Hormone Modulates Protein Dynamics to Regulate Plant Growth COMMENTARY Yonglun Zenga,1, Jinbo Shena,1, Baiying Lia,1, and Liwen Jianga,B,2

COMMENTARY

Hormone modulates protein dynamics to regulate plant growth COMMENTARY Yonglun Zenga,1, Jinbo Shena,1, Baiying Lia,1, and Liwen Jianga,b,2

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 polariza- tion, 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 deliv- ered into the trans-Golgi network (TGN)/early endosomes in plants (5). Ubiquitinated PM proteins are further sorted into the intralumenal vesicles of multi- Fig. 1. Plant endosomal trafficking machinery and the mode of GA action on PIN vesicular bodies, previously identified as a prevacuolar dynamics in plants. (A) Known endosomal trafficking machinery in plant cells. The compartment (6), by the endosomal sorting complex 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 required for transport machinery for vacuolar degrada- is regulated by endocytosis, endosomal recycling, and vacuolar degradation. tion (7). Alternatively, PM proteins without a ubiquitin (B) Cellular mechanism and key molecular components of the GA-dependent tag (or after removal of ubiquitin by a deubiquitinating trafficking of PINs. The GA signaling pathway branches at the level of DELLA enzyme) are recycled back to the PM from the TGN or proteins via their interaction with PFD proteins and the downstream CLASP1, which regulates retromer activity and redirects PIN traffic from the vacuolar pathway to recycling endosome (RE) (1, 8). In plants, numerous PM the PM. COPI, coat protein I; COPII, coat protein II; EE, early endosome; ESCRT, proteins undergo endocytosis and endosomal recycling, endosomal sorting complex required for transport; GID: gibberellin-insensitive 1; with the PIN-FORMED (PINs) transporters for the plant MVB/PVC/LE, multivesicular body/prevacuolar compartment/late endosome; PIN, hormone auxin being the most studied (9). Polarized PM PIN auxin efflux carriers; TF, transcription factor. localization of PINs has a profound developmental im- portance and is tightly regulated by multiple endosomal back to the PM via the GNOM-positive putative RE trafficking routes, including endocytosis, endosomal re- or through a retromer-dependent recycling route (4, cycling, and vacuolar degradation. PINs are internalized 8–10). Notably, several major phytohormones, such via clathrin-mediated endocytosis and then recycled as auxin itself (9), cytokinins, and gibberellic acid

aSchool of Life Sciences, Centre for Cell and Developmental Biology and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China; and bThe Chinese University of Hong Kong Shenzhen Research Institute, Shenzhen 518057, China Author contributions: Y.Z., J.S., and B.L. analyzed data; and Y.Z., J.S., B.L., and L.J. wrote the paper. The authors declare no conflict of interest. Published under the PNAS license. See companion article 10.1073/pnas.1721760115. 1Y.Z., J.S., and B.L. contributed equally to this work. 2To whom correspondence should be addressed. Email: [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.1802175115 PNAS Latest Articles | 1of3 Downloaded by guest on September 28, 2021 (GA), have been reported to regulate the abundance and polar the regulation of PIN2 transcription and does not require de novo distribution of PINs by modulating their endocytosis and endosomal protein synthesis. The authors further tested the specificity of GA recycling to achieve feedback regulation or hormone cross-talk with effects on PIN2 vacuolar degradation using a more sensitive eval- auxin signaling (11–13). However, the precise mechanisms underly- uation method under dark conditions vs. conditions in a previous ing hormone action on PIN trafficking and recycling remain elusive. study (13) and showed that the vacuolar delivery of several other In PNAS, Salanenka et al. (14) have delved into the cellular and PM proteins, and not only PIN2, is also enhanced in the GA- molecular mechanisms by which GA redirects polar trafficking of deficient conditions. These observations indicate that GA utilizes PINs in regulating plant growth and development. a nontranscriptional mechanism to balance the vacuolar traffick- In nature, plants have a sessile lifestyle and are constantly ing of PM cargos and their recycling back to the PM. adapting to developmental and environmental changes. To Early studies indicated that the polar localization of PINs at the trigger and govern diverse physiological processes during devel- PM is modulated by SNX1-bearing endosomes (10). The SNX1- opment, as well as in response to stresses, plants produce low- containing endosomes are associated with microtubules (MTs) via abundance bioactive signaling compounds: hormones. GA, similar the interaction of SNX1 with cytoplasmic linker-associated protein to auxin, mediates the ubiquitination and degradation of nuclear (CLASP) (15), which stabilizes MT activity. However, whether growth repressors through an intracellular receptor, effectively SNX1 and CLASP-dependent protein retrieval is required for GA regulating plant growth and development. In sufficient amounts, action on PIN2 trafficking is under investigation. To answer this bioactive GA binds to GA-receptor gibberellin-insensitive 1 (GID1) unresolved question, Salanenka et al. (14) performed genetic and and promotes its interaction with the nuclear repressor DELLAs and pharmacological analysis. Surprisingly, GA action on PIN2 recycling the E3 ubiquitin ligase complex SCFSLY1, leading to the ubiquitination and degradation of DELLAs. The reduction of DELLAs then In PNAS, Salanenka et al. have delved into the releases transcription factors and activates the expression of GA- responsive genes. Simultaneously, the cochaperone tubulin folding cellular and molecular mechanisms by which GA prefoldins (PFDs) complex is released from DELLAs, allowing PFDs redirects polar trafficking of PINs in regulating to translocate from the nucleus into the cytoplasm and making them plant growth and development. presumably available for regulating cytoskeletal functions (Fig. 1B). Besides its predominant roles in specified signaling pathways, ex- is less sensitive in an snx1 mutant and another retromer complex tensive studies have revealed that GA also interacts synergistically mutant, pat3-3. As SNX1 and CLASP have been shown to interact with the auxin signaling pathway to orchestrate a wide-range of co- physically and genetically to modulate PIN2 endosomal trafficking herent growth decisions. In roots, auxin activates the transcription of (15), the authors included a clasp1 mutant to investigate the po- the rate-limiting factors in GA biosynthesis while enhancing the deg- tential function of CLASP on GA-mediated PIN2 trafficking. Sim- radation of the DELLA repressors to reinforce GA responses, ensur- clasp1 ing proper cell expansion and tissue differentiation. ilar to SNX1 and the retromer components, the mutant Conversely, GA also modulates auxin-regulated organ forma- exhibited less sensitivity to PAC-mediated PIN2 depletion from tion and gravitropism through regulating PIN distribution. With the PM and GA application was insufficient to restore PIN2 at the insufficient GA signaling, the polarized PM-localized PINs are PM. These results suggest that both the retromer complex and reduced dramatically by virtue of vacuolar degradation (12, 13). CLASP activity are required for GA-dependent redirection of the With GA accumulated, PINs enrich at the PM with a decrement in PIN2 trafficking to the PM. To assess the GA effect on the SNX1- vacuolar trafficking (13) (Fig. 1B). However, the cellular mechanisms positive endosomal compartment as well as CLASP distribution, and key molecular machinery mediating the GA-dependent traffick- the authors performed various chemical treatments. Surprisingly, ing of auxin transporters have remained obscure. abnormally enlarged SNX1-GFP bodies were observed upon To address this issue, Salanenka et al. (14) have performed live- PAC treatments. Cell edge-localized CLASP1 distribution also cell imaging and utilized photoconvertible PIN2 to dissect the changes significantly in PAC-treated roots in a similar pattern upon GA-targeted membrane trafficking routes that regulate PIN abun- oryzalin application. To strengthen the point that the effects of GA on dances at the PM. Interestingly, depletion of PIN2-Dendra from PIN2 trafficking depend upon intact and dynamic MTs, Salanenka the PM was not affected by GA availability, ruling out the possi- et al. (14) performed drug and genetic interferences on MTs. Both bility that GA inhibits endocytosis of PINs. Notably, green signal oryzalin and taxol, drugs that affect MT polymerization and stabili- recovery of PIN2-Dendra at the PM is abundant in GA-optimal zation, respectively, caused a decreaseinPIN2incidenceatthePM, conditions but severely reduced upon treatment with the GA syn- and the effects were irreversible by GA. To avoid the side effect of thesis inhibitor paclobutrazol (PAC), while fluorescence recovery drug treatments, a katanin 1 (ktn1)mutantthatisknowntobedefective after photobleaching analysis also shows strong decrements of in the formation of cortical MT arrays was used for the genetic analysis. PM-localized PIN2-mCherry recovery in GA-deficient conditions, In agreement with the drug treatments, GA was not able to restore suggesting that the GA signaling modulates processes balancing PIN2 incidence at the PM in ktn1 roots pretreated with PAC. Genetic PIN2 vacuolar degradation and recycling back to the PM. and pharmacological analyses suggest that GA balances PIN2 endo- To further investigate whether the regulation of PIN2 recycling somal recycling and vacuolar degradation through modulating the and degradation is dependent on transcription or de novo protein retromer complex and its associated protein CLASP1 as well as MTs. synthesis, Salanenka et al. (14) show that PIN2 transcription was Finally, Salanenka et al. (14) assessed which GA signaling not affected by PAC or GA treatments, while PIN2 promoter ac- mechanism acts upstream of MTs and retromer to regulate PIN traf- tivity showed no difference between GA-sufficient or -deficient ficking. Both quintuple della knockout mutants and the dominant- conditions. In combination with PAC, cycloheximide treatment negative DELLA mutant gaiΔ17 were ineffective in restoring PIN2 of PIN2-GFP exhibits additional decrement of PIN2 at the PM, at the PM upon GA application. Because PFDs were shown to but the effects can be reversed by GA. These results indicate that interact with DELLAs and are known to control MT folding and GA-dependent incremental PIN2 delivery to the PM is not due to dynamics in higher organisms, the authors tested PFD mutants

2of3 | www.pnas.org/cgi/doi/10.1073/pnas.1802175115 Zeng et al. Downloaded by guest on September 28, 2021 under GA sufficient and insufficient conditions. The mutant pfd5pfd6 (11). Cytokinin and GA affect plant development in an antagonis- exhibited a similar behavior as the dominant-negative DELLA mu- tic manner, which is tightly controlled at the biosynthetic and tant, showing ineffective restoration of PIN2 proteins at the PM upon signal transductional levels. It remains elusive, but worthwhile, GA treatment in the PAC-pretreated roots. These results indicate the to investigate further whether cytokinin, GA, and other hormones requirement of properly functioning DELLA and its interactor PFDs cross-talking with auxin may share and compete in relevant path- for GA-mediated PIN2 trafficking. ways on endosomal trafficking for the recycling and vacuolar degra- In summary, the work by Salanenka et al. (14) elaborates a dation of PINs. On the other hand, the aforementioned effect of GA mechanism by which GA regulates PM incidence of PIN proteins on vacuolar degradation and recycling of PIN2 depend on Brefeldin and other cargos by controlling the MT-dependent endosomal A (BFA)-sensitive endosomes (13); herein, PIN2 is transported to the recycling pathway via SNX1 and its interaction partner CLASP. apical membrane of epidermal cells in a predominantly BFA- This GA effect is mediated by canonical components of the GA insensitive manner (14, 16). Since BFA also affects conventional signaling, the DELLA proteins and their interactor PFDs, which are post-Golgi protein sorting, PIN trafficking routes are possibly mediated released from the nucleus following GA-mediated DELLA degra- by a different population of endosomes under different conditions. dation, thus being free to regulate microtubular functions (Fig. The unresolved mechanisms of polar PIN distribution rekindle interest 1B). Since the GA-DELLA pathway has been typically associated in the mysterious identity of the REs and their possible employment in with regulation of transcription in the nucleus, these new findings recycling of PINs. The enlarged size of SNX-GFP–labeled endosomal highlight a nontranscriptional branch of this signaling module. compartments upon GA induction observed in this study might serve They also shed new light on the nontranscriptional spatial-temporal as a breakthrough point for answering this question. It will be greatly regulation of membrane protein trafficking and function by plant rewarding to further clarify the identity of organelles in the endosomal hormones. In recent years, evidence has accumulated to substan- pathway in terms of hormone-regulated polarized trafficking in plants. tiate the effects and roles of plant hormones on endocytic trafficking. While auxin and SA affect PIN trafficking by inhibiting bulk Acknowledgments transport depending on clathrin-mediated endocytosis (9), cyto- Our research is supported by grants from the Research Grants Council of Hong Kong kinin specifically regulates the vacuolar degradation of PIN1 in an (Grants CUHK14130716, CUHK14102417, C4011-14R, C4012-16E, C4002-17G, actin-dependent manner, with enigmatic underlying mechanisms and AoE/M-05/12).

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