Transcriptomic Analyses Of Seed Germination Under Salt Stress In Populus Euphratica And P. Pruinosa

Soil salinity is one of major abiotic stresses limiting seed germination,growth and development of plants and has been a severe problem that restricts crop production and even restoration of the ecological environment.It is important to understand the regulation mechanism of salt tolerance in plants for effective reclamation and utilization of soil and improvement adaptability and yield of crop.Seed germination is highly related not only to the seedling survival rate but also to subsequent vegetative growth.Populus euphratica and P.pruinosa,naturally distributed in the extreme environment of desert regions of western and extraordinarily well adapted to the local stresses,are closely related species.For the two species with different adaptability to salt stress,they are ideal model species for studying mechanisms of plants’ adaptation to extreme environments.To examine reaction dynamics of adaptation during seed germination under salt stress of two species,we performed a comprehensive analysis of transcriptomes from seeds sampled from different time points of germination process under salt stress.Two sets of stress experiments were performed under Na Cl and compound salts,which were extracted from surface soils of original habitats.These two-dimensional datasets provide a comprehensive view on the differences in genetic basis of ecological adaptation between the two species and dynamics of gene expression and biochemical processes underpinning seed germination and salt tolerance.The main results are summarized as follows.(1)To elucidate the threshold salinity of the two species under salt treatments and suitable germination habitat,seed germination experiment was conducted under different concentrations(0.2%,0.4%,0.6%,0.8%,1.0%,1.2%,1.4% and 1.6%)of Na Cl treatment in P.euphratica and P.pruinosa.The results showed that germination percentage of the two species decreased with increasing salinity.The average germination percentage and the threshold salinity of P.pruinosa was higher than those of P.euphratica under Na Cl stress.Under 1.0% Na Cl stress,the sampling points of seed were at 0h,4h,12 h,24h,48 h and 72 h,6 time points,during seed germination process,totally 36 samples for transcriptome sequence.Based on Spearman correlation analysis,seed samples collected at 0h,4h and 12 h,clustered together,while those collected at 24 h,48h and 72 h clustered into the other group.We therefore defined 0h,4h and 12 h as early phase,24 h as middle phase and 48 h to 72 h as late phase of the germination process.Our analyses identified 12,831 differentially expressed genes(DEGs)for seed germination processes and 8,071 DEGs for salt tolerance in the two species.Functional enrichment analyses revealed that the strongly expressed genes in early phase were related to “structural constituent of cytoskeleton”,“cellular structure restoration”,“proteasome” and “oxidative phosphorylation” pathways while the highly expressed genes in middle phase were associated with “flavonoid biosynthesis” and the pathways that enriched in early phase.In addition,the significantly upregulated genes in last phase were typified by abundant functions including photosynthesis,energy production,hormonal regulation and transcription factors regulation pathways.In the comparison of salt tolerance between the two species,the flavonoid and brassinosteroid pathways were significantly enriched.More specifically,in the flavonoid pathway,FLS and F3’5’H exhibited significant differential expressions between two species.In the brassinosteroid pathway,the expression levels of DWF4,BR6OX2 and ROT3 were notably higher in P.pruinosa than in P.euphratica.Therefore,P.pruinosa was found to have higher gene expressions in the anti-oxidantanand flavonoid biosyntheses and other related pathways and therefore higher salt-tolerant than P.euphratica.To further confirm this conclusion,the content of ROS and the activities of SOD,POD and CAT in P.euphratica and P.pruinosa were determined in 0.4%,0.8% and 1.0% Na Cl.Five selected DEGs were verified by q RT-PCR.(2)We further examined how compound salt from natural habitats of two species affected seed germination and gene expressions of P.euphratica and P.pruinosa.We found that compound salt had less harmfulness on seed germination than Na Cl.We compared seed germination and gene expression of the two species under the compound salt and the Na Cl treatment conditions.The results showed that salt tolerance of two species were different with increasing compound salt stress.We sampled the seedlings with the same time point of germination for transcriptome substitution between two different salt treatments.We further sampled the seedlings with the same time point of germination for transcriptome analyses.DEGs were identified between adjacent time points of seed germination process.There were 6,885,1,670,341,2,051 and 1,338 DEGs identified in P.euphratica,respectively.Then hierarchical clustering analysis of total DEGs in P.euphratica shown that the expression patterns could be divide into early,middle and late phases,containing 3,654,2,213 and 4,167 DEGs,respectively.Similarly,8,488,2,925,3,693,85 and 82 DEGs were identified in P.pruinose,respectively.There were 3,115,3,697 and 5,129 DEGs in three phases of seed germination,respectively.The DEGs in early phase were enriched in “proteasome”,“ribosome” and “oxidative phosphorylation” pathway in the two species.The late phase exhibited the enrichment of several amino-acids metabolisms and “phenylpropanoid biosynthesis” pathway in both P.euphratica and P.pruinosa.A total of 8,876 DEGs were identified between two species at the same time points.Of these DEGs,4565,2,070,2,545,378,2,006 and 1,910 DEGs were identified at each time point,respectively.On the basis of function enrichment,the ribosome related genes were found to express in P.pruinose longer than P.euphratica.The result suggested that these genes might account for salt tolerance between the two species under compound salt.In addition,P.euphratica was found to be significantly different from P.pruinose in the responding pathways and genes expression level under compound salt stress.For instance,genes enriched in “Galactose metabolism” and “Linolemic acid metabolism”pathways were highly expressed in P.pruinosa,while genes related to“Phenylpropanoid biosynthesis” and “Valine degradation” were highly expressed in P.euphratica.These differences are suggested to affect the types and proportions of secondary metabolites accumulated in the two species and indicate that the salt tolerance mechanisms of the two poplars have diverged.Overall,our findings together suggest that P.euphratica and P.pruinosa can tolerate high salinity and P.pruinosa shows a higher salt tolerance than P.euphratica at the three seed germination phases.We further illuminated the transcript dynamics and highlighted gene expression differences between the two species.In addition,these differences similarly occur when different salts were stressed.We further show the enrichment functions of these genes with different expressions,which may be genetic bases of the different salt tolerances of the two species.Qverall,the results provide an important theoretical basis for further understanding the molecular mechanism of P.euphratica and P.pruinosa in response to salt stress during seed germination,and are of great significance to improve the environmental adaptability of plants and improthe yield and growth of crops.