Calcium-dependent protein kinase CDPK16 phosphorylates serine-856 of glutamate receptor-like GLR3.6 protein leading to salt-responsive root growth in Arabidopsis.

Calcium-permeable channels in the plasma membrane play vital roles in plant growth, development, and response to environmental stimuli. Arabidopsis possesses 20 glutamate receptor-like proteins that share similarities with animal ionotropic glutamate receptors and mediate Ca2+ influx in plants. Calcium-dependent protein kinases (CDPKs) phosphorylate serine (Ser)-860 of glutamate receptor-like (GLR)3.7 protein, which interacts with 14-3-3ω and plays an essential role in salt and abscisic acid response in Arabidopsis by modulating Ca2+ signaling. However, the significance of CDPK- mediated phosphorylation status of Ser residues of GLR3.6 with regard to the functioning of GLR3.6 remains to be elucidated. In this study, we performed an in vitro kinase assay using CDPK16 and peptides containing the 14-3-3ω interacting domain of GLR3.6. We showed that Ser861/862 of GLR3.6 are required for the interaction with 14-3-3ω and that Ser856 of GLR3.6 is specifically phosphorylated by CDPK16 but not by CDPK3 and CDPK34. In addition, the expression of GLR3.6 was quickly downregulated by salt stress, and plants of glr3.6 mutants and GLR3.6-overexpression lines presented shorter and longer root lengths, respectively, under normal growth conditions than Col. Overexpression of the GLR3.6-Ser856 to Ala mutation resulted in a less sensitive phenotype in response to salt stress similar to glr3.6. Our results indicated that the Ser861/862 residues of GLR3.6 are required for interaction with 14-3-3ω. Additionally, the phosphorylation status of Ser856 residue of GLR3.6, which is mediated specifically by CDPK16, regulates root growth in normal and salt stress and conditions.

Impacts of Mn, Fe, and Oxidative Stressors on MnSOD Activation by AtMTM1 and AtMTM2 in Arabidopsis.

It has been reported that the mitochondrial carrier family proteins of AtMTM1 and AtMTM2 are necessary for manganese superoxide dismutase (MnSOD) activation in Arabidopsis, and are responsive to methyl viologen (MV)-induced oxidative stress. In this study, we showed that MnSOD activity was enhanced specifically by Mn treatments. By using AtMnSOD-overexpressing and AtMnSOD-knockdown mutant plants treated with the widely used oxidative stressors including MV, NaCl, H2O2, and tert-butyl hydroperoxide (t-BH), we revealed that Arabidopsis MnSOD was crucial for root-growth control and superoxide scavenging ability. In addition, it has been reported that E. coli MnSOD activity is inhibited by Fe and that MTM1-mutated yeast cells exhibit elevated Fe content and decreased MnSOD activity, which can be restored by the Fe2+-specific chelator, bathophenanthroline disulfonate (BPS). However, we showed that BPS inhibited MnSOD activity in AtMTM1 and AtMTM2 single- and double-mutant protoplasts, implying that altered Fe homeostasis affected MnSOD activation through AtMTM1 and AtMTM2. Notably, we used inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis to reveal an abnormal Fe/Mn ratio in the roots and shoots of AtMTM1 and AtMTM2 mutants under MV stress, indicating the importance of AtMTM1 in roots and AtMTM2 in shoots for maintaining Fe/Mn balance.

Guard Cell-Specific Pectin METHYLESTERASE53 Is Required for Abscisic Acid-Mediated Stomatal Function and Heat Response in Arabidopsis.

Pectin is a major component of the plant cell wall, forming a network that contributes to cell wall integrity and flexibility. Pectin methylesterase (PME) catalyzes the removal of methylester groups from the homogalacturonan backbone, the most abundant pectic polymer, and contributes to intercellular adhesion during plant development and different environmental stimuli stress. In this study, we identified and characterized an Arabidopsis type-II PME, PME53, which encodes a cell wall deposited protein and may be involved in the stomatal lineage pathway and stomatal functions. We demonstrated that PME53 is expressed explicitly in guard cells as an abscisic acid (ABA)-regulated gene required for stomatal movement and thermotolerance. The expression of PME53 is significantly affected by the stomatal differentiation factors SCRM and MUTE. The null mutation in PME53 results in a significant increase in stomatal number and susceptibility to ABA-induced stomatal closure. During heat stress, the pme53 mutant highly altered the activity of PME and significantly lowered the expression level of the calmodulin AtCaM3, indicating that PME53 may be involved in Ca2+-pectate reconstitution to render plant thermotolerance. Here, we present evidence that the PME53-mediated de-methylesterification status of pectin is directed toward stomatal development, movement, and regulation of the flexibility of the guard cell wall required for the heat response.

Significance of AtMTM1 and AtMTM2 for Mitochondrial MnSOD Activation in Arabidopsis.

The manganese (Mn) tracking factor for mitochondrial Mn superoxide dismutase (MnSOD) has been annotated as yMTM1 in yeast, which belongs to the mitochondrial carrier family. We confirmed that Arabidopsis AtMTM1 and AtMTM2 are functional homologs of yMTM1 as they can revive yeast MnSOD activity in yMTM1-mutant cells. Transient expression of AtMnSOD-3xFLAG in the AtMTM1 and AtMTM2-double mutant protoplasts confirmed that AtMTM1 and AtMTM2 are required for AtMnSOD activation. Our study revealed that AtMnSOD interacts with AtMTM1 and AtMTM2 in the mitochondria. The expression levels of AtMTM1, AtMTM2, and AtMnSOD respond positively to methyl viologen (MV) and metal stress. AtMTM1 and AtMTM2 are involved in Mn and Fe homeostasis, root length, and flowering time. Transient expression of chloroplast-destined AtMnSOD revealed that an evolutionarily conserved activation mechanism, like the chloroplastic-localized MnSOD in some algae, still exists in Arabidopsis chloroplasts. This study strengthens the proposition that AtMTM1 and AtMTM2 participate in the AtMnSOD activation and ion homeostasis.

Coordination of ABA and Chaperone Signaling in Plant Stress Responses.

The abscisic acid (ABA) and chaperone signaling pathways are the central regulators of plant stress defense. Despite their significance and potential overlap, these systems have been described separately. In this review, we summarize information about mechanisms by which the ABA and chaperone signaling pathways might be coregulated. The central factors that join the ABA and chaperone signaling systems are the SWI/SNF chromatin-remodeling proteins, which are involved in stress memory. A benefit from coordination is that the signals sensed through both the ABA and chaperone signaling systems are perceived and stored via chromatin-remodeling factors. For improving plant stress resistance, we propose new bioengineering strategies, which we term 'bioengineering memory'.

Pectin Methylesterases: Cell Wall Remodeling Proteins Are Required for Plant Response to Heat Stress.

Heat stress (HS) is expected to be of increasing worldwide concern in the near future, especially with regard to crop yield and quality as a consequence of rising or varying temperatures as a result of global climate change. HS response (HSR) is a highly conserved mechanism among different organisms but shows remarkable complexity and unique features in plants. The transcriptional regulation of HSR is controlled by HS transcription factors (HSFs) which allow the activation of HS-responsive genes, among which HS proteins (HSPs) are best characterized. Cell wall remodeling constitutes an important component of plant responses to HS to maintain overall function and growth; however, little is known about the connection between cell wall remodeling and HSR. Pectin controls cell wall porosity and has been shown to exhibit structural variation during plant growth and in response to HS. Pectin methylesterases (PMEs) are present in multigene families and encode isoforms with different action patterns by removal of methyl esters to influencing the properties of cell wall. We aimed to elucidate how plant cell walls respond to certain environmental cues through cell wall-modifying proteins in connection with modifications in cell wall machinery. An overview of recent findings shed light on PMEs contribute to a change in cell-wall composition/structure. The fine-scale modulation of apoplastic calcium ions (Ca2+) content could be mediated by PMEs in response to abiotic stress for both the assembly and disassembly of the pectic network. In particular, this modulation is prevalent in guard cell walls for regulating cell wall plasticity as well as stromal aperture size, which comprise critical determinants of plant adaptation to HS. These insights provide a foundation for further research to reveal details of the cell wall machinery and stress-responsive factors to provide targets and strategies to facilitate plant adaptation.

Pectin methylesterase is required for guard cell function in response to heat.

Pectin is an important cell wall polysaccharide required for cellular adhesion, extension, and plant growth. The pectic methylesterification status of guard cell walls influences the movement of stomata in response to different stimuli. Pectin methylesterase (PME) has a profound effect on cell wall modification, especially on the degree of pectic methylesterification during heat response. The Arabidopsis thaliana PME34 gene is highly expressed in guard cells and in response to the phytohormone abscisic acid. The genetic data highlighted the significant role of PME34 in heat tolerance through the regulation of stomatal movement. Thus, the opening and closure of stomata is mediated by changes in response to a given stimulus, could require a specific cell wall modifying enzyme to function properly.

PECTIN METHYLESTERASE34 Contributes to Heat Tolerance through Its Role in Promoting Stomatal Movement.

Pectin, a major component of the primary cell wall, is synthesized in the Golgi apparatus and exported to the cell wall in a highly methylesterified form, then is partially demethylesterified by pectin methylesterases (PMEs; EC 3.1.1.11). PME activity on the status of pectin methylesterification profoundly affects the properties of pectin and, thereby, is critical for plant development and the plant defense response, although the roles of PMEs under heat stress (HS) are poorly understood. Functional genome annotation predicts that at least 66 potential PME genes are contained in Arabidopsis (Arabidopsis thaliana). Thermotolerance assays of PME gene T-DNA insertion lines revealed two null mutant alleles of PME34 (At3g49220) that both consistently showed reduced thermotolerance. Nevertheless, their impairment was independently associated with the expression of HS-responsive genes. It was also observed that PME34 transcription was induced by abscisic acid and highly expressed in guard cells. We showed that the PME34 mutation has a defect in the control of stomatal movement and greatly altered PME and polygalacturonase (EC 3.2.1.15) activity, resulting in a heat-sensitive phenotype. PME34 has a role in the regulation of transpiration through the control of the stomatal aperture due to its cell wall-modifying enzyme activity during the HS response. Hence, PME34 is required for regulating guard cell wall flexibility to mediate the heat response in Arabidopsis.

Using Silicon Polymer Impression Technique and Scanning Electron Microscopy to Measure Stomatal Aperture, Morphology, and Density.

The number of stomata on leaves can be affected by intrinsic development programming and various environmental factors, in addition the control of stomatal apertures is extremely important for the plant stress response. In response to elevated temperatures, transpiration occurs through the stomatal apertures, allowing the leaf to cool through water evaporation. As such, monitoring of stomata behavior to elevated temperatures remains as an important area of research. The protocol allows analysis of stomatal aperture, morphology, and density through a non-destructive imprint of Arabidopsis thaliana leaf surface. Stomatal counts were performed and observed under a scanning electron microscope.

The Heat Stress Factor HSFA6b Connects ABA Signaling and ABA-Mediated Heat Responses.

Heat stress response (HSR) is a conserved mechanism developed to increase the expression of heat shock proteins (HSPs) via a heat shock factor (HSF)-dependent mechanism. Signaling by the stress phytohormone abscisic acid (ABA) is involved in acquired thermotolerance as well. Analysis of Arabidopsis (Arabidopsis thaliana) microarray databases revealed that the expression of HSFA6b, a class A HSF, extensively increased with salinity, osmotic, and cold stresses, but not heat. Here, we show that HSFA6b plays a pivotal role in the response to ABA and in thermotolerance. Salt-inducible HSFA6b expression was down-regulated in ABA-insensitive and -deficient mutants; however, exogenous ABA application restored expression in ABA-deficient, but not -insensitive plants. Thus, ABA signaling is required for proper HSFA6b expression. A transcriptional activation assay of protoplasts revealed that ABA treatment and coexpression of an ABA signaling master effector, ABA-RESPONSIVE ELEMENT-BINDING PROTEIN1, could activate the HSFA6b promoter. In addition, HSFA6b directly bound to the promoter of DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN2A and enhanced its expression. Analysis of ABA responses in seed germination, cotyledon greening, and root growth as well as salt and drought tolerance in HSFA6b-null, overexpression, and dominant negative mutants revealed that HSFA6b is a positive regulator participating in ABA-mediated salt and drought resistance. Thermoprotection tests showed that HSFA6b was required for thermotolerance acquisition. Our study reveals a network in which HSFA6b operates as a downstream regulator of the ABA-mediated stress response and is required for heat stress resistance. This new ABA-signaling pathway is integrated into the complex HSR network in planta.

Regulation of transcription of nucleotide-binding leucine-rich repeat-encoding genes SNC1 and RPP4 via H3K4 trimethylation.

Plant nucleotide-binding leucine-rich repeat (NB-LRR) proteins serve as intracellular sensors to detect pathogen effectors and trigger immune responses. Transcription of the NB-LRR-encoding Resistance (R) genes needs to be tightly controlled to avoid inappropriate defense activation. How the expression of the NB-LRR R genes is regulated is poorly understood. The Arabidopsis (Arabidopsis thaliana) suppressor of npr1-1, constitutive 1 (snc1) mutant carries a gain-of-function mutation in a Toll/Interleukin1 receptor-like (TIR)-NB-LRR-encoding gene, resulting in the constitutive activation of plant defense responses. A snc1 suppressor screen identified modifier of snc1,9 (mos9), which partially suppresses the autoimmune phenotypes of snc1. Positional cloning revealed that MOS9 encodes a plant-specific protein of unknown function. Expression analysis showed that MOS9 is required for the full expression of TIR-NB-LRR protein-encoding RECOGNITION OF PERONOSPORA PARASITICA 4 (RPP4) and SNC1, both of which reside in the RPP4 cluster. Coimmunoprecipitation and mass spectrometry analyses revealed that MOS9 associates with the Set1 class lysine 4 of histone 3 (H3K4) methyltransferase Arabidopsis Trithorax-Related7 (ATXR7). Like MOS9, ATXR7 is also required for the full expression of SNC1 and the autoimmune phenotypes in the snc1 mutant. In atxr7 mutant plants, the expression of RPP4 is similarly reduced, and resistance against Hyaloperonospora arabidopsidis Emwa1 is compromised. Consistent with the attenuated expression of SNC1 and RPP4, trimethylated H3K4 marks are reduced around the promoters of SNC1 and RPP4 in mos9 plants. Our data suggest that MOS9 functions together with ATXR7 to regulate the expression of SNC1 and RPP4 through H3K4 methylation, which plays an important role in fine-tuning their transcription levels and functions in plant defense.

Chaperonin 20 might be an iron chaperone for superoxide dismutase in activating iron superoxide dismutase (FeSOD).

Activation of Cu/Zn superoxide dismutases (CuZnSODs) is aided by Cu incorporation and disulfide isomerization by Cu chaperone of SOD (CCS). As well, an Fe-S cluster scaffold protein, ISU, might alter the incorporation of Fe or Mn into yeast MnSOD (ySOD2), thus leading to active or inactive ySOD2. However, metallochaperones involved in the activation of FeSODs are unknown. Recently, we found that a chloroplastic chaperonin cofactor, CPN20, could mediate FeSOD activity. To investigate whether Fe incorporation in FeSOD is affected by CPN20, we used inductively coupled plasma mass spectrometry to analyze the ability of CPN20 to bind Fe. CPN20 could bind Fe, and the Fe binding to FeSOD was increased with CPN20 incubation. Thus, CPN20 might be an Fe chaperone for FeSOD activation, a role independent of its well-known co-chaperonin activity.

Oscillation regulation of Ca2+ /calmodulin and heat-stress related genes in response to heat stress in rice (Oryza sativa L.).

The Ca ( 2+) /calmodulin (CaM) signaling pathway mediates the heat stress (HS) response and acquisition of thermotolerance in plants. We showed that the rice CaM1-1 isoform can interpret a Ca ( 2+) signature difference in amplitude, frequency, and temporal-spatial properties in regulating transcription of nucleoplasmic small heat-shock protein gene (sHSPC/N) during HS. Ca ( 2+) and A23187 treatments under HS generated an intense and sustained increase in [Ca ( 2+) ]cyt and accelerated the expression of CaM1-1 and sHSPC/N genes, which suggests that HS-induced apoplastic Ca ( 2+) influx was responsible for the [Ca ( 2+) ]cyt transient and downstream HS signaling. Here, we discuss an emerging paradigm in the oscillation regulation of CaM1-1 expression during HS and highlight the areas that need further investigation.

Models for the mechanism for activating copper-zinc superoxide dismutase in the absence of the CCS Cu chaperone in Arabidopsis.

Copper-zinc superoxide dismutase (CuZnSOD; CSD) is an important antioxidant enzyme for oxidative stress protection. To date, two activation pathways have been identified in many species. One requiring the CCS, Cu chaperone for SOD, to insert Cu and activate CSD (referred to as CCS-dependent pathway), and the other works independently of CCS (referred to as CCS-independent pathway). In our previous study, we suggest an unidentified factor will work with glutathione (GSH) for CSD activation in the absence of the CCS. Here, two models of the CCS-independent mechanism are proposed. The role of the unidentified factor may work as a scaffold protein, which provides a platform for the CSD protein and Cu-GSH to interact, or as a Cu carrier, which itself can bind Cu and interact with CSD proteins. We also suggest that the CSD protein conformation at C-terminal is important in providing a docking site for unidentified factor to access.

Heat shock-induced biphasic Ca(2+) signature and OsCaM1-1 nuclear localization mediate downstream signalling in acquisition of thermotolerance in rice (Oryza sativa L.).

We investigated heat shock (HS)-triggered Ca(2+) signalling transduced by a Ca(2+) sensor, calmodulin (CaM), linked to early transcriptome changes of HS-responsive genes in rice. We observed a biphasic [Ca(2+) ](cyt) signature in root cells that was distinct from that in epicotyl and leaf cells, which showed a monophasic response after HS. Treatment with Ca(2+) and A23187 generated an intense and sustained increase in [Ca(2+) ](cyt) in response to HS. Conversely, treatment with Ca(2+) chelator, L-type Ca(2+) channel blocker and CaM antagonist, but not intracellular Ca(2+) release inhibitor, strongly inhibited the increased [Ca(2+) ](cyt) . HS combined with Ca(2+) and A23187 accelerated the expression of OsCaM1-1 and sHSPC/N genes, which suggests that the HS-induced apoplastic Ca(2+) influx is responsible for the [Ca(2+) ](cyt) response and downstream HS signalling. In addition, the biphasic response of OsCaM1-1 in the nucleus followed the Ca(2+) signature, which may provide the information necessary to direct HS-related gene expression. Overexpression of OsCaM1-1 induced the expression of Ca(2+) /HS-related AtCBK3, AtPP7, AtHSF and AtHSP at a non-inducing temperature and enhanced intrinsic thermotolerance in transgenic Arabidopsis. Therefore, HS-triggered rapid increases in [Ca(2+) ](cyt) , together with OsCaM1-1 expression and its nuclear localization, are important in mediating downstream HS-related gene expression for the acquisition of thermotolerance in rice.

Copper chaperone-dependent and -independent activation of three copper-zinc superoxide dismutase homologs localized in different cellular compartments in Arabidopsis.

Superoxide dismutases (SODs) are important antioxidant enzymes that catalyze the disproportionation of superoxide anion to oxygen and hydrogen peroxide to guard cells against superoxide toxicity. The major pathway for activation of copper/zinc SOD (CSD) involves a copper chaperone for SOD (CCS) and an additional minor CCS-independent pathway reported in mammals. We characterized the CCS-dependent and -independent activation pathways for three CSDs localized in different cellular compartments in Arabidopsis (Arabidopsis thaliana). The main activation pathway for CSD1 in the cytoplasm involved a CCS-dependent and -independent pathway, which was similar to that for human CSD. Activation of CSD2 in chloroplasts depended totally on CCS, similar to yeast (Saccharomyces cerevisiae) CSD. Peroxisome-localized CSD3 via a CCS-independent pathway was similar to nematode (Caenorhabditis elegans) CSD in retaining activity in the absence of CCS. In Arabidopsis, glutathione played a role in CCS-independent activation, as was reported in humans, but an additional factor was required. These findings reveal a highly specific and sophisticated regulation of CSD activation pathways in planta relative to other known CCS-independent activation.

Characterization of copper/zinc and manganese superoxide dismutase in green bamboo (Bambusa oldhamii): Cloning, expression and regulation.

Bamboo is distinguished by its rapid growth, for growth more than 100 cm per day. Because of the rapid growth, tissues have significant ATP requirements, which results in intense reduction of oxygen and thus oxidative stress. For this reason, bamboo may have a special and efficient scavenger system to release the stress during fast cell division and elongation. Here, we investigated superoxide dismutase (SOD, E.C.1.15.1.1), the first line of antioxidant enzymes, in green bamboo (Bambusa oldhamii). The SOD activity profile in this species was complex, with 5 genes and 7 isozymes of CuZnSOD and 4 genes and 1 isozyme of MnSOD. We isolated one of each of the green bamboo CuZnSOD and MnSOD genes, and their activities were stable under a broad range of pH and temperature treatments, even at room temperature for more than 3 days. Bamboo SODs showed developmental and tissue-specific regulation, and both transcript and protein levels were responsive to abscisic acid, UV-B and high-light treatments. The complexity of the cis-elements in promoter regions implied that the regulation mechanisms of SOD might help accomplish the unique fast-growth phenotype of green bamboo.

Heat shock-triggered Ca2+ mobilization accompanied by pectin methylesterase activity and cytosolic Ca2+ oscillation are crucial for plant thermotolerance.

Apoplastic Ca(2+) concentration controls membrane permeability, cell wall stabilization, and cell integrity; however, little is known about its role in thermotolerance in plants. Here, we report that the acquired thermotolerance of etiolated rice seedlings (Oryza sativa) was abolished by an exogenously supplied Ca(2+) chelator, EGTA, related to increased cellular content leakage during heat shock (HS) treatment. Thermotolerance was restored by the addition of Ca(2+) during EGTA incubation. Pectin methylesterase (EC 3.1.1.11), a cell-wall remodeling enzyme, was activated in response to HS, and its elevated activity was related to the recovery of the HS-released Ca(2+) concentration. EGTA interfered with the capability of HS to increase oscillation of [Ca(2+)]cyt content. We assume that heat-activated PME activity is involved in cell-wall-localized Ca(2+). The removal of apoplastic Ca(2+) might participate in HS signaling to induce HS protein expression and cell-wall remodeling to retain plasma membrane integrity, prevent cellular content leakage and confer thermoprotection.

AtHSBP functions in seed development and the motif is required for subcellular localization and interaction with AtHSFs.

In Arabidopsis thaliana, heat shock factor binding protein (AtHSBP) is a negative regulator of the heat shock response (HSR), and defective AtHSBP leads to seed abortion. We found that the wild-type and AtHSBP-knockout plants did not differ in ovule phenotypes at flower position 3, which indicates that the seed abortion occurs after fertilization and during embryogenesis. The conserved residues of the hydrophobic heptad repeat (HR) domains in AtHSBP were mutated and examined for their subcellular localization and interacting ability with heat shock factors (AtHSFs). The HR domains at the C terminus of AtHSBP are important for retaining AtHSBP in the cytoplasm under normal growth conditions and for interacting with AtHSFs, which negatively affects the DNA-binding capacity and transactivation activity of AtHSFs during the HSR.

Recovery of heat shock-triggered released apoplastic Ca2+ accompanied by pectin methylesterase activity is required for thermotolerance in soybean seedlings.

Synthesis of heat shock proteins (HSPs) in response to heat shock (HS) is essential for thermotolerance. The effect of a Ca(2+) chelator, EGTA, was investigated before a lethal HS treatment in soybean (Glycine max) seedlings with acquired thermotolerance induced by preheating. Such seedlings became non-thermotolerant with EGTA treatment. The addition of Ca(2+), Sr(2+) or Ba(2+) to the EGTA-treated samples rescued the seedlings from death by preventing the increased cellular leakage of electrolytes, amino acids, and sugars caused by EGTA. It was confirmed that EGTA did not affect HSP accumulation and physiological functions but interfered with the recovery of HS-released Ca(2+) concentration which was required for thermotolerance. Pectin methylesterase (PME, EC 3.1.1.11), a cell wall remodelling enzyme, was activated in response to HS, and its elevated activity caused an increased level of demethylesterified pectin which was related to the recovery of the HS-released Ca(2+) concentration. Thus, the recovery of HS-released Ca(2+) in Ca(2+)-pectate reconstitution through PME activity is required for cell wall remodelling during HS in soybean which, in turn, retains plasma membrane integrity and co-ordinates with HSPs to confer thermotolerance.