Numerous studies have revealed that the GSK3s proteins are mainly regulated at the post-translational level via modulation of protein stability, protein activity via various modifications (i.e., phosphorylation, acetylation and oxidation), and subcellular localization (Li et al., 2021). A series of GSK3s proteins, such as AtBIN2, AtBIL1, AtBIL2, AtSK11, AtSK12, OsGSK2, and TaSG-D1, is degradated through proteasome-mediated pathway (Li and Nam, 2002; Peng et al., 2008; Yan et al., 2009; Tong et al., 2012; Li et al., 2018; Cheng et al., 2020; Chen et al., 2020b). Moreover, the proteasome-mediated regulation of GSK3s is modulated via a conserved C-terminal TREE motif, and mutations in this motif cause the gain-of-function effects. For example, the mutation in the TREE motif impaired the ubiquitylation-dependent proteasomal degradation of BIN2 mediated by the E3 ubiquitin ligase kink suppressed in bzr1-1D 1 (KIB1). Moreover, homologous proteins of KIB1 are also involved in the BR-induced proteasomal degradation of BIN2, and they function redundantly in the suppression of the GSK3 kinase activity (Zhu et al., 2017). But how KIB1 and its homologous proteins interact with other proteins of the BR signaling remains elusive (Li et al., 2021).

Evidence that the activity of GSK3s is altered by protein modifications including phosphorylation, acetylation, and oxidation is also accumulated. For example, dephosphorylation and phosphorylation of GSK3s, which are the most common ways in the regulation of GSK3s ( Figure 1 ), significantly change the activities of the proteins in various species. The suppressor of bri1 (BSU1) dephosphorylates BIN2 at phospho-tyrosine 200, and deactivates BIN2 kinase activity in the BR signaling transduction (Kim et al., 2009). Similarly, the BIN2 ortholog in rice OsGSK3 (OsSK23) is dephosphorylated by the rice ortholog of BSU1, OsPPKL1 (phosphatase with Kelch-like repeat domains), and affectes BR signaling (Gao et al., 2019). However, the effect of OsPPKL1 on OsGSK3 activity is completely different with that caused by AtBSU1. The dephosphorylation of AtBIN2 kinase resulted in the BIN2 degradation, but in rice, OsPPKL1 stabilized the OsGSK3 protein. The opposite effect on GSK3s protein stabilization may be related with its specific role in different species, which requires further investigation. The bZIP transcription factor, elongated hypocotyl 5 (HY5), binds to BIN2 and enhances the autophosphorylation of BIN2 at the tyrosine 200 residue (Li et al., 2020a). Ribosomal protein S6 kinase 2 (S6K2) phosphorylates BIN2 at serines 187 and 203, and inhibites BIN2 activity in Arabidopsis (Xiong et al., 2017). They also find that the phosphorylation of BIN2 depends on the upstream TOR-S6K2 signaling, which plays a vital role in coordinating plant growth and stress responses. Histone deacetylase 6 (HDA6) can also interact with and deacetylate BIN2 at lysine 189 to repress its kinase activity (Hao et al., 2016). The histone acetylation modification of GSK3s proteins is also identified in rice (Hou et al., 2022). The histone deacetylase1 (OsHDAC1) directly interactes with and deacetylates OsGSK2, and eventually inhibits OsGSK2 activity. The above results suggest the conserved regulation mechanism of GSK3 by acetylation in Arabidopsis and rice. The expression pattern of HDA6 gene when exposed to BR is opposite with that of OsHDAC1 gene, which needs further research. Moreover, it has been found that the interaction of BIN2 and BES1 is oxygen-dependent, and the activity of BIN2 is regulated by the oxidation of several cysteine (Cys) residues including C59, C95, C99, and C162 (Song et al., 2019).

Emerging evidence has indicated that regulation of the subcellular localization of GSK3s significantly influences their biological functions in plants. Plasma membrane recruitment of GSK3s modulates BR signaling in Arabidopsis and rice. In Arabidopsis, several proteins including octopus (OPS), tetratricopeptide thioredoxin-like 3 (TTL3), and brassinosteroid signaling kinase 3 (BSK3), interact with and recruit BIN2 to the plasma membrane, and prevent BIN2 inhibitory role in the nucleus in the BR signaling pathway. Among these proteins, TTL3 affects BIN2 subcellular localization and promotes the degradation of BIN2 (Anne et al., 2015; Amorim-Silva et al., 2019; Ren et al., 2019). As mentioned above, OsPPKL1 dephosphorylated and stabilized OsGSK3 in the cytoplasm to modulate BR signaling (Gao et al., 2019). We find that TTL3 not only influences BIN2 stability, but also affects the BIN2 subcellular localization. OsPPKL1 not only influences BIN2 through protein modifications, but also alters the BIN2 subcellular localization. These results together indicates that the upstream proteins can regulate GSK3s proteins in more ways than one. In rice, a calmodulin binding protein grain width 5 (GW5) can also physically interact with and recruit OsGSK32 to the plasma membrane, thus resulting in accumulation of unphosphorylated OsBZR1 and dwarf and low-tillering (DLT) to promote BR signaling (Liu et al., 2017). Moreover, GSK3s subcellular localization mediates plant transition from the salt stress response to growth recovery. BIN2 is mainly locialized in the nucleus to regulate BR signaling and plant growth under salt stresses. In the recovery phase, BIN2 was recruited to the plasma membrane by the salt-triggered calcium sensors salt overly sensitive 3 (SOS3) and SOS3-like calcium binding protein 8 (SCaBP8) to regulate the salt stress response (Li et al., 2020b). Taking the above aspects into account, we can conclude that the altered subcellular localization of GSK3s proteins contributes to realize GSK3s proteins functions in the balance between plant growth and stress response. GSK3s subcellular localization also influences stomatal patterning. Stomatal cell lineage is an archetypal example of asymmetric cell division (ACD). The scaffold membrane protein polar localization during asymmetric division and redistribution (POLAR) can confine BIN2 and AtSK12 to the cytosol to drive ACD. After ACDs, BIN2 and AtSK12 phosphorylate and dissociate from POLAR (Houbaert et al., 2018).

Like upstream regulation, GSK3s also intensively modulate downstream substrates through phosphorylation to accomplish their functions in regulating plant development and stress responses. GSK3s can phosphorylate a series of substrates including transcription factors, cofactors, kinases, scaffold proteins, cytoskeleton proteins, cyclins, metabolic enzymes, and components of the ubiquitin-proteasome system (UPS). The role of GSK3s in modulating proteins including kinases, scaffold proteins, cytoskeleton proteins, cyclins, metabolic enzymes, and components of the ubiquitin-proteasome system (UPS), has been extensively discussed in several excellent recent reviews (Youn and Kim, 2015; Li et al., 2021; Mao et al., 2021; Zolkiewicz and Gruszka, 2022). In this section, we mainly focus on the discoveries about effects of GSK3s on the transcription factors and cofactors, which play essential roles in modulating stress responsive gene expression.

GSK3s-mediated phosphorylation of the transcription factors and cofactors, eventually affects the protein stability, localization, and activity of these substrates. Recent reports have revealed that GSK3-mediated phosphorylation not only promotes the degradation of the substrates but also stabilizes downstream targets. During stomatal patterning, BIN2 and MAPKs phosphorylate and promote the degradation of speechless (SPCH), which is a bHLH transcription factor in the nucleus required for stomatal initiation (Gudesblat et al., 2012). Phosphorylation and degradation of phytochrome interacting factor 3 (PIF3) and 4 (PIF4) are facilitated by BIN2 to control skotomorphogenesis and hypocotyl elongation, respectively (Bernardo-García et al., 2014; Ling et al., 2017). In plants, GSK3s also stabilizes transcription factors through phosphorylation. A series of transcription factors, such as myeloblastosis family transcription factor-like (MYBL2; a MYB transcription factor), ATBS1-interacting factor 2 (AIF2; a bHLH transcription factor), abscisic acid insensitive 5 (ABI5; a bZIP transcription factor), upbeat 1 (UPB1; a bHLH transcription factor), TINY (AP2 family transcription factor) and RD26 (NAC family transcription factor), are phosphorylated and stabilized by BIN2 (Ye et al., 2012; Hu and Yu, 2014; Kim et al., 2017; Jiang et al., 2019; Xie et al., 2019; Li et al., 2020c). However, the underlying molecular mechanisms of substrates degradation or accumulation associated with BIN2-catalyzed phosphorylation have not been investigated.GSK3s-mediated regulation of subcellular localization in substrates is ubiquitous in plants. BIN2-catalyzed phosphorylation of BZR1/BZR2 promotes their binding to the 14-3-3 proteins, resulting in cytoplasmic retention and inhibition of BR-regulated gene expression (Gampala et al., 2007). In rice, phosphorylation of OsBZR1 and transcription factor ovate family protein 8 (OFP8) by OsGSK2 also results in their nuclear export (Yang et al., 2016). Furthermore, GSK3s-mediated phosphorylation can restrict the nuclear localization of their substrates. For example, the bHLH transcription factor enhancer of glabra 3 (EGL3) is phosphorylated by BIN2, thus remaining nuclear localization and participating in the BR-regulated root epidermal cell patterning (Cheng et al., 2014).

In addition, GSK3s have been early documented to affect the activity of transcription factors or cofactors. For example, phosphorylation of auxin response factors ARF7 and ARF19 by BIN2 suppresses their interaction with the AUX/IAAs repressors and enhances auxin signaling export in enhancing lateral root development (Cho et al., 2014). AtSK11 and AtSK12 phosphorylate the WD40 motif-containing transcriptional cofactor transparent testa glabra 1 (TTG1), influencing the interaction between TTG1 and a MYB domain transcription factor transparent testa 2 (TT2), which eventually influence the carbon partitioning between various parts of developing seed (Li et al., 2018). GSK3s also affect the DNA-binding ability or transcriptional activity of transcription factors, such as BES1, BZR1, ARF2, rice growth regulating factor GRF4 (reduced), golden 2-like 1 (GLK1) and cesta CES (enhanced) (Vert and Chory, 2006; Vert et al., 2008; Khan et al., 2014; Che et al., 2015; Duan et al., 2015; Zhang et al., 2021). The transcription factors or cofactors targeted by GSK3s proteins regulate the expression of downstream plant stress responsive genes, and eventually allow GSK3s to participate in plant response to environmental stresses.

Soil salinization is a growing problem for agriculture worldwide. It is crucial to uncover the key components of the plant salt tolerance network. Recent studies have shown that GSK3s play important roles in regulating plant salinity stress responses. In Arabidopsis, overexpression of AtGSK1 enhances plant resistance to sodium chloride (NaCl) stress, and induces the expression of some NaCl stress-responsive genes including the Ca²⁺-binding protein (AtCP1), the desiccation 29A (RD29A) and the chalcone synthase gene (CHS1), suggesting that AtGSK1 is involved in the signal transduction pathway of NaCl stress responses (Piao et al., 2001). BIN2 is found to function as a molecular switch between plant salt stress response and growth through phosphorylating and affecting the activity of calcium sensors, such as SOS3, SCaBP8, and SOS2 (Li et al., 2020b). Heat shock proteins (HSPs), a group of highly conserved chaperone protein, are involved in the regulation of plant responses to salinity. HSP90 regulates the activity of the BIN2 kinase by modulating its subcellular localization (Samakovli et al., 2014). The co-chaperone heat shock protein (HSP)70-HSP90 organizing protein 1 (HOP1) and 2 (HOP2) affect HSP90-BIN2 interaction and are involved in plant salt tolerance by affecting BR signaling (Zhang et al., 2022). In rice, knockout mutants of OsGSK1 showed enhanced tolerance to salt stress (Koh et al., 2007). Heterologous overexpression of a GSK3 gene from soybean GmBIN2 enhanced plant tolerance to salt in Arabidopsis. GSK3-like kinase GmSK2-8 is strongly induced in soybean under salt stress. GmSK2-8 interactes with two nodulation signaling pathway 1 (GmNSP1) proteins GmNSP1a and GmNSP1b, which are key transcription factors involved in legume-rhizobium symbiosis. GmSK2-8 phosphorylates the GmNSP1a protein, and thus suppresses nodule formation under salt stress (He et al., 2021). It is reported that the GSK3 family shows altered expression in response to salt stress treatments in Solanum tuberosum L. Overexpression of StSK21 provides enhanced sensitivity to salt stress in Arabidopsis (Huang et al., 2021). In addition, the response of the GSK3 family to salt stress is also identified in cotton (Wang et al., 2018b). In barley (Hordeum vulgare L.), the RNAi-mediated silencing of the target HvGSK1.1 gene enhances the BR-dependent signaling, and generates plants with improved agricultural traits under salt stress conditions (Kloc et al., 2020). Researches show that the wheat (Triticum aestivum L.) TaGSK1 gene is induced by NaCl stress, and expresses more strongly in salt-stress resistant lines than in salt-stress sensitive lines (Chen et al., 2003). From the above results we can see that some of the GSK3s proteins function as positive regulators of plant salt tolerance such as in soybean and wheat, while the GSK3s proteins reported in rice, potato, barley negatively regulate plant salt tolerance. In addition, different members in the Arabidopsis and cotton GSK3s family proteins show different response to the salt treatment. These results indicate that molecular mechanism of the GSK3s proteins in response to salt stress in different species or different members in the protein family is specific.

Drought stress causes a decline in the quantity of crop yields, and has become more accentuated recently due to climatic change. The GSK3 proteins are involved in drought stress tolerance. The stress-responsive NAC transcription factor RD26 participates in the interaction between growth and drought stress signaling by the phosphorylation of BIN2. BIN2 directly interacted with and phosphorylated RD26, which is required for RD26 transcriptional activation on drought-responsive genes (Jiang et al., 2019). Arabidopsis stress-inducible AP2/ERF transcription factor TINY positively regulates drought responses by activating drought-responsive genes. The BR negative regulator BIN2 phosphorylates and stabilizes TINY, which provides a mechanism for BR-mediated down-regulation of TINY to prevent activation of stress responses (Xie et al., 2019).

GSK3s are also involved in osmotic, low temperature and low phosphate stresses. AtSK11 and AtSK12 are involved in the mild osmotic stress (-0.4 MPa) response in Arabidopsis thaliana. They negatively regulate the induction of root growth in response to mild osmotic stress (Dong et al., 2020). Plant U-box 40 protein (PUB40) is a ubiquitin E3 ligase and mediates the proteasomal degradation of BZR1 in Arabidopsis. The interaction between PUB40 and BZR1 influences root tolerance to the low phosphate stress. BIN2 phosphorylates and stabilizes PUB40 to promote BZR1 degradation and enhances the interaction between PUB40 and BZR1, thus reducing plant tolerance to the low phosphate stress (Kim et al., 2019). Moreover, BIN2 is also regulated by the E3 ubiquitin ligase KIB1 as is mentioned above (Zhu et al., 2017). These results indicate that the E3 ubiquitin ligases play pivotal roles in the BIN2-mediated regulation of BR signaling pathway transduction. BR signaling and downstream transcriptional cascades are reported to be involved in regulating plant cold tolerance (Khan et al., 2014; Eremina et al., 2016; Li et al., 2017). As repressors of BR signaling, BIN2 and its homologs mediate the phosphorylation of BZR1 and inducer of CBF expression 1 (ICE1) to facilitate their degradation, and thus affect downstream transcriptional cascades related to cold stress response (Ye et al., 2019). BIN2 negatively regulates chilling tolerance in tomato. BIN2 also regulates the accumulation of BZR1, which controls the expression of ABA biosynthesis gene 9-cis-epoxycarotenoid dioxygenase 1 (NCED1) in tomato. These results demonstrate that BR signaling positively regulates chilling tolerance via ABA biosynthesis in tomato (An et al., 2023). Moreover, cesta (CES) is a bHLH transcription factor of BR signaling and affects plant cold tolerance (Eremina et al., 2016). CES degradation is also promoted by BIN2-mediated phosphorylation, but whether and how this protein turnover is associated in vivo with cold stress response should be further explored.

Biotic stresses including bacteria, fungi, oomycetes, viruses and insects, wreak havoc on agricultural products worldwide and increase the risk of starvation in many areas. Advancing researches have shown that GSK3s play multifaceted roles in plant responses to various kinds of biotic stresses and is tightly regulated during plant response to pathogen infection (Wrzaczek et al., 2007). It is reported that the GSK3-like kinase ASKα is identified as a positive regulator of plant immune signaling (Stampfl et al., 2016). Loss of ASKα attenuates, whereas its overexpression enhances, diverse pattern-triggered immunity (PTI) responses, which is the first layer of plant immunity against pathogenic microbes. The bacterial pathogen Pseudomonas syringae glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the oxidative pentose phosphate pathway and is phosphorylated by ASKα. Rice black-streaked dwarf virus (RBSDV), a double-stranded RNA virus, causes acute growth abnormalities in plants and results in serious yield losses in cereal crops. Plants over-expressing OsGSK2 display milder symptoms than the control, suggesting a positive role of OsGSK2 in suppressing RBSDV infection in rice (He et al., 2020). Geminiviruses transmitted by whiteflies cause severe developmental abnormalities in plants. The C4 protein encoded by geminiviruses induces abnormal cell division that determines viral symptoms. The tomato leaf curl Yunnan virus (TLCYnV) C4 protein interacts with and affects the subcellular localization of Nicotiana benthamiana GSK3 protein NbSKη, eventually impairing GSK3-mediated degradation of cell division associated protein NbCycD1;1 (Mei et al., 2018; Mei et al., 2020). In Medicago sativa, the GSK3-like kinase MsK1 is important for innate immunity and limits the severity of infection caused by virulent bacterial pathogen P. syringae. MsK1 activity is downregulated by the elicitor cellulase. Cellulase treatment also triggeres the degradation of the MsK1 protein (Wrzaczek et al., 2007). In Lotus japonicu, nodulation is mainly formed through the symbiotic nitrogen-fixing bacterium Mesorhizobium loti infection. Studies show that lotus SHAGGY-like kinase 1 (LSK1) is required to suppress nodulation (Garagounis et al., 2019; Solovou et al., 2021). Ralstonia solanacearum is a devastating soil-borne bacterium that causes wilting disease in over 200 economically-important plant species. The GSK3/SHAGGY-like kinase CaSK23 negatively regulates plant response to R. solanacearum attack in Capsicum annuum (Qiu et al., 2018). We also find that BIN2 negatively regulates plant defence against Verticillium dahliae in Arabidopsis and cotton, consistent with others reports on the regulatory function of GSK3s proteins (Song et al., 2021).

Autophagy is a highly conserved quality control mechanism in which harmful or unwanted cellular components are delivered into lytic vacuoles for recycling, and can promote plant resistance to various stresses (Signorelli et al., 2019; Yang et al., 2020). The GSK3-like kinase is one of the key regulators of autophagy (Nolan et al., 2017). The dominant suppressor of KAR 2 (DSK2) is a ubiquitin receptor protein that targets BES1 to the autophagy pathway by interacting with ATG8, a ubiquitin-like protein that directs autophagosome formation and cargo recruitment. BIN2 can phosphorylate DSK2 and promote DSK2-ATG8 interaction, which ultimately targets BES1 for degradation (Nolan et al., 2017). Reactive oxygen species (ROS) play a key role in the acclimation process of plants to abiotic and biotic stresses (Choudhury et al., 2017). Researchers have found that the interaction of BIN2 and BES1 is dependent on oxygen, which can directly modify BIN2 (Song et al., 2019). In Arabidopsis, AtASKα regulates plant stress tolerance by activating glc-6-phosphate dehydrogenase (G6PD) responsible for maintaining cellular redox balance. Plants overexpressing ASKα have low levels of ROS in stress responses and are more tolerant to salt stress (Dal Santo et al., 2012; Stampfl et al., 2016). The target of rapamycin (TOR) is an atypical Ser/Thr protein kinase that is evolutionally conserved among yeasts, plants, and mammals. TOR signaling is involved in plant adaptation to nutrient deficiency and various abiotic stresses (Fu et al., 2020). The TOR downstream effector S6K2 can phosphorylate BIN2 protein, suggesting that BIN2 acts as a downstream effector of TOR signaling (Xiong et al., 2017). The egulatory-associated protein of TOR 1B (RAPTOR1B) is an important component of plant TOR complex. In the absence of BR, the BIN2 kinase directly phosphorylates and inhibits the activity of RAPTOR1B. Furthermore, autophagy is negatively regulated by TOR. Phosphorylation of RAPTOR1B by BIN2 thus activates the autophagy pathway. In the presence of BR, the inhibition effect of BIN2 on RAPTOR1B is attenuated, and results in increased TOR activity and ATG13a phosphorylation, and decreased autophagy activity (Liao et al., 2022). These results suggest that BIN2 functions as a key hub in the crosstalk between TOR and BR signaling pathways. Further studies are required to fully elucidate whether and how BIN2 interacts with other components of TOR signaling pathway.