Preliminary Research On The Physiological And Molecular Mechanisms Of Low Nitrogen Tolerance In Cucumber

Cucumber( Cucumis sativus L.) is one of the most important horticulture crops in fac ility cultivation, which als o pr efer to nitr ate. In or der to increase cucumber yield, f armers tends to apply nitrogen( N) blindly, and it caus es environmental pollution, excess ive content of nitr ate in fruit and other iss ues. Applying N fer tilizer sc ientif ic ally and breeding a cucumber cultivar w ith high capability for low N toler ance might be eff ective ways to solve above-mentioned problems. Study of the molecular mechanism of low N tolerance and identif ic ation of genes r elated to low N toler ance in cucumber w ill pr ovide theor etical r efer ence for pollution-free vegetable br eeding.To incr ease our understanding of the adaption for low N tolerance in cucumber, D0328 with a high capability for low N toler ance and D0422 w ith a low capability f or low N tolerance were used as exper imental mater ials in the pres ent study. The phys iological and molecular response patterns of two cucumber cultivars w ith differ ent capacities f or low N toler ance were compar ed by us ing is otopic trac ing and other methods. The mechanism of low N toler ance in cucumber was investigated based on the information of express ion pr of iles of low N tolerance related genes, and techniques such as met abolis m analys is and transgenic technology. The function of low N toler ance related gene was also analyzed. In addition, in order to quic kly and accurately determine the status of N in cucumber production and timely fertilization and fer tilizer controlling, candidate marker genes for detection of low N status in cucumber cultivation were screened by r eal- time f luorescence quantitative( q RT-PCR) in this study. The main r esults are as follows:(1) D0328 and D0422 were treated under low and high N conditions res pectively. Phys iological indexes and express ion levels of genes related N uptake, ass imilation, and remobilization were detected and analyzed at thr ee key per iods of growth and development(seedling, vining, and full fruit stages). The res ults showed that N uptake and ass imilation capac ities was similar between two cucumber cultivars under low N toler ance, while N remobilization capability of D0328 was stronger than that of D0422; the results in isotope trac ing exper iment fur ther demonstr ated that N remobilization effic iency of D0328 at full fruit stage was higher that of D0422 at the same time. These results revealed that D0328 might have higher N remobilization eff iciency than D0422, while N uptake and assimilation might not be related to low N tolerance in cucumber.(2) Source leaves of D0328 and D0422, which gr ew under low and high N condition at full fruit stage, were used for expr ession prof iling analys is of low N toler ance related genes. In this study, a total of 767(461 up-r egulated and 306 down-r egulated) and 533(304 up-regulated and 229 down-regulated) diff erentially express ed genes were identif ied in D0328 and D0422, respectively. Among these diff erentially express ed genes, 499(293 up-r egulated and 206 down-regulated) and 265( 136 up-regulated and 129 down-r egulated) genes were spec if ic ally expr essed in D0328 and D0422, and 268 genes were co-expr essed between two cultivars. Go analys is results showed that the biologic al process es that s ignif ic antly changed in D0328 were metabolic, organonitrogen compound biosynthetic, car boxylic ac id biosynthetic, organic ac id biosynthetic, and alpha-amino ac id biosynthetic processes. Other metabolic pr ocesses, such as cellular amino ac id biosynthetic, alpha-amino ac id metabolic, glutamine f amily amino ac id metabolic and biosynthetic, aspar tate family amino ac id biosynthetic, and aspartate family amino ac id metabolic processes, were als o signif ic antly changed. In pathway analys is, some of these genes w ith spec if ic up-r egulation expr ess ion patter n in D0328 wer e enr ic hed f or pathways with links to biosynthes is of amino ac ids, carbon metabolism, and carbon f ixation in photosynthetic organisms. Amino acid deter mination r esults indicated that the patterns of content changes of tryptophan(Trp), which is an auxin synthes is of im portant precursor substance, were diff erent in source leaves of D0328 and D0422 at full fruit stage. Compar ed with high N, Trp content in D0328 increased s ignif icantly under low N toler ance, while that in D0422 was signif icantly decreased at the same time. In addition, m RNA for CsGH3.17, an auxin biosynthes is gene, accumulated to a s ignif icantly higher level in source leaves of D0328 under low N conditions than high N conditions, wher eas Cs GH3.17 expr ess ion diff ered minimally between low and high N conditions in D0422. To confir m this hypothes is further, I AA content of D0328 and D0422 was measur ed under low and high N conditions. Under high N conditions, D0328 and D0422 exhibited s imilar I AA contents, while D0328 had higher I AA content than D0422 under low N conditions. I AA content of D0328 was increased with a s ignif icant higher ratio under low N tolerance than that of D0422. Further study showed that N r emobilization eff iciency of cucumber old leaves was increas ed by auxin biosynthes is promoter naphthaleneacetic acid(NAA) treatment, while it was repressed by antiauxins α-(p-chlorophenoxy) isobutyric acid(PCIB) treatment. Taken together, the results of this study revealed that auxin pr omotes N remobilization in cucumber leaf dur ing reproductive stage, and this positive regulation is associated with low N tolerance in cucumber.(3) In the expr ession prof iling res ults mentioned above, eight NAC tr anscr iption factor family genes wer e identif ied, which included f ive(f our up-regulated and one down-r egulated) genes were spec if ically expr essed in D0328, one gene was down-regulated in D0422, and two genes were up-regulated in D0328 and D0422. The numbers of encoding amino ac id of eight genes were between 238 and 432. I n amino ac id sequence, there was a NAM domain an d a conserved domain WKAT GXD(I/R), and their pr otein structur es contained f ive sub domains with high s imilar ity. According to the previous report, Cs NAC036 was selected for subs equent gene function analys is. The full- length CDS s equence of CsNA C036 w ith 912 bp was c loned by PCR. The function of Cs NAC036 was identif ied further. The r esults revealed that, the root length of w ild type was 5.96±0.1605 cm under high N condition, while the root length of w ild type was reduced by 0.11 times(5.29±0. 0951 cm) under low N condition; the root length of transgenic plants did not change under low and high N condition. I n addition, the root length of transgenic plants was longer than that of wild type under both low and high N conditions. This result r evealed that overexpress ion of Cs NAC036 might enhanc e the capability for low nitrogen tolerance in Arabidopsis.(4) Cucumber chalcone synthase gene( Cs CHS) was identif ied as a marker gene for low N detection in cucumber by RT-qP CR based on the r esults of gene express io n prof iling analys is. Cs CHS was f irst identif ied by Solexa s equenc ing w ith a cons istent up-regulation expr ess ion pattern by low N tolerance in two cucumber cultivars with diff erent capac ities for low N toler ance. RT-q PCR results further showed that Cs CHS had cons istent up-r egulation expr ess ion pattern and stable expr ession level by low N toler ance in another s ix cucumber cultivars w ith different ecotypes and differ ent stages and suggested that CsCHS could was us ed to identify the status of N def ic iency in cucumber. In order to validate this detection system, the express ion of Cs CHS was analyzed in diff erent NO3- concentr ations(1 m M, 3 mM, 5 m M, 7 m M and 14 mM NO3-), and the r esults r evealed that NO3- concentrations below 5 m M could be identif ied as low N tolerance in cucumber plants, and it also illustr ated that 5 mM NO3- is a cr itic al concentration for low N tolerance in cucumber. Sequence analys is of Cs CHS promoter showed that bes ides TATA-boxes and CAAT-boxes, which existed as essential promoter elements for promoter recognition, r egulatory elements homologous to those identif ied in hormones or light-r espons ive(regulation) genes were found in the CsCHS pr omoter r egion such as ABREs, Aux RR-core, and light-r espons ive elements such as GATA- motif. Other elements were also identif ied, inc luding MBS, W- box, and WRKY71 OS, among others. These elements existing in the CsCHS promoter may be involved in the low N toler ance in cucumber. Taken together, the r esults obtained in this study r evealed that Cs CHS is an impor tant marker gene for low N toler ance in cucumber. Us ing the express ion infor mation of Cs CHS, one can readily evaluate the NO3- fertilizer status of cucumber plants.