Effect Of Aerenchyma Formation Related Genes On Growth And Nitrogen Utilization In Rice

Rice(Oryza sativa L.)generally grows in flooding paddy soil where ammonium(NH4+)is the dominant form of nitrogen(N).Rice roots can secrete oxygen(O2)which is produced by shoot photosynthesis and transport to roots into the rhizosphere by aerenchyma.The abundant oxygen on the root surface can oxidize the NH4+ into nitrate(NO3-)by nitrifying bacteria.It has been reported that the development of root aerenchyma was positively related with the nitrification activity in rhizosphere.In general,the more the developed aerenchyma abundance in the roots,the higher the nitrifying capacity and larger numbers of ammonium-oxidizing bacteria(AOB)and NO3-concentration.The nitrifying capacity of rhizosphere affects the form of N acquired by rice roots,and thus the nitrogen use efficiency(NUE)and yield.However,it was not known for the relationship between aerenchyma abundance and growth and N uptake and utilization at the molecular level in rice.In model plant Arabidopsis,the three genes,Lesion Simulating Disease 1(AtLSD1),Enhanced Disease Susceptibility(AtEDS)and Phytoalexin deficient 4(AtPAD4)are involved in regulating the aerenchyma formation.AtLSD1 could affect the ethylene biosynthesis by regulating the expression of its downstream genes AtEDS and AtPAD4,and ethylene content could control the programmed cell death(PCD)and further aerenchyma formation.In this study,we isolated four genes with high amino acids homology respectively to AtLSDl,AtEDS and AtPAD4,and named them OsLSD1.1,OsLSD2,OsEDS and OsPAD as aerenchyma formation related genes in rice.We selected two rice varieties that had larger difference in aerenchyma development and NUE for dissecting the effect of enhanced aeration on growth and N acquisition.We conducted the experiments for the expression patterns and bioinformatics prediction of aerenchyma formation related genes in the rice varieties.We further evaluated the preliminary function of these genes on rice growth and N utilization by generating and evaluating the transgenic lines of overexpression and RNAi-knockdown.At the last part,we characterized the physiological function of OsLSD1.1 in regulating aerenchyma formation and N utilization in rice.The main results were as follows:1.Increasing aeration in the culture solution could significantly decrease aerenchyma formation in rice roots and increase the numbers of adventitious roots,larger total root length and root surface.The additive aeration increased total N uptake,together with up-regulated the expression of several nitrate and ammonium transporter genes,and nitrate reductase and ammonium assimilation genes in the rice roots.The genes expression and total N accumulation in Nongken57(NK57)that had the less developed aerenchyma formation was changed more significantly than Yangdao6(YD6)by aeration.Such effect of aeration on aerenchyma abundance and total N accumulation was also dependent on the forms of N supply.When rice was supplied with nitrate or nitrate and ammonium mixture,total N accumulation in shoot and root of both rice varieties were increased by extra aeration.When ammonium was the only form of supplied N,the aeration increased total N accumulation in roots of both varieties,and only in shoot of YD6 variety.2.The corresponding gene expression patterns supported the different effects of aeration on root growth and N accumulation in the two rice varieties.Expression of aerenchyma formation related genes(OsLSD1.1,OsLSD2,OsEDS and OsPAD)was altered by the extra aeration and such influence was different in two rice varieties.Interestingly,the supplied N form also affected the aeration induced expression of the aerenchyma related genes.Among the four candidate genes,OsLSD1.1 showed the largest effect by the N forms and aerationin both rice varieties.3.Constitutively overexpression driven by 35S promoter or RNAi-knockdown of OsLSD2,OsEDS and OsPAD in rice did not obviously change architecture of rice,in particular,the height and numbers of tillers.The overexpression of OsLSD2 in rice could increase the chlorophyll content in leaf sheath,and OsEDS overexpression could increase total N content in brown rice under sufficient N supply condition.4.Bioinformatics analysis showed that OsLSD1.1-encoded protein in rice had the highest homology with AtLOL which positively regulates the aerenchyma formation in Arabidopsis.OsLSD1.1 was mainly expressed in rice shoot,especially in culm and sheath(CS),the first and second leaf blades from the top.The expression level of OsLSD1.1 in roots was much lower than that in shoot,and its expression became strong gradually from root tips to root base.Expression of OsLSD1.1 gene in the second leaf from the top was strongly affected by N supply condition,light intensity and duration.The nutrient deficiency and salt stress up-regulated the expression of OsLSD1.1.5.The analysis of oslsdl.l knockout mutants,RNAi-knockdown mutants and OsLSD1.1 over-expression lines of rice showed that altering OsLSDl.1 expression changed in different rice varieties had different effect on rice growth and N nutrition.Over expression and RNAi of OsLSD1.1 in Nipponbare did not change the rice height and numbers of tiller but changed chloropgyll and total N content.In high yielding cultivar,cv.Wuyujing 7,overexpression of OsLSD1.1 could significantly increase the aerenchyma formation,water content and increase rice height and biomass;up-regulated expression of oxidation-reduction related genes;increased chloropgyll and total N content and related genes on photosynthesis pathway;increased content of iron(Fe)and decreased content of zinc(Zn),calcium(Ca)and magnesium(Mg).Overexpression of OsLSD1.1 gene significantly affected ions-binding related genes in gene chip data.Under different environmental stress conditions such as shorter light duration,nitrate supply and polyethylene glycol(PEG)simulating the water stress,the above difference between wild type and OsLSD1.1 over-expression transgenic rice became larger.