Cross-talks between Ca<Superscript>2+</Superscript>/CaM and H<Subscript>2</Subscript>O<Subscript>2</Subscript> in abscisic acid-induced antioxidant defense in leaves of maize plants exposed to water stress

Here we examined whether Ca²⁺/Calmodulin (CaM) is involved in abscisic acid (ABA)-induced antioxidant defense and the possible relationship between CaM and H₂O₂ in ABA signaling in leaves of maize (Zea mays L.) plants exposed to water stress. An ABA-deficient mutant vp5 and its wild type were used for the experimentation. We found that water stress enhanced significantly the contents of CaM and H₂O₂, and the activities of chloroplastic and cytosolic superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and the gene expressions of the CaM1, cAPX, GR1 and SOD4 in leaves of wild-type maize. However, the increases mentioned above were almost arrested in vp5 plants and in the wild-type plants pretreated with ABA biosynthesis inhibitor tungstate (T), suggesting that ABA is required for water stress-induced H₂O₂ production, the enhancement of CaM content and antioxidant defense. Besides, we showed that the up-regulation of water stress-induced antioxidant defense was almost completely blocked by pretreatment with Ca²⁺ inhibitors, CaM antagonists and reactive oxygen (ROS) manipulators. Moreover, the analysis of time course of CaM and H₂O₂ production under water stress showed that the increase in CaM content preceded that of H₂O₂. These results suggested that Ca²⁺/CaM and H₂O₂ were involved in the ABA-induced antioxidant defense under water stress, and the increases of Ca²⁺/CaM contents triggered H₂O₂ production, which inversely affected the contents of CaM. Thus, a cross-talk between Ca²⁺/CaM and H₂O₂ may play a pivotal role in the ABA signaling.     

Proteomic analysis of the response to high-salinity stress in <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Physcomitrella patens is well known because of its importance in the study of plant systematics and evolution. The tolerance of P. patens for high-salinity environments also makes it an ideal candidate for studying the molecular mechanisms by which plants respond to salinity stresses. We measured changes in the proteome of P. patens gametophores that were exposed to high-salinity (250, 300, and 350 mM NaCl) using two-dimensional gel electrophoresis (2-DE) via liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sixty-five protein spots were significantly altered by exposure to the high-salinity environment. Among them, 16 protein spots were down-regulated and 49 protein spots were up-regulated. These proteins were associated with a variety of functions, including energy and material metabolism, protein synthesis and degradation, cell defense, cell growth/division, transport, signal transduction, and transposons. Specifically, the up-regulated proteins were primarily involved in defense, protein folding, and ionic homeostasis. In summary, we outline several novel insights into the response of P. patens to high-salinity; (1) HSP70 is likely to play a significant role in protecting proteins from denaturation and degradation during salinity stress, (2) signaling proteins, such as 14-3-3 and phototropin, may work cooperatively to regulate plasma membrane H(+)-ATPase and maintain ion homeostasis, (3) an increase in photosynthetic activity may contribute to salinity tolerance, and (4) ROS scavengers were up-regulated suggesting that the antioxidative system may play a crucial role in protecting cells from oxidative damage following exposure to salinity stress in P. patens.     

Photosynthetic CO<Subscript>2</Subscript>/H<Subscript>2</Subscript>O gas exchange and dynamics of carbohydrates content in maize leaves under drought

We studied the temporal sequence of changes in the photosynthetic CO₂/H₂O gas exchange intensity, as well as leaf water status, contents of soluble carbohydrates, starch, proline, pigments, and MDA, in maize seedlings (Zea mays L., cv. Luchistaya) under adaptation to increasing water deficit. The duration of drought was 2, 3, 5, and 6 days. Withholding water from maize plants caused gradual increase in the intensity of water deficit: from mild (2 or 3 days) to moderate (5 days) and nearly severe (6 days) water stress. After 6 days, relative leaf water content decreased by 19.8% as compared to the control. On the second day after the onset of drought, slight reduction in the photosynthetic CO₂/H₂O gas exchange intensity of the treated plants was observed. After 6 days, photosynthesis and transpiration of leaves synchronously reduced almost threefold due to stomatal closure. The progressive soil drought had substantial impact on the carbohydrate metabolism. After 2 days of water deficit, the content of reducing sugars and sucrose increased slightly, whereas after 6 days, it increased ten and four times, respectively. After 2, 3, and 5 days of drought, the starch content declined slightly; however, under severe drought (6 days), it increased by 30% as compared to the control. Simultaneously with the increase in the content of soluble sugars, proline content increased significantly and it was the highest on the sixth day of drought. At all stages of water deficit, the proline content increased more significantly than the content of reducing carbohydrates and sucrose. Under increasing water deficit (5 and 6 days), the content of MDA was found to rise. At the initial drought stage (2 or 3 days) and under severe water deficit (6 days), no significant changes in the pigment content were observed. Thus, at the initial stages of progressive drought, in the leaves of this maize cultivar, a decline in photosynthetic activity proceeded simultaneously with accumulation of reducing sugars, sucrose, and proline. The results obtained showed that, at the first stages of adaptation of maize seedlings to drought, the changes in carbohydrate and proline metabolism have been observed, which have increased upon further plant dehydration.     

ZmHSP16.9, a cytosolic class I small heat shock protein in maize (Zea mays), confers heat tolerance in transgenic tobacco

Various organisms produce HSPs in response to high temperature and other stresses. The function of heat shock proteins, including small heat shock protein (sHSP), in stress tolerance is not fully explored. To improve our understanding of sHSPs, we isolated ZmHSP16.9 from maize. Sequence alignments and phylogenetic analysis reveal this to be a cytosolic class I sHSP. ZmHSP16.9 expressed in root, leaf and stem tissues under 40 °C treatment, and was up-regulated by heat stress and exogenous H?O?. Overexpression of ZmHSP16.9 in transgenic tobacco conferred tolerance to heat and oxidative stresses by increased seed germination rate, root length, and antioxidant enzyme activities compared with WT plants. These results support the positive role of ZmHSP16.9 in response to heat stress in plant. KEY MESSAGE: The overexpression of ZmHSP16.9 enhanced tolerance to heat and oxidative stress in transgenic tobacco.     

Differentially expressed proteins associated with drought tolerance in bananas (<Emphasis Type="Italic">Musa</Emphasis> spp.)

Bananas are one of the most important fruits in tropical and subtropical regions worldwide. Each year, banana plantations expand, but the areas available are mostly dry lands. The establishment of strategies for obtaining drought tolerant cultivars depends on understanding of biological responses at genetic, molecular and biochemical levels. Proteomics is a powerful tool for functional characterization of the response of plants to abiotic stress and little is known about drought tolerance in Musa spp. Therefore, the aim of this study was to identify proteins related to drought tolerance in two contrasting banana genotypes, Prata Anã and BRS Tropical, susceptible and tolerant to drought, respectively. Proteins were extracted from rhizomes of bananas grown under greenhouse conditions with control, irrigated and water deficit regimes. The differential protein expression pattern was established by two-dimensional (2-D) electrophoresis and spots analyzed in nano Q-Tof Micro UPLC. Twenty-three differentially expressed proteins were found in the tolerant genotype (BRS Tropical) under water deficit, with proteins involved in metabolism, defense and transport. Proteins were classified according to known function and biosynthetic pathways. Signaling proteins in response to water stress, especially for the biological function of growth and development of plants cells, were also encountered, whereas heat shock proteins played a significant role. This is the first report of proteomic analysis for drought tolerance in ‘Pome’ and ‘Silk-type’ bananas containing the ‘B’ genome. Our work provides insights into Musa spp. response to drought and data for further studies regarding molecular mechanisms, which determine how Musa spp. cells better overcome environmental perturbations.     

Quantitative proteomic analysis of short photoperiod and low-temperature responses in bark tissues of peach (<Emphasis Type="Italic">Prunus persica</Emphasis> L. Batsch)

In the temperate climate of the northern hemisphere, winter survival of woody plants is determined by the ability to acclimate to freezing temperatures and to undergo a period of dormancy. Cold acclimation in many woody plants is initially induced by short photoperiod and low, non-freezing temperatures. These two factors (5°C and short photoperiod) were used to study changes in the proteome of bark tissues of 1-year-old peach trees. Difference in-gel electrophoresis technology, a gel-based approach involving the labeling of proteins with different fluorescent dyes, was used to conduct a quantitative assessment of changes in the peach bark proteome during cold acclimation. Using this approach, we were able to identify differentially expressed proteins and to assign them to a class of either ‘temperature-responsive’ or ‘photoperiod-responsive’ proteins. The most significant factor affecting the proteome appeared to be low temperature, while the combination of low temperature and short photoperiod was shown to act either synergistically or additively on the expression of some proteins. Fifty-seven protein spots on gels were identified by mass spectrometry. They included proteins involved in carbohydrate metabolism (e.g., enolase, malate dehydrogenase, etc), defense or protective mechanisms (e.g., dehydrin, HSPs, and PR-proteins), energy production and electron transport (e.g., adenosine triphosphate synthases and lyases), and cytoskeleton organization (e.g., tubulins and actins). The information derived from the analysis of the proteome is discussed as a function of the two treatment factors: low temperature and short photoperiod. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">NnHSP17.5</Emphasis>, a cytosolic class II small heat shock protein gene from <Emphasis Type="Italic">Nelumbo nucifera</Emphasis>, contributes to seed germination vigor and seedling thermotolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

In plants, small heat shock proteins (sHSPs) are unusually abundant and diverse proteins involved in various abiotic stresses, but their functions in seed vigor remain to be fully explored. In this study, we report the isolation and functional characterization of a sHSP gene, NnHSP17.5, from sacred lotus (Nelumbo nucifera Gaertn.) in seed germination vigor and seedling thermotolerance. Sequence alignment and phylogenetic analysis indicate that NnHSP17.5 is a cytosolic class II sHSP, which was further supported by the cytosolic localization of the NnHSP17.5-YFP fusion protein. NnHSP17.5 was specifically expressed in seeds under normal conditions, and was strongly up-regulated in germinating seeds upon heat and oxidative stresses. Transgenic Arabidopsis seeds ectopically expressing NnHSP17.5 displayed enhanced seed germination vigor and exhibited increased superoxide dismutase activity after accelerated aging treatment. In addition, improved basal thermotolerance was also observed in the transgenic seedlings. Taken together, this work highlights the importance of a plant cytosolic class II sHSP both in seed germination vigor and seedling thermotolerance.     

Tolerance to soil water stress by <Emphasis Type="Italic">Oryza sativa</Emphasis> cv. IR20 was improved by expression of <Emphasis Type="Italic">Wsi18</Emphasis> gene locus from <Emphasis Type="Italic">Oryza nivara</Emphasis>

Wild rice genotypes are rich in genetic diversity. This has potential to improve agronomic rice by allele mining for superior traits. Late embryogenesis abundant (LEA) proteins are often associated with desiccation tolerance and stress signalling. In the present study, a group 3 LEA gene, Wsi18 from the wild rice Oryza nivara was expressed under its own inducible promoter element in stress susceptible cultivated indica rice (cv. IR20). The resulting transgenic plants cultivated in a greenhouse showed enhanced tolerance to soil water deficit. Transgenic plants had higher grain yield, plant survival rate, and shoot relative water content compared to wild type (WT) IR20. Cell membrane stability index, proline and soluble sugar content were also greater in transgenic than WT plants under water stress. These results demonstrate the potential for improving SWS tolerance in agronomically important rice cultivar by incorporating Wsi18 gene from a wild rice O. nivara.     

Proteomic analysis of cold stress-responsive proteins in <Emphasis Type="Italic">Thellungiella</Emphasis> rosette leaves

Low temperature is one of the most severe environmental factors that impair plant growth and agricultural production. To investigate how Thellungiella halophila, an Arabidopsis-like extremophile, adapts to cold stress, a comparative proteomic approach based on two-dimensional electrophoresis was adopted to identify proteins that changed in abundance in Thellungiella rosette leaves during short term (6 h, 2 and 5 days) and long term (24 days) exposure to cold stress. Sixty-six protein spots exhibited significant change at least at one time point and maximal cold stress induced-proteome change was found in long-term cold stress group while the minimal change was found in 6-h cold treatment group. Fifty protein spots were identified by mass spectrometry analysis. The identified proteins mainly participate in photosynthesis, RNA metabolism, defense response, energy pathway, protein synthesis, folding and degradation, cell wall and cytoskeleton and signal transduction. These proteins might work cooperatively to establish a new homeostasis under cold stress. Nearly half of the identified cold-responsive proteins were associated with various aspects of chloroplast physiology suggesting that the cold stress tolerance of T. halophila is achieved, at least partly, by regulation of chloroplast function. All protein spots involved in RNA metabolism, defense response, protein synthesis, folding and degradation were found to be upregulated markedly by cold treatment, indicating enhanced RNA metabolism, defense and protein metabolism may play crucial roles in cold tolerance mechanism in T. halophila.     

Proteome analysis of leaves from the resurrection plant <Emphasis Type="Italic">Boea hygrometrica</Emphasis> in response to dehydration and rehydration

Resurrection plants differ from other species in their unique ability to survive desiccation. In order to understand the mechanisms of desiccation tolerance, proteome studies were carried out using leaves of the resurrection plant Boea hygrometrica to reveal proteins that were differentially expressed in response to changes in relative water content. This opportunity was afforded by the rare ability of excised B. hygrometrica leaves to survive and resume metabolism following desiccation in a manner similar to intact plants. From a total of 223 proteins that were reproducibly detected and analyzed, 35% showed increased abundance in dehydrated leaves, 5% were induced in rehydrated leaves and 60% showed decreased or unchanged abundance in dehydrated and rehydrated leaves. Since the induction kinetics fall into clearly defined patterns, we suggest that programmed regulation of protein expression triggered by changes of water status. Fourteen dehydration responsive proteins were analyzed by mass spectrometry. Eight proteins were classified as playing a role in reactive oxygen species scavenging, photosynthesis and energy metabolism. In agreement with these findings, glutathione content and polyphenol oxidase activity were found to increase upon dehydration and rapid recovery of photosynthesis was observed.     

Proteomic identification of differentially expressed proteins in Arabidopsis in response to methyl jasmonate

Jasmonates (JAs) are the well characterized fatty acid-derived cyclopentanone signals involved in the plant response to biotic and abiotic stresses. JAs have been shown to regulate many aspects of plant metabolism, including glucosinolate biosynthesis. Glucosinolates are natural plant products that function in defense against herbivores and pathogens. In this study, we applied a proteomic approach to gain insight into the physiological processes, including glucosinolate metabolism, in response to methyl jasmonate (MeJA). We identified 194 differentially expressed protein spots that contained proteins that participated in a wide range of physiological processes. Functional classification analysis showed that photosynthesis and carbohydrate anabolism were repressed after MeJA treatment, while carbohydrate catabolism was up-regulated. Additionally, proteins related to the JA biosynthesis pathway, stress and defense, and secondary metabolism were up-regulated. Among the differentially expressed proteins, many were involved in oxidative tolerance. The results indicate that MeJA elicited a defense response at the proteome level through a mechanism of redirecting growth-related metabolism to defense-related metabolism.     

Responses of wild watermelon to drought stress: accumulation of an ArgE homologue and citrulline in leaves during water deficits

Wild watermelon from the Botswana desert had an ability to survive under severe drought conditions by maintaining its water status (water content and water potential). In the analysis by two-dimensional electrophoresis of leaf proteins, seven spots were newly induced after watering stopped. One with the molecular mass of 40 kilodaltons of the spots was accumulated abundantly. The cDNA encoding for the protein was cloned based on its amino-terminal sequence and the amino acid sequence deduced from the determined nucleotide sequences of the cDNA exhibited homology to the enzymes belong to the ArgE/DapE/Acy1/Cpg2/YscS protein family (including acetylornithine deacetylase, carboxypeptidase and aminoacylase-1). This suggests that the protein is involved in the release of free amino acid by hydrolyzing a peptidic bond. As the drought stress progressed, citrulline became one of the major components in the total free amino acids. Eight days after withholding watering, although the lower leaves wilted significantly, the upper leaves still maintained their water status and the content of citrulline reached about 50% in the total free amino acids. The accumulation of citrulline during the drought stress in wild watermelon is an unique phenomenon in C3-plants. These results suggest that the drought tolerance of wild watermelon is related to (1) the maintenance of the water status and (2) a metabolic change to accumulate citrulline.     

Proteomics Analysis of Drought Stress-Responsive Proteins in <Emphasis Type="Italic">Hippophae rhamnoides</Emphasis> L.

Hippophae rhamnoides L. is uniquely capable of growing well under extreme environmental conditions such as water deficit, low temperature, and high altitude. Such tolerance invokes much interest in understanding the biology of this plant species and its utilization potential. In this study, analysis of drought stress-responsive proteins in H. rhamnoides was conducted wherein greenhouse-grown seedlings were subjected to drought stress. By using proteomic techniques, proteins, extracted from leaves, were analyzed using two-dimensional electrophoresis and MALDI-TOF MS. Altogether, 55 proteins exhibited changes in abundance under stress. Of these, 13 proteins were identified, including three that disappeared under drought (a putative ABC transporter ATP-binging protein, a heat shock protein HslU, and a hypothetical protein XP-515578), seven that were up-regulated (three large subunits of rubisco, a hypothetical protein DSM3645–23351, a putative acyl-CoA dehydrogenase, a nesprin-2, and a J-type co-chaperone HSC20), and three that were only detected under drought (a probable nitrogen regulation protein (NtrX), a 4-hydroxyphenylpyruvate dioxygenase, and an unnamed protein product). These proteins may function in β-oxidation pathways in mitochondria, across membranes transport, abnormal protein removal, or prevent protein aggregation arrest, cell division, cytoskeleton stabilization, iron–sulfur cluster assembly, nitrogen metabolism regulation, and antioxidant substance biosynthesis. Four proteins (J-type co-chaperone Hsc20, a putative ABC transporter ATP-binging protein, NtrX, and HslU) were deemed as new discoveries in higher plants, and their functions were predicted either from their conserved domains or homologies to other organisms. These results provide new insights into our understanding of the mechanism of drought tolerance in plants.     

Expression analysis of nine small heat shock protein genes from Tamarix hispida in response to different abiotic stresses and abscisic acid treatment

Heat shock proteins (HSPs) play important roles in protecting plants against environmental stresses. Furthermore, small heat shock proteins (sHSPs) are the most ubiquitous HSP subgroup with molecular weights ranging from 15 to 42 kDa. In this study, nine sHSP genes (designated as ThsHSP1–9) were cloned from Tamarix hispida. Their expression patterns in response to cold, heat shock, NaCl, PEG and abscisic acid (ABA) treatments were investigated in the roots and leaves of T. hispida by real-time RT-PCR analysis. The results showed that most of the nine ThsHSP genes were expressed at higher levels in roots than in leaves under normal growth condition. All of ThsHSP genes were highly induced under conditions of cold (4 °C) and different heat shocks (36, 40, 44, 48 and 52 °C). Under NaCl stress, all nine ThsHSPs genes were up-regulated at least one stress time-point in both roots and leaves. Under PEG and ABA treatments, the nine ThsHSPs showed various expression patterns, indicating a complex regulation pathway among these genes. This study represents an important basis for the elucidation of ThsHSP gene function and provides essential information that can be used for stress tolerance genetic engineering in future studies.     

Comparative proteomic analysis of <Emphasis Type="Italic">Phalaenopsis</Emphasis> leaves in the vegetative and flowering phase

Phalaenopsis, an epiphytic crassulacean acid metabolism (CAM) plant, requires moderate variations of day/night temperatures for flowering. In this study, changes in chlorophyll content, chlorophyll fluorescence, sugar components, titratable acidity and soluble protein content in Phalaenopsis leaves during flowering were observed. Comparative proteomic analysis of Phalaenopsis leaves in the vegetative and flowering phase was performed for the first time using iTRAQ (isobaric tags for relative and absolute quantification). A total of 126 proteins were differentially expressed in Phalaenopsis leaves. Analysis of potential functions revealed that the major categories of predicted function of the up-regulated proteins were protein destination (27 %), photosynthesis (15.9 %), primary metabolism (14.3 %) and defense (12.7 %) in the flowering phase, while the major categories of predicted function of the down-regulated proteins were protein destination (33.3 %), primary metabolism (20.6 %), transportation (14.3 %) and signal transduction (11.1 %). Proteome profile analysis indicated that the proteome changes were consistent with changes in sugar and protein metabolites. Some novel proteins were differentially expressed, most of which were identified as signaling proteins, including 14-3-3 proteins, fibrillin, rapid alkalinization factors (RALF), the Ras-related protein RABB1c, calreticulin and calmodulin. Histone, importin alpha, multidrug resistance proteins and the ABC transporters were also differentially expressed. These results provide insights into the mechanisms that regulate flowering in complex flowering plants.