Involvement of a class III peroxidase and the mitochondrial protein TSPO in oxidative burst upon treatment of moss plants with a fungal elicitor

Production of apoplastic reactive oxygen species (ROS), or oxidative burst, is among the first responses of plants upon recognition of microorganisms. It requires peroxidase or NADPH oxidase (NOX) activity and factors maintaining cellular redox homeostasis. Here, PpTSPO1 involved in mitochondrial tetrapyrrole transport and abiotic (salt) stress tolerance was tested for its role in biotic stress in Physcomitrella patens, a nonvascular plant (moss). The fungal elicitor chitin caused an immediate oxidative burst in wild-type P. patens but not in the previously described ΔPrx34 mutants lacking the chitin-responsive secreted class III peroxidase (Prx34). Oxidative burst in P. patens was associated with induction of the oxidative stress-related genes AOX, LOX7, and NOX, and also PpTSPO1. The available ΔPpTSPO1 knockout mutants overexpressed AOX and LOX7 constitutively, produced 2.6-fold more ROS than wild-type P. patens, and exhibited increased sensitivity to a fungal necrotrophic pathogen and a saprophyte. These results indicate that Prx34, which is pivotal for antifungal resistance, catalyzes ROS production in P. patens, while PpTSPO1 controls redox homeostasis. The capacity of TSPO to bind harmful free heme and porphyrins and scavenge them through autophagy, as shown in Arabidopsis under abiotic stress, seems important to maintenance of the homeostasis required for efficient pathogen defense.     

A novel Arabidopsis thaliana protein is a functional peripheral-type benzodiazepine receptor

A key element in the regulation of mammalian steroid biosynthesis is the 18 kDa peripheral-type benzodiazepine receptor (PBR), which mediates mitochondrial cholesterol import. PBR also possess an affinity to the tetrapyrrole metabolite protoporphyrin. The bacterial homolog to the mammalian PBR, the Rhodobacter TspO (CrtK) protein, was shown to be involved in the bacterial tetrapyrrole metabolism. Looking for a similar mitochondrial import mechanism in plants, protein sequences from Arabidopsis and several other plants were found with significant similarities to the mammalian PBR and to the Rhodobacter TspO protein. A PBR-homologous Arabidopsis sequence was cloned and expressed in E. coli. The recombinant gene product showed specific high affinity benzodiazepine ligand binding. Moreover, the protein applied to E. coli protoplasts caused an equal benzodiazepine-stimulated uptake of cholesterol and protoporphyrin IX. These results suggest that the PBR like protein is involved in steroid import and is directing protoporphyrinogen IX to the mitochondrial site of protoheme formation.     

Tetrapyrrole‐based drought stress signalling

Tetrapyrroles such as chlorophyll and heme play a vital role in primary plant metabolic processes such as photosynthesis and respiration. Over the past decades, extensive genetic and molecular analyses have provided valuable insights into the complex regulatory network of the tetrapyrrole biosynthesis. However, tetrapyrroles are also implicated in abiotic stress tolerance, although the mechanisms are largely unknown. With recent reports demonstrating that modified tetrapyrrole biosynthesis in plants confers wilting avoidance, a component physiological trait to drought tolerance, it is now timely that this pathway be reviewed in the context of drought stress signalling. In this review, the significance of tetrapyrrole biosynthesis under drought stress is addressed, with particular emphasis on the inter‐relationships with major stress signalling cascades driven by reactive oxygen species (ROS) and organellar retrograde signalling. We propose that unlike the chlorophyll branch, the heme branch of the pathway plays a key role in mediating intracellular drought stress signalling and stimulating ROS detoxification under drought stress. Determining how the tetrapyrrole biosynthetic pathway is involved in stress signalling provides an opportunity to identify gene targets for engineering drought‐tolerant crops.     

Activation of SnRK2 by Raf-like kinase ARK represents a primary mechanism of ABA and abiotic stress responses

The Raf-like protein kinase abscisic acid (ABA) and abiotic stress-responsive Raf-like kinase (ARK) previously identified in the moss Physcomitrium (Physcomitrella) patens acts as an upstream regulator of subgroup III SNF1-related protein kinase2 (SnRK2), the key regulator of ABA and abiotic stress responses. However, the mechanisms underlying activation of ARK by ABA and abiotic stress for the regulation of SnRK2, including the role of ABA receptor-associated group A PP2C (PP2C-A), are not understood. We identified Ser1029 as the phosphorylation site in the activation loop of ARK, which provided a possible mechanism for regulation of its activity. Analysis of transgenic P. patens ark lines expressing ARK-GFP with Ser1029-to-Ala mutation indicated that this replacement causes reductions in ABA-induced gene expression, stress tolerance, and SnRK2 activity. Immunoblot analysis using an anti-phosphopeptide antibody indicated that ABA treatments rapidly stimulate Ser1029 phosphorylation in the wild type (WT). The phosphorylation profile of Ser1029 in ABA-hypersensitive ppabi1 lacking protein phosphatase 2C-A (PP2C-A) was similar to that in the WT, whereas little Ser1029 phosphorylation was observed in ABA-insensitive ark missense mutant lines. Furthermore, newly isolated ppabi1 ark lines showed ABA-insensitive phenotypes similar to those of ark lines. Therefore, ARK is a primary activator of SnRK2, preceding negative regulation by PP2C-A in bryophytes, which provides a prototype mechanism for ABA and abiotic stress responses in plants.

Phosphorylation in the activation loop of the Raf-like kinase ARK is critical for SNF1-related protein kinase2 regulation during abscisic acid responses in the moss Physcomitrium (Physcomitrella) patens.     

AtSec20 is involved in osmotic stress tolerance and AtSec20 mutation unaffects the integrity of intracellular organelles and the anterograde biosynthetic trafficking

The soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play essential roles in intracellular trafficking. However, few experimental data have clarified their roles in the stress responses and the early secretary pathway in Arabidopsis. The AtSec20 gene encodes a protein that is homologous to yeast Sec20p and mammalian BNIP1, which are involved in the Golgi-to-ER retrograde trafficking in yeast and mammalian cells. In this study, AtSec20 is found to be required for the responses to salt stress, osmotic stress and gibberellin (GA) during seed germination and early seedling establishment. Mutation of AtSec20 unaffects the morphology of intracellular organelles, such as endoplasmic reticulum (ER), trans-Golgi network, and peroxisome, and vacuolar protein trafficking is normal in sec20 mutants. Collectively, these results imply that the AtSec20 is involved in abiotic stress tolerance, potentially via roles in retrograde vesicle fusion process in Arabidopsis.     

Possible plant mitochondria involvement in cell adaptation to drought stress. A case study: durum wheat mitochondria

Although plant cell bioenergetics is strongly affected by abiotic stresses, mitochondrial metabolism under stress is still largely unknown. Interestingly, plant mitochondria may control reactive oxygen species (ROS) generation by means of energy-dissipating systems. Therefore, mitochondria may play a central role in cell adaptation to abiotic stresses, which are known to induce oxidative stress at cellular level. With this in mind, in recent years, studies have been focused on mitochondria from durum wheat, a species well adapted to drought stress. Durum wheat mitochondria possess three energy-dissipating systems: the ATP-sensitive plant mitochondrial potassium channel (PmitoK(ATP)); the plant uncoupling protein (PUCP); and the alternative oxidase (AOX). It has been shown that these systems are able to dampen mitochondrial ROS production; surprisingly, PmitoK(ATP) and PUCP (but not AOX) are activated by ROS. This was found to occur in mitochondria from both control and hyperosmotic-stressed seedlings. Therefore, the hypothesis of a 'feed-back' mechanism operating under hyperosmotic/oxidative stress conditions was validated: stress conditions induce an increase in mitochondrial ROS production; ROS activate PmitoK(ATP) and PUCP that, in turn, dissipate the mitochondrial membrane potential, thus inhibiting further large-scale ROS production. Another important aspect is the chloroplast/cytosol/mitochondrion co-operation in green tissues under stress conditions aimed at modulating cell redox homeostasis. Durum wheat mitochondria may act against chloroplast/cytosol over-reduction: the malate/oxaloacetate antiporter and the rotenone-insensitive external NAD(P)H dehydrogenases allow cytosolic NAD(P)H oxidation; under stress this may occur without high ROS production due to co-operation with AOX, which is activated by intermediates of the photorespiratory cycle.     

AtPP2CG1, a protein phosphatase 2C, positively regulates salt tolerance of Arabidopsis in abscisic acid-dependent manner

AtPP2CG1 (Arabidopsis thaliana protein phosphatase 2C G Group 1) was predicted as an abiotic stress candidate gene by bioinformatic analysis in our previous study. The gene encodes a putative protein phosphatase 2C that belongs to Group G of PP2C. There is no report of Group G genes involved in abiotic stress so far. Real-time RT-PCR analysis showed that AtPP2CG1 expression was induced by salt, drought, and abscisic acid (ABA) treatment. The expression levels of AtPP2CG1 in the ABA synthesis-deficient mutant abi2-3 were much lower than that in WT plants under salt stress suggesting that the expression of AtPP2CG1 acts in an ABA-dependent manner. Over-expression of AtPP2CG1 led to enhanced salt tolerance, whereas its loss of function caused decreased salt tolerance. These results indicate that AtPP2CG1 positively regulates salt stress in an ABA-dependent manner. Under salt treatment, AtPP2CG1 up-regulated the expression levels of stress-responsive genes, including RD29A, RD29B, DREB2A and KIN1. GUS activity was detected in roots, leaves, stems, flower, and trichomes of AtPP2CG1 promoter-GUS transgenic plants. AtPP2CG1 protein was localized in nucleus and cytoplasm via AtPP2CG1:eGFP and YFP:AtPP2CG1 fusion approaches.     

Yeast 5-aminolevulinate synthase provides additional chlorophyll precursor in transgenic tobacco

Synthesis of the tetrapyrrole precursor 5-aminolevulinate (ALA) in plants starts with glutamate and is a tRNA-dependent pathway consisting of three enzymatic steps localized in plastids. In animals and yeast, ALA is formed in a single step from succinyl CoA and glycine by aminolevulinate synthase (ALA-S) in mitochondria. A gene encoding a fusion protein of yeast ALA-S with an amino-terminal transit sequence for the small subunit of ribulose bisphosphate carboxylase was introduced into the genome of wild-type tobacco and a chlorophyll-deficient transgenic line expressing glutamate 1-semi-aldehyde aminotransferase (GSA-AT) antisense RNA. Expression of ALA-S in the GSA-AT antisense transgenic line provided green-pigmented co-transformants similar to wild-type in chlorophyll content, while transformants derived from wild-type plants did not show phenotypical changes. The capacity to synthesize ALA and chlorophyll was increased in transformed plants, indicating a contribution of ALA-S to the ALA supply for chlorophyll synthesis. ALA-S activity was detected in plastids of the transformants. Preliminary evidence is presented that succinyl CoA, the substrate for ALA-S, can be synthesized and metabolized in plastids. The transgenic plants formed chlorophyll in the presence of gabaculine, an inhibitor of GSA-AT. Steady-state RNA and protein levels and, consequently, the enzyme activity of GSA-AT were reduced in plants expressing ALA-S. In analogy to the light-dependent ALA synthesis attributed to feedback regulation, a mechanism at the level of intermediates or tetrapyrrole end-products is proposed, which co-ordinates the need for heme and chlorophyll precursors and restricts synthesis of ALA by regulating GSA-AT gene expression. The genetically engineered tobacco plants containing the yeast ALA-S activity demonstrate functional complementation of the catalytic activity of the plant ALA-synthesizing pathway and open strategies for producing tolerance against inhibitors of the C5 pathway.     

Abiotic stress response in the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>: evidence for an evolutionary alteration in signaling pathways in land plants

The mechanisms plants use to adapt to abiotic stress have been widely studied in a number of seed plants. Major research has been focused on the isolation of stress-responsive genes as a means to understand the molecular events underlying the adaptation process. To study stress-related gene regulation in the moss Physcomitrella patens we have isolated two cDNAs showing homology to highly conserved small hydrophobic proteins from different seed plants. The corresponding genes are up-regulated by dehydration, salt, sorbitol, cold and the hormone abscisic acid, indicating overlapping pathways are involved in the control of these genes. Based on the molecular characterization of the moss homologs we propose that signaling pathways in response to abiotic stress may have been altered during the evolution of land plants.Abbreviation ABA Abscisic acid - EST Expressed sequence tag     

烟草磷转运蛋白基因NtPHT2;1的克隆和表达分析

植物对磷酸盐的吸收与利用主要依靠磷转运蛋白,其中PHT2家族编码的低亲和磷转运蛋白主要负责植物在正常供磷条件下磷酸盐的吸收、转运与再利用。为了探究低亲和磷转运蛋白基因NtPHT2;1在烟草转运磷酸盐中的作用和表达模式,本研究以普通烟草K326的cDNA为模板,克隆得到NtPHT2;1,对该基因进行生物信息学分析和蛋白质的亚细胞定位,并通过荧光定量PCR技术对该基因在低磷等非生物胁迫下的基因表达模式进行分析。结果表明：（1）NtPHT2;1基因的全长为1 764 bp,编码587个氨基酸。（2）亚细胞定位结果表明,NtPHT2;1蛋白定位于叶绿体上。（3）同源性比对发现,NtPHT2;1蛋白与辣椒CaPHT2;1蛋白的同源性最高达到91.00%。（4）启动子分析表明,NtPHT2;1启动子含有参与调控植物衰老、逆境胁迫相关的顺式作用元件。（5）组织表达模式分析表明,NtPHT2;1在叶片中的表达量最高,新叶中的表达量比老叶中的高;在低磷诱导条件下,该基因的表达量与正常条件相比差异不显著。（6）不同非生物胁迫下的表达模式表明,在盐胁迫和干旱胁迫下,该基因的表达量显著降低。研究认为,NtPHT2;1基因主要是负责烟株正常生长发育条件下磷酸盐的转运与利用。     

Tetrapyrrole regulation of nuclear gene expression

Tetrapyrroles are the structural backbone of chlorophyll and heme, and are essential for primary photochemistry, light harvesting, and electron transport. The biochemistry of their synthesis has been studied extensively, and it has been suggested that some of the tetrapyrrole biochemical intermediates can affect nuclear gene expression. In this review, tetrapyrrole biosynthesis, which occurs in the chloroplast, and its regulation will be covered. An analysis of the intracellular location of tetrapyrrole intermediates will also be included. The focus will be on tetrapyrrole intermediates that have been suggested to affect gene expression. These include Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethyl ester. Recent evidence also suggests a specific signaling role for the H subunit of Mg-chelatase, an enzyme that catalyzes the insertion of Mg into the tetrapyrrole ring. Since gene expression studies have been done in plants and green algae, our discussion will be limited to these organisms. This revised version was published online in June 2006 with corrections to the Cover Date.     

Arabidopsis mutants deregulated in RCI2A expression reveal new signaling pathways in abiotic stress responses

To uncover new pathways involved in low-temperature signal transduction, we screened for mutants altered in cold-induced expression of RCI2A, an Arabidopsis gene that is not a member of the CBF/DREB1 regulon and is induced not only by low temperature but also by abscisic acid (ABA), dehydration (DH) and NaCl. This was accomplished by generating a line of Arabidopsis carrying a transgene consisting of the RCI2A promoter fused to the firefly luciferase coding sequence. A number of mutants showing low or high RCI2A expression in response to low temperature were identified. These mutants also displayed deregulated RCI2A expression in response to ABA, DH or NaCl. Interestingly, however, they were not altered in stress-induced expression of RD29A, a CBF/DREB1-target gene, suggesting that the mutations affect signaling intermediates of CBF/DREB1-independent regulatory pathways. Several mutants showed alterations in their tolerance to freezing, DH or salt stress, as well as in their ABA sensitivity, which indicates that the signaling intermediates defined by the corresponding mutations play an important role in Arabidopsis tolerance to abiotic stresses. Based on the mutants identified, we discuss the involvement of CBF/DREB1-independent pathways in modulating stress signaling.     

Characterization and molecular cloning of a serine hydroxymethyltransferase 1 (OsSHM1) in rice

Serine hydroxymethyltransferase(SHMT) is important for one carbon metabolism and photorespiration in higher plants for its participation in plant growth and development,and resistance to biotic and abiotic stresses. A rice serine hydroxymethyltransferase gene, Os SHM1, an ortholog of Arabidopsis SHM1, was isolated using map-based cloning. The osshm1 mutant had chlorotic lesions and a considerably smaller,lethal phenotype under natural ambient CO2 concentrations,but could be restored to wild type with normal growth under elevated CO2levels(0.5% CO2), showing a typical photorespiratory phenotype. The data from antioxidant enzymes activity measurement suggested that osshm1 was subjected to signi fi cant oxidative stress. Also, Os SHM1 was expressed in allorgans tested(root, culm, leaf, and young panicle) but predominantly in leaves. Os SHM1 protein is localized to the mitochondria. Our study suggested that molecular function of the Os SHM1 gene is conserved in rice and Arabidopsis.     

A novel proteomic approach reveals a role for Mg-protoporphyrin IX in response to oxidative stress

The presence of genes encoding organellar proteins in different cellular compartments necessitates a tight coordination of expression by the different genomes of the eukaryotic cell. This coordination of gene expression is achieved by organelle-to-nucleus communication. Stress-induced perturbations of the tetrapyrrole pathway trigger large changes in nuclear gene expression. In order to investigate whether the tetrapyrrole Mg-ProtoIX itself is an important part of plastid-to-nucleus communication, we used an affinity column containing Mg-ProtoIX covalently linked to an Affi-Gel matrix. The proteins that bound to Mg-ProtoIX were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis combined with nano liquid chromatography-mass spectrometry (MS)/MS. Thus, we present a novel proteomic approach to address the mechanisms involved in cellular signaling and we identified interactions between Mg-ProtoIX and a large number of proteins associated with oxidative stress responses. Our approach revealed an interaction between Mg-ProtoIX and the heat shock protein 90-type protein, HSP81-2 suggesting that a regulatory complex including HSP90 proteins and tetrapyrroles controlling gene expression is evolutionarily conserved between yeast and plants. In addition, our list of putative Mg-ProtoIX-binding proteins demonstrated that binding of tetrapyrroles does not depend on a specific amino acid motif but possibly on a specific fold of the protein.