Project description:Drought is one of the most important environmental fluctuations affecting tree growth and survival. Therefore, understanding of physiological and transcriptomic responses of trees to this stress factor will make important contributions to forest health and productivity. Here, we report comparative physiological and microarray based transcriptome analysis between drought resistant (N.62.191) and drought-sensitive (N.03.368.A) black poplar genotypes under well-watered (WWP), moderate drought (MD), severe drought (SD) and post drought re-watering (PDR) conditions. In the study, sensitive genotype exhibited a drought escape strategy with lower leaf water potential, higher reactive oxygen production, complete leaf abscission and subsequent terminal shoot necrosis under drought stress. On the other hand, resistant genotype had a dehydration tolerance indicating highly delayed leaf abscission under drought and fast growing capacity during re-watering conditions. Gene ontology enrichment analysis attributed drought susceptibility of black poplar to significant up-regulation of genes functional in transcription regulation (AP2/ERF, NAC and WRKY), cell wall modification (Expansins), fatty acid metabolism (enoyl-ACP reductase, lipid transport protein particle), protein degradation (endopeptidases), ethylene synthesis (1-aminocyclopropane-1-carboxylate) and riboflavin synthesis (GTP cyclohydrolase II) under drought stress. Transcriptomic comparison indicated significant down-regulation of photosynthesis, electron transport and carbohydrate metabolism related genes under drought stress in sensitive genotype. Although, similar reduction in carbohydrate metabolism was also recorded for resistant genotype, genes related with photosynthesis and electron transport systems were not down regulated even under SD for this genotype. Resistant genotype specific up-regulation of small heat shock proteins (sHSP) and bark storage proteins revealed importance of protein protection and nitrogen remobilization under drought stress, respectively. This is the first study associating BSP production to delayed leaf abscission and drought tolerance in trees.

Project description:Drought is one of the most important environmental fluctuations affecting tree growth and survival. Therefore, understanding of physiological and transcriptomic responses of trees to this stress factor will make important contributions to forest health and productivity. Here, we report comparative physiological and microarray based transcriptome analysis between drought resistant (N.62.191) and drought-sensitive (N.03.368.A) black poplar genotypes under well-watered (WWP), moderate drought (MD), severe drought (SD) and post drought re-watering (PDR) conditions. In the study, sensitive genotype exhibited a drought escape strategy with lower leaf water potential, higher reactive oxygen production, complete leaf abscission and subsequent terminal shoot necrosis under drought stress. On the other hand, resistant genotype had a dehydration tolerance indicating highly delayed leaf abscission under drought and fast growing capacity during re-watering conditions. Gene ontology enrichment analysis attributed drought susceptibility of black poplar to significant up-regulation of genes functional in transcription regulation (AP2/ERF, NAC and WRKY), cell wall modification (Expansins), fatty acid metabolism (enoyl-ACP reductase, lipid transport protein particle), protein degradation (endopeptidases), ethylene synthesis (1-aminocyclopropane-1-carboxylate) and riboflavin synthesis (GTP cyclohydrolase II) under drought stress. Transcriptomic comparison indicated significant down-regulation of photosynthesis, electron transport and carbohydrate metabolism related genes under drought stress in sensitive genotype. Although, similar reduction in carbohydrate metabolism was also recorded for resistant genotype, genes related with photosynthesis and electron transport systems were not down regulated even under SD for this genotype. Resistant genotype specific up-regulation of small heat shock proteins (sHSP) and bark storage proteins revealed importance of protein protection and nitrogen remobilization under drought stress, respectively. This is the first study associating BSP production to delayed leaf abscission and drought tolerance in trees. For Microarray experiment total RNA was isolated from the leaves randomly selected from two balck poplar seedlings (two biological replicates) for resistant and sensitive genotypes at well watered period (WWP), moderate drought (MD), severe drought (SD) and post drought rewatering (PDR) periods. For each water availability regime total isolated RNA was loaded onto two Affymetrix poplar Gene Chips for each genotype. Totally 16 Affymetrix poplar GeneChips (2 genotypes × 4 water availability regimes × 2 biological replicates) were used for transcriptional analysis.

Project description:Drought stress is a major problem around the world and although progress in understanding how vegetable crops and model plants adapt to drought have been made, there is still little information about how fruit crops deal with moderate drought stress. In this study, we investigated the response of two apple genotypes: a drought-sensitive genotype (M26) and a drought-tolerant genotype (MBB). Our results of the morphology, physiology and biochemistry under moderate drought stress, indicated that relative water content (RWC) and leaf area (LA) were not significant changes in two genotypes. However, it had larger leaf mass per area (LMA), and accumulated higher free proline (CFP), soluble sugars (CSS) and malonaldehyde (MDA) in the leaves. Thus, it appears that the MBB genotype could produce more osmosis-regulating substances. Phosphoproteomic was analyzed from leaves of both genotypes under moderate drought stress using the isobaric tags for relative and absolute quantification (iTRAQ) technology. A total of 595 unique phosphopeptides, 682 phosphorylated sites and 446 phosphoproteins were quantitatively analyzed in the two genotypes. Motif analyses of the phosphorylation sites showed that six motifs including [PxsP], [sP], [sD], [Rxxs], [sxP] and [sxs] were enriched. We identified 12 and 48 PLSC phosphoproteins in M26 and MBB, respectively. Among these, 9 PLSC phosphoproteins were common to both genotypes, perhaps indicating a partial overlaps of the mechanisms to moderate drought stress. Gene ontology analyses revealed that the PLSC phosphoproteins present a unique combination of metabolism, transcription, translation and protein processing, suggesting that the response in apple to moderate drought stress encompasses a new homeostasis of major cellular processes. The basic trend was an increase in protein abundance related to drought and organic substance upon moderate drought stress between two genotypes. These increases were higher in the drought-tolerant genotype (MBB) than in the drought-sensitive genotype (M26). The 23 differentially expressed mRNA encoding phosphoproteins were analysis by quantitative real-time PCR (qRT-PCR). Our study is the first to address the phosphoproteome of a major fruit crop, apple rootstocks, in response to moderate drought stress, and provide insights into the molecular regulation mechanisms of apple rootstock under moderate drought stress.

Project description:Six months old seed grown under well-watered greenhouse conditions were used in the current study. The seedlings of the two wild emmer wheat genotypes were vernalized on a moist germination paper (Hofman Manufacturing, Inc, Jefferson, OR, USA) for three weeks in the dark at 4oC, followed by three days acclimation at 24oC. Seedlings were then transplanted into 5L pots containing a mixture of pure quartz sand and peat (4:1 v/v), supplemented with a slow release fertilizer (2 g/L, Osmocote® Standard 14-14-14, Scotts-Sierra Horticulture, Marysville, OH, USA) and by a weekly application of 100 ml/pot of 0.5X Murashigi and Skoog growth solution (Sigma Chemical Co., St Louis, MO, USA). Pots were placed in a screen-house under natural winter conditions (December to February; 5-18°C) in Haifa, Israel (Mt. Carmel; 35o01′ E, 32o45′ N; 480 m above sea level) for 10 weeks and irrigated daily by a drip irrigation system. Three weeks prior to application of terminal drought stress, the pots were transferred to a controlled environment greenhouse (22/18 oC; 12 h day/12 h night) in order to prevent rainfall during the drought experiment. Five pots per genotype served as biological replicates, plants of three pots of each genotype/treatment were used for transcriptome study whereas two additional pots were used for quantitative PCR analysis (altogether five biological replicated of were used for quantitative real time PCR). Three individual plants were grown in each pot, one plant was used for measurements of leaf relative water content (RWC) (Barrs and Weatherley 1968), whereas the other two plants were used for sampling of flag leaf tissue for RNA extraction. Terminal drought stress (D) was applied at inflorescence emergence stage (Zadoks 50-60), (Zadoks et al. 1974), after emergence of 1-2 spikes in all biological replicates of both genotypes. The time from transplanting to inflorescence emergence stage was not significantly different between genotypes (70 days in the S genotype and 72 days in the R genotype). Drought stress application initiated after irrigation with excess amount of water in order to assure that all pots start the experiment at the same soil water capacity. Drought stress was imposed by withholding water for eight days until stress symptoms (i.e. leaf rolling and wilting symptoms) were visible in plants of both genotypes. Stress symptoms were more visible in the S genotype, however, leaf relative water content (RWC), measured after eight days of drought stress was low but was not significantly different between the two genotypes (49.68%±1.48 in the R genotype and 53.34%±1.83 in the S genotype). The well-watered control (C) plants were irrigated daily by ample amount of water. Flag leaf tissues of drought-stressed plants and well-watered control plants were harvested, immediately frozen in liquid nitrogen, and stored at -80ºC for RNA extraction. ****[PLEXdb(http://www.plexdb.org) has submitted this series at GEO on behalf of the original contributor, Krugman Tamar. The equivalent experiment is TA33 at PLEXdb.] genotype: Drought resistant (R) Y12-3 - treated or untreated: Well-watered(3-replications); genotype: Drought resistant (R) Y12-3 - treated or untreated: terminal drought(3-replications); genotype: Drought susceptible (S) A24-39 - treated or untreated: Well-watered(3-replications); genotype: Drought susceptible (S) A24-39 - treated or untreated: terminal drought(3-replications)

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Gene regulation network of drought escape, avoidance and tolerance strategies in black poplar

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