Identification of a Hydrolyzable Tannin,Oenothein B,as an Aluminum-Detoxifying Ligand in a Highly Aluminum-Resistant Tree,Eucalyptus camaldulensis

Eucalyptus camaldulensis is a tree species in the Myrtaceae that exhibits extremely high resistance to aluminum (Al). To explore a novel mechanism of Al resistance in plants, we examined the Al-binding ligands in roots and their role in Al resistance of E. camaldulensis. We identified a novel type of Al-binding ligand, oenothein B, which is a dimeric hydrolyzable tannin with many adjacent phenolic hydroxyl groups. Oenothein B was isolated from root extracts of E. camaldulensis by reverse-phase high-performance liquid chromatography and identified by nuclear magnetic resonance and mass spectrometry analyses. Oenothein B formed water-soluble or -insoluble complexes with Al depending on the ratio of oenothein B to Al and could bind at least four Al ions per molecule. In a bioassay using Arabidopsis (Arabidopsis thaliana), Al-induced inhibition of root elongation was completely alleviated by treatment with exogenous oenothein B, which indicated the capability of oenothein B to detoxify Al. In roots of E. camaldulensis, Al exposure enhanced the accumulation of oenothein B, especially in EDTA-extractable forms, which likely formed complexes with Al. Oenothein B was localized mostly in the root symplast, in which a considerable amount of Al accumulated. In contrast, oenothein B was not detected in three Al-sensitive species, comprising the Myrtaceae tree Melaleuca bracteata, Populus nigra, and Arabidopsis. Oenothein B content in roots of five tree species was correlated with their Al resistance. Taken together, these results suggest that internal detoxification of Al by the formation of complexes with oenothein B in roots likely contributes to the high Al resistance of E. camaldulensis.Aluminum (Al) toxicity is a major factor that limits plant growth in acid soils and affects approximately 30% of the total ice-free land area of the world (von Uexküll and Mutert, 1995). Although Al in soils exist in nonphytotoxic silicate or oxide forms at neutral pH, it is solubilized into a phytotoxic form, mainly as Al³⁺, at a pH of less than 5 (Kinraide, 1991; Kochian, 1995). The accumulation of Al in root tips causes rapid inhibition of root elongation, which is a characteristic symptom of Al toxicity in plants (Delhaize and Ryan, 1995; Ma, 2007). In general, plants exhibit an inhibition of root elongation as early as 30 to 120 min after exposure to excessive Al (Barceló and Poschenrieder, 2002). Inhibition of root elongation leads to decreased water and nutrient uptake and, eventually, to restriction of growth of the whole plant.Plants have evolved different levels of Al resistance mediated by two distinct classes of mechanisms (Kochian et al., 2004; Ma, 2007). One strategy is the exclusion of Al from the root tips (exclusion mechanism), and the other is tolerance to Al that enters the root tips (internal tolerance mechanism). The secretion of organic acid anions from roots in response to exposure to Al is the best-documented mechanism for Al exclusion. Organic acid anions (i.e. malate, citrate, and oxalate) can form a complex with Al in the rhizosphere and thereby prevent Al from entering the root tips. The genes encoding transporters for the Al-induced secretion of malate and citrate have been identified and characterized in several plant species (Ryan et al., 2011; Delhaize et al., 2012). Organic acid anions also play a role in the detoxification of Al that enters the roots by means of internal formation of complexes with Al (Ma et al., 1998). However, findings in recent studies increasingly suggest that the Al resistance of some plant species and cultivars cannot be explained solely by these two functions of organic acid anions (Wenzl et al., 2001, 2002; Piñeros et al., 2005; Zheng et al., 2005; Famoso et al., 2010). In addition to organic acid anions, flavonoid-type phenolics (Kidd et al., 2001), phenolic compounds (Ofei-Manu et al., 2001), cyclic hydroxamates (Poschenrieder et al., 2005), and proanthocyanidins (Osawa et al., 2011) in roots or root exudates are proposed as potential organic ligands for Al. The mechanisms by which these additional ligands confer Al resistance remain poorly understood.Eucalyptus camaldulensis is an evergreen tree belonging to the Myrtaceae family and is cultivated in tropical and subtropical regions of the world on account of its superior growth, broad adaptability, and multipurpose wood properties. E. camaldulensis can grow in acid soils and even in acid sulfate soils, where the pH is often lower than 3.5 and the Al concentration in the soil solution often reaches the millimolar level (van Breemen and Pons, 1978). Indeed, seedlings of this species show no inhibition of root elongation and plant growth when exposed to 1 mm Al for 20 d under hydroponic conditions (Tahara et al., 2005). Such Al resistance is considerably higher than that reported for a variety of herbaceous crops and model plants in studies of Al resistance mechanisms; such plants exhibit an inhibition of root elongation at 1 to 50 μm Al (Wenzl et al., 2001). Although our understanding of Al resistance mechanisms in some crops and model plants has improved recently, that for extremely Al-resistant species such as E. camaldulensis is limited.In E. camaldulensis, citrate secretion from roots and its content in the root tips are increased by exposure to Al, suggesting that citrate may contribute to its Al resistance (Tahara et al., 2008a). However, the amounts of organic acid anions, including citrate, secreted from roots and contained within the root tips are lower than those of more sensitive species (Tahara et al., 2008a). Therefore, the high Al resistance of E. camaldulensis cannot be explained only by the presence of organic acid anions. Roots of E. camaldulensis can accumulate large amounts of Al (11 mg g⁻¹ dry weight) with no symptoms of Al toxicity (Tahara et al., 2005), suggesting the existence of additional mechanisms for internal tolerance. In this study, we investigated the presence of novel Al-binding ligands other than organic acid anions in E. camaldulensis roots and identified a hydrolyzable tannin, oenothein B, as a novel type of Al-binding ligand. We also examined the role of the ligand in the internal Al tolerance of E. camaldulensis.     

Natural Variation of Arabidopsis Root Architecture Reveals Complementing Adaptive Strategies to Potassium Starvation

Root architecture is a highly plastic and environmentally responsive trait that enables plants to counteract nutrient scarcities with different foraging strategies. In potassium (K) deficiency (low K), seedlings of the Arabidopsis (Arabidopsis thaliana) reference accession Columbia (Col-0) show a strong reduction of lateral root elongation. To date, it is not clear whether this is a direct consequence of the lack of K as an osmoticum or a triggered response to maintain the growth of other organs under limiting conditions. In this study, we made use of natural variation within Arabidopsis to look for novel root architectural responses to low K. A comprehensive set of 14 differentially responding root parameters were quantified in K-starved and K-replete plants. We identified a phenotypic gradient that links two extreme strategies of morphological adaptation to low K arising from a major tradeoff between main root (MR) and lateral root elongation. Accessions adopting strategy I (e.g. Col-0) maintained MR growth but compromised lateral root elongation, whereas strategy II genotypes (e.g. Catania-1) arrested MR elongation in favor of lateral branching. K resupply and histochemical staining resolved the temporal and spatial patterns of these responses. Quantitative trait locus analysis of K-dependent root architectures within a Col-0 × Catania-1 recombinant inbred line population identified several loci each of which determined a particular subset of root architectural parameters. Our results indicate the existence of genomic hubs in the coordinated control of root growth in stress conditions and provide resources to facilitate the identification of the underlying genes.The ability of plants to actively respond to nutrient scarcity with changes in root architecture is a fascinating phenomenon. Advances in root research and breeding efforts that focus on the enhancement of root traits have been recognized as principal goals to ensure those high yields necessary to feed an ever-growing human population (Hammer et al., 2009; Den Herder et al., 2010). Indeed, understanding the adaptations of root systems to environmental factors has been pointed out as a key issue in modern agriculture (Den Herder et al., 2010).Potassium (K) is the quantitatively most important cation for plant growth, as it serves as the major osmoticum for cell expansion (Leigh and Wyn Jones, 1984; Amtmann et al., 2006). Moreover, K is essential for many cellular and tissue processes, such as enzymatic activity, transport of minerals and metabolites, and regulation of stomatal aperture (Amtmann et al., 2006). Even in fertilized fields, rapid K uptake by plants can lead to K shortage in the root environment, especially early in the growth season. Root adaptations to K deficiency (low K) take place at the physiological (Armengaud et al., 2004; Shin and Schachtman, 2004; Alemán et al., 2011), metabolic (Armengaud et al., 2009a), and morphological levels. In a classic study, Drew (1975) showed an increase in overall lateral root (LR) growth of barley seedlings, even when K was supplied only to parts of the root system. Conversely, a typical response of Arabidopsis (Arabidopsis thaliana) Columbia (Col-0) seedlings to low K is the drastic reduction of LR elongation (Armengaud et al., 2004; Shin and Schachtman, 2004). Conflicting data have been published on the effect of low K on main root (MR) growth in the same species, ranging from no effect (Shin and Schachtman, 2004) to impaired MR elongation (Jung et al., 2009; Kim et al., 2010). Some components involved in K starvation responses have been identified, such as jasmonates (Armengaud et al., 2004, 2010), reactive oxygen species (Shin and Schachtman, 2004), and ethylene (Jung et al., 2009). However, the molecular identity of a root K sensor acting at the base of the signaling cascade is so far unknown.Genetic variation within species is a useful resource to dissect the genetic components determining phenotypes (Koornneef et al., 2004; Trontin et al., 2011; Weigel, 2012). Natural variation within Arabidopsis has been the basis for many studies on plant morphology, physiology, and development as well as stress response (Alonso-Blanco et al., 2009; Weigel, 2012). Natural variation of root traits such as primary root length (Mouchel et al., 2004; Loudet et al., 2005; Sergeeva et al., 2006), LR length (Loudet et al., 2005), and total root size (Fitz Gerald et al., 2006) have pinpointed genomic regions underlying the phenotypic variation via mapping of quantitative trait loci (QTLs) as a first step toward the identification of novel regulatory genes (Mouchel et al., 2004). This strategy has also been applied to environmental responses, such as growth responses to phosphate starvation (Reymond et al., 2006; Svistoonoff et al., 2007). However, despite their importance for plant growth and their strong effect on overall root architecture, root responses to K deficiency have not been genetically dissected.Here, we show that Arabidopsis accessions follow different strategies to adapt to K starvation. We present the quantification of a comprehensive set of root architectural parameters of Arabidopsis grown in K-sufficient and K-deficient media and the identification of genetic loci, each of which determines the response of a distinct subset of root architectural parameters to K starvation.     

Capturing Arabidopsis Root Architecture Dynamics with root-fit Reveals Diversity in Responses to Salinity

The plant root is the first organ to encounter salinity stress, but the effect of salinity on root system architecture (RSA) remains elusive. Both the reduction in main root (MR) elongation and the redistribution of the root mass between MRs and lateral roots (LRs) are likely to play crucial roles in water extraction efficiency and ion exclusion. To establish which RSA parameters are responsive to salt stress, we performed a detailed time course experiment in which Arabidopsis (Arabidopsis thaliana) seedlings were grown on agar plates under different salt stress conditions. We captured RSA dynamics with quadratic growth functions (root-fit) and summarized the salt-induced differences in RSA dynamics in three growth parameters: MR elongation, average LR elongation, and increase in number of LRs. In the ecotype Columbia-0 accession of Arabidopsis, salt stress affected MR elongation more severely than LR elongation and an increase in LRs, leading to a significantly altered RSA. By quantifying RSA dynamics of 31 different Arabidopsis accessions in control and mild salt stress conditions, different strategies for regulation of MR and LR meristems and root branching were revealed. Different RSA strategies partially correlated with natural variation in abscisic acid sensitivity and different Na⁺/K⁺ ratios in shoots of seedlings grown under mild salt stress. Applying root-fit to describe the dynamics of RSA allowed us to uncover the natural diversity in root morphology and cluster it into four response types that otherwise would have been overlooked.Salt stress is known to affect plant growth and productivity as a result of its osmotic and ionic stress components. Osmotic stress imposed by salinity is thought to act in the early stages of the response, by reducing cell expansion in growing tissues and causing stomatal closure to minimize water loss. The build-up of ions in photosynthetic tissues leads to toxicity in the later stages of salinity stress and can be reduced by limiting sodium transport into the shoot tissue and compartmentalization of sodium ions into the root stele and vacuoles (Munns and Tester, 2008). The effect of salt stress on plant development was studied in terms of ion accumulation, plant survival, and signaling (Munns et al., 2012; Hasegawa, 2013; Pierik and Testerink, 2014). Most studies focus on traits in the aboveground tissues, because minimizing salt accumulation in leaf tissue is crucial for plant survival and its productivity. This approach has led to the discovery of many genes underlying salinity tolerance (Munns and Tester, 2008; Munns et al., 2012; Hasegawa, 2013; Maathuis, 2014). Another way to estimate salinity stress tolerance is by studying the rate of main root (MR) elongation of seedlings transferred to medium supplemented with high salt concentration. This is how Salt Overly Sensitive mutants were identified, being a classical example of genes involved in salt stress signaling and tolerance (Hasegawa, 2013; Maathuis, 2014). The success of this approach is to be explained by the important role that the root plays in salinity tolerance. Roots not only provide anchorage and ensure water and nutrient uptake, but also act as a sensory system, integrating changes in nutrient availability, water content, and salinity to adjust root morphology to exploit available resources to the maximum capacity (Galvan-Ampudia et al., 2013; Gruber et al., 2013). Understanding the significance of environmental modifications of root system architecture (RSA) for plant productivity is one of the major challenges of modern agriculture (de Dorlodot et al., 2007; Den Herder et al., 2010; Pierik and Testerink, 2014).The RSA of dicotyledonous plants consists of an embryonically derived MR and lateral roots (LRs) that originate from xylem pole pericycle cells of the MR, or from LRs in the case of higher-order LRs. Root growth and branching is mainly guided through the antagonistic action of two plant hormones: auxin and cytokinins (Petricka et al., 2012). Under environmental stress conditions, the synthesis of abscisic acid (ABA), ethylene, and brassinosteroids is known to be induced and to modulate the growth of MRs and LRs (Achard et al., 2006; Osmont et al., 2007; Achard and Genschik, 2009; Duan et al., 2013; Geng et al., 2013). In general, lower concentrations of salt were observed to slightly induce MR and LR elongation, whereas higher concentrations resulted in decreased growth of both MRs and LRs (Wang et al., 2009; Zolla et al., 2010). The reduction of growth is a result of the inhibition of cell cycle progression and a reduction in root apical meristem size (West et al., 2004). However, conflicting results were presented for the effect of salinity on lateral root density (LRD; Wang et al., 2009; Zolla et al., 2010; Galvan-Ampudia and Testerink, 2011). Some studies suggest that mild salinity enhances LR initiation or emergence events, thereby affecting patterning, whereas other studies imply that salinity arrests LR development. The origin of those contradictory observations could be attributable to studying LR initiation and density at single time points, rather than observing the dynamics of LR development, because LR formation changes as a function of root growth rate (De Smet et al., 2012). The dynamics of LR growth and development were characterized previously for the MR region formed before the salt stress exposure, identifying the importance of ABA in early growth arrest of postemerged LRs in response to salt stress (Duan et al., 2013). The effect of salt on LR emergence and initiation was found to differ for MR regions formed prior and subsequent to salinity exposure (Duan et al., 2013), consistent with LR patterning being determined at the root tip (Moreno-Risueno et al., 2010). Yet the effect of salt stress on the reprogramming of the entire RSA on a longer timescale remains elusive.Natural variation in Arabidopsis (Arabidopsis thaliana) is a great source for dissecting the genetic components underlying phenotypic diversity (Trontin et al., 2011; Weigel, 2012). Genes underlying phenotypic plasticity of RSA to environmental stimuli were also found to have high allelic variation leading to differences in root development between different Arabidopsis accessions (Rosas et al., 2013). Supposedly, genes responsible for phenotypic plasticity of the root morphology to different environmental conditions are under strong selection for adaptation to local environments. Various populations of Arabidopsis accessions were used to study natural variation in ion accumulation and salinity tolerance (Rus et al., 2006; Jha et al., 2010; Katori et al., 2010; Roy et al., 2013). In addition, a number of studies focusing on the natural variation in RSA have been published, identifying quantitative trait loci and allelic variation for genes involved in RSA development under control conditions (Mouchel et al., 2004; Meijón et al., 2014) and nutrient-deficient conditions (Chevalier et al., 2003; Gujas et al., 2012; Gifford et al., 2013; Kellermeier et al., 2013; Rosas et al., 2013). Exploring natural variation not only expands the knowledge of genes and molecular mechanisms underlying biological processes, but also provides insight on how plants adapt to challenging environmental conditions (Weigel, 2012) and whether the mechanisms are evolutionarily conserved. The early growth arrest of newly emerged LRs upon exposure to salt stress was observed to be conserved among the most commonly used Arabidopsis accessions Columbia-0 (Col-0), Landsberg erecta, and Wassilewskija (Ws; Duan et al., 2013). By studying salt stress responses of the entire RSA and a wider natural variation in root responses to stress, one could identify new morphological traits that are under environmental selection and possibly contribute to stress tolerance.In this work, we not only identify the RSA components that are responsive to salt stress, but we also describe the natural variation in dynamics of salt-induced changes leading to redistribution of root mass and different root morphology. The growth dynamics of MRs and LRs under different salt stress conditions were described by fitting a set of quadratic growth functions (root-fit) to individual RSA components. Studying salt-induced changes in RSA dynamics of 31 Arabidopsis accessions revealed four major strategies conserved among the accessions. Those four strategies were due to differences in salt stress sensitivity of individual RSA components (i.e. growth rates of MRs and LRs, and increases in the number of emerged LRs). This diversity in root morphology responses caused by salt stress was observed to be partially associated with differences in ABA, but not ethylene sensitivity. In addition, we observed that a number of accessions exhibiting a relatively strong inhibition of LR elongation showed a smaller increase in the Na⁺/K⁺ ratio in shoot tissue after exposure to salt stress. Our results imply that different RSA strategies identified in this study reflect diverse adaptations to different soil conditions and thus might contribute to efficient water extraction and ion compartmentalization in their native environments.     

The Arabidopsis Endosomal Sorting Complex Required for Transport III Regulates Internal Vesicle Formation of the Prevacuolar Compartment and Is Required for Plant Development

We have established an efficient transient expression system with several vacuolar reporters to study the roles of endosomal sorting complex required for transport (ESCRT)-III subunits in regulating the formation of intraluminal vesicles of prevacuolar compartments (PVCs)/multivesicular bodies (MVBs) in plant cells. By measuring the distributions of reporters on/within the membrane of PVC/MVB or tonoplast, we have identified dominant negative mutants of ESCRT-III subunits that affect membrane protein degradation from both secretory and endocytic pathways. In addition, induced expression of these mutants resulted in reduction in luminal vesicles of PVC/MVB, along with increased detection of membrane-attaching vesicles inside the PVC/MVB. Transgenic Arabidopsis (Arabidopsis thaliana) plants with induced expression of ESCRT-III dominant negative mutants also displayed severe cotyledon developmental defects with reduced cell size, loss of the central vacuole, and abnormal chloroplast development in mesophyll cells, pointing out an essential role of the ESCRT-III complex in postembryonic development in plants. Finally, membrane dissociation of ESCRT-III components is important for their biological functions and is regulated by direct interaction among Vacuolar Protein Sorting-Associated Protein20-1 (VPS20.1), Sucrose Nonfermenting7-1, VPS2.1, and the adenosine triphosphatase VPS4/SUPPRESSOR OF K⁺ TRANSPORT GROWTH DEFECT1.Endomembrane trafficking in plant cells is complicated such that secretory, endocytic, and recycling pathways are usually integrated with each other at the post-Golgi compartments, among which, the trans-Golgi network (TGN) and prevacuolar compartment (PVC)/multivesicular body (MVB) are best studied (Tse et al., 2004; Lam et al., 2007a, 2007b; Müller et al., 2007; Foresti and Denecke, 2008; Hwang, 2008; Otegui and Spitzer, 2008; Robinson et al., 2008; Richter et al., 2009; Ding et al., 2012; Gao et al., 2014). Following the endocytic trafficking of a lipophilic dye, FM4-64, the TGN and PVC/MVB are sequentially labeled and thus are defined as the early and late endosome, respectively, in plant cells (Lam et al., 2007a; Chow et al., 2008). While the TGN is a tubular vesicular-like structure that may include several different microdomains and fit its biological function as a sorting station (Chow et al., 2008; Kang et al., 2011), the PVC/MVB is 200 to 500 nm in size with multiple luminal vesicles of approximately 40 nm (Tse et al., 2004). Membrane cargoes destined for degradation are sequestered into these tiny luminal vesicles and delivered to the lumen of the lytic vacuole (LV) via direct fusion between the PVC/MVB and the LV (Spitzer et al., 2009; Viotti et al., 2010; Cai et al., 2012). Therefore, the PVC/MVB functions between the TGN and LV as an intermediate organelle and decides the fate of membrane cargoes in the LV.In yeast (Saccharomyces cerevisiae), carboxypeptidase S (CPS) is synthesized as a type II integral membrane protein and sorted from the Golgi to the lumen of the vacuole (Spormann et al., 1992). Genetic analyses on the trafficking of CPS have led to the identification of approximately 17 class E genes (Piper et al., 1995; Babst et al., 1997, 2002a, 2002b; Odorizzi et al., 1998; Katzmann et al., 2001) that constitute the core endosomal sorting complex required for transport (ESCRT) machinery. The evolutionarily conserved ESCRT complex consists of several functionally different subcomplexes, ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III and the ESCRT-III-associated/Vacuolar Protein Sorting4 (VPS4) complex. Together, they form a complex protein-protein interaction network that coordinates sorting of cargoes and inward budding of the membrane on the MVB (Hurley and Hanson, 2010; Henne et al., 2011). Cargo proteins carrying ubiquitin signals are thought to be passed from one ESCRT subcomplex to the next, starting with their recognition by ESCRT-0 (Bilodeau et al., 2002, 2003; Hislop and von Zastrow, 2011; Le Bras et al., 2011; Shields and Piper, 2011; Urbé, 2011). ESCRT-0 recruits the ESCRT-I complex, a heterotetramer of VPS23, VPS28, VPS37, and MVB12, from the cytosol to the endosomal membrane (Katzmann et al., 2001, 2003). The C terminus of VPS28 interacts with the N terminus of VPS36, a member of the ESCRT-II complex (Kostelansky et al., 2006; Teo et al., 2006). Then, cargoes passed from ESCRT-I and ESCRT-II are concentrated in certain membrane domains of the endosome by ESCRT-III, which includes four coiled-coil proteins and is sufficient to induce the membrane invagination (Babst et al., 2002b; Saksena et al., 2009; Wollert et al., 2009). Finally, the ESCRT components are disassociated from the membrane by the adenosine triphosphatase (ATPase) associated with diverse cellular activities (AAA) VPS4/SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1 (SKD1) before releasing the internal vesicles (Babst et al., 1997, 1998).Putative homologs of ESCRT-I–ESCRT-III and ESCRT-III-associated components have been identified in plants, except for ESCRT-0, which is only present in Opisthokonta (Winter and Hauser, 2006; Leung et al., 2008; Schellmann and Pimpl, 2009). To date, only a few plant ESCRT components have been studied in detail. The Arabidopsis (Arabidopsis thaliana) AAA ATPase SKD1 localized to the PVC/MVB and showed ATPase activity that was regulated by Lysosomal Trafficking Regulator-Interacting Protein5, a plant homolog of Vps Twenty Associated1 Protein (Haas et al., 2007). Expression of the dominant negative form of SKD1 caused an increase in the size of the MVB and a reduction in the number of internal vesicles (Haas et al., 2007). This protein also contributes to the maintenance of the central vacuole and might be associated with cell cycle regulation, as leaf trichomes expressing its dominant negative mutant form lost the central vacuole and frequently contained multiple nuclei (Shahriari et al., 2010). Double null mutants of CHARGED MULTIVESICULAR BODY PROTEIN, chmp1achmp1b, displayed severe growth defects and were seedling lethal. This may be due to the mislocalization of plasma membrane (PM) proteins, including those involved in auxin transport such as PINFORMED1, PINFORMED2, and AUXIN-RESISTANT1, from the vacuolar degradation pathway to the tonoplast of the LV (Spitzer et al., 2009).Plant ESCRT components usually contain several homologs, with the possibility of functional redundancy. Single mutants of individual ESCRT components may not result in an obvious phenotype, whereas knockout of all homologs of an ESCRT component by generating double or triple mutants may be lethal to the plant. As a first step to carry out systematic analysis on each ESCRT complex in plant cells, here, we established an efficient analysis system to monitor the localization changes of four vacuolar reporters that accumulate either in the lumen (LRR84A-GFP, EMP12-GFP, and aleurain-GFP) or on the tonoplast (GFP-VIT1) of the LV and identified several ESCRT-III dominant negative mutants. We reported that ESCRT-III subunits were involved in the release of PVC/MVB’s internal vesicles from the limiting membrane and were required for membrane protein degradation from secretory and endocytic pathways. In addition, transgenic Arabidopsis plants with induced expression of ESCRT-III dominant negative mutants showed severe cotyledon developmental defects. We also showed that membrane dissociation of ESCRT-III subunits was regulated by direct interaction with SKD1.     

Bisphosphonate Inhibitors Reveal a Large Elasticity of Plastidic Isoprenoid Synthesis Pathway in Isoprene-Emitting Hybrid Aspen

Recently, a feedback inhibition of the chloroplastic 1-deoxy-d-xylulose 5-phosphate (DXP)/2-C-methyl-d-erythritol 4-phosphate (MEP) pathway of isoprenoid synthesis by end products dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP) was postulated, but the extent to which DMADP and IDP can build up is not known. We used bisphosphonate inhibitors, alendronate and zoledronate, that inhibit the consumption of DMADP and IDP by prenyltransferases to gain insight into the extent of end product accumulation and possible feedback inhibition in isoprene-emitting hybrid aspen (Populus tremula × Populus tremuloides). A kinetic method based on dark release of isoprene emission at the expense of substrate pools accumulated in light was used to estimate the in vivo pool sizes of DMADP and upstream metabolites. Feeding with fosmidomycin, an inhibitor of DXP reductoisomerase, alone or in combination with bisphosphonates was used to inhibit carbon input into DXP/MEP pathway or both input and output. We observed a major increase in pathway intermediates, 3- to 4-fold, upstream of DMADP in bisphosphonate-inhibited leaves, but the DMADP pool was enhanced much less, 1.3- to 1.5-fold. In combined fosmidomycin/bisphosphonate treatment, pathway intermediates accumulated, reflecting cytosolic flux of intermediates that can be important under strong metabolic pull in physiological conditions. The data suggested that metabolites accumulated upstream of DMADP consist of phosphorylated intermediates and IDP. Slow conversion of the huge pools of intermediates to DMADP was limited by reductive energy supply. These data indicate that the DXP/MEP pathway is extremely elastic, and the presence of a significant pool of phosphorylated intermediates provides an important valve for fine tuning the pathway flux.Isoprenoids constitute a versatile class of compounds fulfilling major physiological functions. They are formed by two pathways in plants, the mevalonate (MVA) pathway in the cytosol (Gershenzon and Croteau, 1993) and the 1-deoxy-d-xylulose 5-phosphate (DXP)/2-C-methyl-d-erythritol 4-phosphate (MEP) pathway in plastids (Gershenzon and Croteau, 1993; Jomaa et al., 1999; Li and Sharkey, 2013b). The MVA pathway is primarily responsible for the synthesis of sesquiterpenes (C15), triterpenes (C30) including brassinosteroids, and even larger molecules such as dolichols (Bick and Lange, 2003; Li and Sharkey, 2013b; Rajabi Memari et al., 2013; Rosenkranz and Schnitzler, 2013). The DXP/MEP pathway is responsible for the synthesis of the simplest isoprenoids, isoprene and 2-methyl-3-buten-2-ol (C5), monoterpenes (C10), diterpenes (C20) including gibberellins and phytol residue of chlorophylls, and tetraterpenes (C40) including carotenoids (Rodríguez-Concepción and Boronat, 2002; Roberts, 2007).Given that in plants both pathways produce ultimately the same substrates, dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP), the pertinent question is to what extent the two pathways can exchange metabolites (Rodríguez-Concepción and Boronat, 2002). There is evidence of a certain exchange of IDP between cytosolic and plastidic compartments, although the contribution of IDP from one compartment to the pathway flux in the other seems to be relatively minor (Schwender et al., 2001; Rodríguez-Concepción and Boronat, 2002; Bick and Lange, 2003). Some studies have further demonstrated that the exchange of IDP is fully bidirectional (De-Eknamkul and Potduang, 2003; Rodríguez-Concepción, 2006), whereas other studies suggest that IDP export from plastids to cytosol operates with a greater efficiency than the opposite transport (Hemmerlin et al., 2003; Laule et al., 2003). However, although the overall intercompartmental exchange of isoprenoid substrates to pathway flux in the given compartment might seem minor under nonstressed conditions, the importance of cross talk among the pathways might increase under stress conditions that specifically inhibit isoprenoid synthesis in one pathway. In fact, the DXP/MEP pathway is strongly linked to photosynthetic metabolism, and therefore, inhibition of photosynthesis under stressful conditions such as heat stress or drought or photoinhibition could inhibit the synthesis of isoprenoids when they are most needed to fulfill their protective function (Loreto and Schnitzler, 2010; Niinemets, 2010; Possell and Loreto, 2013). There is some evidence demonstrating a certain cooperativity among the two isoprenoid synthesis pathways under conditions leading to a reduction of the activity of one of them (Piel et al., 1998; Jux et al., 2001; Page et al., 2004; Rodríguez-Concepción, 2006), but the capacity for such a replacement of function and regulation is poorly understood.Recent studies using genetically modified plants accumulating end products of the DXP/MEP pathway or using natural variation in product accumulation have demonstrated the existence of a potentially important feedback regulation of the DXP/MEP pathway flux by primary end products of the pathway (Banerjee et al., 2013; Ghirardo et al., 2014; Wright et al., 2014). In particular, binding of DMADP and perhaps IDP to DXP synthase, the first enzyme in the DXP/MEP pathway, leads to downregulation of the pathway flux when the end products cannot be used, such as under stress conditions. However, the strength of such a feedback regulation can be importantly modified by accumulation of phosphorylated intermediates of the pathway, such that DMADP and IDP do not accumulate. Previous studies have demonstrated that there is a certain pool of phosphorylated intermediates in vivo, and that this pool can strongly increase under certain conditions, including experimental and genetic modification of DXP/MEP pathway input and output (Li et al., 2011; Rasulov et al., 2011; Li and Sharkey, 2013a; Ghirardo et al., 2014; Wright et al., 2014).It has further been shown that 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (ME-cDP) is the metabolite accumulating in the plastids, and this accumulation can buffer DMADP and IDP changes in the case of varying DXP/MEP pathway input and consumption (Li and Sharkey, 2013a; Wright et al., 2014). A significant part of ME-cDP might even escape to cytosol, implying the existence of another interesting link between cytosolic and chloroplastic processes in isoprenoid synthesis (Wright et al., 2014). Furthermore, as ME-cDP is an important signaling molecule eliciting a number of gene expression responses (Xiao et al., 2012), accumulation of ME-cDP in plastids and flux to cytosol and further to the nucleus is particularly interesting from the perspective of long-term regulation of isoprenoid synthesis, and suggests a coordination of cellular stress responses by the plastidial isoprenoid synthesis pathway.Isoprene-emitting species constitute an exciting model system where a very large DXP/MEP pathway flux goes to isoprene synthesis under physiological conditions (Li and Sharkey, 2013b; Sharkey et al., 2013). In isoprene-emitting species, there is a concomitant use of the primary substrate DMADP between the plastidic synthesis of isoprene and isoprenoids with a larger molecular size, such as phytol residue of chlorophyll (C20) and carotenoids (C40; Ghirardo et al., 2014; Rasulov et al., 2014), and different from nonemitting species, isoprene emitters seem to support a much larger pool of DMADP without the onset of feedback inhibition (Ghirardo et al., 2014; Wright et al., 2014). However, it is poorly understood how inhibition of one branch of the pathway (isoprene versus larger isoprenoids) affects the other, to what extent it can lead to accumulation of phosphorylated intermediates, how it affects the overall pathway flux through the feedback regulation, and what is the possible role of cytosolic import and export of intermediates. These are all relevant questions to gain insight into the control of the partitioning of pathway flux between isoprene and larger isoprenoids and to understand the biological role of isoprene emission.Studies using metabolic inhibitors to deconvolute the factors involved in pathway regulation and understand the biological role of isoprene have so far used inhibitors that block the early steps of the corresponding pathways. In particular, fosmidomycin, a specific inhibitor of DXP reductoisomerase, the enzyme responsible for the synthesis of MEP from DXP, has been used to inhibit the DXP/MEP pathway (Loreto and Velikova, 2001; Sharkey et al., 2001; Loreto et al., 2004). In addition, lovastatin (mevinolin), the inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase that controls the MVA pathway flux, has been used to study the cooperativity of the two pathways (e.g. Laule et al., 2003; Mansouri and Salari, 2014). However, these inhibitors are not suitable to understand how end product accumulation can alter the pathway flux.Bisphosphonates constitute a promising class of inhibitors that could be particularly apt for studies on the effects of the inhibition of the end points of the pathway. They have been demonstrated to inhibit cytosolic farnesyl diphosphate (FDP) synthase activity (Oberhauser et al., 1998; Cromartie et al., 1999; van Beek et al., 1999; Bergstrom et al., 2000; Burke et al., 2004), аs well as geranyl diphosphate (GDP) and geranylgeranyl diphosphate (GGDP) synthase activities (Oberhauser et al., 1998; Cromartie et al., 1999; Kloer et al., 2006; No et al., 2012; Lindert et al., 2013). To our knowledge, bisphosphonates have not been used to study the effects of end product accumulation on the pathway flux in isoprene-emitting species, with the exception of one study that investigated the development of isoprene emission capacity through leaf ontogeny (Rasulov et al., 2014).A limitation with any inhibitor study could be a certain nonspecificity, inhibition of additional nondesired reactions, but so far there are no data on such nonspecificity of bisphosphonates. However, there is evidence that diphosphate and its analogs are inhibitors of any ferredoxin (Fd)-dependent reaction (Forti and Meyer, 1969; Bojko and Więckowski, 1999). This could be potentially relevant given that DXP/MEP pathway-reducing steps, at the level of 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate (HMBDP) synthase (HDS) and reductase (HDR), directly accept electrons from Fd in light (Eisenreich et al., 2001; Seemann et al., 2006; Li and Sharkey, 2013a). In addition to the DXP/MEP pathway, inhibition at the level of Fd could also affect photosynthetic reactions and thereby alter energy supply for the DXP/MEP pathway.In this study, we have investigated the effects of inhibition of the initial and final steps of the DXP/MEP pathway by fosmidomycin and bisphosphonate inhibitors alendronate and zoledronate in a strong isoprene emitter hybrid aspen (Populus tremula × Populus tremuloides). Alendronate is a highly specific inhibitor of GDP (Lange et al., 2001; Burke et al., 2004) and FDP synthases (Bergstrom et al., 2000; Burke et al., 2004), and a less specific inhibitor of GGDP synthase (Szabo et al., 2002). Zoledronate operates similarly to alendronate, but is a much stronger inhibitor, being operationally active in concentrations several orders of magnitude less than alendronate (Lange et al., 2001; Henneman et al., 2011; Wasko, 2011). A unique in vivo method was used to study dynamic changes in DMADP and phosphorylated intermediate pool sizes (Rasulov et al., 2009a, 2011; Li et al., 2011), and different inhibitors were applied alone or in sequence to study the regulation of the pathway flux in conditions when the flux out of the pathway or into the pathway is curbed and when both the input and the output are curbed. Dynamic model calculations were used to quantitatively evaluate the significance of the cytosolic intermediate input into chloroplastic isoprenoid synthesis under different conditions of the DXP/MEP pathway entrance and exit, and to evaluate the possible nonspecific inhibition of other steps controlling DXP/MEP pathway flux. The study demonstrates the important regulation of DXP/MEP pathway input and output under conditions of end product accumulation and partial cooperativity among chloroplastic and cytosolic isoprenoid synthesis pathways.     

Freeze-Thaw Stress: Effects of Temperature on Hydraulic Conductivity and Ultrasonic Activity in Ten Woody Angiosperms

Freeze-thaw events can affect plant hydraulics by inducing embolism. This study analyzed the effect of temperature during the freezing process on hydraulic conductivity and ultrasonic emissions (UE). Stems of 10 angiosperms were dehydrated to a water potential at 12% percentage loss of hydraulic conductivity (PLC) and exposed to freeze-thaw cycles. The minimal temperature of the frost cycle correlated positively with induced PLC, whereby species with wider conduits (hydraulic diameter) showed higher freeze-thaw-induced PLC. Ultrasonic activity started with the onset of freezing and increased with decreasing subzero temperatures, whereas no UE were recorded during thawing. The temperature at which 50% of UE were reached varied between −9.1°C and −31.0°C across species. These findings indicate that temperatures during freezing are of relevance for bubble formation and air seeding. We suggest that species-specific cavitation thresholds are reached during freezing due to the temperature-dependent decrease of water potential in the ice, while bubble expansion and the resulting PLC occur during thawing. UE analysis can be used to monitor the cavitation process and estimate freeze-thaw-induced PLC.Xylem embolism is a limiting factor for plant survival and distribution (Choat et al., 2012; Charrier et al., 2013). Two major factors can induce embolism in the xylem of plants: drought and freeze stress. Freeze-thaw-induced embolism is caused by bubbles formed during freezing that then expand on thawing (Lemoine et al., 1999; Hacke and Sperry, 2001; Cruiziat et al., 2002; Tyree and Zimmermann, 2002). As wider conduits contain more gas and form larger bubbles, which expand at less negative tension, conduit diameter and xylem sap tension are critical for the formation of freeze-thaw-induced embolism (Davis et al., 1999; Pittermann and Sperry, 2003). Accordingly, Mayr and Sperry (2010) observed a loss of conductivity only when samples were under critical tension during thawing. Under drought stress, tension in the xylem sap increases the sensitivity to embolism generated by successive freeze-thaw cycles (Mayr et al., 2003, 2007).Ultrasonic emissions (UE) analysis can be used to detect cavitation events in wood. It is unclear how well related UE are to cavitation events, as they are extracted from continuous acoustic emissions and depend on set definitions. However, UE analysis has been proven effective for monitoring drought-induced embolism in the laboratory (Pena and Grace, 1986; Salleo and Lo Gullo, 1986; Borghetti et al., 1993; Salleo et al., 2000) as well as in field experiments (Ikeda and Ohtsu, 1992; Jackson et al., 1995; Jackson and Grace, 1996; Hölttä et al., 2005; Ogaya and Penuelas, 2007). In a cavitating conduit, signals are probably produced by the disruption of the water column and subsequent tension relaxation of cell walls.UE have also been detected during freezing events, but the origin of these signals was less clear. In some cases, UE were observed during thawing, which are thus probably related to embolism formation according to the classic thaw-expansion hypothesis (Mayr and Sperry, 2010); however, all species studied have produced UE on freezing, which cannot yet be explained (Raschi et al., 1989; Kikuta and Richter, 2003; Mayr et al., 2007; Mayr and Sperry, 2010; Mayr and Zublasing, 2010). The low solubility of gases in ice prompted the idea that air bubbles expulsed from the ice structure produce UE near the ice-liquid interface (Sevanto et al., 2012). As the water potential of ice is strongly temperature dependent, the minimum temperature during freezing might be a relevant factor. Numerous studies have analyzed UE patterns during freeze-thaw cycles in conifers (Mayr et al., 2007; Mayr and Sperry, 2010; Mayr and Zublasing, 2010) or angiosperms (Weiser and Wallner, 1988; Kikuta and Richter, 2003), but few of them measured embolism concomitantly. Percentage loss of hydraulic conductivity (PLC) was only measured in a few studies and only in conifers (Mayr et al., 2007; Mayr and Sperry, 2010).In this study, we analyzed the effect of freeze-thaw cycles on the hydraulic conductivity and ultrasonic activity in 10 angiosperm species. We hypothesized that (1) the extent of freeze-thaw-induced embolism depends on xylem anatomy (related to conduit diameter) and minimal temperature (related to the water potential of ice); (2) ultrasonic activity is also influenced by anatomy and temperature; and (3) PLC and UE are positively correlated. PLC was measured in 10 angiosperm species after freeze-thaw cycles at different minimal temperatures (−10 to −40°C). Furthermore, UE were recorded during a freeze-thaw cycle down to −40°C.     

The Evolution of Mechanisms Driving the Stomatal Response to Vapor Pressure Deficit

Stomatal responses to vapor pressure deficit (VPD) are a principal means by which vascular land plants regulate daytime transpiration. While much work has focused on characterizing and modeling this response, there remains no consensus as to the mechanism that drives it. Explanations range from passive regulation by leaf hydration to biochemical regulation by the phytohormone abscisic acid (ABA). We monitored ABA levels, leaf gas exchange, and water status in a diversity of vascular land plants exposed to a symmetrical, mild transition in VPD. The stomata in basal lineages of vascular plants, including gymnosperms, appeared to respond passively to changes in leaf water status induced by VPD perturbation, with minimal changes in foliar ABA levels and no hysteresis in stomatal action. In contrast, foliar ABA appeared to drive the stomatal response to VPD in our angiosperm samples. Increased foliar ABA level at high VPD in angiosperm species resulted in hysteresis in the recovery of stomatal conductance; this was most pronounced in herbaceous species. Increased levels of ABA in the leaf epidermis were found to originate from sites of synthesis in other parts of the leaf rather than from the guard cells themselves. The transition from a passive regulation to ABA regulation of the stomatal response to VPD in the earliest angiosperms is likely to have had critical implications for the ecological success of this lineage.Plants continuously regulate transpiration by controlling the aperture of the stomatal pores on the surface of the leaf. The principal atmospheric determinant of stomatal aperture is the humidity of the air, which can be expressed as the vapor pressure difference between the leaf and the atmosphere. Stomatal responses to atmospheric vapor pressure deficit (VPD) have been well characterized across the diversity of vascular plant species (Darwin, 1898; Lange et al., 1971; Turner et al., 1984; Franks and Farquhar, 1999; Oren et al., 1999; Brodribb and McAdam, 2011; Mott and Peak, 2013), with stomata typically closing at high VPD and opening at low VPD. This comprehensive characterization has allowed for the development of highly effective empirical and mechanistic models of leaf gas exchange that provide robust predictions of the responses of transpiration to changes in VPD (Buckley et al., 2003; Katul et al., 2009; Damour et al., 2010; Medlyn et al., 2011). Despite the success of this modeling, the mechanism for the stomatal response to VPD remains poorly understood (Damour et al., 2010). Different hypotheses range from one extreme, whereby stomata respond passively through changes in leaf water content induced by the VPD or humidity perturbation (Lange et al., 1971; Mott and Peak, 2013), to the other extreme, whereby stomata close uniquely in response to the phytohormone abscisic acid (ABA; Xie et al., 2006; Bauer et al., 2013).From the earliest recognition that stomata open and close by changes in guard cell turgor (Heath, 1938), there have been many attempts to link the passive changes in water status that occur during VPD or humidity transitions with stomatal responses to VPD or humidity (Lange et al., 1971; Mott and Peak, 2013). Studies have suggested that changes in atmospheric water content passively drive stomatal responses by changing bulk leaf water status, which in turn changes guard cell turgor (Oren et al., 1999), or alternatively by changing guard cell turgor directly (Mott and Peak, 2013). Models based on these entirely passive processes are highly effective in predicting steady-state stomatal conductance (g_s) in response to changes in VPD or humidity in angiosperms (Mott and Peak, 2013).While hydraulic models provide robust predictions of steady-state g_s, they are less effective at predicting the dynamic responses of stomata to short-term perturbations, particularly with respect to the wrong-way responses that typically occur as transients (Buckley, 2005), as well as feed-forward behavior (Farquhar, 1978; Bunce, 1997; Franks et al., 1997; Tardieu and Simonneau, 1998; Ocheltree et al., 2014; compare with Mott and Peak, 2013). Although some of these models provide a pathway for incorporating the effect of ABA (Buckley, 2005), a lack of knowledge of ABA dynamics or action makes it difficult to integrate the influence of this active regulator of guard cell aperture into models. The stomatal behavior of single gene mutants (most notably the ABA synthesis and signaling mutants of Arabidopsis) strongly supports a role for ABA in mediating standard stomatal responses to changes in VPD. The stomata of these mutants are known to have less pronounced responses to a reduction in relative humidity compared with wild-type plants (Xie et al., 2006). Recently, molecular work has shown that guard cells express many of the genes required to synthesize ABA (Okamoto et al., 2009; Bauer et al., 2013), with molecular proxies for ABA level also indicating that the biochemical activity of ABA in the guard cell may increase following short-term exposure of leaves to a reduction in relative humidity (Waadt et al., 2014). These findings suggest a role for ABA in regulating stomatal responses to VPD and have led some to the conclusion that ABA synthesized autonomously by the guard cells is the predominant mechanism for stomatal responses to increased VPD (Bauer et al., 2013).Although the experimental evidence from molecular studies presents an argument for the role of ABA in the responses of stomata to changes in VPD, very few studies have quantified changes in ABA level in response to VPD. It is well established that ABA levels in leaves and guard cells can increase following the imposition of turgor loss or water stress (Pierce and Raschke, 1980; Harris et al., 1988; Harris and Outlaw, 1991). However, only a few studies have reported increases in foliar ABA level in response to high VPD (Bauerle et al., 2004; Giday et al., 2013), and none have investigated whether these observed dynamic changes or differences in ABA level were functionally relevant for stomatal control. In addition, no study has quantified the levels of ABA in guard cells during a transition in VPD.Here, we investigate the relative importance of ABA for the stomatal response to VPD in whole plants, sampled from across the vascular land plant lineage. We provide, to our knowledge, the first functional assessment of changes in ABA levels driving stomatal responses to VPD as well as critically investigate the recent suggestion that stomatal responses to VPD are driven by an autonomous guard cell synthesis of ABA.     

The Role of a Potassium Transporter OsHAK5 in Potassium Acquisition and Transport from Roots to Shoots in Rice at Low Potassium Supply Levels

In plants, K transporter (KT)/high affinity K transporter (HAK)/K uptake permease (KUP) is the largest potassium (K) transporter family; however, few of the members have had their physiological functions characterized in planta. Here, we studied OsHAK5 of the KT/HAK/KUP family in rice (Oryza sativa). We determined its cellular and tissue localization and analyzed its functions in rice using both OsHAK5 knockout mutants and overexpression lines in three genetic backgrounds. A β-glucuronidase reporter driven by the OsHAK5 native promoter indicated OsHAK5 expression in various tissue organs from root to seed, abundantly in root epidermis and stele, the vascular tissues, and mesophyll cells. Net K influx rate in roots and K transport from roots to aerial parts were severely impaired by OsHAK5 knockout but increased by OsHAK5 overexpression in 0.1 and 0.3 mm K external solution. The contribution of OsHAK5 to K mobilization within the rice plant was confirmed further by the change of K concentration in the xylem sap and K distribution in the transgenic lines when K was removed completely from the external solution. Overexpression of OsHAK5 increased the K-sodium concentration ratio in the shoots and salt stress tolerance (shoot growth), while knockout of OsHAK5 decreased the K-sodium concentration ratio in the shoots, resulting in sensitivity to salt stress. Taken together, these results demonstrate that OsHAK5 plays a major role in K acquisition by roots faced with low external K and in K upward transport from roots to shoots in K-deficient rice plants.Potassium (K) is one of the three most important macronutrients and the most abundant cation in plants. As a major osmoticum in the vacuole, K drives the generation of turgor pressure, enabling cell expansion. In the vascular tissue, K is an important participant in the generation of root pressure (for review, see Wegner, 2014 [including his new hypothesis]). In the phloem, K is critical for the transport of photoassimilates from source to sink (Marschner, 1996; Deeken et al., 2002; Gajdanowicz et al., 2011). In addition, enhancing K absorption and decreasing sodium (Na) accumulation is a major strategy of glycophytes in salt stress tolerance (Maathuis and Amtmann, 1999; Munns and Tester, 2008; Shabala and Cuin, 2008).Plants acquire K through K-permeable proteins at the root surface. Since available K concentration in the soil may vary by 100-fold, plants have developed multiple K uptake systems for adapting to this variability (Epstein et al., 1963; Grabov, 2007; Maathuis, 2009). In a classic K uptake experiment in barley (Hordeum vulgare), root K absorption has been described as a high-affinity and low-affinity biphasic transport process (Epstein et al., 1963). It is generally assumed that the low-affinity transport system (LATS) in the roots mediates K uptake in the millimolar range and that the activity of this system is insensitive to external K concentration (Maathuis and Sanders, 1997; Chérel et al., 2014). In contrast, the high-affinity transport system (HATS) was rapidly up-regulated when the supply of exogenous K was halted (Glass, 1976; Glass and Dunlop, 1978).The membrane transporters for K flux identified in plants are generally classified into three channels and three transporter families based on phylogenetic analysis (Mäser et al., 2001; Véry and Sentenac, 2003; Lebaudy et al., 2007; Alemán et al., 2011). For K uptake, it was predicted that, under most circumstances, K transporters function as HATS, while K-permeable channels mediate LATS (Maathuis and Sanders, 1997). However, a root-expressed K channel in Arabidopsis (Arabidopsis thaliana), Arabidopsis K Transporter1 (AKT1), mediates K absorption over a wide range of external K concentrations (Sentenac et al., 1992; Lagarde et al., 1996; Hirsch et al., 1998; Spalding et al., 1999), while evidence is accumulating that many K transporters, including members of the K transporter (KT)/high affinity K transporter (HAK)/K uptake permease (KUP) family, are low-affinity K transporters (Quintero and Blatt, 1997; Senn et al., 2001), implying that functions of plant K channels and transporters overlap at different K concentration ranges.Out of the three families of K transporters, cation proton antiporter (CPA), high affinity K/Na transporter (HKT), and KT/HAK/KUP, CPA was characterized as a K⁺(Na⁺)/H⁺ antiporter, HKT may cotransport Na and K or transport Na only (Rubio et al., 1995; Uozumi et al., 2000), while KT/HAK/KUP were predicted to be H⁺-coupled K⁺ symporters (Mäser et al., 2001; Lebaudy et al., 2007). KT/HAK/KUP were named by different researchers who first identified and cloned them (Quintero and Blatt, 1997; Santa-María et al., 1997). In plants, the KT/HAK/KUP family is the largest K transporter family, including 13 members in Arabidopsis and 27 members in the rice (Oryza sativa) genome (Rubio et al., 2000; Mäser et al., 2001; Bañuelos et al., 2002; Gupta et al., 2008). Sequence alignments show that genes of this family share relatively low homology to each other. The KT/HAK/KUP family was divided into four major clusters (Rubio et al., 2000; Gupta et al., 2008), and in cluster I and II, they were further separated into A and B groups. Genes of cluster I or II likely exist in all plants, cluster III is composed of genes from both Arabidopsis and rice, while cluster IV includes only four rice genes (Grabov, 2007; Gupta et al., 2008).The functions of KT/HAK/KUP were studied mostly in heterologous expression systems. Transporters of cluster I, such as AtHAK5, HvHAK1, OsHAK1, and OsHAK5, are localized in the plasma membrane (Kim et al., 1998; Bañuelos et al., 2002; Gierth et al., 2005) and exhibit high-affinity K uptake in the yeast Saccharomyces cerevisiae (Santa-María et al., 1997; Fu and Luan, 1998; Rubio et al., 2000) and in Escherichia coli (Horie et al., 2011). Transporters of cluster II, like AtKUP4 (TINY ROOT HAIRS1, TRH1), HvHAK2, OsHAK2, OsHAK7, and OsHAK10, could not complement the K uptake-deficient yeast (Saccharomyces cerevisiae) but were able to mediate K fluxes in a bacterial mutant; they might be tonoplast transporters (Senn et al., 2001; Bañuelos et al., 2002; Rodríguez-Navarro and Rubio, 2006). The function of transporters in clusters III and IV is even less known (Grabov, 2007).Existing data suggest that some KT/HAK/KUP transporters also may respond to salinity stress (Maathuis, 2009). The cluster I transporters of HvHAK1 mediate Na influx (Santa-María et al., 1997), while AtHAK5 expression is inhibited by Na (Rubio et al., 2000; Nieves-Cordones et al., 2010). Expression of OsHAK5 in tobacco (Nicotiana tabacum) BY2 cells enhanced the salt tolerance of these cells by accumulating more K without affecting their Na content (Horie et al., 2011).There are only scarce reports on the physiological function of KT/HAK/KUP in planta. In Arabidopsis, mutation of AtKUP2 (SHORT HYPOCOTYL3) resulted in a short hypocotyl, small leaves, and a short flowering stem (Elumalai et al., 2002), while a loss-of-function mutation of AtKUP4 (TRH1) resulted in short root hairs and a loss of gravity response in the root (Rigas et al., 2001; Desbrosses et al., 2003; Ahn et al., 2004). AtHAK5 is the only system currently known to mediate K uptake at concentrations below 0.01 mm (Rubio et al., 2010) and provides a cesium uptake pathway (Qi et al., 2008). AtHAK5 and AtAKT1 are the two major physiologically relevant molecular entities mediating K uptake into roots in the range between 0.01 and 0.05 mm (Pyo et al., 2010; Rubio et al., 2010). AtAKT1 may contribute to K uptake within the K concentrations that belong to the high-affinity system described by Epstein et al. (1963).Among all 27 members of the KT/HAK/KUP family in rice, OsHAK1, OsHAK5, OsHAK19, and OsHAK20 were grouped in cluster IB (Gupta et al., 2008). These four rice HAK members share 50.9% to 53.4% amino acid identity with AtHAK5. OsHAK1 was expressed in the whole plant, with maximum expression in roots, and was up-regulated by K deficiency; it mediated high-affinity K uptake in yeast (Bañuelos et al., 2002). In this study, we examined the tissue-specific localization and the physiological functions of OsHAK5 in response to variation in K supply and to salt stress in rice. By comparing K uptake and translocation in OsHAK5 knockout (KO) mutants and in OsHAK5-overexpressing lines with those in their respective wild-type lines supplied with different K concentrations, we found that OsHAK5 not only mediates high-affinity K acquisition but also participates in root-to-shoot K transport as well as in K-regulated salt tolerance.     

Spore Density Determines Infection Strategy by the Plant Pathogenic Fungus Plectosphaerella cucumerina

Necrotrophic and biotrophic pathogens are resisted by different plant defenses. While necrotrophic pathogens are sensitive to jasmonic acid (JA)-dependent resistance, biotrophic pathogens are resisted by salicylic acid (SA)- and reactive oxygen species (ROS)-dependent resistance. Although many pathogens switch from biotrophy to necrotrophy during infection, little is known about the signals triggering this transition. This study is based on the observation that the early colonization pattern and symptom development by the ascomycete pathogen Plectosphaerella cucumerina (P. cucumerina) vary between inoculation methods. Using the Arabidopsis (Arabidopsis thaliana) defense response as a proxy for infection strategy, we examined whether P. cucumerina alternates between hemibiotrophic and necrotrophic lifestyles, depending on initial spore density and distribution on the leaf surface. Untargeted metabolome analysis revealed profound differences in metabolic defense signatures upon different inoculation methods. Quantification of JA and SA, marker gene expression, and cell death confirmed that infection from high spore densities activates JA-dependent defenses with excessive cell death, while infection from low spore densities induces SA-dependent defenses with lower levels of cell death. Phenotyping of Arabidopsis mutants in JA, SA, and ROS signaling confirmed that P. cucumerina is differentially resisted by JA- and SA/ROS-dependent defenses, depending on initial spore density and distribution on the leaf. Furthermore, in situ staining for early callose deposition at the infection sites revealed that necrotrophy by P. cucumerina is associated with elevated host defense. We conclude that P. cucumerina adapts to early-acting plant defenses by switching from a hemibiotrophic to a necrotrophic infection program, thereby gaining an advantage of immunity-related cell death in the host.Plant pathogens are often classified as necrotrophic or biotrophic, depending on their infection strategy (Glazebrook, 2005; Nishimura and Dangl, 2010). Necrotrophic pathogens kill living host cells and use the decayed plant tissue as a substrate to colonize the plant, whereas biotrophic pathogens parasitize living plant cells by employing effector molecules that suppress the host immune system (Pel and Pieterse, 2013). Despite this binary classification, the majority of pathogenic microbes employ a hemibiotrophic infection strategy, which is characterized by an initial biotrophic phase followed by a necrotrophic infection strategy at later stages of infection (Perfect and Green, 2001). The pathogenic fungi Magnaporthe grisea, Sclerotinia sclerotiorum, and Mycosphaerella graminicola, the oomycete Phytophthora infestans, and the bacterial pathogen Pseudomonas syringae are examples of hemibiotrophic plant pathogens (Perfect and Green, 2001; Koeck et al., 2011; van Kan et al., 2014; Kabbage et al., 2015).Despite considerable progress in our understanding of plant resistance to necrotrophic and biotrophic pathogens (Glazebrook, 2005; Mengiste, 2012; Lai and Mengiste, 2013), recent debate highlights the dynamic and complex interplay between plant-pathogenic microbes and their hosts, which is raising concerns about the use of infection strategies as a static tool to classify plant pathogens. For instance, the fungal genus Botrytis is often labeled as an archetypal necrotroph, even though there is evidence that it can behave as an endophytic fungus with a biotrophic lifestyle (van Kan et al., 2014). The rice blast fungus Magnaporthe oryzae, which is often classified as a hemibiotrophic leaf pathogen (Perfect and Green, 2001; Koeck et al., 2011), can adopt a purely biotrophic lifestyle when infecting root tissues (Marcel et al., 2010). It remains unclear which signals are responsible for the switch from biotrophy to necrotrophy and whether these signals rely solely on the physiological state of the pathogen, or whether host-derived signals play a role as well (Kabbage et al., 2015).The plant hormones salicylic acid (SA) and jasmonic acid (JA) play a central role in the activation of plant defenses (Glazebrook, 2005; Pieterse et al., 2009, 2012). The first evidence that biotrophic and necrotrophic pathogens are resisted by different immune responses came from Thomma et al. (1998), who demonstrated that Arabidopsis (Arabidopsis thaliana) genotypes impaired in SA signaling show enhanced susceptibility to the biotrophic pathogen Hyaloperonospora arabidopsidis (formerly known as Peronospora parastitica), while JA-insensitive genotypes were more susceptible to the necrotrophic fungus Alternaria brassicicola. In subsequent years, the differential effectiveness of SA- and JA-dependent defense mechanisms has been confirmed in different plant-pathogen interactions, while additional plant hormones, such as ethylene, abscisic acid (ABA), auxins, and cytokinins, have emerged as regulators of SA- and JA-dependent defenses (Bari and Jones, 2009; Cao et al., 2011; Pieterse et al., 2012). Moreover, SA- and JA-dependent defense pathways have been shown to act antagonistically on each other, which allows plants to prioritize an appropriate defense response to attack by biotrophic pathogens, necrotrophic pathogens, or herbivores (Koornneef and Pieterse, 2008; Pieterse et al., 2009; Verhage et al., 2010).In addition to plant hormones, reactive oxygen species (ROS) play an important regulatory role in plant defenses (Torres et al., 2006; Lehmann et al., 2015). Within minutes after the perception of pathogen-associated molecular patterns, NADPH oxidases and apoplastic peroxidases generate early ROS bursts (Torres et al., 2002; Daudi et al., 2012; O’Brien et al., 2012), which activate downstream defense signaling cascades (Apel and Hirt, 2004; Torres et al., 2006; Miller et al., 2009; Mittler et al., 2011; Lehmann et al., 2015). ROS play an important regulatory role in the deposition of callose (Luna et al., 2011; Pastor et al., 2013) and can also stimulate SA-dependent defenses (Chaouch et al., 2010; Yun and Chen, 2011; Wang et al., 2014; Mammarella et al., 2015). However, the spread of SA-induced apoptosis during hyperstimulation of the plant immune system is contained by the ROS-generating NADPH oxidase RBOHD (Torres et al., 2005), presumably to allow for the sufficient generation of SA-dependent defense signals from living cells that are adjacent to apoptotic cells. Nitric oxide (NO) plays an additional role in the regulation of SA/ROS-dependent defense (Trapet et al., 2015). This gaseous molecule can stimulate ROS production and cell death in the absence of SA while preventing excessive ROS production at high cellular SA levels via S-nitrosylation of RBOHD (Yun et al., 2011). Recently, it was shown that pathogen-induced accumulation of NO and ROS promotes the production of azelaic acid, a lipid derivative that primes distal plants for SA-dependent defenses (Wang et al., 2014). Hence, NO, ROS, and SA are intertwined in a complex regulatory network to mount local and systemic resistance against biotrophic pathogens. Interestingly, pathogens with a necrotrophic lifestyle can benefit from ROS/SA-dependent defenses and associated cell death (Govrin and Levine, 2000). For instance, Kabbage et al. (2013) demonstrated that S. sclerotiorum utilizes oxalic acid to repress oxidative defense signaling during initial biotrophic colonization, but it stimulates apoptosis at later stages to advance necrotrophic colonization. Moreover, SA-induced repression of JA-dependent resistance not only benefits necrotrophic pathogens but also hemibiotrophic pathogens after having switched from biotrophy to necrotrophy (Glazebrook, 2005; Pieterse et al., 2009, 2012).Plectosphaerella cucumerina ((P. cucumerina, anamorph Plectosporum tabacinum) anamorph Plectosporum tabacinum) is a filamentous ascomycete fungus that can survive saprophytically in soil by decomposing plant material (Palm et al., 1995). The fungus can cause sudden death and blight disease in a variety of crops (Chen et al., 1999; Harrington et al., 2000). Because P. cucumerina can infect Arabidopsis leaves, the P. cucumerina-Arabidopsis interaction has emerged as a popular model system in which to study plant defense reactions to necrotrophic fungi (Berrocal-Lobo et al., 2002; Ton and Mauch-Mani, 2004; Carlucci et al., 2012; Ramos et al., 2013). Various studies have shown that Arabidopsis deploys a wide range of inducible defense strategies against P. cucumerina, including JA-, SA-, ABA-, and auxin-dependent defenses, glucosinolates (Tierens et al., 2001; Sánchez-Vallet et al., 2010; Gamir et al., 2014; Pastor et al., 2014), callose deposition (García-Andrade et al., 2011; Gamir et al., 2012, 2014; Sánchez-Vallet et al., 2012), and ROS (Tierens et al., 2002; Sánchez-Vallet et al., 2010; Barna et al., 2012; Gamir et al., 2012, 2014; Pastor et al., 2014). Recent metabolomics studies have revealed large-scale metabolic changes in P. cucumerina-infected Arabidopsis, presumably to mobilize chemical defenses (Sánchez-Vallet et al., 2010; Gamir et al., 2014; Pastor et al., 2014). Furthermore, various chemical agents have been reported to induce resistance against P. cucumerina. These chemicals include β-amino-butyric acid, which primes callose deposition and SA-dependent defenses, benzothiadiazole (BTH or Bion; Görlach et al., 1996; Ton and Mauch-Mani, 2004), which activates SA-related defenses (Lawton et al., 1996; Ton and Mauch-Mani, 2004; Gamir et al., 2014; Luna et al., 2014), JA (Ton and Mauch-Mani, 2004), and ABA, which primes ROS and callose deposition (Ton and Mauch-Mani, 2004; Pastor et al., 2013). However, among all these studies, there is increasing controversy about the exact signaling pathways and defense responses contributing to plant resistance against P. cucumerina. While it is clear that JA and ethylene contribute to basal resistance against the fungus, the exact roles of SA, ABA, and ROS in P. cucumerina resistance vary between studies (Thomma et al., 1998; Ton and Mauch-Mani, 2004; Sánchez-Vallet et al., 2012; Gamir et al., 2014).This study is based on the observation that the disease phenotype during P. cucumerina infection differs according to the inoculation method used. We provide evidence that the fungus follows a hemibiotrophic infection strategy when infecting from relatively low spore densities on the leaf surface. By contrast, when challenged by localized host defense to relatively high spore densities, the fungus switches to a necrotrophic infection program. Our study has uncovered a novel strategy by which plant-pathogenic fungi can take advantage of the early immune response in the host plant.     

Acclimation of Leaf Nitrogen to Vertical Light Gradient at Anthesis in Wheat Is a Whole-Plant Process That Scales with the Size of the Canopy

Vertical leaf nitrogen (N) gradient within a canopy is classically considered as a key adaptation to the local light environment that would tend to maximize canopy photosynthesis. We studied the vertical leaf N gradient with respect to the light gradient for wheat (Triticum aestivum) canopies with the aims of quantifying its modulation by crop N status and genetic variability and analyzing its ecophysiological determinants. The vertical distribution of leaf N and light was analyzed at anthesis for 16 cultivars grown in the field in two consecutive seasons under two levels of N. The N extinction coefficient with respect to light (b) varied with N supply and cultivar. Interestingly, a scaling relationship was observed between b and the size of the canopy for all the cultivars in the different environmental conditions. The scaling coefficient of the b-green area index relationship differed among cultivars, suggesting that cultivars could be more or less adapted to low-productivity environments. We conclude that the acclimation of the leaf N gradient to the light gradient is a whole-plant process that depends on canopy size. This study demonstrates that modeling leaf N distribution and canopy expansion based on the assumption that leaf N distribution parallels that of the light is inappropriate. We provide a robust relationship accounting for vertical leaf N gradient with respect to vertical light gradient as a function of canopy size.In cereals, as in many crop species, nitrogen (N) nutrition is a major determinant in the elaboration of grain yield and quality (Lemaire and Millard, 1999; Lawlor, 2002; Hikosaka, 2005). N is involved in both meristematic and photosynthetic activities, with consequences on plant architecture and carbon acquisition and in fine on grain yield and protein concentration. Beside the total amount of N absorbed by the crop, the allocation of N among plant organs plays a key role in determining crop productivity and quality (Grindlay, 1997; Dreccer et al., 1998; Hikosaka, 2005).Light interception and leaf N content are the two main factors governing carbon assimilation at the leaf scale (Evans, 1989). For various species, both light and leaf N attenuate with cumulative leaf area index counted from the top of the canopy (Field, 1983; Hirose and Werger, 1987). Leaf N vertical gradients have been regarded as an adaptive response to the local light environment, maximizing canopy photosynthesis and N utilization efficiency (Hirose and Werger, 1987; Hikosaka et al., 1994; Drouet and Bonhomme, 1999), as N is largely contained in the assimilatory enzyme Rubisco. Theoretical studies indicated that leaf N maximizes canopy photosynthesis when it parallels the light gradient (i.e. when the light [K_L] and N [K_N] extinction coefficients are equal), considering that the leaf N gradient is “optimal” in accordance with the “optimization theory” (Field, 1983; Hirose and Werger, 1987; Anten et al., 1995b).Factors other than the photosynthetic photon flux density (PPFD) might be responsible for the observed leaf N distribution. For instance, the acropetal gradients of leaf age (Hikosaka et al., 1994; Hikosaka, 2005) and light composition (Rousseaux et al., 1999) are known to strengthen the leaf N gradient. However, the impact of each of these factors has been shown to be much less than that of the PPFD gradient (Werger and Hirose, 1991; Pons and de Jong-van Berkel, 2004), although for the grass species Brachypodium pinnatum other factors than light might be involved (Pons et al., 1993). At the molecular level, the process could be driven by the import of compounds such as cytokinins transported in the transpiration stream (Pons et al., 2001; Boonman et al., 2007). Although the actual N distribution usually follows the light gradient, in all studies it is less steep than the calculated optimal N profile maximizing canopy photosynthesis (Pons et al., 1989; Yin et al., 2003). Possible reasons for this discrepancy have been discussed in detail by Kull (2002). Sink-source relations and in particular the demand for N could modulate the light-leaf N relationship (Dreccer et al., 1998), but conflicting results have been reported regarding the effect of N availability on the light-leaf N relationship. While some authors found no effect of N availability (Sinclair and Shiraiwa, 1993; Milroy et al., 2001), others found that the N gradient relative to light (i.e. K_L/K_N) was steeper under low N (Hikosaka et al., 1994; Grindlay et al., 1995; Lötscher et al., 2003) or that the response of the light-leaf N relationship to N availability depended on the developmental stage (Dreccer et al., 2000). Interspecific differences in the light-leaf N relationship have also been reported and were related to differences in phenotypic plasticity (Aerts, 1996) or plant architecture (leaf stature and branching pattern; Anten et al., 1995a; Lötscher et al., 2003).Since canopy photosynthesis is dependent upon the leaf N gradient, it has been suggested that the pattern of leaf N distribution could be responsible for part of the genetic variability associated with the negative correlation between grain yield and protein concentration reported for various crop species (Dreccer et al., 1998). In wheat (Triticum aestivum), N accumulated before anthesis contributes 30% to 70% of grain N (Mi et al., 2000; Kichey et al., 2007). The efficiency of N translocation from the lower to the upper leaves may increase with the steepness of the N gradient, with only a negligible effect on canopy carbon gain integrated over the whole grain-filling period. This hypothesis is consistent with experimental studies based on a range of genotypes showing that, at a given grain yield level, grain protein concentration is positively related to the efficiency of N translocation either from the lower to the upper leaves (Wang et al., 2005) or from the leaves to the grains (Monaghan et al., 2001; Jukanti and Fischer, 2008). Only a few studies have investigated the intraspecific variability of the light-N relationship at the intraspecific level (Shiraiwa and Sinclair, 1993; Bindraban, 1999; Bertheloot et al., 2008; van Oosterom et al., 2010). For wheat, published analyses of the genetic variability of the light-leaf N relationship were limited to only two to five genotypes, and no genetic differences were found (Bindraban, 1999; Bertheloot et al., 2008).This paper focuses on the genetic variability of the vertical leaf N gradient with respect to light for wheat. Three main issues were investigated. What is the effect of N supply on the vertical distribution of leaf N? Does the distribution of leaf N with respect to light differ among genotypes? If the adjustment of leaf N to the light gradient varies with both the genotype and N supply, could this genetic and environmental variability have a unique ecophysiological determinant (driving variable)?These questions were addressed using 16 genotypes (Supplemental Table S1) covering a wide range of variation for N use efficiency (i.e. grain dry mass yield per unit of available mineral N from the soil and fertilizer), for grain protein concentration (Le Gouis et al., 2000; Foulkes et al., 2006; Gaju et al., 2011) and for the deviation from the negative correlation between grain yield and protein concentration (Oury et al., 2003). The 16 genotypes were grown in the field under two conditions of N supply (N− and N+ for low- and high-N treatments, respectively) in order to modulate crop N status at Clermont-Ferrand (CF) in France in two consecutive seasons (experiments CF07 and CF08). In addition, four of the 16 cultivars representing the variability observed for N utilization and N uptake efficiency were grown in the field under two conditions of N supply at Sutton Bonington (SB) in the United Kingdom in one season (experiment SB07). The distribution of leaf N was analyzed at anthesis. The first reason for this is that the distribution of both light and leaf N within the canopy is relatively stable from this phenological stage until almost the end of grain filling (Bertheloot et al., 2008). Whereas the canopy green area index (GAI) decreases dramatically during the grain-filling period, the structure of the canopy affecting light interception does not change significantly during that period. Both the vertical light and N distributions down the canopy are unchanged during most of the grain-filling period; therefore, the K_N-to-K_L ratio is constant during that period (Bertheloot et al., 2008). Similarly, Archontoulis et al. (2011) showed that K_N-to-K_L ratio is not modified during the vegetative and reproductive stages for field-gown sunflower (Helianthus annuus) crops. Therefore, as most of the final grain yield results from carbon assimilated after anthesis (Bidinger et al., 1977; Gebbing and Schnyder, 1999), the N distribution at anthesis is very relevant in terms of carbon assimilation and grain yield in wheat. A second reason is that the number and potential size of grains are determined around anthesis, which therefore appears as a critical stage in the formation of grain yield. A better understanding of the ecophysiological determinants of leaf N gradient at this phenological stage could consequently be crucial for improving wheat productivity and quality (Dreccer et al., 1998).     

S-Nitrosylation of Ascorbate Peroxidase Is Part of Programmed Cell Death Signaling in Tobacco Bright Yellow-2 Cells

Nitric oxide (NO) is a small redox molecule that acts as a signal in different physiological and stress-related processes in plants. Recent evidence suggests that the biological activity of NO is also mediated by S-nitrosylation, a well-known redox-based posttranslational protein modification. Here, we show that during programmed cell death (PCD), induced by both heat shock (HS) or hydrogen peroxide (H₂O₂) in tobacco (Nicotiana tabacum) Bright Yellow-2 cells, an increase in S-nitrosylating agents occurred. NO increased in both experimentally induced PCDs, although with different intensities. In H₂O₂-treated cells, the increase in NO was lower than in cells exposed to HS. However, a simultaneous increase in S-nitrosoglutathione (GSNO), another NO source for S-nitrosylation, occurred in H₂O₂-treated cells, while a decrease in this metabolite was evident after HS. Consistently, different levels of activity and expression of GSNO reductase, the enzyme responsible for GSNO removal, were found in cells subjected to the two different PCD-inducing stimuli: low in H₂O₂-treated cells and high in the heat-shocked ones. Irrespective of the type of S-nitrosylating agent, S-nitrosylated proteins formed upon exposure to both of the PCD-inducing stimuli. Interestingly, cytosolic ascorbate peroxidase (cAPX), a key enzyme controlling H₂O₂ levels in plants, was found to be S-nitrosylated at the onset of both PCDs. In vivo and in vitro experiments showed that S-nitrosylation of cAPX was responsible for the rapid decrease in its activity. The possibility that S-nitrosylation induces cAPX ubiquitination and degradation and acts as part of the signaling pathway leading to PCD is discussed.Nitric oxide (NO) is a gaseous and diffusible redox molecule that acts as a signaling compound in both animal and plant systems (Pacher et al., 2007; Besson-Bard et al., 2008). In plants, NO has been found to play a key role in several physiological processes, such as germination, lateral root development, flowering, senescence, stomatal closure, and growth of pollen tubes (Beligni and Lamattina, 2000; Neill et al., 2002; Correa-Aragunde et al., 2004; He et al., 2004; Prado et al., 2004; Carimi et al., 2005). In addition, NO has been reported to be involved in plant responses to both biotic and abiotic stresses (Leitner et al., 2009; Siddiqui et al., 2011) and in the signaling pathways leading to programmed cell death (PCD; Delledonne et al., 1998; de Pinto et al., 2006; De Michele et al., 2009; Lin et al., 2012; Serrano et al., 2012).The cellular environment may greatly influence the chemical reactivity of NO, giving rise to different biologically active NO-derived compounds, collectively named reactive nitrogen species, which amplify and differentiate its ability to activate physiological and stress-related processes. Many of the biological properties of NO are due to its high affinity with transition metals of metalloproteins as well as its reactivity with reactive oxygen species (ROS; Hill et al., 2010). However, recent evidence suggests that protein S-nitrosylation, due to the addition of NO to reactive Cys thiols, may act as a key mechanism of NO signaling in plants (Wang et al., 2006; Astier et al., 2011). NO is also able to react with reduced glutathione (GSH), the most abundant cellular thiol, thus producing S-nitrosoglutathione (GSNO), which also acts as an endogenous trans-nitrosylating agent. GSNO is also considered as a NO store and donor and, as it is more stable than NO, acts as a long-distance NO transporter through the floematic flux (Malik et al., 2011). S-Nitrosoglutathione reductase (GSNOR), which is an enzyme conserved from bacteria to humans, has been suggested to play a role in regulating S-nitrosothiols (SNO) and the turnover of S-nitrosylated proteins in plants (Liu et al., 2001; Rusterucci et al., 2007).A number of proteins involved in metabolism, stress responses, and redox homeostasis have been identified as potential targets for S-nitrosylation in Arabidopsis (Arabidopsis thaliana; Lindermayr et al., 2005). During the hypersensitive response (HR), 16 proteins were identified to be S-nitrosylated in the seedlings of the same species (Romero-Puertas et al., 2008); in Citrus species, S-nitrosylation of about 50 proteins occurred in the NO-mediated resistance to high salinity (Tanou et al., 2009).However, while the number of candidate proteins for S-nitrosylation is increasing, the functional significance of protein S-nitrosylation has been explained only in a few cases, such as for nonsymbiotic hemoglobin (Perazzolli et al., 2004), glyceraldehyde 3-phosphate dehydrogenase (Lindermayr et al., 2005; Wawer et al., 2010), Met adenosyltransferase (Lindermayr et al., 2006), and metacaspase9 (Belenghi et al., 2007). Of particular interest are the cases in which S-nitrosylation involves enzymes controlling ROS homeostasis. For instance, it has been reported that S-nitrosylation of peroxiredoxin IIE regulates the antioxidant function of this enzyme and might contribute to the HR (Romero-Puertas et al., 2007). It has also been shown that in the immunity response, S-nitrosylation of NADPH oxidase inactivates the enzyme, thus reducing ROS production and controlling HR development (Yun et al., 2011).Recently, S-nitrosylation has also been shown to be involved in PCD of nitric oxide excess1 (noe1) rice (Oryza sativa) plants, which are mutated in the OsCATC gene coding for catalase (Lin et al., 2012). In these plants, which show PCD-like phenotypes under high-light conditions, glyceraldehyde 3-phosphate dehydrogenase and thioredoxin are S-nitrosylated. This suggests that the NO-dependent regulation of these proteins is involved in plant PCD, similar to what occurs in animal apoptosis (Sumbayev, 2003; Hara et al., 2005; Lin et al., 2012). The increase in hydrogen peroxide (H₂O₂) after exposure to high light in noe1 plants is responsible for the production of NO required for leaf cell death induction (Lin et al., 2012). There is a strict relationship between H₂O₂ and NO in PCD activation (Delledonne et al., 2001; de Pinto et al., 2002); however, the mechanism of this interplay is largely still unknown (for review, see Zaninotto et al., 2006; Zhao, 2007; Yoshioka et al., 2011). NO can induce ROS production and vice versa, and their reciprocal modulation in terms of intensity and timing seems to be crucial in determining PCD activation and in controlling HR development (Delledonne et al., 2001; Zhao, 2007; Yun et al., 2011).In previous papers, we demonstrated that heat shock (HS) at 55°C and treatment with 50 mm H₂O₂ promote PCD in tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells (Vacca et al., 2004; de Pinto et al., 2006; Locato et al., 2008). In both experimental conditions, NO production and decrease in cytosolic ascorbate peroxidase (cAPX) were observed as early events in the PCD pathway, and cAPX decrease has been suggested to contribute to determining the redox environment required for PCD (de Pinto et al., 2006; Locato et al., 2008).In this study, the production of nitrosylating agents (NO and GSNO) in the first hours of PCD induction by HS or H₂O₂ treatment in tobacco BY-2 cells and their role in PCD were studied. The possibility that S-nitrosylation could be a first step in regulating cAPX activity and turnover as part of the signaling pathway leading to PCD was also investigated.