Modulation of abscisic acid signaling via endosomal TOL proteins.

The phytohormone abscisic acid (ABA) functions in the control of plant stress responses, particularly in drought stress. A significant mechanism in attenuating and terminating ABA signals involves regulated protein turnover, with certain ABA receptors, despite their main presence in the cytosol and nucleus, subjected to vacuolar degradation via the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Collectively our findings show that discrete TOM1-LIKE (TOL) proteins, which are functional ESCRT-0 complex substitutes in plants, affect the trafficking for degradation of core components of the ABA signaling and transport machinery. TOL2,3,5 and 6 modulate ABA signaling where they function additively in degradation of ubiquitinated ABA receptors and transporters. TOLs colocalize with their cargo in different endocytic compartments in the root epidermis and in guard cells of stomata, where they potentially function in ABA-controlled stomatal aperture. Although the tol2/3/5/6 quadruple mutant plant line is significantly more drought-tolerant and has a higher ABA sensitivity than control plant lines, it has no obvious growth or development phenotype under standard conditions, making the TOL genes ideal candidates for engineering to improved plant performance.

Recent advances in cellular degradation and nuclear control of leaf senescence.

Senescence is the final stage of plant growth and development, and is a highly regulated process at the molecular, cellular, and organismal levels. When triggered by age, hormonal, or environmental cues, plants actively adjust their metabolism and gene expression to execute the progression of senescence. Regulation of senescence is vital for the reallocation of nutrients to sink organs, to ensure reproductive success and adaptations to stresses. Identification and characterization of hallmarks of leaf senescence are of great importance for understanding the molecular regulatory mechanisms of plant senescence, and breeding future crops with more desirable senescence traits. Tremendous progress has been made in elucidating the genetic network underpinning the metabolic and cellular changes in leaf senescence. In this review, we focus on three hallmarks of leaf senescence - chlorophyll and chloroplast degradation, loss of proteostasis, and activation of senescence-associated genes (SAGs), and discuss recent findings of the molecular players and the crosstalk of senescence pathways.

ESCRT-III component OsSNF7.2 modulates leaf rolling by trafficking and endosomal degradation of auxin biosynthetic enzyme OsYUC8 in rice.

The endosomal sorting complex required for transport (ESCRT) is highly conserved in eukaryotic cells and plays an essential role in the biogenesis of multivesicular bodies and cargo degradation to the plant vacuole or lysosomes. Although ESCRT components affect a variety of plant growth and development processes, their impact on leaf development is rarely reported. Here, we found that OsSNF7.2, an ESCRT-III component, controls leaf rolling in rice (Oryza sativa). The Ossnf7.2 mutant rolled leaf 17 (rl17) has adaxially rolled leaves due to the decreased number and size of the bulliform cells. OsSNF7.2 is expressed ubiquitously in all tissues, and its protein is localized in the endosomal compartments. OsSNF7.2 homologs, including OsSNF7, OsSNF7.3, and OsSNF7.4, can physically interact with OsSNF7.2, but their single mutation did not result in leaf rolling. Other ESCRT complex subunits, namely OsVPS20, OsVPS24, and OsBRO1, also interact with OsSNF7.2. Further assays revealed that OsSNF7.2 interacts with OsYUC8 and aids its vacuolar degradation. Both Osyuc8 and rl17 Osyuc8 showed rolled leaves, indicating that OsYUC8 and OsSNF7.2 function in the same pathway, conferring leaf development. This study reveals a new biological function for the ESCRT-III components, and provides new insights into the molecular mechanisms underlying leaf rolling.

A plant-unique ESCRT component, FYVE4, regulates multivesicular endosome biogenesis and plant growth.

During evolution, land plants generated unique proteins that participate in endosomal sorting and multivesicular endosome (MVE) biogenesis, many of them with specific phosphoinositide-binding capabilities. Nonetheless, the function of most plant phosphoinositide-binding proteins in endosomal trafficking remains elusive. Here, we analysed several Arabidopsis mutants lacking predicted phosphoinositide-binding proteins and first identified fyve4-1 as a mutant with a hypersensitive response to high-boron conditions and defects in degradative vacuolar sorting of membrane proteins such as the borate exporter BOR1-GFP. FYVE4 encodes a plant-unique, FYVE domain-containing protein that interacts with SNF7, a core component of ESCRT-III (Endosomal Sorting Complex Required for Transport III). FYVE4 affects the membrane association of the late-acting ESCRT components SNF7 and VPS4, and modulates the formation of intraluminal vesicles (ILVs) inside MVEs. The critical function of FYVE4 in the ESCRT pathway was further demonstrated by the strong genetic interactions with SNF7B and LIP5. Although the fyve4-1, snf7b and lip5 single mutants were viable, the fyve4-1 snf7b and fyve4-1 lip5 double mutants were seedling lethal, with strong defects in MVE biogenesis and vacuolar sorting of ubiquitinated membrane proteins. Taken together, we identified FYVE4 as a novel plant endosomal regulator, which functions in ESCRTing pathway to regulate MVE biogenesis.

DGK5ß-derived phosphatidic acid regulates ROS production in plant immunity by stabilizing NADPH oxidase.

In plant immunity, phosphatidic acid (PA) regulates reactive oxygen species (ROS) by binding to respiratory burst oxidase homolog D (RBOHD), an NADPH oxidase responsible for ROS production. Here, we analyze the influence of PA binding on RBOHD activity and the mechanism of RBOHD-bound PA generation. PA binding enhances RBOHD protein stability by inhibiting vacuolar degradation, thereby increasing chitin-induced ROS production. Mutations in diacylglycerol kinase 5 (DGK5), which phosphorylates diacylglycerol to produce PA, impair chitin-induced PA and ROS production. The DGK5 transcript DGK5ß (but not DGK5α) complements reduced PA and ROS production in dgk5-1 mutants, as well as resistance to Botrytis cinerea. Phosphorylation of S506 residue in the C-terminal calmodulin-binding domain of DGK5ß contributes to the activation of DGK5ß to produce PA. These findings suggest that DGK5ß-derived PA regulates ROS production by inhibiting RBOHD protein degradation, elucidating the role of PA-ROS interplay in immune response regulation.

Crossing paths: recent insights in the interplay between autophagy and intracellular trafficking in plants.

Autophagy fulfills a crucial role in plant cellular homeostasis by recycling diverse cellular components ranging from protein complexes to whole organelles. Autophagy cargos are shuttled to the vacuole for degradation, thereby completing the recycling process. Canonical autophagy requires the lipidation and insertion of ATG8 proteins into double-membrane structures, termed autophagosomes, which engulf the cargo to be degraded. As such, the autophagy pathway actively contributes to intracellular membrane trafficking. Yet, the autophagic process is not fully considered a bona fide component of the canonical membrane trafficking pathway. However, recent findings have started to pinpoint the interconnection between classical membrane trafficking pathways and autophagy. This review details the latest advances in our comprehension of the interplay between these two pathways. Understanding the overlap between autophagy and canonical membrane trafficking pathways is important to illuminate the inner workings of both pathways in plant cells.

Microscopic Imaging of Endosomal Trafficking of ABA Receptors.

The abscisic acid (ABA) is a key hormone for stress tolerance. The balance between growth/development and stress responses is crucial for the optimal course of plant life meaning that plants need to control the timing and extent of ABA pathway activation. In this regard, protein turnover regulation by means of both the ubiquitin-proteasome system (UPS) and non-26S proteasome endomembrane trafficking pathways, plays a critical role in the regulation of ABA signaling activation and deactivation. Over the last few years, the ubiquitination of ABA receptors PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) at the plasma membrane by the RING between RING fingers (RBR)-type E3 ligase RING FINGER OF SEED LONGEVITY1 (RSL1) triggering their internalization through the clathrin-mediated endocytosis (CME) pathway, followed by their endosomal trafficking and delivery to the vacuole for degradation, was reported. For this process, the direct role of some components of the endosomal sorting complex required for transport (ESCRT) machinery, that is, FYVE DOMAIN-CONTAINING PROTEIN 1 (FYVE1)/FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FREE1) and VACUOLAR PROTEIN SORTING23A (VPS23A) members of ESCRT-I complex, and ALG-2 INTERACTING PROTEIN-X (ALIX) associated protein of ESCRT-III, was reported. In this chapter, we will detail two methods for imaging endosomal trafficking of ABA receptor proteins by confocal microscopy: (a) colocalization of GFP-PYL4 (also known as RCAR10) and CLATHRIN LIGHT CHAIN 2 (CLC2)-mOrange in clathrin-coated vesicles in Nicotiana benthamiana leaf cells and (b) localization of GFP-PYL4 into Wortmannin (WM)-enlarged late endosomes in Arabidopsis thaliana root cells.

Ubiquitin-independent proteasome system is required for degradation of Arabidopsis COPPER TRANSPORTER 2.

Plants have evolved sophisticated mechanisms to adjust to deficiency or excess of nutrients. Membrane transport proteins play a central role in nutrient uptake from soil. In Arabidopsis thaliana, the COPPER TRANSPORTOR (COPT) family encodes high-affinity copper transporters. COPT2 is transcriptionally regulated in response to changing levels of cellular copper. However, little is known about whether COPT2 activity is subject to multiple levels of regulation. Here, we showed that the plasma membrane-/endoplasmic reticulum-resident COPT2 protein is degraded in response to excess copper. Confocal microscopy analysis together with pharmacological treatment with a vesicle trafficking inhibitor or vacuolar ATPase inhibitor indicated that copper-mediated downregulation of COPT2 is unlikely to be controlled by endosomal recycling and vacuolar system. However, COPT2 protein is stabilized by proteasome inhibition. Through site-directed mutagenesis, we found that COPT2 cannot be ubiquitinated, and lysine residues at the C-terminus is dispensable for copper-induced degradation of COPT2 but required for copper acquisition. Altogether, our findings reveal that unlike many metal transporters in Arabidopsis, COPT2 is a substrate of ubiquitin-independent proteasomal degradation but not of vacuolar proteases. These findings highlight the mechanistic diversity and complexity of plasma membrane transporter degradation.

Analysis of Membrane Proteins Transport from Endosomal Compartments to Vacuoles.

Endocytosis and endosomal trafficking to vacuoles play important roles in regulating the homeostasis of plasma membrane (PM) proteins in plant cells. FREE1 (FYVE domain protein required for endosomal sorting 1) is a plant-unique component of the ESCRT (endosomal sorting complex required for transport) machinery. In free1 mutant plants, PIN-FORMED 2 (PIN2)-GFP was found to mislocalize from the PM to the tonoplast. In this chapter, we describe a detailed protocol for studying vacuolar sorting and degradation of PIN2-GFP by using T-DNA insertional mutants, dexamethasone (DEX) inducible RNAi lines, and other tools, including Fei-Mao (FM) dye staining and dark treatment. By using these methods, we illustrate the endosomal trafficking and vacuolar degradation of PIN2-GFP in plants.

Degradation of Abscisic Acid Receptors Through the Endosomal Pathway.

Turnover of membrane proteins or soluble proteins associated to plasma membrane involves clathrin-mediated endocytosis (CME), endosomal trafficking, and vacuolar degradation. Thus, endocytic and endosomal trafficking regulate numerous physiological processes, including mineral transport, hormone signaling, and pathogen response. Abscisic acid (ABA) signaling is triggered upon ABA perception by PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR), which are soluble proteins that can associate to membrane by interaction with members of the C2-domain ABA-related (CAR) protein family and the RING finger of seed longevity (RSL1) E3 ubiquitin ligase. Half-life of PYR/PYL/RCAR ABA receptors is regulated by ubiquitination and degradation in different subcellular compartments. In particular, pharmacological, genetic, and cell biology approaches have been used to study the different steps that encompass from CME to receptor degradation in the vacuole. In this chapter, we will focus on (1) coimmunoprecipitation (co-IP) assays of clathrin heavy chain (CHC) subunits together with HA-tagged PYL4 ABA receptor and (2) analysis of PYL4 delivery to the vacuole using the TMD23-Ub marker.

The Asparagine-Rich Protein NRP Facilitates the Degradation of the PP6-type Phosphatase FyPP3 to Promote ABA Response in Arabidopsis.

The phytohormone abscisic acid (ABA) plays critical roles in abiotic stress responses and plant development. In germinating seeds, the phytochrome-associated protein phosphatase, FyPP3, negatively regulates ABA signaling by dephosphorylating the transcription factor ABI5. However, whether and how FyPP3 is regulated at the posttranscriptional level remains unclear. Here, we report that an asparagine-rich protein, NRP, interacts with FyPP3 and tethers FyPP3 to SYP41/61-positive endosomes for subsequent degradation in the vacuole. Upon ABA treatment, the expression of NRP was induced and NRP-mediated FyPP3 turnover was accelerated. Consistently, ABA-induced FyPP3 turnover was abolished in an nrp null mutant. On the other hand, FyPP3 can dephosphorylate NRP in vitro, and overexpression of FyPP3 reduced the half-life of NRP in vivo. Genetic analyses showed that NRP has a positive role in ABA-mediated seed germination and gene expression, and that NRP is epistatic to FyPP3. Taken together, our results identify a new regulatory circuit in the ABA signaling network, which links the intracellular trafficking with ABA signaling.

The Multivesicular Body and Autophagosome Pathways in Plants.

In eukaryotic cells, the endomembrane system consists of multiple membrane-bound organelles, which play essential roles in the precise transportation of various cargo proteins. In plant cells, vacuoles are regarded as the terminus of catabolic pathways whereas the selection and transport of vacuolar cargoes are mainly mediated by two types of organelles, multivesicular bodies (MVBs) also termed prevacuolar compartments (PVCs) and autophagosomes. MVBs are single-membrane bound organelles with intraluminal vesicles and mediate the transport between the trans-Golgi network (TGN) and vacuoles, while autophagosomes are double-membrane bound organelles, which mediate cargo delivery to the vacuole for degradation and recycling during autophagy. Great progress has been achieved recently in identification and characterization of the conserved and plant-unique regulators involved in the MVB and autophagosome pathways. In this review, we present an update on the current knowledge of these key regulators and pay special attention to their conserved protein domains. In addition, we discuss the possible interplay between the MVB and autophagosome pathways in regulating vacuolar degradation in plants.

Ultramicroscopy reveals that senescence induces in-situ and vacuolar degradation of plastoglobules in aging watermelon leaves.

The dynamics of plastoglobules in chloroplasts in aging watermelon leaves were examined by means of transmission electron microscopy, with the aim to understand the intracellular sites for the degradation of plastoglobules in response to leaf senescence. Plastoglobules in chloroplasts in aging leaves with 40% loss of chlorophyll increased drastically in number and size in comparison with young and mature leaves. As senescence advanced, plastoglobules underwent degradation within chloroplasts, or were secreted outside chloroplasts. There were two distinct types of secretion. One type was that chloroplasts protruded to form plastoglobule-containing vesicles and, as the vesicles were detached from chloroplasts, plastoglobules were carried outside chloroplasts. The other type was that plastoglobules squeezed out through the chloroplast envelope into cytoplasm. Lipid droplets were present in the vacuole and underwent degradation therein. Lipid droplets in the vacuole shared similar ultramicroscopic appearance with plastoglobules in chloroplasts, indicating that plastoglobules were engulfed and degraded by the vacuole after they were secreted outside chloroplasts. These results suggested that senescence induces both in-situ and vacuolar degradation of plastoglobules in aging watermelon leaves.