baginifolia) have been determined [96, 99] (Fig.  7,  a

andb). The details of how 14-3-3 proteins affect their

interaction partners are either unknown or incom-

plete, which leads to ambiguity in the interpretation

of experimental data and contradictions. For example,

neither the phosphorylated residue (Ser229) in SPS

from S. oleracea originally identified by Toroser etal.

[98], nor the inhibitory effect of 14-3-3 upon binding

to the enzyme have been confirmed in later studies.

The regulatory mechanism has been most fully de-

scribed for the two classical 14-3-3 partner proteins–

NR [124] and PMA [99], but for many other proteins,

the details of interaction with 14-3-3 remain unknown.

Thus, there are no data on the 14-3-3 binding sites in

GAPN, ACS, GS1, and transcription factors WRI1, VP1,

and EmBP1, as well as on the kinases that phosphor-

ylate them. Based on the general concepts of 14-3-3

interaction with partner proteins, it might be possible

to fully describe the regulatory mechanism if the fol-

lowing information is revealed: (i)  14-3-3 recognition

site in the partner protein; (ii)  kinase that phosphory-

lates it; (iii)  effect of 14-3-3 binding on the activity and

conformation of the partner protein in vitro; (iv) func-

tional consequences of this interaction in vivo. Only

by answering all these questions, researchers will be

able to resolve existing contradictions and clarify the

mechanisms of 14-3-3-regulated processes in plants.

Is yeast two-hybrid assay applicable to study-

ing 14-3-3 interactions with partner proteins? Yeast

two-hybrid assay has been repeatedly used in the stud-

ies of 14-3-3 proteins. The screening of cDNA libraries

has revealed tens and hundreds of protein–protein

interactions [171, 214]. However, the method may not

be suitable for testing specific binary interactions due

to specific features of 14-3-3 interaction with partner

proteins. The binding of 14-3-3 requires phosphory-

lation of the partner protein at a specific motif by a

specific kinase, which might exist in plants only. The

formed protein complexes might not localize to the

nucleus, for example, if the partner protein is located

in the membrane or near it. These features complicate

the use of yeast two-hybrid assay and limit its appli-

cations in the studies of protein–protein interactions

of 14-3-3 isoforms. For example, the conclusion about

the absence of direct binding between 14-3-3 and

GORK channels based on the negative results of yeast

two-hybrid assay is questionable, as in the same study,

specific binding of these two proteins was demon-

strated by the pull-down assay [169]. Therefore, yeast

two-hybrid assay should be used with great caution

and, preferably, in combination with other methods

when studying protein interactions involving 14-3-3

proteins.

Can plant 14-3-3 proteins regulate the function-

ing of partner proteins through the chaperone-like

activity or by influencing the LLPS? A number of

mechanisms by which the binding to 14-3-3 changes

the biological function of partner proteins have been

described for both plant and mammalian 14-3-3 pro-

teins (Fig.3,a-e). However, the chaperone-like activity

and effect on LLPS have been found only for mamma-

lian 14-3-3 proteins [117-119] (Fig. 4, b and c). Since

plant 14-3-3 proteins are structurally similar to their

mammalian homologs and have the same biological

significance, it is reasonable to assume that similar

mechanisms can be found in plants as well. In plants,

LLPS has been described for several important phys-

iological processes, including formation of molecular

condensates in the nucleus during regulation of flow-

ering and initiation of transcriptional response in the

phytochrome phyB signaling [215]. 14-3-3 proteins are

directly involved in the latter two processes and may

play a role in the occurring LLPS events.

Are 14-3-3 proteins present in chloroplasts and

mitochondria and how do they get there? 14-3-3

proteins are typically located in the cytosol and nu-

cleus and lack a signal sequence for their import into

chloroplasts [216]. However, several early studies have

shown that plant 14-3-3 proteins can localize to the

chloroplast stroma [44,  106]. Moreover, it has been

shown that 14-3-3 isoforms can interact with and reg-

ulate the activity of several chloroplast proteins, such

as GS2 [129], starch synthase [44], and chloroplast

and mitochondrial ATP synthases [217]. It remains un-

known how 14-3-3 proteins get into the double-mem-

brane organelles and what functions they perform

there. These issues, addressed mainly in early works,

need to be reconsidered and investigated using mod-

ern methods of biochemical and structural analysis.

The problems of phenotypic analysis of plants

mutant for 14-3-3 proteins. Phenotypic analysis of

mutants could provide opportunities for identification

and characterization of specific functions of 14-3-3 iso-

forms in plants. The knockouts, overexpression, and

heterologous expression of 14-3-3 proteins in differ-

ent plant species have been extensively investigated

[18,  25,  37-45,  169], however, the studies on 14-3-3 mu-

tants are often unsystematic and incomplete. The lack

of systematic approach is evidenced by the fact that

there are no published studies where single mutants

for all 13 isoforms in A. thaliana have been pheno-

typically characterized. The isoforms for studying

are often chosen randomly; an isoform of one plant

can be expressed in another plant. The studies are

often focused on a particular trait of mutant plants

WORLD OF PLANT 14-3-3 PROTEINS S25

BIOCHEMISTRY (Moscow) Vol. 90 Suppl. 1 2025

and ignore the others (there are very few or no stud-

ies of the “one isoform-many traits” or “one trait-all

isoforms” type). As noted above, the main phenotyp-

ic manifestations of 14-3-3 mutations are changes in

plant growth, timing of transition to flowering, toler-

ance to salt stress, and starch accumulation, i.e., traits

important in agriculture. We assume that the authors

of such studies reported only prominent and omitted

less obvious effects of mutations. Interpretation of

results of knockout studies can be significantly com-

plicated by the compensatory effect of other 14-3-3

isoforms, which certainly occurs in the case of fairly

similar 14-3-3 proteins. The scope and fundamental

significance of studies on the reverse genetics of plant

14-3-3 proteins could be greatly increased by using

a systematic approach and comprehensive analysis

of 14-3-3 mutants.

Contributions. I. A. S. and N. N. S. conceived the

idea of critically summarizing current literature and

discussed the plan; I. A. S. analyzed the literature,

wrote the review, and prepared tables and illustra-

tions with input from N. N. S.; I. A. S., and N. N. S. edit-

ed the manuscript.

Funding. The work was in part supported by the

Russian Science Foundation (project no.24-74-00091).

Ethics approval and consent to participate.

This work does not contain any studies involving hu-

man and animal subjects.

Conflict of interest. The authors of this work de-

clare that they have no conflicts of interest.

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