The Arabidopsis Chloroplast Stromal N-Terminome: Complexities of Amino-Terminal Protein Maturation and Stability

Protein amino (N) termini are prone to modifications and are major determinants of protein stability in bacteria, eukaryotes, and perhaps also in chloroplasts. Most chloroplast proteins undergo N-terminal maturation, but this is poorly understood due to insufficient experimental information. Consequently, N termini of mature chloroplast proteins cannot be accurately predicted. This motivated an extensive characterization of chloroplast protein N termini in Arabidopsis (Arabidopsis thaliana) using terminal amine isotopic labeling of substrates and mass spectrometry, generating nearly 14,000 tandem mass spectrometry spectra matching to protein N termini. Many nucleus-encoded plastid proteins accumulated with two or three different N termini; we evaluated the significance of these different proteoforms. Alanine, valine, threonine (often in N-α-acetylated form), and serine were by far the most observed N-terminal residues, even after normalization for their frequency in the plastid proteome, while other residues were absent or highly underrepresented. Plastid-encoded proteins showed a comparable distribution of N-terminal residues, but with a higher frequency of methionine. Infrequent residues (e.g. isoleucine, arginine, cysteine, proline, aspartate, and glutamate) were observed for several abundant proteins (e.g. heat shock proteins 70 and 90, Rubisco large subunit, and ferredoxin-glutamate synthase), likely reflecting functional regulation through their N termini. In contrast, the thylakoid lumenal proteome showed a wide diversity of N-terminal residues, including those typically associated with instability (aspartate, glutamate, leucine, and phenylalanine). We propose that, after cleavage of the chloroplast transit peptide by stromal processing peptidase, additional processing by unidentified peptidases occurs to avoid unstable or otherwise unfavorable N-terminal residues. The possibility of a chloroplast N-end rule is discussed.Following synthesis, most proteins undergo various N-terminal (Nt) protein modifications, including removal of the Nt Met and signal peptide, N-terminal α-acetylation (NAA), ubiquitination, and acylations. These Nt modifications play an important role in the regulation of cellular functions. The N terminus of proteins has also been shown to be a major determinant of protein stability in bacteria (Varshavsky, 2011), eukaryotes (Graciet et al., 2009), mitochondria, and perhaps in plastids/chloroplasts (Apel et al., 2010; Nishimura et al., 2013; van Wijk, 2015). The role of the N terminus in protein stability is conceptualized in the N-end rule, which states that certain amino acids, when exposed at the N terminus of a protein, act as triggers for degradation (Bachmair et al., 1986; Dougan et al., 2012; Tasaki et al., 2012; Gibbs et al., 2014).Most of the approximately 3,000 plastid proteins are nucleus encoded (n-encoded) and are targeted to the plastid through an Nt chloroplast transit peptide (cTP). After import, the cTP is cleaved by the stromal processing peptidase (SPP; Richter and Lamppa, 1998; Trösch and Jarvis, 2011). The consensus site of cTP cleavage by SPP is only loosely defined, and the rules, mechanisms, and enzymes for possible subsequent processing, stabilization, and other posttranslational modifications (PTMs) are not well characterized (for discussion, see van Wijk, 2015). The exact N terminus is unknown for many chloroplast proteins and cannot be accurately predicted, because SPP specificity is not sufficiently understood (Emanuelsson et al., 2000; Zybailov et al., 2008) and probably also because additional Nt processing occurs for many chloroplast proteins (Fig. 1A). The approximately 85 plastid-encoded (p-encoded) proteins typically first undergo cotranslational Nt deformylation, followed by N-terminal Met excision (NME; Giglione et al., 2009; Fig. 1B); both these PTMs are required for normal plastid/chloroplast development and protein stability (Dirk et al., 2001, 2002; Giglione et al., 2003; Meinnel et al., 2006). Both n-encoded and p-encoded proteins can undergo NAA inside the plastid (Zybailov et al., 2008; Fig. 1). Postulated functions of NAA in eukaryotes include the mediation of protein location, assembly, and stability (Jones and O’Connor, 2011; Starheim et al., 2012; Hoshiyasu et al., 2013; Xu et al., 2015), thereby affecting a variety of processes, including drought tolerance in Arabidopsis (Arabidopsis thaliana; Linster et al., 2015).Open in a separate windowFigure 1.Conceptual illustration of Nt maturation of n-encoded and p-encoded proteins. Ac, Acetylated; MAP, Met amino peptidase; NAT, N-acetyltransferase; N-term, N-terminal; PDF, peptide deformylase. A, Nt maturation of n-encoded plastid proteins including removal of cTP by SPP and potential subsequent Nt modifications. B, Nt maturation of p-encoded proteins. *, The removal depends on the penultimate residue, generally following the N-terminal Met Excision (NME) rule; **, N-terminal acetylation typically occurs only for selected residues; “Results”).Typical proteomics work flows generally yield only partial coverage of protein sequences, and it is often difficult to know which peptides represent the true N termini (Nti) or C termini. Systematic identification of Nti or C termini requires specific labeling and enrichment strategies, such as combined fractional diagonal chromatography, developed by Gevaert and colleagues (Staes et al., 2011), and terminal amine isotopic labeling of substrates (TAILS), developed by the group of Overall (Kleifeld et al., 2011; Lange and Overall, 2013). These strategies allow the identification of different Nt proteoforms and were recently also applied to plants (Tsiatsiani et al., 2013; Carrie et al., 2015; Kohler et al., 2015; Zhang et al., 2015) and diatoms (Huesgen et al., 2013). We previously reported on Nti of chloroplast proteins based on tandem mass spectrometry (MS/MS) analysis, but because no Nt enrichment/labeling technique was used, only those that underwent NAA could be considered bona fide Nti (Zybailov et al., 2008). Nt Edman degradation sequencing was systematically carried out for thylakoid lumen proteins (Peltier et al., 2000, 2002) but not for stromal proteins or chloroplast membrane proteins with their Nti exposed to the stroma. The Nti of thylakoid lumen proteins are mostly generated by lumenal peptidases (Hsu et al., 2011; Midorikawa et al., 2014), and the thylakoid lumen contains a different set of peptidases than the stroma; hence, rules for Nt maturation and stability are likely different than those for stroma-exposed proteins.The objective of this study was to systematically determine the Nti of stroma-exposed chloroplast proteins of Arabidopsis (the N-terminome) and to provide a baseline for understanding Nt protein maturation and protein stability in the chloroplast stroma. To that end, we applied the TAILS technique and determined the Nti of approximately 250 chloroplast proteins by mass spectrometry (MS). We observed that many n-encoded plastid proteins accumulated with two or even three different Nt residues, in many cases both with and without NAA. The extent of accumulation of different Nt proteoforms is surprising and will be discussed. The p-encoded proteins generally showed very similar Nt residues as compared with the n-encoded proteins, with the exception of Met. Our data show that small, apolar, or hydroxylated residues (Ala, Val, Ser, and Thr) are the most frequent Nt residues of stromal proteins, whereas other residues are strictly avoided or are only present for very specific proteins likely to aid in their function. Chloroplast protein degradation products were also detected, with enrichment for peptides generated by cleavage between Arg and Thr residues. We present testable hypotheses for understanding Nt processing and maturation, stability, and a possible N-end rule in chloroplast stroma.     

Pepper Heat Shock Protein 70a Interacts with the Type III Effector AvrBsT and Triggers Plant Cell Death and Immunity

Heat shock proteins (HSPs) function as molecular chaperones and are essential for the maintenance and/or restoration of protein homeostasis. The genus Xanthomonas type III effector protein AvrBsT induces hypersensitive cell death in pepper (Capsicum annuum). Here, we report the identification of the pepper CaHSP70a as an AvrBsT-interacting protein. Bimolecular fluorescence complementation and coimmunoprecipitation assays confirm the specific interaction between CaHSP70a and AvrBsT in planta. The CaHSP70a peptide-binding domain is essential for its interaction with AvrBsT. Heat stress (37°C) and Xanthomonas campestris pv vesicatoria (Xcv) infection distinctly induce CaHSP70a in pepper leaves. Cytoplasmic CaHSP70a proteins significantly accumulate in pepper leaves to induce the hypersensitive cell death response by Xcv (avrBsT) infection. Transient CaHSP70a overexpression induces hypersensitive cell death under heat stress, which is accompanied by strong induction of defense- and cell death-related genes. The CaHSP70a peptide-binding domain and ATPase-binding domain are required to trigger cell death under heat stress. Transient coexpression of CaHSP70a and avrBsT leads to cytoplasmic localization of the CaHSP70a-AvrBsT complex and significantly enhances avrBsT-triggered cell death in Nicotiana benthamiana. CaHSP70a silencing in pepper enhances Xcv growth but disrupts the reactive oxygen species burst and cell death response during Xcv infection. Expression of some defense marker genes is significantly reduced in CaHSP70a-silenced leaves, with lower levels of the defense hormones salicylic acid and jasmonic acid. Together, these results suggest that CaHSP70a interacts with the type III effector AvrBsT and is required for cell death and immunity in plants.The heat shock protein HSP70 is a ubiquitous essential protein chaperone and one of the most abundant and diverse heat stress proteins in plants. HSP70s are induced by environmental stresses and are required for plants to cope with heat. HSP70s are involved in protein folding, synthesis, translocation, and macromolecular assemblies such as microtubules (Mayer et al., 2001; Hartl and Hayer-Hartl, 2002). HSP70s protect cells from heat stress by preventing protein aggregation and by facilitating the refolding of denatured proteins. Protein stability can decrease under heat stress conditions and expose hydrophobic patches that cause the aggregation of denatured proteins. HSP70s bind to hydrophobic patches of partially unfolded proteins in an ATP-dependent manner and prevent protein aggregation (Mayer and Bukau, 2005). The modular HSP70 structure consists of a N-terminal ATPase domain and a C-terminal peptide-binding domain that contains a β-sandwich subdomain with a peptide-binding cleft and an α-helical latch-like segment (Zhu et al., 1996; Hartl and Hayer-Hartl, 2002).HSP70s are involved in microbial pathogenesis, cell death responses, and immune responses. Diverse RNA viruses induce HSP70 expression in Arabidopsis (Arabidopsis thaliana; Whitham et al., 2003). Cytoplasmic HSP70s enhance the infection of Nicotiana benthamiana by Tobacco mosaic virus, Potato virus X, Cucumber mosaic virus, and Watermelon mosaic virus (Chen et al., 2008). Recently, the coat protein of Tomato yellow leaf curl virus was suggested to recruit host plant HSP70 during virus infection (Gorovits et al., 2013). HSP70s appear to be involved in regulating viral reproduction, protein folding, and movement, which ultimately promotes viral infection (Boevink and Oparka, 2005; Hafrén et al., 2010). The Pseudomonas syringae effector protein Hopl1 directly binds and manipulates host HSP70, which promotes bacterial virulence (Jelenska et al., 2010). The cytosolic/nuclear heat shock cognate 70 (HSC70) chaperone, which is highly homologous to HSP70 (Tavaria et al., 1996), regulates Arabidopsis immune responses together with SGT1 (for the suppressor of the G2 allele of S-phase kinase-associated protein1 [skp1]; Noël et al., 2007). Cytoplasmic HSP70 is required for the Phytophthora infestans INF1-mediated hypersensitive response (HR) and nonhost resistance to Pseudomonas cichorii in N. benthamiana (Kanzaki et al., 2003). HSP70 is proposed to be involved in both positive and negative regulation of cell death. Selective HSP70 depletion from human cell lines activates a tumor-specific death program that is independent of known caspases and p53 tumor-suppressor protein (Nylandsted et al., 2000), whereas HSP70 promotes tumor necrosis factor-mediated apoptosis by binding IkB kinase γ and impairing nuclear factor-κB signaling in Cos-1 cells (Ran et al., 2004). In N. benthamiana, HSP70 is required for tabtoxinine-β-lactam-induced cell death (Ito et al., 2014). However, HSP70 expression is shown to decrease the cell death triggered by salicylic acid (SA) in Nicotiana tabacum protoplasts (Cronjé et al., 2004). Overexpression of mitochondrial HSP70 suppresses heat- and hydrogen peroxide (H₂O₂)-induced programmed cell death in rice (Oryza sativa; Qi et al., 2011).The genus Xanthomonas YopJ-like AvrBsT protein activates effector-triggered immunity (ETI) in Arabidopsis Pitztal 0 plants (Cunnac et al., 2007). AvrBsT is a member of the YopJ/AvrRxv family identified in Xanthomonas campestris pv vesicatoria (Xcv; Lewis et al., 2011). AvrBsT alters phospholipid signaling and activates defense responses in Arabidopsis (Kirik and Mudgett, 2009). AvrBsT is an acetyltransferase that acetylates Arabidopsis ACETYLATED INTERACTING PROTEIN1 (ACIP1), a microtubule-associated protein required for plant immunity (Cheong et al., 2014). Xcv strain Bv5-4a secretes the AvrBsT type III effector protein that induces hypersensitive cell death and strong defense responses in pepper (Capsicum annuum) and N. benthamiana (Orth et al., 2000; Escolar et al., 2001; Kim et al., 2010). AvrBsT-induced HR-like cell death in pepper is likely part of the typical ETI-mediated defense response cascade (Jones and Dangl, 2006; Eitas et al., 2008; Eitas and Dangl, 2010). AvrBsT overexpression in Arabidopsis triggers plant cell death and defense signaling, leading to both disease and defense responses to diverse microbial pathogens (Hwang et al., 2012). Type III effectors such as Hopl1 and AvrBsT are used to identify unknown components of plant defense cascades (Nomura et al., 2006; Block et al., 2008; Jelenska et al., 2010; Kim et al., 2014) that modulate host innate immunity to achieve disease resistance. The pepper SGT1 was identified recently as a host interactor of AvrBsT (Kim et al., 2014). Pepper SGT1 has features of a cochaperone (Shirasu and Schulze-Lefert, 2003), interacts with AvrBsT, and promotes hypersensitive cell death associated with the pepper receptor-like cytoplasmic protein kinase1 (PIK1) phosphorylation cascade.In this study, we used a yeast (Saccharomyces cerevisiae) two-hybrid screen to identify the pepper HSP70a (CaHSP70a) as an interacting partner of the Xanthomonas spp. type III effector AvrBsT. Coimmunoprecipitation and bimolecular fluorescence complementation (BiFC) analyses verify that CaHSP70a interacts with AvrBsT in planta. Transient CaHSP70a overexpression in pepper leaves enhances heat stress sensitivity and leads to a cell death response. Cytoplasmic localization of the AvrBsT-CaHSP70a complex strongly elevates cell death. CaHSP70a expression is rapidly and strongly induced by avrBsT (for avirulent Xcv Dukso1 [Ds1]) infection in pepper. CaHSP70a silencing enhances susceptibility to Xcv infection, attenuates the reactive oxygen species (ROS) burst and cell death response, reduces SA and jasmonic acid (JA) levels, and disrupts expression of the defense response genes C. annuum pathogenesis-related protein1 (CaPR1; Kim and Hwang, 2000), CaPR10 (Choi et al., 2012), and CaDEF1 (for defensin; Do et al., 2004). Taken together, this study demonstrates that CaHSP70a is a target of the Xanthomonas spp. type III effector AvrBsT and acts as a positive regulator of plant cell death and immunity signaling.     

Probing Arabidopsis Chloroplast Diacylglycerol Pools by Selectively Targeting Bacterial Diacylglycerol Kinase to Suborganellar Membranes

Diacylglycerol (DAG) is an intermediate in metabolism of both triacylglycerols and membrane lipids. Probing the steady-state pools of DAG and understanding how they contribute to the synthesis of different lipids is important when designing plants with altered lipid metabolism. However, traditional methods of assaying DAG pools are difficult, because its abundance is low and because fractionation of subcellular membranes affects DAG pools. To manipulate and probe DAG pools in an in vivo context, we generated multiple stable transgenic lines of Arabidopsis (Arabidopsis thaliana) that target an Escherichia coli DAG kinase (DAGK) to each leaflet of each chloroplast envelope membrane. E. coli DAGK is small, self inserts into membranes, and has catalytic activity on only one side of each membrane. By comparing whole-tissue lipid profiles between our lines, we show that each line has an individual pattern of DAG, phosphatidic acid, phosphatidylcholine, and triacylglycerol steady-state levels, which supports an individual function of DAG in each membrane leaflet. Furthermore, conversion of DAG in the leaflets facing the chloroplast intermembrane space by DAGK impairs plant growth. As a result of DAGK presence in the outer leaflet of the outer envelope membrane, phosphatidic acid accumulation is not observed, likely because it is either converted into other lipids or removed to other membranes. Finally, we use the outer envelope-targeted DAGK line as a tool to probe the accessibility of DAG generated in response to osmotic stress.Diacylglycerol (DAG) is a central metabolite in plant lipid metabolism. Its glycerol backbone is modified with two acyl chains. If a third acyl chain is added, triacylglycerol (TAG) is formed, whereas if a head group is added, it is converted into polar lipids such as a galactolipid. In green tissues, the majority of DAG is used as an intermediate in galactolipid synthesis, because the extensive thylakoid membrane system consists of approximately 85% galactolipids (Block et al., 1983). Although under normal conditions the galactolipids are exclusively chloroplastic, in Arabidopsis (Arabidopsis thaliana), the DAG used to make galactolipids is derived from assembly pathways in both the chloroplast and the endoplasmic reticulum (ER; Benning, 2009). In both pathways, the bulk of the fatty acids are synthesized in the chloroplast stroma (Browse et al., 1986) in the following order of abundance: 18:1, 16:0, and 18:0 (Wallis and Browse, 2002).In the chloroplast pathway, these fatty acids are directly attached to a glycerol-3-P, generating first lyso-phosphatidic acid (l-PtdOH) and then phosphatidic acid (PtdOH) in the inner leaflet of the chloroplast inner envelope (Fig. 1; Frentzen et al., 1983). The acyltransferases involved are specific to the extent that the sn-2 position of the glycerol backbone predominantly receives a 16:0 fatty acid. PtdOH is then used directly for phosphatidylglycerol (PtdGro) synthesis (Babiychuk et al., 2003) or converted to DAG by a PtdOH phosphatase (Joyard and Douce, 1977). The PtdOH phosphatase activity is known to be associated with the inner envelope, though which leaflet is obscured by the fact that DAG can efficiently flip across membranes (Hamilton et al., 1991) and the actual enzyme has not been unambiguously identified and located (Nakamura et al., 2007). However, the leaflet associations of two other enzymes that use DAG in the inner envelope have been established. MGD1, which uses DAG to synthesize the most abundant galactolipid, monogalactosyldiacylglycerol (MGDG), is on the outer leaflet of the inner envelope membrane (Xu et al., 2005), while SQD2, which uses DAG to generate the less abundant sulfolipid, sulfoquinovosyldiacylglycerol (SQDG), is located on the inner leaflet of the inner envelope membrane (Tietje and Heinz, 1998). Also associated with the inner envelope membrane are a number of fatty acid desaturases, including FAD4, FAD5, FAD6, FAD7, and FAD8 (Joyard et al., 2010). Two of these are specific, generating lipids with signature desaturations: FAD4 desaturates only the 16:0 fatty acid of PtdGro, giving plastidic PtdGro a distinct 16:1 Δ3 trans moiety (Browse et al., 1985; Gao et al., 2009), and FAD5 desaturates primarily the 16:0 fatty acid of MGDG, producing 16:1 Δ7 cis (Kunst et al., 1989). The remaining desaturases are less specific, with little preference for head group or acyl tail. They further desaturate 16:1 or 18:1 in the cis conformation to 16:2 or 18:2 (FAD6; Browse et al., 1989) and on to 16:3 or 18:3 (FAD7 and FAD8; Wallis and Browse, 2002). The combined actions of these FADs result in the highly desaturated fatty acid profiles seen for most chloroplast lipids.Open in a separate windowFigure 1.Overview of DAG pools in the chloroplast envelope membranes. Processes that are known to have activity feeding into or withdrawing from DAG pools in the chloroplast envelope membranes are shown. Enzymes are indicated, and their substrates and products are connected with black arrows. However, for space reasons, not all reactants are shown. Membrane leaflets are indicated, and enzymes with known membrane topology are displayed correctly, while those without known topology are displayed in the center of the appropriate membrane. The acyl group preferred by each l-PtdOH acyltransferase is given in parentheses. Proposed processes transporting lipids from the ER to the chloroplast are shown with dashed arrows. Enzymes are as follows: 1, ATS1; 2, ATS2; 3, lipid phosphate phosphatase γ; 4, MGD1; 5, SQD2; 6, cytosolic phospholipases; 7, MGD2 or MGD3; 8, SFR2; 9, acyl-CoA:glycerol-3-P acyltransferase; 10, l-PtdOH acyltransferase; 11, PtdOH phosphatase; 12, cytidine diphosphate-choline:DAG cholinephosphotransferase; 13, TGD4; and 14, TGD1, TGD 2, TGD3 lipid transport complex. OE, Chloroplast outer envelope membrane; IE, chloroplast inner envelope membrane; ACP, acyl carrier protein. [See online article for color version of this figure.]In unstressed plants, DAG seems to be used primarily in the inner chloroplast envelope. However, several conditions are known to cause extensive DAG use in the chloroplast outer envelope. During phosphate deprivation, MGD2 and MDG3 synthesize MGDG from DAG on the outer leaflet of the outer envelope membrane (Kobayashi et al., 2009). The DAG backbones are probably supplied from the phosphatidylcholine (PtdCho) pool by phospholipase activity, which was shown to be simultaneously up-regulated (Andersson et al., 2004; Nakamura et al., 2005). DAG is also generated during freezing stress by a galactolipid:galactolipid galactosyltransferase named Sensitive to FReezing2 (SFR2). This enzyme transfers the galactosyl head group of MGDG onto another MGDG, giving rise to digalactosyldiacylglycerol (DGDG) and DAG (Moellering et al., 2010). The DAG is subsequently sequestered into a lipid droplet by formation of TAG by an as yet unidentified enzyme.In the ER pathway, fatty acids synthesized in the chloroplast stroma are exported through a still poorly defined mechanism to the ER and activated to acyl-CoAs. Acyltransferases sequentially catalyze formation of l-PtdOH and PtdOH from glycerol-3-P and acyl-CoAs. Again, the acyltransferase working on the sn-2 position of the glycerol backbone is specific, but unlike the chloroplast isoform, it prefers an 18:1 carbon fatty acid (Frentzen et al., 1983). Newly generated PtdOH can be converted to PtdGro or phosphatidyl inositol (PtdIns) (Collin et al., 1999) or hydrolyzed to DAG (Shimojima et al., 2009). DAG can then be further metabolized to TAG and PtdCho. PtdCho acyl groups (18:1/18:1 and 18:1/16:0) are desaturated sequentially by desaturases FAD2 (Okuley et al., 1994) and FAD3 (Browse et al., 1993). These desaturases prefer PtdCho as substrate. The acyl chains modified on PtdCho are transferred to other ER lipids, including DAG, as a result of continual acyl editing of the PtdCho pool (Bates et al., 2012). Furthermore, PtdOH and many of the other extraplastidic phospholipids can be converted to DAG by action of phospholipases (Shimojima et al., 2009). These have as yet partially defined roles in response to stress or recycling of membrane lipids (Testerink and Munnik, 2005).Glycerolipid precursors generated by de novo synthesis, acyl editing, and possibly stress conditions in the ER are transported to the chloroplast by a mechanism that is likely to involve at least two putative lipid transporters: trigalactosyldiacylglycerol4 (TGD4) in the chloroplast outer envelope membrane and the TGD1, TGD2, and TGD3 complex in the inner envelope membrane (Wang and Benning, 2012). The actual lipid species transported remains unclear, but PtdCho, lyso-phosphatidylcholine, PtdOH, and DAG have been discussed in the literature (Andersson and Dörmann, 2009). The DAG moieties are then fully incorporated into all plastidic lipids except PtdGro, presumably using the same pathways that metabolize plastidic DAG, described above. Because of the preference of chloroplast and ER sn-2 acyltransferases for 16 or 18 carbon fatty acids, respectively, the origin of the DAG moieties can be distinguished by positional analysis of the acyl groups on the glycerol backbone (Roughan and Slack, 1982). In Arabidopsis, the chloroplast and ER lipid synthesis pathways contribute nearly equally to mature chloroplast lipids (Browse et al., 1986; Mongrand et al., 1998). Thus, the DAG pools described so far in the chloroplast inner and outer envelope membranes are each of dual origin.A challenge for the analysis of the different DAG pools is that this compound is not a bilayer-forming lipid and thus does not accumulate stably to high levels. Furthermore, during any lengthy fractionation procedure, its levels can be expected to alter, as DAG-modifying enzymes exist in multiple membranes. Moreover, because DAG is quickly metabolized and may have efficient transport systems (Dong et al., 2012), it is difficult to confirm whether metabolizing enzymes are accessing the same or separate DAG pools.To probe different DAG pools of chloroplast membranes in vivo, we have generated a series of stable transgenic Arabidopsis lines in which we target an Escherichia coli DAG kinase (DAGK) to each leaflet of the chloroplast envelope membranes. The basic utility of this approach was previously shown by targeting a DAGK to the chloroplast in tobacco (Nicotiana tabacum) using a single construct fusing the bacterial protein to the Rubisco small subunit N-terminal peptide (Fritz et al., 2007). Here, we present a full phenotypic analysis of these lines, determining which chloroplast membranes show steady-state alterations of DAG and PtdOH levels predicted by ectopic DAGK activity. We further determine the accessibility of DAG pools generated on the outer leaflet of the chloroplast outer envelope membrane during osmotic stress. Having this system established in Arabidopsis will allow characterization of DAG pools in multiple lipid mutant lines.     

Trafficking of Vacuolar Proteins: The Crucial Role of Arabidopsis Vacuolar Protein Sorting 29 in Recycling Vacuolar Sorting Receptor

The retromer is involved in recycling lysosomal sorting receptors in mammals. A component of the retromer complex in Arabidopsis thaliana, vacuolar protein sorting 29 (VPS29), plays a crucial role in trafficking storage proteins to protein storage vacuoles. However, it is not known whether or how vacuolar sorting receptors (VSRs) are recycled from the prevacuolar compartment (PVC) to the trans-Golgi network (TGN) during trafficking to the lytic vacuole (LV). Here, we report that VPS29 plays an essential role in the trafficking of soluble proteins to the LV from the TGN to the PVC. maigo1-1 (mag1-1) mutants, which harbor a knockdown mutation in VPS29, were defective in trafficking of two soluble proteins, Arabidopsis aleurain-like protein (AALP):green fluorescent protein (GFP) and sporamin:GFP, to the LV but not in trafficking membrane proteins to the LV or plasma membrane or via the secretory pathway. AALP:GFP and sporamin:GFP in mag1-1 protoplasts accumulated in the TGN but were also secreted into the medium. In mag1-1 mutants, VSR1 failed to recycle from the PVC to the TGN; rather, a significant proportion was transported to the LV; VSR1 overexpression rescued this defect. Moreover, endogenous VSRs were expressed at higher levels in mag1-1 plants. Based on these results, we propose that VPS29 plays a crucial role in recycling VSRs from the PVC to the TGN during the trafficking of soluble proteins to the LV.     

Cooperative Protein Folding by Two Protein Thiol Disulfide Oxidoreductases and ERO1 in Soybean

Most proteins produced in the endoplasmic reticulum (ER) of eukaryotic cells fold via disulfide formation (oxidative folding). Oxidative folding is catalyzed by protein disulfide isomerase (PDI) and PDI-related ER protein thiol disulfide oxidoreductases (ER oxidoreductases). In yeast and mammals, ER oxidoreductin-1s (Ero1s) supply oxidizing equivalent to the active centers of PDI. In this study, we expressed recombinant soybean Ero1 (GmERO1a) and found that GmERO1a oxidized multiple soybean ER oxidoreductases, in contrast to mammalian Ero1s having a high specificity for PDI. One of these ER oxidoreductases, GmPDIM, associated in vivo and in vitro with GmPDIL-2, was unable to be oxidized by GmERO1a. We therefore pursued the possible cooperative oxidative folding by GmPDIM, GmERO1a, and GmPDIL-2 in vitro and found that GmPDIL-2 synergistically accelerated oxidative refolding. In this process, GmERO1a preferentially oxidized the active center in the a′ domain among the a, a′, and b domains of GmPDIM. A disulfide bond introduced into the active center of the a′ domain of GmPDIM was shown to be transferred to the active center of the a domain of GmPDIM and the a domain of GmPDIM directly oxidized the active centers of both the a or a′ domain of GmPDIL-2. Therefore, we propose that the relay of an oxidizing equivalent from one ER oxidoreductase to another may play an essential role in cooperative oxidative folding by multiple ER oxidoreductases in plants.In eukaryotes, many secretory and membrane proteins fold via disulfide bond formation in the endoplasmic reticulum (ER). Seed storage proteins of major crops, such as wheat, corn, rice, and beans, which are important protein sources for humans and domestic animals, are synthesized in the ER of the endosperm or cotyledon. A number of seed storage proteins fold by the formation of intramolecular disulfide bonds (oxidative folding) and are transported to and accumulate in protein bodies (Kermode and Bewley, 1999; Jolliffe et al., 2005). In contrast to normally folded proteins, misfolded and unfolded proteins are retained in the ER and degraded by an ER-associated degradation or vacuolar system (Smith et al., 2011; Pu and Bassham, 2013). Therefore, quick and efficient oxidative folding of nascent seed storage proteins is needed for their accumulation in protein bodies.During this process, protein disulfide isomerase (PDI; EC 5.3.4.1) and other ER protein thiol disulfide oxidoreductases (ER oxidoreductases) are thought to catalyze the formation and isomerization of disulfide bonds in nascent proteins (Hatahet and Ruddock, 2009; Feige and Hendershot, 2011; Lu and Holmgren, 2014). After phylogenetic analysis of the Arabidopsis genome, 10 classes of ER oxidoreductases (classes I–X) were identified (Houston et al., 2005). Among them, class I ER oxidoreductase, a plant PDI ortholog, has been studied in a wide variety of plants. Class I ER oxidoreductases have two catalytically active domains a and a′, containing active centers composed of Cys-Gly-His-Cys and two catalytically inactive domains b and b′. An Arabidopsis ortholog of class I ER oxidoreductases is required for proper seed development and regulates the timing of programmed cell death by chaperoning and inhibiting Cys proteases (Andème Ondzighi et al., 2008). OaPDI, a PDI from Oldenlandia affinis, a coffee family (Rubiaceae) plant, is involved in the folding of knotted circular proteins (Gruber et al., 2007). The rice ortholog (PDIL1-1) was suggested to be involved in the maturation of the major seed storage protein glutelin (Takemoto et al., 2002). Furthermore, rice PDIL1-1 plays a role in regulatory activities for various proteins that are essential for the synthesis of grain components as determined by analysis of a T-DNA insertion mutant (Satoh-Cruz et al., 2010).The oxidative refolding ability of class I ER oxidoreductases was confirmed in recombinant soybean (GmPDIL-1) and wheat proteins produced by an Escherichia coli expression system established from cDNAs (Kamauchi et al., 2008; Kimura et al., 2015).Class II and III ER oxidoreductases have an a–b–b′–a′ domain structure. Class II ER oxidoreductases have an acidic amino acid-rich sequence in the N-terminal region ahead of the a domain. Recombinant soybean (GmPDIL-2) and wheat class II ER oxidoreductases have oxidative refolding activities similar to that of class I (Kamauchi et al., 2008; Kimura et al., 2015). Class III ER oxidoreductases contain the nonclassical redox-center Cys-X-X-Ser/Cys motifs, as opposed to the more traditional CGHC sequence, in the a and a′ domains. Recombinant soybean (GmPDIL-3) and wheat proteins lack oxidative refolding activity in vitro (Iwasaki et al., 2009; Kimura et al., 2015). Class IV ER oxidoreductases are unique to plants and have an a–a′–ERp29 domain structure, which is homologous to the C-terminal domain of mammalian ERp29 (Demmer et al., 1997).Recombinant soybean class IV ER oxidoreductases (GmPDIS-1 and GmPDIS-2) and wheat class IV ER oxidoreductase possess an oxidative refolding activity that is weaker than that of classes I and II (Wadahama et al., 2007; Kimura et al., 2015). Class V ER oxidoreductases are plant orthologs of mammalian P5 and have an a–a′–b domain structure. A rice class V ER oxidoreductase, consisting of PDIL2 and PDIL3, plays an important role in the accumulation of the seed storage protein Cys-rich 10-kD prolamin (crP10; Onda et al., 2011). Recombinant soybean class V ER oxidoreductase, GmPDIM and wheat class V ER oxidoreductase possess an oxidative refolding activity similar to that of class IV (Wadahama et al., 2008; Kimura et al., 2015). In the soybean, GmPDIL-1, GmPDIL-2, GmPDIM, GmPDIS-1, and GmPDIS-2 were found to associate transiently with a seed storage precursor protein, proglycinin, in the ER of the cotyledon by coimmunoprecipitation experiments, suggesting that multiple ER oxidoreductases are involved in the folding of the nascent proglycinin.The disulfide bond in the active center of ER oxidoreductases is reduced as a result of catalyzing disulfide bond formation in an unfolded protein. The reduced active center of PDI was discovered to be oxidized again by ER oxidoreductin-1 (Ero1p) in yeast (Frand and Kaiser, 1998; Pollard et al., 1998). Ero1p orthologs are present universally in eukaryotes. Yeast and flies have a single copy of the ERO1 gene, which is essential for survival (Frand and Kaiser, 1998; Pollard et al., 1998; Tien et al., 2008). Mammals have two genes encoding Ero1-α (Cabibbo et al., 2000) and Ero1-β (Pagani et al., 2000) that function as major disulfide donors to nascent proteins in the ER, but are not critical for survival (Zito et al., 2010). Domain a of yeast PDI is the most favored substrate of yeast Ero1p (Vitu et al., 2010), whereas a′ of human PDI is specifically oxidized by human Ero1-α (Chambers et al., 2010) and Ero1-β (Wang et al., 2011). Electrons from Cys residues of the active centers of PDI are transferred to oxygen by Ero1 (Tu and Weissman, 2004; Sevier and Kaiser, 2008). The reaction mechanisms of yeast Ero1p and human Ero1s have been intensively investigated; their regulation by PDI has been extensively studied as well (Tavender and Bulleid, 2010; Araki and Inaba, 2012; Benham et al., 2013; Ramming et al., 2015). Only rice Ero1 (OsERO1) has been identified as a plant ortholog of Ero1p (Onda et al., 2009). OsERO1 is necessary for disulfide bond formation in rice endosperm. The formation of native disulfide bonds in the major seed storage protein proglutelin was demonstrated to depend upon OsERO1 by RNAi knockdown experiments. However, no plant protein thiol disulfide oxidoreductases that are oxidized by a plant Ero1 ortholog have been identified to date.In this study, we show that multiple soybean ER oxidoreductases can be activated by a soybean Ero1 ortholog (GmERO1a). In addition, we propose a synergistic mechanism by which GmPDIM and GmPDIL-2 cooperatively fold unfolded proteins using oxidizing equivalents provided by GmERO1 in vitro.     

Phosphatidylinositol 4-Phosphate Negatively Regulates Chloroplast Division in Arabidopsis

Chloroplast division is performed by the constriction of envelope membranes at the division site. Although constriction of a ring-like protein complex has been shown to be involved in chloroplast division, it remains unknown how membrane lipids participate in the process. Here, we show that phosphoinositides with unknown function in envelope membranes are involved in the regulation of chloroplast division in Arabidopsis thaliana. PLASTID DIVISION1 (PDV1) and PDV2 proteins interacted specifically with phosphatidylinositol 4-phosphate (PI4P). Inhibition of phosphatidylinositol 4-kinase (PI4K) decreased the level of PI4P in chloroplasts and accelerated chloroplast division. Knockout of PI4Kβ2 expression or downregulation of PI4Kα1 expression resulted in decreased levels of PI4P in chloroplasts and increased chloroplast numbers. PI4Kα1 is the main contributor to PI4P synthesis in chloroplasts, and the effect of PI4K inhibition was largely abolished in the pdv1 mutant. Overexpression of DYNAMIN-RELATED PROTEIN5B (DRP5B), another component of the chloroplast division machinery, which is recruited to chloroplasts by PDV1 and PDV2, enhanced the effect of PI4K inhibition, whereas overexpression of PDV1 and PDV2 had additive effects. The amount of DRP5B that associated with chloroplasts increased upon PI4K inhibition. These findings suggest that PI4P is a regulator of chloroplast division in a PDV1- and DRP5B-dependent manner.     

Hitching a Ride on Vesicles: Cauliflower Mosaic Virus Movement Protein Trafficking in the Endomembrane System

The transport of a viral genome from cell to cell is enabled by movement proteins (MPs) targeting the cell periphery to mediate the gating of plasmodesmata. Given their essential role in the development of viral infection, understanding the regulation of MPs is of great importance. Here, we show that cauliflower mosaic virus (CaMV) MP contains three tyrosine-based sorting signals that interact with an Arabidopsis (Arabidopsis thaliana) μA-adaptin subunit. Fluorophore-tagged MP is incorporated into vesicles labeled with the endocytic tracer N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide. The presence of at least one of the three endocytosis motifs is essential for internalization of the protein from the plasma membrane to early endosomes, for tubule formation, and for CaMV infection. In addition, we show that MP colocalizes in vesicles with the Rab GTPase AtRAB-F2b, which is resident in prevacuolar late endosomal compartments that deliver proteins to the vacuole for degradation. Altogether, these results demonstrate that CaMV MP traffics in the endocytic pathway and that virus viability depends on functional host endomembranes.Membrane trafficking is essential in eukaryotic cells. Cellular membranes serve as a delivery system for newly synthesized proteins such as transporters and receptors exiting the endoplasmic reticulum after proper folding. They then transit through the Golgi complex, reaching the plasma membrane (PM) or the tonoplast via intermediate endomembrane compartments. Receptors and transporters returning from the PM are either recycled or targeted to the vacuole for degradation. Delivery and recycling sorting pathways overlap in the trans-Golgi network (TGN)/early endosome (EE), an intermediate compartment for both exocytosis and endocytosis (Reyes et al., 2011). In plant systems, the endoplasmic reticulum and PM provide membrane continuity between cells through the connections made by plasmodesmata (PD), cytoplasmic channels that regulate traffic in the symplasm (Maule et al., 2011).The selective transport of macromolecules between different compartments of the endomembrane system is mediated by coat proteins promoting the generation of small cargo-trafficking coated vesicles (Spang, 2008). The recognition and recruitment of cargo proteins are mediated by so-called adaptor complexes (AP complexes [AP-1–AP-4]; Robinson, 2004) one of which, AP-1, is localized on the TGN/EE and endosomes, whereas AP-2 is in the PM. The μ-subunit of AP complexes is devoted to cargo protein selection via a specific and well-characterized interaction with a Tyr-sorting signal, YXXΦ, where Φ is a bulky hydrophobic residue and X is any amino acid (Bonifacino and Dell’Angelica, 1999). YXXΦ motifs are present in the cytoplasmic tail of many proteins integral to the PM and TGN/EE and have been found in the movement proteins (MPs) of some viruses (Laporte et al., 2003; Haupt et al., 2005). Plant viruses are obligate parasites that exploit host components to move within the cell and from cell to cell into the vascular system for systemic invasion of the host. Virus movement, which requires the passage of macromolecules through PD connections, is mediated by one or more virus-encoded MPs with the help of the host cytoskeleton and/or endomembranes (Harries et al., 2010). While most MPs act to increase the size exclusion limit of PD to facilitate the passage of the viral nucleoprotein complex, other MPs are assembled in tubules that pass inside highly modified PD and transport encapsidated particles through their lumen.Here, we focus on this second group of tubule-forming MPs and examine the intracellular trafficking of cauliflower mosaic virus (CaMV) MP. The MP encoded by CaMV forms tubules guiding encapsidated virus particle cell-to-cell transport via an indirect MP-virion interaction (Stavolone et al., 2005; Sánchez-Navarro et al., 2010). However, how CaMV MP (and the other tubule-forming MPs) targets the PM and forms tubules remains to be elucidated. Tubule-forming MPs do not require an intact cytoskeleton for PM targeting (Huang et al., 2000; Pouwels et al., 2002) and/or tubule formation (Laporte et al., 2003). However, suppression of tubule formation upon treatment with brefeldin A (BFA), a specific inhibitor of secretion or endocytosis, suggests the involvement of the endomembrane system in correct functioning of some tubule-forming MPs (Huang et al., 2000; Laporte et al., 2003). In this study, we examined the three Tyr-sorting motifs in CaMV MP and show that each of the three domains interacts directly with subunit μ of an Arabidopsis (Arabidopsis thaliana) AP complex. Mutations in these domains revert in the viral context to maintain CaMV viability. MP is found in endosomal compartments labeled by AtRAB-F2b (ARA7) and N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4-64). The presence of at least one functional YXXΦ domain is essential for the localization of MP to endosomes and for tubule assembly but is not required for MP targeting to the PM. We provide several lines of evidence to show CaMV MP trafficking in the endocytic pathway. Our findings are discussed in the light of the recent demonstration that the TGN/EE functions as a major hub controlling secretory and endocytic pathways in plants.     

A Critical Role of Lyst-Interacting Protein5, a Positive Regulator of Multivesicular Body Biogenesis,in Plant Responses to Heat and Salt Stresses

Multivesicular bodies (MVBs) are unique endosomes containing vesicles in the lumen and play critical roles in many cellular processes. We have recently shown that Arabidopsis (Arabidopsis thaliana) Lyst-Interacting Protein5 (LIP5), a positive regulator of the Suppressor of K⁺ Transport Growth Defect1 (SKD1) AAA ATPase in MVB biogenesis, is a critical target of the mitogen-activated protein kinases MPK3 and MPK6 and plays an important role in the plant immune system. In this study, we report that the LIP5-regulated MVB pathway also plays a critical role in plant responses to abiotic stresses. Disruption of LIP5 causes compromised tolerance to both heat and salt stresses. The critical role of LIP5 in plant tolerance to abiotic stresses is dependent on its ability to interact with Suppressor of K⁺ Transport Growth Defect1. When compared with wild-type plants, lip5 mutants accumulate increased levels of ubiquitinated protein aggregates and NaCl under heat and salt stresses, respectively. Further analysis using fluorescent dye and MVB markers reveals that abiotic stress increases the formation of endocytic vesicles and MVBs in a largely LIP5-dependent manner. LIP5 is also required for the salt-induced increase of intracellular reactive oxygen species, which have been implicated in signaling of salt stress responses. Basal levels of LIP5 phosphorylation by MPKs and the stability of LIP5 are elevated by salt stress, and mutation of MPK phosphorylation sites in LIP5 reduces the stability and compromises the ability to complement the lip5 salt-sensitive mutant phenotype. These results collectively indicate that the MVB pathway is positively regulated by pathogen/stress-responsive MPK3/6 through LIP5 phosphorylation and plays a critical role in broad plant responses to biotic and abiotic stresses.Multivesicular bodies (MVBs) are a subset of late endosomes that contain intraluminal vesicles generated when the limiting membrane of the endosome invaginates and buds into its own lumen. MVBs perform a variety of functions and, as a result, can have different compositions and morphologies. The most well-established role of MVBs in all eukaryotic cells is as a degradation route in the endocytic pathway that allows protein-containing intraluminal vesicles to be delivered into and degraded upon fusion with lysosomes or vacuoles (Reyes et al., 2011; Contento and Bassham, 2012). The route acts as a mechanism for removing damaged proteins as well as proteins that require down-regulation or clearing from the plasma membrane as part of a regulatory process. Those proteins retained in the limiting membrane of MVBs, on the other hand, can be delivered to the membrane of lysosomes or vacuoles or sorted back to the plasma membrane or other cellular compartments (Reyes et al., 2011; Contento and Bassham, 2012).The protein machinery involved in MVB biogenesis has been well studied in yeast and other eukaryotic organisms. A majority of proteins required for protein sorting into MVBs are components of three distinct protein complexes named ESCRT-I, ESCRT-II, and ESCRT-III (for Endosomal Sorting Complexes Required for Transport; Winter and Hauser, 2006). These complexes are recruited to endosomal membranes and function in sorting cargo and the formation of intraluminal vesicles. Ubiquitinated membrane proteins are first recognized by specific ubiquitin-binding proteins that also recruit ESCRT-I components from the cytoplasm. ESCRT-II and ESCRT-III complexes are then recruited and transiently assembled on the endosomal membrane for cargo sorting, concentration, and intraluminal vesicle formation. Unlike ESCRT-I and ESCRT-II, which are stable protein complexes, ESCRT-III proteins are monomers in the cytoplasm and only form complexes on the endosomal membrane. Disassembly of ESCRT-III, however, is not spontaneous but, rather, requires catalysis by the Vacuolar protein sorting 4p (Vps4p)/Suppressor of K⁺ Transport Growth Defect1 (SKD1) AAA ATPase together with its positive regulator Vacuolar protein sorting20-associated1 (Vta1)/LIP5 in an ATP-dependent reaction (Babst et al., 1998; Fujita et al., 2004; Scott et al., 2005; Azmi et al., 2006; Lottridge et al., 2006). In yeast and mammalian cells, both Vps4p/SKD1 and Vta1/LIP5 are critical players of MVB biogenesis (Yeo et al., 2003; Shiflett et al., 2004; Ward et al., 2005; Azmi et al., 2006).As sessile organisms, plants are constantly exposed to a wide range of biotic and abiotic stresses and, through evolution, have developed a battery of complicated adaptive mechanisms. Studies over the past decade have provided increasing evidence for the association of vesicle trafficking with plant responses to both biotic and abiotic stresses. Plant immune responses to biotic stresses consists of two interconnected branches: pattern-triggered immunity and effector-triggered immunity, which are conferred by pattern-recognition receptors and RESISTANCE (R) proteins, respectively. A number of pattern-recognition receptors, such as Arabidopsis (Arabidopsis thaliana) Flagellin-sensitive2 and R proteins, become associated with late endosomes/MVBs upon pattern and effector recognition, respectively, and there is strong evidence for a critical role of the association with vesicles in plant disease resistance (Choi et al., 2013; Spallek et al., 2013). In the penetration resistance of cereal plants against powdery mildew fungal pathogens, which is conferred by local cell wall appositions (papillae), electron and confocal microscopy detected trafficking molecules through late endosomes/MVBs for delivering defense-related materials to papillae, thereby executing a timely and localized defense response to invading pathogens (An et al., 2006a, 2006b; Meyer et al., 2009; Böhlenius et al., 2010; Nielsen et al., 2012). Similar relocalization of defense-related molecules, such as the PENTRATION RESISTANCE3 ATP-binding cassette transporter for cell surface defense in response to conserved pathogen elicitors, has also been observed in Arabidopsis (Underwood and Somerville, 2013). There is also evidence for a role of late endosomes/MVBs in plant abiotic stress responses (Jou et al., 2004, 2006; Ho et al., 2010; Xia et al., 2013). Generally speaking, however, while there is a large body of evidence for a critical role of general vesicle trafficking in plant stress responses, there has been only a limited number of studies that address specifically the roles and regulation of MVBs in plant responses to biotic and abiotic stresses. Studies on the role of MVBs in plant immune responses have been largely through microscopic analysis of the accumulation of the late endosomes in response to pathogen infection or elicitor treatment. Genetic analysis of the role of MVBs in plant stress responses has not been straightforward, because mutants for genes essential for MVB biogenesis are often lethal (Haas et al., 2007; Spitzer et al., 2009). While constitutive MVB biogenesis is known to be essential in many cellular processes, it remains to be determined whether there are specific pathogen/stress-responsive pathways for increased MVB biogenesis during plant stress responses.In Arabidopsis, disruption of the SKD1 gene is lethal, and expression of an ATPase-deficient mutant SKD1 causes alterations in the endosomal system and ultimately cell death (Haas et al., 2007). Arabidopsis LIP5 interacts strongly with SKD1 and increases in vitro the ATPase activity of SKD1 by 4- to 5-fold (Haas et al., 2007). However, disruption of LIP5 in Arabidopsis causes no major phenotypic alterations under normal growth conditions, indicating that the basal level of the SKD1 ATPase activity without stimulation by LIP5 is sufficient for plant growth and development (Haas et al., 2007). Recently, we reported the identification of Arabidopsis LIP5 as an interacting protein and a substrate of the pathogen-responsive mitogen-activated protein kinases (MAPKs) MPK6/MPK3 (Wang et al., 2014). Functional analysis with lip5 transfer DNA (T-DNA) insertion mutants indicates that LIP5 plays a critical role in pathogen-induced MVB trafficking and in basal resistance to Pseudomonas syringae strains (Wang et al., 2014). The critical role of LIP5 in the plant immune system is dependent on its ability to interact with SKD1. Further analysis reveals that LIP5 is expressed at low levels in healthy plants, but its protein levels can be substantially elevated through phosphorylation by the pathogen-responsive MPK cascade. Mutation of MPK phosphorylation sites in LIP5 does not affect its interaction with SKD1 but reduces its stability and, as a result, compromises its ability to complement the basal resistance of the lip5 mutants. These results provide genetic evidence for a critical role of induced MVB biogenesis in plant basal resistance and establish an important mechanism for the regulation of vesicle trafficking during plant-pathogen interactions (Wang et al., 2014).In this study, we report that the LIP5-regulated MVB pathway is also a critical cellular process during plant responses to abiotic stresses. Disruption of LIP5 causes compromised tolerance to both heat and salt stresses. The critical role of LIP5 in plant tolerance to abiotic stresses is again dependent on its ability to interact with SKD1. When compared with wild-type plants, lip5 mutants accumulate increased levels of ubiquitinated protein aggregates, suggesting a possible role of LIP5-regulated MVB trafficking as a critical route for the degradation of heat-damaged proteins. Compromised salt tolerance of the lip5 mutants was associated with an increased accumulation of cellular NaCl but reduced levels of cellular reactive oxygen species (ROS), which have been implicated in the signaling of salt stress responses (Kaye et al., 2011; Xie et al., 2011). The roles of LIP5 and its phosphorylation by MPK3/6 in plant responses to heat and salt stresses were also investigated. These results collectively provide important insights into the role and regulation of pathogen- and stress-responsive MVB biogenesis in broad plant stress responses.