Functional Characterization Of OsSAE1a In Regulation Of Phosphate And Nitrogen Utilization In Rice

Phosphorus(P)and nitrogen(N)are essential macronutrients for plants growth and development,and vital for plant growth and development.However,the phosphate(Pi)availability and nitrogen concentration in soil are not only very low,but also limiting factors for plants growth and agricultural production.SUMOylation(Small ubiquitin-like modifier)is a post-translational modification,has important functions in diverse biological processes in plants.Such as DNA repair,nuclear transport,transcriptional regulation,ion channel activity and nutrient response by influencing stability,cellular localization,activity of target proteins,and nutrient response.In Arabidopsis,the sumoylation is increased in the activities of PHR1 and NIA1/2,PHR1is involved in regulating the genes in the phosphate signal ways and NIA1/2 control nitrogen assimilation.In addition,OsSIZ1 is involved in the regulation of the responses to phosphate and nitrogen in rice.In this study,two genes in the SUMOylation,OsSAE1a and OsSIZ2 were identified.The expression pattern of OsSAE1a was detected by using qRT-PCR.Functionally characterized in rice growth and development,and its functions in P and N absorption,translocation and distribution were analyzed by using transgenic rice plants with overexpressed(OsSAE1a-Ox)or suppressed(OsSAE1a-Ri)expression of OsSAE1a.In addition,the functions of OsSIZ2 in N absorption,translocation and distribution were analyzed by using wild type,ossiz2 and OsSIZ2-Ri plants.The possible relationships between OsSAE1a and SUMO E3 ligase OsSIZl/2 in the utilization of nitrogen were analyzed.The main results acquired are listed as follow:1.OsSAE1a is localized on chromosome 11,consisted of eleven exons and ten introns.The open reading frame of OsSAE1a is 987 bp in length,which is predicted to encode 328 amino acids.2.The expression pattern of OsSAE1a is detected by searching rice chip database and qRT-PCR.OsSAE1a was constitutively expressed in different organs throughout the whole growth period,and the expression profile at the grain filling stage was higher than that at the vegetative stage.OsSAE1a was not very responsible to low phosphate in root and shoot and not affected by low nitrogen and different nitrogen forms.3.Transgenic rice plants with overexpressed(OsSAE1a-Ox)or suppressed expression(OsSAE1a-Ri)of OsSAE1a were obtained by transforming the constructs into mature embryos developed from seeds of wild type(’Nipponbare’)via Agrobacterium tumefaciens.At the seedling stage,OsSAE1a-Ri led to a phenotype with stunted growth and decreased biomass,while,OsSAE1a-Ox led to no significant difference;OsSAE1a-Ox and OsSAE1a-Ri resulted in impaired pollen fertility at the flowering stage,and reduced plant height,panicle length and seed setting rate at the mature stage.These results indicated that OsSAE1a is involved in rice growth and development.4.OsSAE1a was involved in the regulation of Pi absorption and translocation and distribution as evidenced by measuring the accumulation of phosphorus under hydroponic and pot experiments.Compared with wild type,under Pi-replete condition,the Pi concentrations in the shoot and root of OsSAE1a-Ri plants were increased 36%and 38%,respectively;and the total P concentrations in the shoot and root of OsSAE1a-Ri plants were increased 19%and 16%,respectively.While the Pi and total P concentrations were increased 24%and 32%in the shoots,decreased 38%and 62%in the roots of OsSAE1a-Ri plants under Pi-deficiency condition,respectively.The Pi and total P concentrations in the shoots and roots of OsSAE1a-Ox plants were no significant difference compared with wild type under both under Pi-sufficiency and-deficiency conditions.These results suggested that knockdown of OsSAE1 might promote Pi uptake and translocation from roots to shoots.32P-labelled Pi uptake measurements proved that the uptake rates in OsSAE1a-Ri plants under+Pi condition and in OsSAE1a-Ox/-Ri plants under-Pi condition were significant increased 127%and 45%/34%compared with wild type.The shoot to root ratio of 32P in Ox and Ri plants were had no significant difference compared with wild type under both+Pi and-Pi conditions.These results indicated that OsSAE1a was involved in Pi acquisition and translocation.At harvest stage,compared with wild type,total P concentrations of OsSAE1a-Ox and-Ri were decreased 24%and 20%in leaf sheath,increased 37%and 40%in culm and 29%and 57%in panicle axis under+Pi condition.Compared with wild type,under-Pi condition,total P concentrations of OsSAE1a-Ox and-Ri were increased 32%and 34%in leaf blade,18%in leaf sheath,50%and 35%in culm.These results suggested that OsSAE1a was involved in Pi distribution.5.OsSAE1a was involved in Pi signaling pathway by analyzed the relative expression of the genes which are involved in Pi regulatory system in the roots of OsSAE1a transgenic plants and wild type by qRT-PCR.Compared with wild type,under+Pi condition,only OsPT8 was up-regulated in the roots of OsSAE1a-Ri plants;the expression levels of Phtl family gene OsPT8,SPX family genes OsSPX2/3/4,OsPHO1;1/1;2 were up-regulated in the roots of OsSAE1a-Ox plants.Under+Pi condition,OsPT2/8,OsIPS1,OsPHO2,OsSPX1/2/4/5,OsPHO1;1/1;2,OsPHR2 and OsMYB2P-1wete up-regulated in the roots of OsSAE1a-Ri plants;and only OsPT2,OsSPX2/5,OsPHO2 and OsPHR2 were up-regulated in the roots of OsSAE1a-Ox plants.These results indicated that under+P condition,the up-regulated expression of OsPT8 might the main reason that caused increased acquisition of Pi in OsSAE1a-Ri plants.Under-Pi condition,the changes of Pi and total P concentrations in OsSAE1a-Ri plants may be caused by many genes work together;OsPT8 may might mainly promote acquisition of Pi and OsPT2,OsPH01;1/1;2 might promote Pi from roots to shoots.6.OsSAE1a was involved in N absorption and translocation and distribution as evidenced by carried out hydroponic,15N labeled NH4+uptake rate analysis and pot experiments.Compared with wild type,both+N and-N conditions,the total N concentrations in shoots of OsSAE1a-Ox/-Ri plants were increased 40%/26%and 43%/47%,respectively.Suggested that OsSAE1a might affect N uptake and translocation from roots to shoots.15N labeled NH4+ analysis further proved OsSAE1a promoted N absorption and translocation from roots to shoots.Compared with wild type,the total N concentrations in lower leaf blades of OsSAE1a-Ox and-Ri plants were decreased 21%and 23%;while increased 43%/35%in flag leaf blades and 34%and 32%in the seeds at harvest stage.These results indicated that OsSAE1a was involved in N translocation and distribution between vegetative organs and reproductive organs.7.Compared with wild type,under+N condition,the expression of OsNRT2.1/2.3,OsNAR2.1 and OsAMTl;2 were up-regulated in the roots of OsSAE1a-Ox plants;while only OsNRT2.3 and the partner protein genes OsNAR2.1/2.2 were up-regulated in the roots of OsSAE1a-Ri plants.Under-N condition,the expression of OsNRT2.1/2.3,OsNPF2.4,OsNAR2.1/2.2,OsAMT1;2 and OsAMT1;3 were up-regulated in the roots of both OsSAE1a-Ox plants;the expression of OsNRT2s,OsNPF2.4,OsNAR2.2,OsAMT1;2 and OsAMT1;3 were also up-regulated in the roots of OsSAE1a-Ri plants.These results indicated that under+N condition,OsNRTs and OsATT1s played important roles in increasing total N concentrations in OsSAE1a-Ox plants;while OsNRT2.3 and its partner protein genes OsNARs might the main causes which led to promote acquisition of N concentrations in OsSAE1a-Ri plants.Under+N condition,both OsNRT2s and OsAMT1s played important roles in increasing total N concentrations in both OsSAE1a-Ox and-Ri plants.8.The results in this thesis and other dates which had been reported by our group were shown that OsSAE1a and OsSIZ1 were involved in the regulation of the responses to phosphate and nitrogen in rice.In addition,OsSIZ2 was involved in phosphate response.On the basis of these we further investigate whether OsSIZ1 was also involved in regulating N absorption and translocation and distribution as evidenced by carried out hydroponic,15N labeled NH4+ uptake rate analysis and pot experiments.Compared with wild type,mutation or suppressed expression of OsSIZ2 led to total N concentrations in roots were decreased and increased under+N and-N conditions,respectively.This result suggested that mutation or suppressed expression of OsSIZ2 inhibited the absorption of nitrogen under+N condition and promoted N absorption under-N condition.15N labeled NH4+analysis proved mutation or suppressed expression of OsSIZ2 promoted N absorption,and inhibited N translocation from roots to shoots under-N condition.Mutation of OsSIZ2 led to the total N concentrations in the leaf blades,leaf sheaths,and panicle axis were increased,while suppressed expression of OsSIZ2 increased the total N concentrations not only in the leaf blades,leaf sheaths,and panicle axis,but also in the culms and grains.These results indicated that OsSIZ2 was involved in regulating N distribution at reproductive growth stage.In conclusion,1)OsSAE1a plays important roles in rice growth and development,especially at the lateral growth stage;2)OsSAE1a may participates in P and N absorption and utilization;3)OsSAE1a is involved in the regulation of N absorption,translocation and distribution by changing the expression of downstream genes.Both OsSAE1a and OsSIZ2 were involved in the regulation of N absorption,translocation and distribution,whether there are some possible relationships between them need to further study.