A positive feedback regulation of SnRK1 signaling by autophagy in plants

SnRK1,an evolutionarily conserved heterotrimeric kinase complex that acts as a key metabolic sensor in maintaining energy homeostasis in plants,is an important upstream activator of autophagy that serves as a cellular degradation mechanism for the healthy growth of plants.However,whether and how the autophagy pathway is involved in regulating SnRK1 activity remains unknown.In this study,we identified a clade of plant-specific and mitochondria-localized Fcs-like zinc finger(FLZ)proteins as currently unknown ATG8-interacting partners that actively inhibit SnRK1 signaling by repressing the T-loop phosphorylation of the catalyticαsubunits of SnRK1,thereby negatively modulating autophagy and plant tolerance to energy deprivation caused by long-term carbon starvation.Interestingly,these AtFLZs are transcriptionally repressed by low-energy stress,and AtFLZ proteins undergo a selective autophagy-dependent pathway to be delivered to the vacuole for degradation,thereby constituting a positive feedback regulation to relieve their repression of SnRK1 signaling.Bioinformatic analyses show that the ATG8-FLZ-SnRK1 regulatory axis first appears in gymnosperms and seems to be highly conserved during the evolution of seed plants.Consistent with this,depletion of ATG8-interacting ZmFLZ14 confers enhanced tolerance,whereas overexpression of ZmFLZ14 leads to reduced tolerance to energy deprivation in maize.Collectively,our study reveals a previously unknown mechanism by which autophagy contributes to the positive feedback regulation of SnRK1 signaling,thereby enabling plants to better adapt to stressful environments.

ABI5–FLZ13 module transcriptionally represses growth-related genes to delay seed germination in response to ABA

The bZIP transcription factor ABSCISIC ACID INSENSITIVE5(ABI5)is a master regulator of seed germination and post-germinative growth in response to abscisic acid(ABA),but the detailed molecularmechanism by which it represses plant growth remains unclear.In this study,we used proximity labeling to map the neighboring proteome of ABI5 and identified FCS-LIKE ZINC FINGER PROTEIN 13(FLZ13)as a novel ABI5 interaction partner.Phenotypic analysis of flz13 mutants and FLZ13-overexpressing lines demonstrated that FLZ13 acts as a positive regulator of ABA signaling.Transcriptomic analysis revealed that both FLZ13 and ABI5 downregulate the expression of ABA-repressed and growth-related genes involved in chlorophyll biosynthesis,photosynthesis,and cell wall organization,thereby repressing seed germination and seedling establishment in response to ABA.Further genetic analysis showed that FLZ13 and ABI5 function together to regulate seed germination.Collectively,our findings reveal a previously uncharacterized transcriptional regulatorymechanismby which ABA mediates inhibition of seed germination and seedling establishment.

HY5-HDA9 Module Transcriptionally Regulates Plant Autophagy in Response to Light-to-Dark Conversion and Nitrogen Starvation

Light is arguably one of the most important environmental factors that determines virtually all aspects of plant growth and development,but the molecular link between light signaling and the autophagy pathway has not been elucidated in plants.In this study,we demonstrate that autophagy is activated during light-to-dark conversion though transcriptional upregulation of autophagy-related genes(ATGs).We showed that depletion of the ELONGATED HYPOCOTYL 5(HY5),a key component of light signaling,leads to enhanced autophagy activity and resistance to extended darkness and nitrogen starvation treatments,contributing to higher expression oiATGs.HY5 interacts with and recruits HISTONE DEACETYLASE 9(HDA9)to ATG5 and ATG8e loci to repress their expression by deacetylation of the Lys9 and Lys27 of histone 3.Furthermore,we found that both darkness and nitrogen depletion induce the degradation of HY5 via 26S proteasome and the concomitant disassociation of HDA9 from ATG5 and ATG8e loci,leading to their depression and thereby activated autophagy.Genetic analysis further confirmed that HY5 and HDA9 act synergistically and function upstream of the autophagy pathway.Collectively,our study unveils a previously unknown transcriptional and epigenetic network that regulates autophagy in response to light-to-dark conversion and nitrogen starvation in plants.

Biogenesis of Plant Prevacuolar Multivesicular Bodies

Plant prevacuolar compartments （PVCs）, or multivesicular bodies （MVBs）, are single membrane-bound organelles that play important roles in mediating protein trafficking to vacuoles in the secretory pathway. PVC/MVB also serves as a late endosome in the endocytic pathway in plants. Since the plant PVC was iden- tified as an MVB more than 10 years ago,-great progress has been made toward the understanding of PVC/ MVB function and biogenesis in plants. In this review, we first summarize previous research into the iden- tification and characterization of plant PVCs/MVBs, and then highlight recent advances on the mechanisms underlying intraluminal vesicle formation and maturation of plant PVCs/MVBs. In addition, we discuss the possible crosstalk that appears to occur between PVCs/MVBs and autophagosomes during autophagy in plants. Finally, we list some open questions and present future perspectives in this field.

Fast-Suppressor Screening for New Components in Protein Trafficking,Organelle Biogenesis and Silencing Pathway in Arabidopsis thaliana Using DEX-Inducible FREE1-RNAi Plants

Membrane trafficking is essential for plant growth and responses to external signals.The plant unique FYVE domain-containing protein FREE1 is a component of the ESCRT complex（endosomal sorting complex required for transport）.FREE1 plays multiple roles in regulating protein trafficking and organelle biogenesis including the formation of intraluminal vesicles of multivesicular body（MVB）,vacuolar protein transport and vacuole biogenesis,and autophagic degradation.FREE1 knockout plants show defective MVB formation,abnormal vacuolar transport,fragmented vacuoles,accumulated autophagosomes,and seedling lethality.To further uncover the underlying mechanisms of FREE1 function in plants,we performed a forward genetic screen for mutants that suppressed the seedling lethal phenotype of FREE1-RNAi transgenic plants.The obtained mutants are termed as suppressors of free1（sof）.To date,229 putative sof mutants have been identified.Barely detecting of FREE1 protein with M3 plants further identified 84 FREE1-related suppressors.Also145 mutants showing no reduction of FREE1 protein were termed as RNAi-related mutants.Through next-generation sequencing（NGS）of bulked DNA from F2 mapping population of two RNAi-related sof mutants,FREE1-RNAi T-DNA inserted on chromosome 1 was identified and the causal mutation of putative sof mutant is being identified similarly.These FREE1-and RNAi-related sof mutants will be useful tools and resources for illustrating the underlying mechanisms of FREE1 function in intracellular trafficking and organelle biogenesis,as well as for uncovering the new components involved in the regulation of silencing pathways in plants.

ESCRT-dependent vacuolar sorting and degradation of the auxin biosynthetic enzyme YUC1 flavin monooxygenase

YUC flavin monooxygenases catalyze the ratelimiting step of auxin biosynthesis. Here we report the vacuolar targeting and degradation of GFP-YUC1. GFP-YUC1 fusion expressed in Arabidopsis protoplasts or transgenic plants was primarily localized in vacuoles. Surprisingly, we established that GFP-YUC1, a soluble protein, was sorted to vacuoles through the ESCRT pathway, which has long been recognized for sorting and targeting integral membrane proteins. We further show that GFP-YUC1 was ubiquitinated and in this form GFP-YUC1 was targeted for degradation, a process that was also stimulated by elevated auxin levels. Our findings revealed a molecular mechanism of GFP-YUC1 degradation and demonstrate that the ESCRT pathway can recognize both soluble and integral membrane proteins as cargoes.

Protein trafficking in plant cells:tools and markers

Eukaryotic cells consist of numerous membrane-bound organelles,which compartmentalize cellular materials to fulfil a variety of vital functions.In the post-genomic era,it is widely recognized that identification of the subcellular organelle localization and transport mechanisms of the encoded proteins are necessary for a fundamental understanding of their biological functions and theorganization of cellular activity.Multiple experimental approaches are now available to determine the subcellular localizations and dynamics of proteins.In this review,we provide an overview of the current methods and organelle markers for protein subcellular localization and trafficking studies in plants,with a focus on the organelles of the endomembrane system.We also discuss the limitations of each method in terms of protein colocalization studies.

SINAT E3 ligases regulate the stability of the ESCRT component FREE1 in response to iron deficiency in plants

The endosomal sorting complex required for transport (ESCRT) machinery is an ancient, evolutionarily conserved membrane remodeling complex that is es-sential for multivesicular body (MVB) biogenesis in eu-karyotes. FYVE DOMAIN PROTEIN REQUIRED FOR EN-DOSOMAL SORTING 1 (FREE1), which was previously identified as a plant-specific ESCRT component, modu-lates MVB-mediated endosomal sorting and autophagic degradation. Although the basic cellular functions of FREE1 as an ESCRT component have been described, the regulators that control FREE1 turnover remain unknown. Here, we analyzed how FREE1 homeostasis is mediated by the RING-finger E3 ubiquitin ligases, SINA of Arabi-dopsis thaliana (SINATs), in response to iron deficiency. Under iron-deficient growth conditions, SINAT1-4 were induced and ubiquitinated FREE1, thereby promoting its degradation and relieving the repressive effect of FREE1 on iron absorption. By contrast, SINAT5, another SINAT member that lacks ubiquitin ligase activity due to the absence of the RING domain, functions as a protector protein which stabilizes FREE1. Collectively, our findings uncover a hitherto unknown mechanism of homeostatic regulation of FREE1, and demonstrate a unique regu-latory SINAT–FREE1 module that subtly regulates plant response to iron deficiency stress.

The roles of endomembrane trafficking in plant abiotic stress responses

Endomembrane trafficking is a fundamental cellular process in all eukaryotic cells and its regulatory mechanisms have been extensively studied.In plants,the endomembrane trafficking system needs to be constantly adjusted to adapt to the ever-changing environment.Evidence has accumulated supporting the idea that endomembrane trafficking is tightly linked to stress signaling pathways to meet the demands of rapid changes in cellular processes and to ensure the correct delivery of stress-related cargo molecules.However,the underlying mechanisms remain unknown.In this review,we summarize the recent findings on the functional roles of both secretory trafficking and endocytic trafficking in different types of abiotic stresses.We also highlight and discuss the unique properties of specific regulatory molecules beyond their conventional functions in endosomal trafficking during plant growth under stress conditions.

植物细胞自噬基因的功能与转录调控机制

自噬(autophagy)是真核生物长期进化形成的一种高度保守的细胞内物质降解和周转途径,通过形成双层膜结构的自噬体将包裹其中的待降解大分子物质,如受损伤的蛋白质、蛋白质复合物和细胞器,运送至液泡或溶酶体进行降解并产生可循环利用的降解产物。细胞自噬在植物生长发育和环境应答等过程中发挥重要作用。在拟南芥(Arabidopsisthaliana)和水稻(Oryza sativa)等模式植物中已鉴定到40多个自噬基因,并发现其中多个基因在植物叶片衰老、种子成熟等发育阶段以及营养饥饿、干旱和病原菌侵染等逆境胁迫响应过程中显著上调表达,但具体的转录激活或抑制机制有待阐明。该文综述了自噬基因在植物生长发育和胁迫应答过程中的功能与转录调控网络。

HY5-HDA9 Module Transcriptionally Regulates Plant Autophagy in Response to Light-to-Dark Conversion and Nitrogen Starvation

(Molecular Plant 13,515-531;March 2020)After publication of our original manuscript,we became aware of errors in Figure 4.During the preparation of Figure 4C in this article as originally published,we inadvertently duplicated the image of hda9-1(MS-N)as that of the hda9-1-C(MS+N).Also,in Figure 4H,the image of pUBQ10:GFP-ATG8a/hda9-1(MS+L)was mistakenly a duplicate of pUBQ10:GFP-ATG8a/WT(MS+L)shown in Figure S7C.A corrected version of Figure 4 is shown below.The scientific conclusions of this article have not been affected by this correction.The authors apologize for not detecting this error prior to publication and for any inconvenience that may have been caused.

AtBRO1 Functions in ESCRT-Ⅰ Complex to Regulate Multivesicular Body Protein Sorting

Dear Editor Membrane proteins destined for degradation in eukaryotic cells are tagged by ubiquitin （Ub） for further sorting by the endosomat sorting complex required for transport （ESCRT） machinery into the intraluminal vesicles （tLVs） of multivesicular bodies （MVBs） or prevacuolar compartments （PVCs） for subsequent lysosomal/vacuolar degradation （Henne et al., 2011; Schmidt and Teis, 2012）. The ESCRT machinery comprises a cascade of several distinct complexes （ESCRTs -0, -Ⅰ, -Ⅱ, and -Ⅲ, and Vps4 complex）.