A Wheat SIMILAR TO RCD-ONE Gene Enhances Seedling Growth and Abiotic Stress Resistance by Modulating Redox Homeostasis and Maintaining Genomic Integrity

Plant growth inhibition is a common response to salinity. Under saline conditions, Shanrong No. 3 (SR3), a bread wheat (Triticum aestivum) introgression line, performs better than its parent wheat variety Jinan 177 (JN177) with respect to both seedling growth and abiotic stress tolerance. Furthermore, the endogenous reactive oxygen species (ROS) was also elevated in SR3 relative to JN177. The SR3 allele of sro1, a gene encoding a poly(ADP ribose) polymerase (PARP) domain protein, was identified to be crucial for both aspects of its superior performance. Unlike RADICAL-INDUCED CELL DEATH1 and other Arabidopsis thaliana SIMILAR TO RCD-ONE (SRO) proteins, sro1 has PARP activity. Both the overexpression of Ta-sro1 in wheat and its heterologous expression in Arabidopsis promote the accumulation of ROS, mainly by enhancing the activity of NADPH oxidase and the expression of NAD(P)H dehydrogenase, in conjunction with the suppression of alternative oxidase expression. Moreover, it promotes the activity of ascorbate-GSH cycle enzymes and GSH peroxidase cycle enzymes, which regulate ROS content and cellular redox homeostasis. sro1 is also found to be involved in the maintenance of genomic integrity. We show here that the wheat SRO has PARP activity; such activity could be manipulated to improve the growth of seedlings exposed to salinity stress by modulating redox homeostasis and maintaining genomic stability.     

Lipid Peroxide-Derived Short-Chain Carbonyls Mediate Hydrogen Peroxide-Induced and Salt-Induced Programmed Cell Death in Plants

Lipid peroxide-derived toxic carbonyl compounds (oxylipin carbonyls), produced downstream of reactive oxygen species (ROS), were recently revealed to mediate abiotic stress-induced damage of plants. Here, we investigated how oxylipin carbonyls cause cell death. When tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells were exposed to hydrogen peroxide, several species of short-chain oxylipin carbonyls [i.e. 4-hydroxy-(E)-2-nonenal and acrolein] accumulated and the cells underwent programmed cell death (PCD), as judged based on DNA fragmentation, an increase in terminal deoxynucleotidyl transferase dUTP nick end labeling-positive nuclei, and cytoplasm retraction. These oxylipin carbonyls caused PCD in BY-2 cells and roots of tobacco and Arabidopsis (Arabidopsis thaliana). To test the possibility that oxylipin carbonyls mediate an oxidative signal to cause PCD, we performed pharmacological and genetic experiments. Carnosine and hydralazine, having distinct chemistry for scavenging carbonyls, significantly suppressed the increase in oxylipin carbonyls and blocked PCD in BY-2 cells and Arabidopsis roots, but they did not affect the levels of ROS and lipid peroxides. A transgenic tobacco line that overproduces 2-alkenal reductase, an Arabidopsis enzyme to detoxify α,β-unsaturated carbonyls, suffered less PCD in root epidermis after hydrogen peroxide or salt treatment than did the wild type, whereas the ROS level increases due to the stress treatments were not different between the lines. From these results, we conclude that oxylipin carbonyls are involved in the PCD process in oxidatively stressed cells. Our comparison of the ability of distinct carbonyls to induce PCD in BY-2 cells revealed that acrolein and 4-hydroxy-(E)-2-nonenal are the most potent carbonyls. The physiological relevance and possible mechanisms of the carbonyl-induced PCD are discussed.In plants, environmental stressors such as extreme temperatures, drought, intense UV-B radiation, and soil salinity can cause tissue damage, growth inhibition, and even death. These detrimental effects are often ascribed to the action of reactive oxygen species (ROS) produced in the stressed plants for the following reasons: (1) various environmental stressors commonly cause the oxidation of biomolecules in plants; and (2) transgenic plants with enhanced antioxidant capacities show improved tolerance to environmental stressors (Suzuki et al., 2014). The production of ROS such as superoxide anion radical and hydrogen peroxide (H₂O₂) is intrinsically associated with photosynthesis and respiration (Foyer and Noctor, 2003; Asada, 2006).Plant cells are equipped with abundant antioxidant molecules such as α-tocopherol, β-carotene, and ascorbic acid and an array of ROS-scavenging enzymes such as superoxide dismutase and ascorbate peroxidase to maintain low intracellular ROS levels. When plants are exposed to severe and prolonged environmental stress, the balance between the production and scavenging of ROS is disrupted and the cellular metabolism reaches a new state of higher ROS production and lower antioxidant capacity. Then, the oxidation of vital biomolecules such as proteins and DNA proceeds, and as a consequence, cells undergo oxidative injury (Mano, 2002). The cause-effect relationship between ROS and tissue injury in plants is thus widely accepted, but the biochemical processes between the generation of ROS and cell death are poorly understood.Increasing evidence shows that oxylipin carbonyls mediate the oxidative injury of plants (Yamauchi et al., 2012; for review, see Mano, 2012; Farmer and Mueller, 2013). Oxylipin carbonyls are a group of carbonyl compounds derived from oxygenated lipids and fatty acids. The production of oxylipin carbonyls in living cells is explained as follows. Lipids in the membranes are constitutively oxidized by ROS to form lipid peroxides (LOOHs; Mène-Saffrané et al., 2007) because they are the most immediate and abundant targets near the ROS production sites. There are two types of LOOH formation reaction from ROS (Halliwell and Gutteridge, 2007). One is the radical-dependent reaction. Highly oxidizing radicals, such as hydroxyl radical (standard reduction potential of the HO^•/H₂O pair, +2.31 V) and the protonated form of superoxide radical (HO₂/H₂O₂, +1.06 V), can abstract a hydrogen atom from a lipid molecule, especially at the central carbon of a pentadiene structure in a polyunsaturated fatty acid, to form a radical. This organic radical rapidly reacts with molecular oxygen, forming a lipid hydroperoxyl radical, which then abstracts a hydrogen atom from a neighboring molecule and becomes a LOOH. The other reaction is the addition of singlet oxygen to a double bond of an unsaturated fatty acid to form an endoperoxide or a hydroperoxide (both are LOOHs). A variety of LOOH species are formed, depending on the source fatty acid and also by the oxygenation mechanism (Montillet et al., 2004). LOOH molecules are unstable, and in the presence of redox catalysts such as transition metal ions or free radicals, they decompose to form various aldehydes and ketones (i.e. oxylipin carbonyls; Farmer and Mueller, 2013). The chemical species of oxylipin carbonyl formed in the cells differ according to the fatty acids and the type of ROS involved (Grosch, 1987; Mano et al., 2014a).More than a dozen species of oxylipin carbonyls are formed in plants (for review, see Mano et al., 2009). Oxylipin carbonyls are constitutively formed in plants under normal physiological conditions, and the levels of certain types of oxylipin carbonyls rise severalfold under stress conditions, detected as increases in the free carbonyl content (Mano et al., 2010; Yin et al., 2010; Kai et al., 2012) and by the extent of the carbonyl modification of target proteins (Winger et al., 2007; Mano et al., 2014b). Among the oxylipin carbonyls, the α,β-unsaturated carbonyls, such as acrolein and 4-hydroxy-(E)-2-nonenal (HNE), have high reactivity and cytotoxicity (Esterbauer et al., 1991; Alméras et al., 2003). They strongly inactivate lipoate enzymes in mitochondria (Taylor et al., 2002) and thiol-regulated enzymes in chloroplasts (Mano et al., 2009) in vitro and cause tissue injury in leaves when they are fumigated (Matsui et al., 2012).The physiological relevance of oxylipin carbonyls has been shown by the observation that the overexpression of different carbonyl-scavenging enzymes commonly confers stress tolerance to transgenic plants (for review, see Mano, 2012). For example, 2-alkenal reductase (AER)-overproducing tobacco (Nicotiana tabacum) showed tolerance to aluminum (Yin et al., 2010), aldehyde dehydrogenase-overproducing Arabidopsis (Arabidopsis thaliana) showed tolerance to osmotic and oxidative stress (Sunkar et al., 2003), and aldehyde reductase-overproducing tobacco showed tolerance to chemical and drought stress (Oberschall et al., 2000). In addition, the genetic suppression of a carbonyl-scavenging enzyme made plants susceptible to stressors (Kotchoni et al., 2006; Shin et al., 2009; Yamauchi et al., 2012; Tang et al., 2014). Under stress conditions, there are positive correlations between the levels of certain carbonyls and the extent of tissue injury (Mano et al., 2010; Yin et al., 2010; Yamauchi et al., 2012). Thus, it is evident that oxylipin carbonyls, downstream products of ROS, are causes of oxidative damage in plant cells.To investigate how oxylipin carbonyls damage cells in oxidatively stressed plants, we here examined the mode of cell death that is induced by oxylipin carbonyls and identified the carbonyl species responsible for the cell death. We observed that oxylipin carbonyls cause programmed cell death (PCD), and our results demonstrated that the oxylipin carbonyls mediate the oxidative stress-induced PCD in tobacco Bright Yellow-2 (BY-2) cultured cells and in roots of tobacco and Arabidopsis plants. We then estimated the relative strengths of distinct carbonyl species to initiate the PCD program. Our findings demonstrate a critical role of the lipid metabolites in ROS signaling.     

Specific Membrane Lipid Composition Is Important for Plasmodesmata Function in Arabidopsis

Plasmodesmata (PD) are nano-sized membrane-lined channels controlling intercellular communication in plants. Although progress has been made in identifying PD proteins, the role played by major membrane constituents, such as the lipids, in defining specialized membrane domains in PD remains unknown. Through a rigorous isolation of “native” PD membrane fractions and comparative mass spectrometry-based analysis, we demonstrate that lipids are laterally segregated along the plasma membrane (PM) at the PD cell-to-cell junction in Arabidopsis thaliana. Remarkably, our results show that PD membranes display enrichment in sterols and sphingolipids with very long chain saturated fatty acids when compared with the bulk of the PM. Intriguingly, this lipid profile is reminiscent of detergent-insoluble membrane microdomains, although our approach is valuably detergent-free. Modulation of the overall sterol composition of young dividing cells reversibly impaired the PD localization of the glycosylphosphatidylinositol-anchored proteins Plasmodesmata Callose Binding 1 and the β-1,3-glucanase PdBG2 and altered callose-mediated PD permeability. Altogether, this study not only provides a comprehensive analysis of the lipid constituents of PD but also identifies a role for sterols in modulating cell-to-cell connectivity, possibly by establishing and maintaining the positional specificity of callose-modifying glycosylphosphatidylinositol proteins at PD. Our work emphasizes the importance of lipids in defining PD membranes.     

Phosphatidic Acid Regulates Microtubule Organization by Interacting with MAP65-1 in Response to Salt Stress in Arabidopsis

Membrane lipids play fundamental structural and regulatory roles in cell metabolism and signaling. Here, we report that phosphatidic acid (PA), a product of phospholipase D (PLD), regulates MAP65-1, a microtubule-associated protein, in response to salt stress. Knockout of the PLDα1 gene resulted in greater NaCl-induced disorganization of microtubules, which could not be recovered during or after removal of the stress. Salt affected the association of MAP65-1 with microtubules, leading to microtubule disorganization in pldα1cells, which was alleviated by exogenous PA. PA bound to MAP65-1, increasing its activity in enhancing microtubule polymerization and bundling. Overexpression of MAP65-1 improved salt tolerance of Arabidopsis thaliana cells. Mutations of eight amino acids in MAP65-1 led to the loss of its binding to PA, microtubule-bundling activity, and promotion of salt tolerance. The pldα1 map65-1 double mutant showed greater sensitivity to salt stress than did either single mutant. These results suggest that PLDα1-derived PA binds to MAP65-1, thus mediating microtubule stabilization and salt tolerance. The identification of MAP65-1 as a target of PA reveals a functional connection between membrane lipids and the cytoskeleton in environmental stress signaling.     

oiwa,a Female Gametophytic Mutant Impaired in a Mitochondrial Manganese-Superoxide Dismutase,Reveals Crucial Roles for Reactive Oxygen Species during Embryo Sac Development and Fertilization in Arabidopsis

Reactive oxygen species (ROS) can function as signaling molecules, regulating key aspects of plant development, or as toxic compounds leading to oxidative damage. In this article, we show that the regulation of ROS production during megagametogenesis is largely dependent on MSD1, a mitochondrial Mn-superoxide dismutase. Wild-type mature embryo sacs show ROS exclusively in the central cell, which appears to be the main source of ROS before pollination. Accordingly, MSD1 shows a complementary expression pattern. MSD1 expression is elevated in the egg apparatus at maturity but is downregulated in the central cell. The oiwa mutants are characterized by high levels of ROS detectable in both the central cell and the micropylar cells. Remarkably, egg apparatus cells in oiwa show central cell features, indicating that high levels of ROS result in the expression of central cell characteristic genes. Notably, ROS are detected in synergid cells after pollination. This ROS burst depends on stigma pollination but precedes fertilization, suggesting that embryo sacs sense the imminent arrival of pollen tubes and respond by generating an oxidative environment. Altogether, we show that ROS play a crucial role during female gametogenesis and fertilization. MSD1 activity seems critical for maintaining ROS localization and important for embryo sac patterning.     

A Developmental Framework for Complex Plasmodesmata Formation Revealed by Large-Scale Imaging of the Arabidopsis Leaf Epidermis

Plasmodesmata (PD) form tubular connections that function as intercellular communication channels. They are essential for transporting nutrients and for coordinating development. During cytokinesis, simple PDs are inserted into the developing cell plate, while during wall extension, more complex (branched) forms of PD are laid down. We show that complex PDs are derived from existing simple PDs in a pattern that is accelerated when leaves undergo the sink–source transition. Complex PDs are inserted initially at the three-way junctions between epidermal cells but develop most rapidly in the anisocytic complexes around stomata. For a quantitative analysis of complex PD formation, we established a high-throughput imaging platform and constructed PDQUANT, a custom algorithm that detected cell boundaries and PD numbers in different wall faces. For anticlinal walls, the number of complex PDs increased with increasing cell size, while for periclinal walls, the number of PDs decreased. Complex PD insertion was accelerated by up to threefold in response to salicylic acid treatment and challenges with mannitol. In a single 30-min run, we could derive data for up to 11k PDs from 3k epidermal cells. This facile approach opens the door to a large-scale analysis of the endogenous and exogenous factors that influence PD formation.     

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.     

β-Galactosyl Yariv Reagent Binds to the β-1,3-Galactan of Arabinogalactan Proteins

Yariv phenylglycosides [1,3,5-tri(p-glycosyloxyphenylazo)-2,4,6-trihydroxybenzene] are a group of chemical compounds that selectively bind to arabinogalactan proteins (AGPs), a type of plant proteoglycan. Yariv phenylglycosides are widely used as cytochemical reagents to perturb the molecular functions of AGPs as well as for the detection, quantification, purification, and staining of AGPs. However, the target structure in AGPs to which Yariv phenylglycosides bind has not been determined. Here, we identify the structural element of AGPs required for the interaction with Yariv phenylglycosides by stepwise trimming of the arabinogalactan moieties using combinations of specific glycoside hydrolases. Whereas the precipitation with Yariv phenylglycosides (Yariv reactivity) of radish (Raphanus sativus) root AGP was not reduced after enzyme treatment to remove α-l-arabinofuranosyl and β-glucuronosyl residues and β-1,6-galactan side chains, it was completely lost after degradation of the β-1,3-galactan main chains. In addition, Yariv reactivity of gum arabic, a commercial product of acacia (Acacia senegal) AGPs, increased rather than decreased during the repeated degradation of β-1,6-galactan side chains by Smith degradation. Among various oligosaccharides corresponding to partial structures of AGPs, β-1,3-galactooligosaccharides longer than β-1,3-galactoheptaose exhibited significant precipitation with Yariv in a radial diffusion assay on agar. A pull-down assay using oligosaccharides cross linked to hydrazine beads detected an interaction of β-1,3-galactooligosaccharides longer than β-1,3-galactopentaose with Yariv phenylglycoside. To the contrary, no interaction with Yariv was detected for β-1,6-galactooligosaccharides of any length. Therefore, we conclude that Yariv phenylglycosides should be considered specific binding reagents for β-1,3-galactan chains longer than five residues, and seven residues are sufficient for cross linking, leading to precipitation of the Yariv phenylglycosides.Arabinogalactan proteins (AGPs) are a type of plant proteoglycans consisting of a Hyp-rich core protein and large arabinogalactan (AG) moieties (Fincher et al., 1983; Nothnagel, 1997). Although there are many molecular species of AGP differentiated by their core proteins, the AG moieties commonly comprise β-1,3-galactan main chains and β-1,6-galactan side chains, to which l-Ara and other auxiliary sugars, such as GlcA, 4-O-methyl-GlcA, l-Fuc, l-Rha, and Xyl, are attached (Fincher et al., 1983; Nothnagel, 1997; Seifert and Roberts, 2007). A commercial product of AGPs prepared from the acacia (Acacia senegal) tree is known as gum arabic and utilized as a food stabilizer. In the Japanese herbal remedy Juzen-Taiho-To, AGs from Astragalus membranaceus are the active ingredient (Majewska-Sawka and Nothnagel, 2000; Kiyohara et al., 2002). In intact plants, AGPs are implicated in various physiological events and serve as extracellular constituents and signaling molecules. For instance, an AGP from stylar transmitting tissue attracts pollen tubes and stimulates their elongation in tobacco (Nicotiana tabacum; Cheung et al., 1995).Yariv phenylglycosides [1,3,5-tri(p-glycosyloxyphenylazo)-2,4,6-trihydroxybenzene] are a group of chemical compounds that were initially developed as carbohydrate antigens for the purification of anti-glycoside antibody and sugar-binding protein (Yariv et al., 1962, 1967a). It then turned out that Yariv phenylglycosides specifically precipitate AGPs (Yariv et al., 1967b; Jermyn and Yeow, 1975). The specific interaction of AGPs with Yariv phenylglycosides forming brown-red precipitate is called Yariv reactivity and has been recognized as an important criterion in the definition of AGPs, even though a number of AGPs do not exhibit Yariv reactivity. Nevertheless, the structure involved in the interaction with Yariv phenylglycoside is presumed to be conserved in many AGPs. The interaction of Yariv phenylglycosides with AGP depends on the glycosyl residues attached to the phenylazotrihydroxybenzene core. In particular, β-glucosyl Yariv phenylglycoside (β-Glc-Yariv) and β-galactosyl Yariv phenylglycoside (β-Gal-Yariv) bind to AGPs, whereas α-glucosyl Yariv and α-galactosyl Yariv (α-Gal-Yariv) do not bind to AGPs (Jermyn and Yeow, 1975; Larkin, 1977, 1978; Nothnagel and Lyon, 1986). Because of the specific interaction with the β-glycosyl Yariv phenylglycosides (β-Yarivs), AGPs were formerly called “β-lectins” (Jermyn and Yeow, 1975; Gleeson and Jermyn, 1979; Nothnagel and Lyon, 1986).The β-Yarivs are useful tools for staining, detection, and quantification of AGPs. Using β-Glc-Yariv, β-lectins were shown to exist in angiosperm, gymnosperm, fern, moss, and liverwort, illustrating the wide distribution of AGPs in the plant kingdom (Jermyn and Yeow, 1975; Clarke et al., 1978). In addition, β-Yarivs are also used as chemical reagents in the purification of AGPs. A nonclassical AGP, xylogen, which is a signaling molecule inducing the differentiation to tracheary elements, has been purified from the culture medium of zinnia (Zinnia elegans) cells by precipitation with β-Glc-Yariv (Motose et al., 2004). As the treatment with β-Yarivs causes the perturbation of various physiological processes in plants, β-Yarivs are reliable cytochemical reagents to explore AGP functions. Application of β-Yarivs to cultured cells of Arabidopsis (Arabidopsis thaliana) induced programmed cell death, demonstrating the involvement of AGPs in the determination of cell fate (Gao and Showalter, 1999). In tobacco cultured cells, the treatment with β-Yarivs has indicated a possible role of AGPs in the orientation of cortical microtubules and the polymerization of F-actin (Sardar et al., 2006).Although Yariv phenylglycosides have been extensively utilized in studies of AGPs over 40 years, the identification of the target structures on AGPs required for β-Yariv reactivity remains elusive (Nothnagel, 1997; Seifert and Roberts, 2007). It has been proposed that β-Yarivs bind to the Hyp-rich core protein, based on the observation that deglycosylation treatment with hydrogen fluoride did not abolish the Yariv reactivity of gum arabic and a tobacco AGP (Akiyama et al., 1984). To the contrary, other reports have asserted the importance of the carbohydrate moieties for Yariv reactivity (Komalavilas et al., 1991). However, with regard to the specific carbohydrate structure required for interaction with β-Yarivs, the results were not always consistent: neither α-l-arabinofuranosyl residues nor β-1,6-galactan side chains were found to be involved in Yariv reactivity of AGPs from Gladiolus spp., radish (Raphanus sativus), and grape (Vitis vinifera; Gleeson and Clarke, 1979; Tsumuraya et al., 1987; Saulnier et al., 1992); partial acid hydrolysis to remove α-l-arabinofuranosyl residues diminished Yariv reactivity of a rose (Rosa spp.) AGP (Komalavilas et al., 1991); and mugwort (Artemisia vulgaris) pollen O-glycans consisting of a β-1,6-galactan core and branched α-l-arabinofuranosyl side chains precipitated with β-Glc-Yariv (Léonard et al., 2005). Accordingly, it has also been suggested that Yariv reactivity depends on the overall physical and chemical properties rather than a specific structural feature of AGPs.In this study, we demonstrate that the peptide component of AGPs is not required for Yariv reactivity. By sequentially trimming the AG moieties of AGPs with sets of specific glycoside hydrolases, we show that β-Gal-Yariv binds to the β-1,3-galactan main chains of radish root AGP. We confirm that β-1,6-galactan side chains are not necessary for Yariv reactivity, we identify β-1,3-galactopentaose (β-1,3-Gal₅) as the smallest carbohydrate structure to interact with β-Gal-Yariv, and we show that β-1,3-galactoheptaose (β-1,3-Gal₇) or longer β-1,3-galactosyl chains are required for the formation of insoluble precipitate with Yariv phenylglycoside. Based on computational modeling, a possible interaction mechanism between β-Gal-Yariv and β-1,3-galactan is suggested.     

Overaccumulation of γ-Glutamylcysteine in a Jasmonate-Hypersensitive Arabidopsis Mutant Causes Jasmonate-Dependent Growth Inhibition

Glutathione (GSH) is essential for many aspects of plant biology and is associated with jasmonate signaling in stress responses. We characterized an Arabidopsis (Arabidopsis thaliana) jasmonate-hypersensitive mutant (jah2) with seedling root growth 100-fold more sensitive to inhibition by the hormone jasmonyl-isoleucine than the wild type. Genetic mapping and genome sequencing determined that the mutation is in intron 6 of GLUTATHIONE SYNTHETASE2, encoding the enzyme that converts γ-glutamylcysteine (γ-EC) to GSH. The level of GSH in jah2 was 71% of the wild type, while the phytoalexin-deficient2-1 (pad2-1) mutant, defective in GSH1 and having only 27% of wild-type GSH level, was not jasmonate hypersensitive. Growth defects for jah2, but not pad2, were also seen in plants grown to maturity. Surprisingly, all phenotypes in the jah2 pad2-1 double mutant were weaker than in jah2. Quantification of γ-EC indicated these defects result from hyperaccumulation of this GSH precursor by 294- and 65-fold in jah2 and the double mutant, respectively. γ-EC reportedly partially substitutes for loss of GSH, but growth inhibition seen here was likely not due to an excess of total glutathione plus γ-EC because their sum in jah2 pad2-1 was only 16% greater than in the wild type. Further, the jah2 phenotypes were lost in a jasmonic acid biosynthesis mutant background, indicating the effect of γ-EC is mediated through jasmonate signaling and not as a direct result of perturbed redox status.Glutathione (GSH) is an essential thiol of most higher organisms, including plants. Primarily found in the reduced form, its roles in maintaining a reduced intracellular state are numerous and well characterized (Foyer and Noctor, 2011; Noctor et al., 2011). Additionally, GSH is involved in detoxifying reactive oxygen species, heavy metal detoxification through phytochelatins, elimination of xenobiotics, and signaling of plant development and stress responses (Rouhier et al., 2008).GSH is synthesized in two steps. The first links Cys to the γ-carboxyl group of Glu through an amide bond catalyzed by γ-glutamylcysteine (γ-EC) synthetase, encoded by the single gene GSH1 in Arabidopsis (Arabidopsis thaliana). Gly is then added by GSH synthetase (GSH-S), also encoded by a single gene (GSH2). GSH is typically present at millimolar levels in plants, and although γ-EC is normally present at only a few percent of this amount, there is evidence that γ-EC has redox activities in Arabidopsis (Pasternak et al., 2008).Insertional knockouts of GSH1 are embryo lethal, and rootmeristemless1, with only 5% of wild-type GSH level, lacks a root apical meristem due to cell cycle arrest (Vernoux et al., 2000; Cairns et al., 2006). Other mutants producing 25% to 50% of wild-type GSH levels grow normally but exhibit defects under various stress conditions. For example, phytoalexin-deficient2-1 (pad2-1) and cadmium sensitive2 mutants are susceptible to pathogens and hypersensitive to Cd, respectively, while regulator of axillary meristems1 causes elevated expression of ASCORBATE PEROXIDASE2 under non-photooxidative-stress conditions (Glazebrook and Ausubel, 1994; Cobbett et al., 1998; Ball et al., 2004).GSH2 null alleles (gsh2-1 and gsh2-2) are also lethal, although plants survive to the early seedling stage (Pasternak et al., 2008). Survival past the embryo stage was attributed to partial complementation of GSH activity by γ-EC, which accumulates to excessive levels in gsh2-1, and the mutant is partially rescued by GSH supplementation. Missense and nonsense GSH2 alleles of membrane trafficking mutants (gsh2-3–gsh2-5) disrupt endoplasmic reticulum (ER) organization and also arrest growth in early seedling development, while a weaker allele (gsh2-6) reached maturity but was smaller than the wild type (Au et al., 2012). A screen for reduced response to Cd also yielded a viable missense mutant of GSH2 (nonresponse or reduced response to Cd2) with approximately 75% of the wild-type GSH level (Jobe et al., 2012).Plant oxidative stress responses involve both redox signaling through GSH and jasmonate hormonal signaling, and gene expression studies have clearly linked these two signaling systems. GSH biosynthesis and metabolism genes are induced by jasmonate, while manipulating GSH level or redox status in various mutants alters expression of genes for jasmonate biosynthesis and signaling (Xiang and Oliver, 1998; Mhamdi et al., 2010; Han et al., 2013). GSH and jasmonate are also associated with protective glucosinolate production in response to insect feeding (Noctor et al., 2011). For example, pad2-1 is deficient in glucosinolates and more susceptible to insects, while several studies have shown jasmonate induces glucosinolates (Brader et al., 2001; Mikkelsen et al., 2003; Sasaki-Sekimoto et al., 2005; Schlaeppi et al., 2008). Liu et al. (2010) isolated jasmonic acid hypersensitive1 (jah1), an Arabidopsis mutant with greater inhibition of root growth than the wild type in the presence of jasmonic acid (JA). The affected gene encodes a cytochrome P450 (CYP82C3) involved in indole glucosinolate production, and this mutant was more susceptible to Botrytis cinerea.The basic mechanism of jasmonate signal transduction and some of the downstream responses emanating from it are now well understood (Browse, 2009; Wasternack and Hause, 2013). However, the mechanisms by which jasmonate and GSH coordinate their activities to mediate oxidative stress and other responses are not known. This study characterized, to our knowledge, a new jasmonate-hypersensitive mutant that accumulates excess γ-EC due to a defect in GSH2, but GSH is only modestly reduced. Results show that elevated γ-EC is deleterious to plant growth through a jasmonate-dependent mechanism.     

The Regulatory Properties of Rubisco Activase Differ among Species and Affect Photosynthetic Induction during Light Transitions

Rubisco’s catalytic chaperone, Rubisco activase (Rca), uses the energy from ATP hydrolysis to restore catalytic competence to Rubisco. In Arabidopsis (Arabidopsis thaliana), inhibition of Rca activity by ADP is fine tuned by redox regulation of the α-isoform. To elucidate the mechanism for Rca regulation in species containing only the redox-insensitive β-isoform, the response of activity to ADP was characterized for different Rca forms. When assayed in leaf extracts, Rubisco activation was significantly inhibited by physiological ratios of ADP to ATP in species containing both α-Rca and β-Rca (Arabidopsis and camelina [Camelina sativa]) or just the β-Rca (tobacco [Nicotiana tabacum]). However, Rca activity was insensitive to ADP inhibition in an Arabidopsis transformant, rwt43, which expresses only Arabidopsis β-Rca, although not in a transformant of Arabidopsis that expresses a tobacco-like β-Rca. ATP hydrolysis by recombinant Arabidopsis β-Rca was much less sensitive to inhibition by ADP than recombinant tobacco β-Rca. Mutation of 17 amino acids in the tobacco β-Rca to the corresponding Arabidopsis residues reduced ADP sensitivity. In planta, Rubisco deactivated at low irradiance except in the Arabidopsis rwt43 transformant containing an ADP-insensitive Rca. Induction of CO₂ assimilation after transition from low to high irradiance was much more rapid in the rwt43 transformant compared with plants containing ADP-sensitive Rca forms. The faster rate of photosynthetic induction and a greater enhancement of growth under a fluctuating light regime by the rwt43 transformant compared with wild-type Arabidopsis suggests that manipulation of Rca regulation might provide a strategy for enhancing photosynthetic performance in certain variable light environments.The activity of Rubisco, the enzyme that catalyzes CO₂ assimilation in photosynthesis, is regulated by Rubisco activase (Rca), a specific catalytic chaperone (Spreitzer and Salvucci, 2002; Portis, 2003). Like other AAA+ ATPases (Snider et al., 2008), Rca uses the energy from ATP hydrolysis to remodel the conformation of its target protein, Rubisco. The conformational changes induced by Rca restore catalytic competence to Rubisco active sites that have been inactivated by the unproductive binding of sugar phosphates, including the substrate ribulose 1,5-bisphosphate (RuBP; Wang and Portis, 1992). Because of the requirement for ATP hydrolysis and the inhibition of activity by ADP (Robinson and Portis, 1988, 1989), Rca adjusts the rate of CO₂ fixation to the rates of electron transport activity via changes in the activation state of Rubisco (Salvucci et al., 1985). As a result of this coordinate regulation, the light response of Rubisco activation closely resembles the light response of CO₂ assimilation, and the levels of RuBP under steady-state conditions are relatively constant over a wide range of irradiance levels (Perchorowicz et al., 1981; Dietz and Heber, 1984).Many plant species express two isoforms of Rca, α and β, that are both active in ATP hydrolysis and Rubisco activation (Shen et al., 1991; Salvucci et al., 2003). In some plant species, these isoforms are the products of an alternative splicing event that generates two polypeptides, which are identical except for a 20- to 30-amino acid extension at the C terminus of the longer α-isoform (Werneke et al., 1989). In cotton (Gossypium hirsutum), soybean (Glycine max), and presumably other plant species, separate genes encode the two isoforms of Rca (Salvucci et al., 2003; Yin et al., 2010). In these species, the amino acid sequences of the overlapping regions of the α- and β-polypeptides are very similar, and the C-terminal extension of the longer α-isoform is similar to the extension produced by alternative splicing (Supplemental Fig. S1).Our current understanding of the role of the two Rca isoforms is based primarily on investigations with Arabidopsis (Arabidopsis thaliana; Zhang and Portis, 1999; Zhang et al., 2001; Wang and Portis, 2006). The C-terminal extension of the α-Rca contains two redox-regulated Cys residues that are modulated by thioredoxin f (Zhang and Portis, 1999). When these residues are oxidized to a disulfide, the affinity for ATP decreases and enzyme activity is more sensitive to inhibition by ADP. Physiological ratios of ADP to ATP significantly inhibit the activity of the Arabidopsis α-Rca when in the oxidized state, but inhibition is much less when this isoform has been reduced by thioredoxin. In contrast, the shorter Arabidopsis β-Rca is not redox regulated and is less sensitive to inhibition by ADP (Zhang and Portis, 1999). Mixing experiments with recombinant Rca have shown that the properties of α-Rca are conferred to the heterooligomer, providing a mechanism for redox regulating the Rca holoenzyme (Zhang et al., 2001). In this way, Rca is similar to the chloroplastic glyceraldehyde 3-P dehydrogenase (GAPDH), which also has both redox-regulated (GAP-B) and non-redox-regulated (GAP-A) forms that differ by a C-terminal extension (Baalmann et al., 1996). Like Rca (Zhang and Portis, 1999; Zhang et al., 2001), redox regulation of two Cys residues in the extension exerts master control over the mixed GAPDH oligomer.Some plant species, including members of the Solanaceae family, as well as maize (Zea mays) and green algae, express only the shorter β-Rca (Salvucci et al., 1987). The β-Rca in these species is not responsive to redox regulation, even though the activation state of Rubisco in these plants is modulated by irradiance (Salvucci and Anderson, 1987) and seems to be associated with the redox status of the chloroplast (Ruuska et al., 2000). With GAPDH, all higher plants appear to have both chloroplastic isoforms, but the non-redox-sensitive form, Gap-A, can be regulated indirectly by thioredoxin through the binding of the small chloroplast protein CP12 (Trost et al., 2006). By analogy, a similar association with CP12 or a CP12-like protein could provide a means of conferring redox sensitivity to β-Rca in species that have only this Rca isoform. However, no association of Rca was observed when the native CP12 complex and other high-molecular-mass species were isolated from tobacco (Nicotiana tabacum) chloroplasts (Carmo-Silva et al., 2011b).In this study, the regulation of β-Rca activity was examined both in vivo and in vitro in plant species that contain only one (i.e. β-) or both (α- and β-) Rca isoforms. The response of enzyme activity to physiological ratios of ADP to ATP was measured for the native Rca in leaf extracts, as well as for recombinant Arabidopsis and tobacco enzymes, to determine the sensitivity of β-Rca to ADP in different species. In addition, experiments were conducted with transgenic Arabidopsis plants containing variants of β-Rca to determine the link between Rca regulation and photosynthetic induction. The results suggest a new strategy for enhancing photosynthetic performance under variable light environments based on altering the regulatory properties of Rca to increase the rate of photosynthetic induction.