Identification And Functional Characterization Of Regulatory Genes Involved In Phosphate Starvation And AM Symbiosis Signalings

Phosphorus （P） is one of the essential macro-nutrients for plant growth and development, and plays important roles in energy transfer, signal transduction, and photosynthesis processes. It is also a structural component of many biologically important macro-molecules, such as nucleic acids, phospholipids and P-containing enzymes. However, since P is readily chelated by cations and precipitated in the soil and the only form of P available for plant uptake is inorganic ortho-phosphate （Pi）, the mobility and availability of P is poor. Plants have evolved a suite of responses to adapt to P-deprived environment, including modification of root architecture, release of acid phosphatase, RNase and organic acids, as well as forming mutualistic symbiotic associations with arbuscular mycorrhizal （AM） fungi.In the past decades, a series of elaborate molecular mechanisms underlying these adaptive responses have been intensively studied, and a molecular regulatory network with regarding to Pi starvation and AM symbiosis has been generated. The nodes in this network, namely the genes involved, are closely related. The alteration in abundance, temporal or spatial expression of one gene may lead to a re-orchestration of the entire network. Although an increasing number of genes involved in Pi starvation and AM symbiosis signaling pathways have been and will be identified in diverse plant species, and the function of the conserved central regulator PHR （PHosphate Starvation Response） that might connect the two signalings have been well elucidated, a lot of work should be done to unravel the complex regulatory mechanism of these signalings. In an attempt to get a better understanding of the Pi starvation and AM symbiosis signaling pathways, we tried to isolate and/or functionally characterize the microRNA （miRNA） and transcription factor encoding genes in the present work. The main results acquired are listed as follows:1. Based on all the mature miRNA sequences in plants, we identified276miRNA genes, which belonging to84families, in tobacco by using its GSS （Genome Survey Sequence） and EST （Expressed Sequence Tag） sequences. We analyzed a series of characteristic parameters of miRNA genes, and found that half of the miRNA families have more than one member; the lengths of the mature miRNAs and precursors are～21and75-114nucleotides, respectively; Ninety percent of the miRNA precursors have a Adnine plus Uridine content ranging from50%-70%; the conserved Pi starvation-induced miR399and miR827are also up-regulated by Pi deprivation.2. Twenty three conserved miRNAs were identified in tomato by miRNA microarray, among which sixteen were responsive to Pi starvation or AM symbiosis or both. The data demonstrated that altered expression of distinct groups of miRNA is an essential component of Pi starvation-induced responses and AM symbiosis, and there are common and specific signalings of the P nutrition and AM symbiosis processes.3. The expression of osa-miR827a （OsmiR827a） as well as its target genes was detected by RT-PCR and histochemical analysis. The results suggest that OsmiR827a is specifically induced by Pi starvation, and its mature fragment might act as a long-distance signal molecule transported from shoot to root through phloem. In addition, we concluded that the mechanism of the negative regulation of miR827on its target（s） might have altered during the divergence of monocots and dicots. Unlike Arabidopsis miR827, which targets AtNLA （Nitrogen Limitation Adaptation; a member of the SPX-RING subfamily）, rice miR827a targets two genes from the SPX-MFS₁subfamily, namely OsSPX7and OsSPX8. Although the amino acid sequence identity of the two plasma membrane located genes is82.35%, and their structures are highly similar, their responses to Pi starvation are opposite.4. The phenotype, Pi concentration and some regular physiological parameters were detected in the Tosl7insertion mutants of OsSPX7and OsSPX8（osspx7-1, osspx7-2and osspx8-1, osspx8-2） under varied Pi supply. Pi toxicity symptoms were observed in osspx7-2under both high Pi （HP） and ammonium nutrition （NH4+） conditions, and the Pi concentration in leaves is6.2and3.8folds as high as that of WT plants, respectively. Under low Pi （LP） condition, the Pi concentration in osspx7-2leaves increased1fold as compared to that in WT. In osspx8-1and osspx8-2, the Pi concentration in leaves showed an increase of40%as compared with that of WT plants under HP and NH4+conditions. Moreover, through detection of the expression of some OsPT （Phosphate Transporter） and Pi responsive genes, as well as comparing with other Pi-overaccumulator, we predicted that OsSPX7and OsSPX8regulate in planta Pi homeostasis partially by repression of OsPT expression. 5. The expression of OsmiR827a was also detected in OsPHR2（PHosphate Starvation Response2） overexpressing plants. The result suggests that OsmiR827a might be a novel target directly regulated by OsPHR2.6. Through succession truncation and targeted point mutation of LePT4promoter, we demonstrated that P1BS （PHR1Binding Sequence） is indispensable for the transcriptional activation of LePT4in response to AM symbiosis. Whereas cis-acting elements, NODCON2GM （responsible for the nodule-and mycorrhiza-specific expression of soybean leghemoglobin lbc3and N23gene） and WRKY710S （responsible for plant defense response）, are not involved.7. Based on the conserved region of Arabidopsis and rice PHR genes, we isolated the full cDNA sequence of the homologous gene in tobacco, NtPHR2, by using RLM-RACE technology. The amino acid sequence alignment and phylogenetic tree analyzes showed that NtPHR2is a novel member of the MYB-CC （Coiled-Coil） transcription factor subfamily, and shares the highest sequence identity with AtPHRl.8. The subcellular localization, expression pattern and the potential protein interaction of NtMYCF1and NtPHR2were analyzed by onion epidermal bombardment experiment, RT-PCR and yeast two hybrid system, respectively. The results suggest that NtMYCF1and NtPHR2localize to the nucleus; the transcript abundance of NtMYCFl in root is increased upon AM symbiosis, while NtPHR2is constitutively expressed in both root and leaf; there might be no interaction between NtMYCF1and NtPHR2protein.