Cloning And Functional Characterization Of Key Genes Pesponse To Nitrate In Fruit Tree Roots

Nitrate is a major source of nitrogen, and it also acts as a signaling molecule that has animpact on gene expression associated with nitrogen uptake, metabolism and organdevelopment. In this study, we have analyzed gene expression changes during this processusing seedlings from peach trees, an important horticultural model plant, and cloned andcharacterized the functional roles of the key genes response to nitrate in fruit tree rootsThe results show that nitrate affects a broad range of genes. Except for carbon metabolism,nitrate affects genes associated with many biological plant processes, such as the biosynthesisof secondary metabolites and plant–pathogen interactions, amongst others. Overall,1,247genes were downregulated,460genes were upregulated by10mmol L^-1nitrate in roots and32,296candidates for new transcripts were found. These results suggest that peach genetranscripts may not have been fully predicted. In this study, expression of many genesinvolved in nitrogen metabolism and signaling (MYB,DOF and GATA transcription factor,NRT, SnRK1and CIPK8/23) was affected by nitrate. Most of these were repressed by10mmol L^-1nitrate in root tissue compared with0.5mmol L^-1nitrate. These results differedfrom earlier studies in other plants that showed some genes did not have significant sequencesimilarity. Future studies will need to characterize the functional roles of the genes withunknown functions, and the results obtained in this work will need to be validated using otherexpression analysis tools.PpDOF1(Genbank accession HM366546) was cloned from peach. The nucleotidesequence of PpDOF1contains a957bp open reading frame encoding a protein of318aminoacids with a mass of35.2kDa, and it had a highly conserved DOF domain. PpDOF1wasclosely related to ppa009811m of the genome sequence for peach became available in2010.Semi-quantitative polymerase chain reaction (SQ-PCR) analysis revealed that this gene wasexpressed in roots, stems and leaves, and levels were greatest in the roots and stems. Whenleaves were exposed to5mmol L^-1N)₃ˉ, the abundance of PpDOF1transcript had increasedafter30min, then subsequently declined. The content of total nitrogen in roots of transgenicplants was higher (14%) than that of the control. Expression of some genes involved innitrogen metabolism and signaling was different compared with that in the control, such as:miR398C,NRT1.8,NRT1.7and NR. This result suggests that may be PpDOF1regulatenitrogen metabolism and signaling. The average number of siliques between the first10cm(counting acropetally) was higher (16) than that of the control (12), and this internode defect appears to be random, because normal-sized internodes were interspersed along thestem.Ovate family protein (OFP), KNOTTED1-like homeobox (KNOX) genes and BEL1-like(BELL) homeodomain proteins play essential roles in inflorescence architecture, Expressionof these genes was different compared with that in the control. This result suggests that maybe PpDOF1regulate the expression of them, and inflorescence development. Expressions of37genes involved in lipid transport and metabolism were different compared with that in thecontrol.SnRK1(sucrose non-fermenting^-1-related protein kinase1) plays an important role in plantcarbon metabolism and development. To understand the mechanism of carbon and nitrogenmetabolism regulated by MhSnRK1from pingyitiancha (Malus hupehensis Rehd. var.pinyiensis Jiang), two transgenic lines (T2-7and T2-9) overexpressing this gene in tomatowere studied. SnRK1activity in the leaves of2transgenic lines was increased by15%^-16%compared with that in the wild-type. The leaf photosynthetic rate in transgenic tomatoes washigher than wild-type. The activity of sucrose synthase breakdown and ADP-glucosepyrophosphorylase was also increased, by approximately25%-36%and44%-48%,respectively, whereas sucrose synthase synthesis and sucrose phosphate synthase activitieswere unchanged. The content of starch in the leaves and red-ripening fruits was higher thanthat of the wild-type. The transgenic fruit ripened¹0days earlier than wild-type. The nitratereductase activity (mg plant^-1h^-1) has no significant difference between the transgenic plantand wild-type, but the N-uptake efficiency and root/shoot ratio in the T2-9line were15%and35%higher than that in the wild-type, respectively. These results suggest that overexpressingMhSnRK1can increase both carbon and nitrogen assimilation rate of the plant as well asregulate the development of fruit.Three full-length nitrate transporter genes, named MhNRT1.5, MhNRT2.1and MhNRT2.5,were cloned from pingyitiancha (Malus hupenensis Rehd.). The genome sequence for applebecame available in2010. The putative amino acid sequences of MhNRT2.1and MhNRT2.5were closely related to MDP0000239537(98.9%sequence identity) and MDP0000266497(98.6%sequence identity), respectively. No sequence was closely related to MhNRT1.5,suggesting that the gene transcripts of apple may be not predicted completely. Quantitativereal-time polymerase chain reaction (QRT-PCR) analysis revealed that these genes wereexpressed in roots, stems and leaves. Expression levels of the three MhNRT genes differedbetween the roots of4-week-old and3-year-old seedlings. The expression of these genes wasinduced over a wide range of nitrate concentrations (0.5^-10mmol L^-1). With the exception ofMhNRT1.5and MhNRT2.5when exposed to10mM nitrate, the abundance of transcripts for each of these MhNRTs was induced initially, with expression peaking at12h, before gradualdownregulation. MhNRT1.5, MhNRT2.1and MhNRT2.5expression patterns suggest that theymay be candidate genes of the inducible nitrate transporter family. Nitrate influx testssuggested that MhNRT2.1, MhNRT2.5and MhNRT1.5may have important roles in nitrateuptake in pingyitiancha roots by both the low-and the high-affinity transport systems. Thecontent of NO₃ˉin the roots of MhNRT2.1transgenic plants was lower than that of the control,but The content of NO₃ˉin the leaves of MhNRT2.1and MhNRT1.5transgenic plants washigher than that of the control.The average number of siliques between the first10cm(counting acropetally) was higher (16) than that of the control (12), but this internode defectappears to be symmetrical.