Different Salt Response Mechanism Of Pumpkins Based On Digital Gene Expression Profiling And Small RNA Sequencing

Salt-affected soils occupy nearly 10 % of the land surface and 50 % of irrigated land across the world. Studies on the mechanism of plant responses to salt stress play an important role to understand the salt tolerance of plants. Previous studies suggested that pumpkin rootstock can improve the salt-tolerance of cucumber, and the key reason is that salt-tolerant pumpkin roots have higher capacity to limit the transport of Na~+ to the shoot. There was significant difference of pumpkin rootstocks on accumulation of Na~+ and the underlying mechanism is not clear. To address this problem, two pumpkins namely N12(Cucurbita maxima Duch.) and N15(Cucurbita moschata Duch.), were used to detect differentially adapted mechanisms under short-term salt stress treatment. The determination of photosynthetic parameters, ABA contents, gene expression analysis, transcriptome sequencing, RNA-seq, and non-invasive micro-test technology(NMT) were used to find the different mechanisms of pumpkin rootstocks on accumulation of Na~+. The results obtained can be utilized in rootstocks improvement and breeding programs. The main results are as follows:1 We studied the accumulation patterns of several pumpkins. Two pumpkins, namely, N12(Cucurbita maxima Duch.) and N15(Cucurbita moschata Duch.), with different sodium accumulation patterns, were selected and used for RNA-seq and small RNA sequencing. Almost 80% of Na~+ was accumulated in N12 shoots, whereas about 50% Na~+ accumulated in N15 roots. N12 accumulated Na~+ in the shoots but better adapt to 100 mM NaCl treatment than N15.2 We studied the response of the two pumpkins on transcriptional level under salt stress. Illumina sequencing was used to determine small RNA populations in root tissues of the two pumpkins after 4 h of salt treatment and control. De nove assembly transcriptome sequences which were 91,995 all-unigenes with an average length of 767 nt was used as reference genome. Sequence alignment using BLAST showed that 68,877 transcripts exhibited gene annotation. There were 834 up-regulated DEGs and 1490 down-regulated DEGs in N12, whereas, 871 up-regulated DEGs and 1826 down-regulated DEGs in N15. In the above results, there were 211 DEGs and 644 DEGs either up-regulated or down-regulated in N12 and N15. Many DEGs exhibited gene annotation, and showed that hormones and transcription factors involved in the pumpkin early response to salt stress, and ABA play an important role in the pumpkin early response to salt stress. These responses were found in both pumpkins suggesting that they exhibited similar regulatory and responsive pathways at the early stage of salt stress.3 We studied the different response on small RNA level of the two pumpkins under salt stress. Illumina sequencing was used to determine small RNA populations in root tissues of the two pumpkins after 4 h of salt treatment and control. De nove assembly transcriptome sequences were used as reference genome. Fifty-eight conserved miRNA families and 33 novel miRNAs were identified in the two pumpkins. Seven miRNAs(six conserved miRNAs and one novel miRNAs) were up-regulated in salt-treated N12 and N15 samples. Most target genes of differentially expressed novel miRNAs were transcription factors and salt stress-responsive proteins, including dehydration-induced protein, cation/H~+ antiporter 18, and CBL-interacting serine/threonine-protein kinase. The differential expression of miRNAs between the two pumpkins under salt stress conditions and their target genes demonstrated that novel mi RNAs play an important role in the response of the two pumpkins to salt stress. The present study initially explored small RNAs in the response of pumpkin to salt stress, and provided valuable information on novel miRNAs and their target genes in Cucurbita.4 We studied the different response of the two pumpkins under salt stress based on the result of sequencing. We have determined photosynthetic parameters and ABA content, analyzed the distribution of Na ~+ by using atomic absorption spectrophotometer and non-invasive micro-test technique of two pumpkins under salt stress. The result showed that ABA concentration in leaves of N12 was rapidly increased and triggered stomatal closure when exposed to salt treatment. Gene expression analysis of sodium transporter indicated that N12 restricted high level Na~+ around the leaf vein instead of leaf mesophyll by high expression of HKT1. ABA concentration in leaf of N15 was quickly increased but could not triggered stomatal closure when exposed to salt treatment.The high expression of HKT1 in steam of N15 restricted Na~+ transport to shoot, but the Na~+ arrived in leaf was transported to leaf mesophyll, and the leaf showed salt injury symptoms after long time salt treatment.