| P. euphratica Oliv. is a valuable tree species used for afforestation on saline and alkaline desert sits in the north-west China, and plays a crucial role to maintain ecological stability in ecologically fragile areas. Recently, the physiological and biochemical mechanisms of the salt tolerance have been extensively investigated in P. euphratica, however, the molecular mechanism of salt tolerance in P. euphratica is not systematicly and thoroughly investigated. Using the Affymetrix poplar genome array, we explored the leaf transcriptome of salt-tolerant Populus euphratica Oliv. and salt-sensitive P. popularis 35-44 (P. popularis) under short term and long term NaCl stress. The aim is to establish a correlation between the gene expression and molecular mechanism of salt tolerance in P. euphratica.1. A modified sodium borate method was adopted to extract the total leaf RNA of P. popularis and P. popularis. This method was effectively to reduce the interference of polyphenolics and polysaccharides during RNA isolation and purification. The follow-up experiments also confirmed that high quality of total RNA could be successfully used in our microarray analysis.2. The transcriptomes of P. euphratica and P. popularis were compared using Affymetrix microarray systems. We found that there were species difference in the expression profiles between the two poplars, which can basically be divided into two categories:natural (non-induced) differences (differences in expression patterns between the poplar species under non-stress conditions) and salt-induced differences (differences in expression profiles before and after salt treatments). The molecular mechanism of salt-tolerant P. euphratica can be inferred according to the differential expressed genes-natural and stress-inducible genes between the salt-tolerant and sensitive poplar. Under non-saline conditions, the transcription abundance of many genes related to stress resistance in P. euphratica is significantly higher than that in P. popularis. They are genes of the transcription factor protein, ion and polysaccharide transporter, signal transduction protein, and anti-oxidative defence components. The data suggest that P. euphratica plants have evolved relevant mechanisms to tolerate saline environments after a long time adaptation to arid and saline desert.3. There are still salt inducible differences in gene expression profiles between P. euphratica and P. popularis. In ST (short-term NaCl)-treated P. euphratica,368 probesets among the 467 differentially expressed genes were up-regulated while the other 99 probesets were down-regulated. Under LT (long-term NaCl) stress conditions,52 probesets were differentially expressed, among which 43 probesets were up-regulated and 9 probesets were down-regulated. For the salt sensitive species, P. popularis, the ST treatment changed the expression of 458 probesets:420 probesets were increased while 38 probesets decreased. The transcription of 3908 probesets were alterd by the LT stress, among which 2047 probesets were up-regulated and 1861 probesets down-regulated. In these differentially expressed genes, we found that some genes were highly associated with the salt tolerance in P. euphratica. These genes are (1) transcriptional regulators genes such as transcription factors (zinc finger protein, wrky51, MYB11, ABF2), phospholipase C, tyrosine phosphatase,14-3-3 and other genes; (2) chloroplast chlorophyll biosynthesis genes; (3) carbohydrate metabolic genes which are involved in glycolysis, Krebs cycle, pentose phosphate and starch synthesis pathway; (4) secondary metabolite (especially osmotic adjustment) synthesis genes like flavonoids, lignin and mannose synthesis genes; (5) ROS metabolism genes like POD, SOD, glutathione-S-transferase, ascorbate reductase, ascorbate peroxidase, hydrogen peroxide lyase and TRX; (6) protective protein genes like heat shock proteins, LEA proteins, cytochrome P450; (7) various transporter genes:nitrogen transporter, cation transporter, sugar transporter, etc. Noteworthy is that these genes were up-regulated in P. euphratica during short-term or long-term salt treatments, but their expression were decreased or remained unchanged in salinised P. popularis. Therefore, we conclude that the salt inducible genes expression is presumably associated with salt tolerance in P. euphratica. Besides the differentially expressed genes that mentioned above, we also found co-upregulated genes in salt-treated plants of the two species. Under ST treatments, transcription of 40 probesets was enhanced in salinied P. euphratica and P. popularis. We extracted the promoter regions of their homolog genes from P. trichocarpa. The bioinformatics analysis show that the promoter of these genes share common cis-acting elements that are responsible for osmotic stress sensing.4. On the basis of poplar transcriptome analysis, we characterized the expression of some important salt-resistant genes in the two poplar species. We found that the gene transcription of ion homeostasis, ROS homeostasis and photosynthesis is closely related to the salt tolerance of P. euphratica.(1) Genes related to ionic homeostasis. We found that genes like Na+/H+ antiporter such as SOS1 and NHD2 and proton pump genes such as PM H+-ATPase were significantly higher expressed in P. euphratica compared to P. popularis. Moreover, their expression was not decreased by the ST and LT stress in P. euphratica. Na+ content in P. euphratica leaves was significantly less than P. popularis under LT treatments. Our microarray data show that P. euphratica maintained high expression of Na+/H+ antiporter and proton pumpss, which are favorable to extrude Na+ to the extracellular space.(2) Genes related to photosynthesis. In P. euphratica, photosynthesis related genes like chlorophyll a/b binding protein, PSB-Q, the chloroplast membrane proteins and electron transport chain genes were up-regulated during ST treatment, and their expression was not down-regulated by the LT treatments. In contrast, the expression of these genes in P. popularis was decreased by LT stress. Therefore, P. euphratica up-regulated photosynthesis related genes to cope with the salt stress, which is beneficial to maintain leaf photosynthesis under saline conditions. The result is consistent to our previous findings in photosynthesis studies.(3) Genes related to ROS homeostasis. After exposure to ST salinity, P. euphratica showed strikingly up-regulated transcription of a variety of anti-oxidant enzymes, including Cu-Zn SOD, CAT, PODs, PRX, TRXs, GRX and GSTs. Compared with P. euphratica, fewer antioxidant enzymes (e.g., APX, anionic POD, TRXs and GRX) were up-regulated in ST-stressed P. popularis. Unlike ST-stressed plants, expression of antioxidant enzymes in P. euphratica was not significantly altered by LT stress. In contrast to P. euphratica, LT salinity significantly affected the transcription of a large number of antioxidant enzymes in P. popularis leaves. NaCl decreased transcription of Cu-Zn SODs, H2O2 lyase, APX, PRXs and some members of POD, TRX, GRX and GST, but enhanced expression of CAT and several members of POD, TRX, GRX and GST. These results indicate the member-specific response to salinity in each family of antioxidant enzymes in P. popularis. P. euphratica rapidly up-regulated expression of antioxidant enzymes upon salt stress, contributing to the control of ROS production and oxidative damage. Confocal analysis of leaves show that P. euphratica strictly limited the level of H2O2 during the LT stress, thus avoiding the salt-induced oxidative damage. In contrast to to P. euphratica, H2O2 burst occurred in LT-stressed P. popularis leaves. This is due to its inability to increase the expression of antioxidant enzymes at the beginning of salt stress, which resulted in an overproduction of ROS in leaf cells, leading to an oxidative damage over a prolonged period of salinity.(4) We used quantitative RT-PCR to evaluate our microarray data. Several representative genes, e.g., HAK1, ATGPX2, APX, POD and K+ channel gene, were verified. Real-time PCR confirmation showed a consistent tendency of gene expression to the microarray data. This indicates that the data derived from Affymetrix poplar genome array was solid and reliable, therefore, the data is sufficiently qualified to continue the subsequent data mining.5. The plasma membrane H+-ATPase plays an important role in ion homeostasis and stress signal transduction in P. euphratica. Our microarray data show that P. euphratica retained a high transcription abundance of the H+-ATPase gene (AHA) under non saline conditions. However, the regulation of H+-ATPase at molecular levels is unkown. We are able to identify different cis-elements related to stress in poplars by comparing the promoter regions of AHAs in the salt tolerant and sensitive poplar species., This can also help us to understand the expression regulation of AHAs upon the salt treatment. The promoter regions of AHAs were cloned from P. euphratica and a salt sensitive species P. canescens, followed by bioinformatics and transient transformation analyses. Bioinformatics analysis show that the promoter structure of PeAHA is rather different to that of PcAHA. Compared to PcAHA, PeAHA is enriched with cis-elements in response to ABA in the promoter region. Moreover, the transient expression results also confirmed that the promoter of PeAHA could response to ABA treatments, but there was no corresponding response in PcAHA promoter.In conclusion, there were marked differences of gene expression between P. euphratica and salt sensitive species. P. euphratica maintained high expression of ion transporters and channels under normal and stressed conditions, which is helpful for the salt-resistant species to control K+/Na+ homeostasis in leaves. This is a mechanism to avoid excessive accumulation of Na+, and reduces its toxicity in leaf cells. P. euphratica can also rapidly increase the expression of ROS metabolism genes to pre(?)ent oxidative damage. This is able to maintain the stability of membrane system and reduces the membrane peroxidation. In addition, P. euphratica is capable of increasing the expression of photosynthesis-related genes, thus contributing to CO2 assimilation under salt stress. The carbohydrates provide an important source of energy for maintaining ionic homeostasis and ROS homeostasis. |
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