Hormone modulates protein dynamics to regulate plant growth

Endosomal traffic in the plant endomembrane system is a fundamental and complex process that controls many essential cellular, developmental, and physiological functions in plants, including cellular polarization, cytokinesis, metal ion homeostasis, pathogen defense, and hormone transport (1). The secretory and endocytic pathways represent two major anterograde protein transport routes for protein delivery into the vacuole in plant cells (Fig. 1A). In the secretory pathway, transportation of newly synthesized soluble vacuolar cargo proteins is mediated by the vacuolar sorting receptors (VSRs) (2). After delivery of the soluble cargos into an intermediate compartment, receptors are recycled by the attachment of conserved sorting nexins (SNXs) and the core subunits of retromer complex (VPS26, VPS29, and VPS35) to the membrane. Nevertheless, the precise localization of the SNXs and the retromer subunits, as well as the identity of the organelles from which VSRs are recycled, remains in debate (3, 4). During endocytosis, plasma membrane (PM) proteins are internalized and delivered into the trans-Golgi network (TGN)/early endosomes in plants (5). Ubiquitinated PM proteins are further sorted into the intralumenal vesicles of multivesicular bodies, previously identified as a prevacuolar compartment (6), by the endosomal sorting complex required for transport machinery for vacuolar degradation (7). Alternatively, PM proteins without a ubiquitin tag (or after removal of ubiquitin by a deubiquitinating enzyme) are recycled back to the PM from the TGN or recycling endosome (RE) (1, 8). In plants, numerous PM proteins undergo endocytosis and endosomal recycling, with the PIN-FORMED (PINs) transporters for the plant hormone auxin being the most studied (9). Polarized PM localization of PINs has a profound developmental importance and is tightly regulated by multiple endosomal trafficking routes, including endocytosis, endosomal recycling, and vacuolar degradation. PINs are internalized via clathrin-mediated endocytosis and then recycled back to the PM via the GNOM-positive putative RE or through a retromer-dependent recycling route (4, 8–10). Notably, several major phytohormones, such as auxin itself (9), cytokinins, and gibberellic acid (GA), have been reported to regulate the abundance and polar distribution of PINs by modulating their endocytosis and endosomal recycling to achieve feedback regulation or hormone cross-talk with auxin signaling (11–13). However, the precise mechanisms underlying hormone action on PIN trafficking and recycling remain elusive. In PNAS, Salanenka et al. (14) have delved into the cellular and molecular mechanisms by which GA redirects polar trafficking of PINs in regulating plant growth and development.

Fig. 1.

Plant endosomal trafficking machinery and the mode of GA action on PIN dynamics in plants. (A) Known endosomal trafficking machinery in plant cells. The plant retromer complex has been postulated to recycle the receptor VSRs from either (i) MVB/PVC/LE) or (ii) TGN/EE. The polar localization of PIN2 at the PM is regulated by endocytosis, endosomal recycling, and vacuolar degradation. (B) Cellular mechanism and key molecular components of the GA-dependent trafficking of PINs. The GA signaling pathway branches at the level of DELLA proteins via their interaction with PFD proteins and the downstream CLASP1, which regulates retromer activity and redirects PIN traffic from the vacuolar pathway to the PM. COPI, coat protein I; COPII, coat protein II; EE, early endosome; ESCRT, endosomal sorting complex required for transport; GID: gibberellin-insensitive 1; MVB/PVC/LE, multivesicular body/prevacuolar compartment/late endosome; PIN, PIN auxin efflux carriers; TF, transcription factor.

In nature, plants have a sessile lifestyle and are constantly adapting to developmental and environmental changes. To trigger and govern diverse physiological processes during development, as well as in response to stresses, plants produce low-abundance bioactive signaling compounds: hormones. GA, similar to auxin, mediates the ubiquitination and degradation of nuclear growth repressors through an intracellular receptor, effectively regulating plant growth and development. In sufficient amounts, bioactive GA binds to GA-receptor gibberellin-insensitive 1 (GID1) and promotes its interaction with the nuclear repressor DELLAs and the E3 ubiquitin ligase complex SCF^SLY1, leading to the ubiquitination and degradation of DELLAs. The reduction of DELLAs then releases transcription factors and activates the expression of GA-responsive genes. Simultaneously, the cochaperone tubulin folding prefoldins (PFDs) complex is released from DELLAs, allowing PFDs to translocate from the nucleus into the cytoplasm and making them presumably available for regulating cytoskeletal functions (Fig. 1B). Besides its predominant roles in specified signaling pathways, extensive studies have revealed that GA also interacts synergistically with the auxin signaling pathway to orchestrate a wide-range of coherent growth decisions. In roots, auxin activates the transcription of the rate-limiting factors in GA biosynthesis while enhancing the degradation of the DELLA repressors to reinforce GA responses, ensuring proper cell expansion and tissue differentiation.

Conversely, GA also modulates auxin-regulated organ formation and gravitropism through regulating PIN distribution. With insufficient GA signaling, the polarized PM-localized PINs are reduced dramatically by virtue of vacuolar degradation (12, 13). With GA accumulated, PINs enrich at the PM with a decrement in vacuolar trafficking (13) (Fig. 1B). However, the cellular mechanisms and key molecular machinery mediating the GA-dependent trafficking of auxin transporters have remained obscure.

To address this issue, Salanenka et al. (14) have performed live-cell imaging and utilized photoconvertible PIN2 to dissect the GA-targeted membrane trafficking routes that regulate PIN abundances at the PM. Interestingly, depletion of PIN2-Dendra from the PM was not affected by GA availability, ruling out the possibility that GA inhibits endocytosis of PINs. Notably, green signal recovery of PIN2-Dendra at the PM is abundant in GA-optimal conditions but severely reduced upon treatment with the GA synthesis inhibitor paclobutrazol (PAC), while fluorescence recovery after photobleaching analysis also shows strong decrements of PM-localized PIN2-mCherry recovery in GA-deficient conditions, suggesting that the GA signaling modulates processes balancing PIN2 vacuolar degradation and recycling back to the PM.

To further investigate whether the regulation of PIN2 recycling and degradation is dependent on transcription or de novo protein synthesis, Salanenka et al. (14) show that PIN2 transcription was not affected by PAC or GA treatments, while PIN2 promoter activity showed no difference between GA-sufficient or -deficient conditions. In combination with PAC, cycloheximide treatment of PIN2-GFP exhibits additional decrement of PIN2 at the PM, but the effects can be reversed by GA. These results indicate that GA-dependent incremental PIN2 delivery to the PM is not due to the regulation of PIN2 transcription and does not require de novo protein synthesis. The authors further tested the specificity of GA effects on PIN2 vacuolar degradation using a more sensitive evaluation method under dark conditions vs. conditions in a previous study (13) and showed that the vacuolar delivery of several other PM proteins, and not only PIN2, is also enhanced in the GA-deficient conditions. These observations indicate that GA utilizes a nontranscriptional mechanism to balance the vacuolar trafficking of PM cargos and their recycling back to the PM.

Early studies indicated that the polar localization of PINs at the PM is modulated by SNX1-bearing endosomes (10). The SNX1-containing endosomes are associated with microtubules (MTs) via the interaction of SNX1 with cytoplasmic linker-associated protein (CLASP) (15), which stabilizes MT activity. However, whether SNX1 and CLASP-dependent protein retrieval is required for GA action on PIN2 trafficking is under investigation. To answer this unresolved question, Salanenka et al. (14) performed genetic and pharmacological analysis. Surprisingly, GA action on PIN2 recycling

In PNAS, Salanenka et al. have delved into the cellular and molecular mechanisms by which GA redirects polar trafficking of PINs in regulating plant growth and development.

is less sensitive in an snx1 mutant and another retromer complex mutant, pat3-3. As SNX1 and CLASP have been shown to interact physically and genetically to modulate PIN2 endosomal trafficking (15), the authors included a clasp1 mutant to investigate the potential function of CLASP on GA-mediated PIN2 trafficking. Similar to SNX1 and the retromer components, the clasp1 mutant exhibited less sensitivity to PAC-mediated PIN2 depletion from the PM and GA application was insufficient to restore PIN2 at the PM. These results suggest that both the retromer complex and CLASP activity are required for GA-dependent redirection of the PIN2 trafficking to the PM. To assess the GA effect on the SNX1-positive endosomal compartment as well as CLASP distribution, the authors performed various chemical treatments. Surprisingly, abnormally enlarged SNX1-GFP bodies were observed upon PAC treatments. Cell edge-localized CLASP1 distribution also changes significantly in PAC-treated roots in a similar pattern upon oryzalin application. To strengthen the point that the effects of GA on PIN2 trafficking depend upon intact and dynamic MTs, Salanenka et al. (14) performed drug and genetic interferences on MTs. Both oryzalin and taxol, drugs that affect MT polymerization and stabilization, respectively, caused a decrease in PIN2 incidence at the PM, and the effects were irreversible by GA. To avoid the side effect of drug treatments, a katanin 1 (ktn1) mutant that is known to be defective in the formation of cortical MT arrays was used for the genetic analysis. In agreement with the drug treatments, GA was not able to restore PIN2 incidence at the PM in ktn1 roots pretreated with PAC. Genetic and pharmacological analyses suggest that GA balances PIN2 endosomal recycling and vacuolar degradation through modulating the retromer complex and its associated protein CLASP1 as well as MTs.

Finally, Salanenka et al. (14) assessed which GA signaling mechanism acts upstream of MTs and retromer to regulate PIN trafficking. Both quintuple della knockout mutants and the dominant-negative DELLA mutant gaiΔ17 were ineffective in restoring PIN2 at the PM upon GA application. Because PFDs were shown to interact with DELLAs and are known to control MT folding and dynamics in higher organisms, the authors tested PFD mutants under GA sufficient and insufficient conditions. The mutant pfd5pfd6 exhibited a similar behavior as the dominant-negative DELLA mutant, showing ineffective restoration of PIN2 proteins at the PM upon GA treatment in the PAC-pretreated roots. These results indicate the requirement of properly functioning DELLA and its interactor PFDs for GA-mediated PIN2 trafficking.

In summary, the work by Salanenka et al. (14) elaborates a mechanism by which GA regulates PM incidence of PIN proteins and other cargos by controlling the MT-dependent endosomal recycling pathway via SNX1 and its interaction partner CLASP. This GA effect is mediated by canonical components of the GA signaling, the DELLA proteins and their interactor PFDs, which are released from the nucleus following GA-mediated DELLA degradation, thus being free to regulate microtubular functions (Fig. 1B). Since the GA-DELLA pathway has been typically associated with regulation of transcription in the nucleus, these new findings highlight a nontranscriptional branch of this signaling module. They also shed new light on the nontranscriptional spatial-temporal regulation of membrane protein trafficking and function by plant hormones. In recent years, evidence has accumulated to substantiate the effects and roles of plant hormones on endocytic trafficking. While auxin and SA affect PIN trafficking by inhibiting bulk transport depending on clathrin-mediated endocytosis (9), cytokinin specifically regulates the vacuolar degradation of PIN1 in an actin-dependent manner, with enigmatic underlying mechanisms (11). Cytokinin and GA affect plant development in an antagonistic manner, which is tightly controlled at the biosynthetic and signal transductional levels. It remains elusive, but worthwhile, to investigate further whether cytokinin, GA, and other hormones cross-talking with auxin may share and compete in relevant pathways on endosomal trafficking for the recycling and vacuolar degradation of PINs. On the other hand, the aforementioned effect of GA on vacuolar degradation and recycling of PIN2 depend on Brefeldin A (BFA)-sensitive endosomes (13); herein, PIN2 is transported to the apical membrane of epidermal cells in a predominantly BFA-insensitive manner (14, 16). Since BFA also affects conventional post-Golgi protein sorting, PIN trafficking routes are possibly mediated by a different population of endosomes under different conditions. The unresolved mechanisms of polar PIN distribution rekindle interest in the mysterious identity of the REs and their possible employment in recycling of PINs. The enlarged size of SNX-GFP–labeled endosomal compartments upon GA induction observed in this study might serve as a breakthrough point for answering this question. It will be greatly rewarding to further clarify the identity of organelles in the endosomal pathway in terms of hormone-regulated polarized trafficking in plants.