Project description:Purpose: Mechanism of gene expression changes in autotetraploid drought resistance variation Methods: Leaves under control and drought treatment of 6h, 12h and 48h were sampled from diploid and the autotetraploid for RNA-seq. Total RNA was extracted from 100mg sample with RNeasy plant mini kit (Omega Biotech, China) and purified with RNase-Free DNase set. Following, cDNA library was constructed and the library concentration and insert size was respectively assessed on the Qubit2.0 (Invitrogen, USA) and Agilent Bioanalyzer 2100 system, and then the effective concentration of the library was accurately quantified through Q-PCR. Prepared high quality libraries were sequenced on Illumina HiSeq X-ten platform to generate paired-end raw reads. qRT–PCR validation was performed using SYBR Green assays. Results: The study findings showed that the autotetraploid sour jujube exhibited a superior drought tolerance and enhanced regrowth potential after dehydration in comparison with the diploid counterpart. The physiological responses gradually triggered important functions in the autotetraploid sour jujube under extreme drought conditions. Furthermore, a comparative transcriptome analysis showed that more differentially expressed genes (DEGs) were detected in autotetraploid after drought stress. Through GO enrichment analysis, many DEGs between the diploid and autotetraploid sour jujube after drought-stress exposure were respectively annotated to the oxidation–reduction process, photosystem, DNA binding transcription factor activity, oxidoreductase activity. Consistently, six reactive oxygen species scavenging-related genes were specifically differentially expressed, positive changes of the genes involved in glutathione metabolism pathways were greater and the lower O2− level and malonaldehyde (MDA) content and higher antioxidant enzymes activity were detected in the autotetraploid under drought-stress conditions. The higher chlorophyll content and differentially enriched genes during photosynthesis suggest that the photosynthetic system in the autotetraploid, having variations in stomatal and cellular characteristics, was enhanced during drought stress. In addition, DEGs in the autotetraploid after stress exposure were significantly enriched in DNA-replication and plant hormone, including auxin, abscisic acid and gibberellin signal-transduction pathways. Under osmotic stress conditions, genes associated with the synthesis and transport of osmotic regulatory substances including anthocyanin biosynthesis were differentially expressed, and the soluble sugar, soluble protein and proline contents were significantly higher in the autotetraploid. Moreover, several genes encoding transcription factors (TFs) including GRAS, Bhlh, MYB, WRKY and NAC were induced specifically or to higher levels in the autotetraploid under drought-stress conditions, and hub genes, LOC107403632, LOC107422279, LOC107434947, LOC107412673 and LOC107432609, related to up-regulated transcription factors in the autotetraploid compared with the diploid were identified. Conclusions: Owing to the whole-genome doubling, many functional genes in the autotetraploid plants were differentially expressed compared with in the diploid during drought stress, resulting in resistance difference.

Project description:In rice, gene expression profiling under different stresses have been carried out earlier through microarrays and SAGE (Kawasaki et al., 2001; Rabbani et al., 2003,;. Yazaki et al., 2003, 2004; Walia et al., 2005; Zhou et al., 2005; Jiao et al., 2005; Zhou, 2007; Wang, et al., 2007)). The results reported so far were mostly derived from different stress treatments, array formats, species, tissue types, and time courses, which make it difficult for direct comparison among studies. Also, most of the stress experiments on expression profiles were conducted under laboratory, growth chamber or field grown plants subjected to stress in laboratory conditions, usually by inducing stress with exogenous compounds such as abscisic acid (ABA) or gibberellin (GA) genes (Yazaki et al., 2003, 2004; Rabbani et al., 2003), salt (Kawasaki et al., 2001; Rabbani et al., 2003; Walia et al., 2005), organ specific gene expression under high salinity (Jiao et al., 2005) and drought (Zhou et al., 2007), comparison with different cultivars (Wang et al., 2007) and other environmental stresses such as cold and drought (Rabbani et al., 2003; Zhou et al., 2005). However, such laboratory, growth chamber grown plants or field grown plants subjected to stress under laboratory conditions significantly differ from the field stress conditions and therefore the resultant plant response may not truly represent the actual stress conditions prevailing on field. This has been extensively reviewed by Mittler, 2006 and the need for utilizing data from multiple stress experiments for developing plants resistant to stress has been focused. The review also focuses on evaluating farmers experiences in stress management and kinds of stress that a plant is subjected prior to taking up research on stress resistances has been of significant importance. In the present study, to bridge the gap between the available data on drought stress and the gene regulation under field drought conditions has been paid attention. The outcome of this data will give new leads for breeders to design strategies to develop plants resistant to drought without much loss in yield. In the present study experiments were conducted under different field capacities using a rainout shelter to minimizing the effects of drought induction due to rains. The microarray analysis is thus expected to throw light on stress responsive gene expression in field drought conditions. Till now, to our knowledge there have been no reports on gene expression in rice under field drought conditions. Further, there is no data available on gene expression profiling during various stages of plant growth in leaf and panicle tissues under different levels of water stress. Here, we have focused on generating stress responsive gene (SRG) expression profiles in field drought conditions through the entire life cycle of rice plant. Optimal drought simulation was achieved by conducting a drought stress experiment and validated by a series of dry runs. The main reason for evaluating gene expression under drought in field condition is as drought itself is not limited to water stress but various other factors, such as heat, humidity, hot winds, and long day lengths effect the gene regulation. All these are likely to trigger common pathways that are also a part of drought stress response and therefore significantly affect the expression profiles. In the present study, field drought stress was initiated from 31 days after germination and transplantation to the field at 100%, 60%, 40% and 15% field capacities designated as A1, A2, A3, and A4. It took 7 days for the onset of the actual drought for the experimental plots to reach to their designated field capacities from 100% FC. Samples were collected from 7 days after drought stress to 33 days after drought stress to evaluate different drought stress regimes based on the developmental stages of the plant. At all the tested stress levels the data points were Hierarchically grouped and clustered sets of genes were examined and an approximate number of clusters to be formed were estimated. Finding a group of genes showing a similar pattern of gene expression by means of expression data from microarray analysis is the basis for identification of genes and pathways controlling a specific trait. We have followed unsupervised classification and clustered the genes hierarchically after normalizing expression data to obtain a set of co-regulated genes. Analysis of data revealed that there are at least 3 to 4 clusters which show differential regulation under each stress regime. The annotated genes falling in these clusters interestingly include a number of novel genes from N22 library along with some known candidate genes for drought tolerance which were earlier identified in our study (Markandeya et al., 2007). Expression pattern of these genes from 7 days after drought stress to 33 days after drought stress with their fold changes were evaluated in the present study. In particular, it is observed that many genes of unknown function are also co-regulated in an interesting pattern along with known genes.

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Ziziphus nummularia cultivar:Ziziphus nummularia

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