烟草氧化胁迫相关基因NtOSA1的克隆表达及亚细胞定位分析

植物类蛋白激酶Abc1家族(activity of bc1complex)在植物生长发育及响应非生物胁迫中起着重要的作用。该研究通过将拟南芥AtOSA1蛋白氨基酸序列与烟草转录组数据进行比对,利用巢式PCR技术克隆得到1个烟草氧化胁迫相关Abc1家族基因NtOSA1,NtOSA1与拟南芥AtOSA1基因具有72.89%的一致性。NtOSA1基因开放阅读框长度为2 283bp,编码760个氨基酸,含有典型的ABC1结构域、一个激酶结构域、叶绿体定位信号肽和两个跨膜结构域。采用实时荧光定量PCR技术,对NtOSA1基因在烟草不同组织以及氧化胁迫、盐胁迫等处理下的表达分析表明:NtOSA1基因的表达具有组织特异性,主要在叶片中表达;NtOSA1基因在H2O2和NaCl处理后表达量上升,均在处理后6h达到最大值,分别为处理前的1.95和2.69倍。亚细胞定位结果表明,NtOSA1蛋白定位在细胞叶绿体上,与预测结果一致。研究表明,NtOSA1基因参与了烟草抗氧化胁迫和盐胁迫的响应。     

Reverse genetic approaches in plants and yeast suggest a role for novel,evolutionarily conserved,selenoprotein-related genes in oxidative stress defense

Oxidation of methionine residues during periods of oxidative stress can lead to loss of protein function. Organisms have developed defense strategies to minimize such damage. The PilB protein, which is involved in pilus formation in the pathogen Neisseria gonorrhoeae, is composed of three functional protein domains (I-III) with putative roles in oxidative stress defense. These domains are evolutionarily conserved and homologs have been discovered in diverse prokaryotes and eukaryotes. Domain III shows similarities to selenoproteins which contain selenium instead of sulfur in a conserved cysteine residue. The substitution of selenium for sulfur alters the redox properties of such proteins. Knock-out mutants were used to elucidate the function of these novel selenoprotein-like domains in yeast and in Arabidopsis thaliana. We show that organisms with non-functional genes for selenoprotein-like polypeptides accumulate higher levels of oxidized methionine residues on exposure to oxidative stress. The behavior of the mutants suggests that these novel selenoprotein-like gene products are part of a ubiquitous detoxification system that interacts with other redox-related proteins such as the thioredoxin-related protein and methionine sulfoxide reductase which are encoded by domains I and II of PilB. These proteins may be encoded by one gene as in the case of several prokaryotes, or by separate genes as in the eukaryotes examined here.     

Characterization of an Abc1 kinase family gene OsABC1-2 conferring enhanced tolerance to dark-induced stress in rice

Leaf senescence is a complex and highly organized process resulting in numerous changes of gene expression and metabolic procedures. However, the exact mechanisms underlying these changes are not well understood. In this study, we reported a rice (Oryza sativa) T-DNA insertion mutant impaired in an Abc1 kinase family gene with a dwarf and pale-green phenotype. The mutant showed reduced pigment content and photosynthetic efficiency and increased superoxide dismutase activity in leaves. The mutated gene, designated OsABC1-2, is expressed primarily in green tissues and/or organs and encodes a protein localized in chloroplast envelope. Expression of the gene was drastically suppressed by dark treatment. Overexpression of the gene in rice enhanced tolerance to prolonged dark-induced stress. Phylogenetic analysis revealed that the plant Abc1 proteins could be divided into three subgroups and OsAbc1-2 resides in a subgroup with potential chloroplast origin. Our results suggest that divergence has occurred among plant Abc1 family and chloroplast Abc1 kinases play potential roles in regulating dark-induced senescence of plants.     

PIC1, an ancient permease in Arabidopsis chloroplasts, mediates iron transport

In chloroplasts, the transition metals iron and copper play an essential role in photosynthetic electron transport and act as cofactors for superoxide dismutases. Iron is essential for chlorophyll biosynthesis, and ferritin clusters in plastids store iron during germination, development, and iron stress. Thus, plastidic homeostasis of transition metals, in particular of iron, is crucial for chloroplast as well as plant development. However, very little is known about iron uptake by chloroplasts. Arabidopsis thaliana PERMEASE IN CHLOROPLASTS1 (PIC1), identified in a screen for metal transporters in plastids, contains four predicted alpha-helices, is targeted to the inner envelope, and displays homology with cyanobacterial permease-like proteins. Knockout mutants of PIC1 grew only heterotrophically and were characterized by a chlorotic and dwarfish phenotype reminiscent of iron-deficient plants. Ultrastructural analysis of plastids revealed severely impaired chloroplast development and a striking increase in ferritin clusters. Besides upregulation of ferritin, pic1 mutants showed differential regulation of genes and proteins related to iron stress or transport, photosynthesis, and Fe-S cluster biogenesis. Furthermore, PIC1 and its cyanobacterial homolog mediated iron accumulation in an iron uptake-defective yeast mutant. These observations suggest that PIC1 functions in iron transport across the inner envelope of chloroplasts and hence in cellular metal homeostasis.     

A cytosolic Arabidopsis D-xylulose kinase catalyzes the phosphorylation of 1-deoxy-D-xylulose into a precursor of the plastidial isoprenoid pathway

Plants are able to integrate exogenous 1-deoxy-D-xylulose (DX) into the 2C-methyl-D-erythritol 4-phosphate pathway, implicated in the biosynthesis of plastidial isoprenoids. Thus, the carbohydrate needs to be phosphorylated into 1-deoxy-D-xylulose 5-phosphate and translocated into plastids, or vice versa. An enzyme capable of phosphorylating DX was partially purified from a cell-free Arabidopsis (Arabidopsis thaliana) protein extract. It was identified by mass spectrometry as a cytosolic protein bearing D-xylulose kinase (XK) signatures, already suggesting that DX is phosphorylated within the cytosol prior to translocation into the plastids. The corresponding cDNA was isolated and enzymatic properties of a recombinant protein were determined. In Arabidopsis, xylulose kinases are encoded by a small gene family, in which only two genes are putatively annotated. The additional gene is coding for a protein targeted to plastids, as was proved by colocalization experiments using green fluorescent protein fusion constructs. Functional complementation assays in an Escherichia coli strain deleted in xk revealed that the cytosolic enzyme could exclusively phosphorylate xylulose in vivo, not the enzyme that is targeted to plastids. xk activities could not be detected in chloroplast protein extracts or in proteins isolated from its ancestral relative Synechocystis sp. PCC 6803. The gene encoding the plastidic protein annotated as "xylulose kinase" might in fact yield an enzyme having different phosphorylation specificities. The biochemical characterization and complementation experiments with DX of specific Arabidopsis knockout mutants seedlings treated with oxo-clomazone, an inhibitor of 1-deoxy-D-xylulose 5-phosphate synthase, further confirmed that the cytosolic protein is responsible for the phosphorylation of DX in planta.     

DNA sequences from Arabidopsis, which encode protein kinases and function as upstream regulators of Snf1 in yeast

Sucrose nonfermenting-1 (Snf1)-related protein kinase-1 (SnRK1) of plants is a global regulator of carbon metabolism through the modulation of enzyme activity and gene expression. It is structurally and functionally related to the yeast protein kinase, Snf1, and to mammalian AMP-activated protein kinase. Two DNA sequences from Arabidopsis thaliana, previously known only by their data base accession numbers of NM_ 125448.3 (protein ID NP_200863) and NM_114393.3 (protein ID NP_566876) each functionally complemented a Saccharomyces cerevisiae elm1 sak1 tos3 triple mutant. This indicates that the Arabidopsis proteins are able to substitute for one of the missing yeast upstream kinases, which are required for activity of Snf1. Both plant proteins were shown to phosphorylate a peptide with the amino acid sequence of the phosphorylation site in the T-loop of SnRK1 and by inference SnRK1 in Arabidopsis. The proteins encoded by NM_125448.3 and NM_114393.3 have been named AtSnAK1 and AtSnAK2 (Arabidopsis thaliana SnRK1-activating kinase), respectively. We believe this is the first time that upstream activators of SnRK1 have been described in any plant species.     

The acceptor availability at photosystem I and ABA control nuclear expression of 2-Cys peroxiredoxin-A in Arabidopsis thaliana

The redox-regulated 2-Cys peroxiredoxin-A (2CPA) promoter, which drives expression of a dominant chloroplast antioxidant enzyme, responds to signals originating from the photosynthetic electron transport downstream of PSI. Modulation of CO(2)- and NO(3)(-) -reduction rates in reporter gene plants expressing glucuronidase under control of the Arabidopsis thaliana 2CPA promoter revealed that promoter activity correlates with the availability of electron acceptors at PSI. The photosynthetic redox-regulation can be simulated by oxidant and antioxidant treatments. Inhibitor studies with PD98059 and staurosporine showed that a mitogen-activated protein kinase kinase transmits the oxidative response, while the antioxidant signal is transmitted by a serine/threonine kinase. Analysis of 2CPA promoter regulation in the abscisic acid (ABA)-biosynthetic mutants aba2 and aba3 and the ABA-insensitive mutants abi1 and abi2 support a regulatory circuitry in which the redox signal cross-talks with the ABA-signaling cascade downstream of ABI1 and ABI2.     

Functional characterization of ARAKIN (ATMEKK1): a possible mediator in an osmotic stress response pathway in higher plants.

The Arabidopsis thaliana ARAKIN (ATMEKK1) gene shows strong homology to members of the (MAP) mitogen-activated protein kinase family, and was previously shown to functionally complement a mating defect in Saccharomyces cerevisiae at the level of the MEKK kinase ste11. The yeast STE11 is an integral component of two MAP kinase cascades: the mating pheromone pathway and the HOG (high osmolarity glycerol response) pathway. The HOG signal transduction pathway is activated by osmotic stress and causes increased glycerol synthesis. Here, we first demonstrate that ATMEKK1 encodes a protein with kinase activity, examine its properties in yeast MAP kinase cascades, then examine its expression under stress in A. thaliana. Yeast cells expressing the A. thaliana ATMEKK1 survive and grow under high salt (NaCl) stress, conditions that kill wild-type cells. Enhanced glycerol production, observed in non-stressed cells expressing ATMEKK1 is the probable cause of yeast survival. Downstream components of the HOG response pathway, HOG1 and PBS2, are required for ATMEKK1-mediated yeast survival. Because ATMEKK1 functionally complements the sho1/ssk2/ssk22 triple mutant, it appears to function at the level of the MEKK kinase step of the HOG response pathway. In A. thaliana, ATMEKK1 expression is rapidly (within 5 min) induced by osmotic (NaCl) stress. This is the same time frame for osmoticum-induced effects on the electrical properties of A. thaliana cells, both an immediate response and adaptation. Therefore, we propose that the A. thaliana ATMEKK1 may be a part of the signal transduction pathway involved in osmotic stress.     

SIS8, a putative mitogen‐activated protein kinase kinase kinase,regulates sugar‐resistant seedling development in Arabidopsis

Sugar signaling pathways have been evolutionarily conserved among eukaryotes and are postulated to help regulate plant growth, development and responses to environmental cues. Forward genetic screens have identified sugar signaling or response mutants. Here we report the identification and characterization of Arabidopsis thaliana sugar insensitive8 (sis8) mutants, which display a sugar‐resistant seedling development phenotype. Unlike many other sugar insensitive mutants, sis8 mutants exhibit wild‐type responses to the inhibitory effects of abscisic acid and paclobutrazol (an inhibitor of gibberellin biosynthesis) on seed germination. Positional cloning of the SIS8 gene revealed that it encodes a putative mitogen‐activated protein kinase kinase kinase (MAPKKK; At1g73660). SIS8mRNA is expressed ubiquitously among Arabidopsis organs. A UDP‐glucosyltransferase, UGT72E1 (At3g50740), was identified as an interacting partner of SIS8 based on a yeast two‐hybrid screen and in planta bimolecular fluorescence complementation. Both SIS8–yellow fluorescent protein (YFP) and UGT72E1–YFP fusion proteins localize to the nucleus when transiently expressed in tobacco leaf cells. T‐DNA insertions in At3g50740 cause a sugar‐insensitive phenotype. These results indicate that SIS8, a putative MAPKKK, is a regulator of sugar response in Arabidopsis and interacts with a UDP‐glucosyltransferase in the nucleus.     

A heterocomplex of iron superoxide dismutases defends chloroplast nucleoids against oxidative stress and is essential for chloroplast development in Arabidopsis

There are three iron superoxide dismutases in Arabidopsis thaliana: FE SUPEROXIDE DISMUTASE1 (FSD1), FSD2, and FSD3. Their biological roles in chloroplast development are unknown. Here, we show that FSD2 and FSD3 play essential roles in early chloroplast development, whereas FSD1, which is found in the cytoplasm, does not. An fsd2-1 fsd3-1 double mutant had a severe albino phenotype on agar plates, whereas fsd2 and fsd3 single knockout mutants had pale green phenotypes. Chloroplast development was arrested in young seedlings of the double mutant. The mutant plants were highly sensitive to oxidative stress and developed increased levels of reactive oxygen species (ROS) during extended darkness. The FSD2 and FSD3 proteins formed a heteromeric protein complex in the chloroplast nucleoids. Furthermore, transgenic Arabidopsis plants overexpressing both the FSD2 and FSD3 genes showed greater tolerance to oxidative stress induced by methyl viologen than did the wild type or single FSD2- or FSD3-overexpressing lines. We propose that heteromeric FSD2 and FSD3 act as ROS scavengers in the maintenance of early chloroplast development by protecting the chloroplast nucleoids from ROS.     

Evidence for a SAL1-PAP chloroplast retrograde pathway that functions in drought and high light signaling in Arabidopsis

Compartmentation of the eukaryotic cell requires a complex set of subcellular messages, including multiple retrograde signals from the chloroplast and mitochondria to the nucleus, to regulate gene expression. Here, we propose that one such signal is a phosphonucleotide (3'-phosphoadenosine 5'-phosphate [PAP]), which accumulates in Arabidopsis thaliana in response to drought and high light (HL) stress and that the enzyme SAL1 regulates its levels by dephosphorylating PAP to AMP. SAL1 accumulates in chloroplasts and mitochondria but not in the cytosol. sal1 mutants accumulate 20-fold more PAP without a marked change in inositol phosphate levels, demonstrating that PAP is a primary in vivo substrate. Significantly, transgenic targeting of SAL1 to either the nucleus or chloroplast of sal1 mutants lowers the total PAP levels and expression of the HL-inducible ASCORBATE PEROXIDASE2 gene. This indicates that PAP must be able to move between cellular compartments. The mode of action for PAP could be inhibition of 5' to 3' exoribonucleases (XRNs), as SAL1 and the nuclear XRNs modulate the expression of a similar subset of HL and drought-inducible genes, sal1 mutants accumulate XRN substrates, and PAP can inhibit yeast (Saccharomyces cerevisiae) XRNs. We propose a SAL1-PAP retrograde pathway that can alter nuclear gene expression during HL and drought stress.     

TaABC1, a member of the activity of bc1 complex protein kinase family from common wheat, confers enhanced tolerance to abiotic stresses in Arabidopsis

Abiotic stresses such as drought, salinity, and low temperature have drastic effects on plant growth and development. However, the molecular mechanisms regulating biochemical and physiological changes in response to stresses are not well understood. Protein kinases are major signal transduction factors among the reported molecular mechanisms mediating acclimation to environmental changes. Protein kinase ABC1 (activity of bc(1) complex) is involved in regulating coenzyme Q biosynthesis in mitochondria in yeast (Saccharomyces cersvisiae), and in balancing oxidative stress in chloroplasts in Arabidopsis thaliana. In the current study, TaABC1 (Triticum aestivum L. activity of bc(1) complex) protein kinase was localized to the cell membrane, cytoplasm, and nucleus. The effects of overexpressing TaABC1 in transgenic Arabidopsis plants on responses to drought, salt, and cold stress were further investigated. Transgenic Arabidopsis overexpressing the TaABC1 protein showed lower water loss and higher osmotic potential, photochemistry efficiency, and chlorophyll content, while cell membrane stability and controlled reactive oxygen species homeostasis were maintained. In addition, overexpression of TaABC1 increased the expression of stress-responsive genes, such as DREB1A, DREB2A, RD29A, ABF3, KIN1, CBF1, LEA, and P5CS, detected by real-time PCR analysis. The results suggest that TaABC1 overexpression enhances drought, salt, and cold stress tolerance in Arabidopsis, and imply that TaABC1 may act as a regulatory factor involved in a multiple stress response pathways.     

Conservation of the salt overly sensitive pathway in rice

The salt tolerance of rice (Oryza sativa) correlates with the ability to exclude Na+ from the shoot and to maintain a low cellular Na+/K+ ratio. We have identified a rice plasma membrane Na+/H+ exchanger that, on the basis of genetic and biochemical criteria, is the functional homolog of the Arabidopsis (Arabidopsis thaliana) salt overly sensitive 1 (SOS1) protein. The rice transporter, denoted by OsSOS1, demonstrated a capacity for Na+/H+ exchange in plasma membrane vesicles of yeast (Saccharomyces cerevisiae) cells and reduced their net cellular Na+ content. The Arabidopsis protein kinase complex SOS2/SOS3, which positively controls the activity of AtSOS1, phosphorylated OsSOS1 and stimulated its activity in vivo and in vitro. Moreover, OsSOS1 suppressed the salt sensitivity of a sos1-1 mutant of Arabidopsis. These results represent the first molecular and biochemical characterization of a Na+ efflux protein from monocots. Putative rice homologs of the Arabidopsis protein kinase SOS2 and its Ca2+-dependent activator SOS3 were identified also. OsCIPK24 and OsCBL4 acted coordinately to activate OsSOS1 in yeast cells and they could be exchanged with their Arabidopsis counterpart to form heterologous protein kinase modules that activated both OsSOS1 and AtSOS1 and suppressed the salt sensitivity of sos2 and sos3 mutants of Arabidopsis. These results demonstrate that the SOS salt tolerance pathway operates in cereals and evidences a high degree of structural conservation among the SOS proteins from dicots and monocots.