拟南芥miR156与miR399在低磷胁迫中的对话机制初探
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摘要
为了进一步研究miR156与miR399在低磷响应过程中是否存在对话机制,采用杂交方法,将35S:MIM156(miR156功能降低拟南芥植株)与miR399f(miR399f超表达拟南芥植株)杂交获得杂交转基因植株35S:MIM156 miR399f。采用原位显色法和根际酸化能力测定法,研究低磷胁迫下35S:MIM156、miR399f及35S:MIM156 miR399f植株根际酸化情况,并测定其花青素和无机磷含量。结果表明,35S:MIM156植株的花青素含量较野生型(WT)植株显著降低,miR399f、35S:MIM156 miR399f植株与WT无显著差异;35S:MIM156植株的酸化能力较WT显著降低,miR399f、35S:MIM156 miR399f植株酸化能力均较WT显著增强;35S:MIM156、miR399f、35S:MIM156 miR399f植株的无机磷含量均较WT显著升高。综合分析,推测在低磷条件下miR399参与了miR156调控的花青素积累和根际酸化过程,miR156和miR399共同参与磷平衡过程。
In order to explore whether the cross talk between miR156 and miR399 exists,the 35 S:MIM156 miR399 f double hybrid transgenic plant was constructed by crossing between 35 S:MIM156(transgenic plant silencing miR156)and miR399 f(transgenic plant overexpressing miR399 f).The rhizosphere acidification phenotype,anthocyanin and inorganic phosphorus contents of 35 S:MIM156 miR399 f,35 S:MIM156 and miR399 f plants were studied by the in-situ coloration method,rhizosphere acidification activity assay and so on.The results showed that compared with WT(wild type),the anthocyan content of 35 S:MIM156 plants significantly decreased,the anthocyan contents of miR399 f and 35 S:MIM156 miR399 f plants were not significantly different from WT;the rhizosphere acidification ability of 35 S:MIM156 significantly decreased,the rhizosphere acidification abilities of the miR399 f and 35 S:MIM156 miR399 f plants significantly increased;the inorganic phosphorus contents of 35 S:MIM156,miR399 f and 35 S:MIM156 miR399 f plants significantly increased.Overall,it was speculated that miR399 was involved in the process of anthocyanin accumulation and rhizosphere acidification processes regulated by miR156,and miR156 and miR399 both took part in phosphate homeostasis process under low phosphorus stress.
In order to explore whether the cross talk between miR156 and miR399 exists,the 35 S:MIM156 miR399 f double hybrid transgenic plant was constructed by crossing between 35 S:MIM156(transgenic plant silencing miR156)and miR399 f(transgenic plant overexpressing miR399 f).The rhizosphere acidification phenotype,anthocyanin and inorganic phosphorus contents of 35 S:MIM156 miR399 f,35 S:MIM156 and miR399 f plants were studied by the in-situ coloration method,rhizosphere acidification activity assay and so on.The results showed that compared with WT(wild type),the anthocyan content of 35 S:MIM156 plants significantly decreased,the anthocyan contents of miR399 f and 35 S:MIM156 miR399 f plants were not significantly different from WT;the rhizosphere acidification ability of 35 S:MIM156 significantly decreased,the rhizosphere acidification abilities of the miR399 f and 35 S:MIM156 miR399 f plants significantly increased;the inorganic phosphorus contents of 35 S:MIM156,miR399 f and 35 S:MIM156 miR399 f plants significantly increased.Overall,it was speculated that miR399 was involved in the process of anthocyanin accumulation and rhizosphere acidification processes regulated by miR156,and miR156 and miR399 both took part in phosphate homeostasis process under low phosphorus stress.
引文
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[15] BAEK D,KIM M C,ChUN H J,et al.Regulation of miR399f transcription by AtMYB2 affects phosphate starvation responses in Arabidopsis[J].Plant Physiol,2013,161(1):362-373.
[16] KIM J,YI H,CHOI G,et al.Functional characterization of phytochrome interacting factor 3 in phytochrome-mediated light signal transduction[J].Plant Cell,2003,15:2399-2407.
[17] SANTI S,SCHMIDT W.Dissecting iron deficiency-induced proton extrusion in Arabidopsis roots[J].New Phytol,2009,183:1072-1084.
[18] AMES B N.Assay of inorganic phosphate,total phosphate and phosphatase[J].Methods Enzymol,1966,8:115-118.
[19] GOU J Y,FELIPPES F,LIU C J,et al.Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPL transcription factor[J].Plant Cell,2011,23:1512-1522.
[20] SUNKAR R,ZHU J K.Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis[J].Plant Cell,2004,16:2001-2019.
[21] YANG L,CONWAY S R,POETHIG R S.Vegetative phase change is mediated by a leaf-derived signal that represses the transcription of miR156[J].Development,2011,138(2):245-249.
[22] SHIKATA M,KOYAMA T,MITSUDA N,et al.Arabidopsis SBP-Box genes SPL10,SPL11 and SPL2 control morphological change in association with shoot maturation in the reproductive phase[J].Plant Cell Physiol,2009,50:2133-2145.
[23] NAVARRO L,DUN OYER P,JAY F,et al.A plant miRNA contributes to antibacterial resistance by repressing auxin signaling[J].Science,2006,312:436-439.
[24] CHIOU T J.The role of microRNAs in sensing nutrient stress[J].Plant Cell Environ,2007,30:323-332.
[2] RAUSCH C,BUCHER M.Molecular mechanisms of phosphate transport in plants[J].Planta,2002,216:23-37.
[3] TICCONI C A,ABEL S.Short on phosphate:Plant surveillance and counter measures[J].Trends Plant Sci,2004,9:548-555.
[4] WANG D,Lü S,JIANG P,et al.Roles,regulation,and agricultural application of plant phosphate transporters[J].Trends Plant Sci,2017,8:817.
[5] BARTEL D P.MicroRNAs:Genomics,biogenesis,mechanism,and function[J].Cell,2004,116:281-297.
[6] CHIOU T J.The role of microRNAs in sensing nutrient stress[J].Plant Cell Environ,2007,30:323-332.
[7] FUJII H,CHIOU T J,LIN S I,et al.A miRNA involved in phosphate-starvation response in Arabidopsis[J].Curr Biol,2005,15(22):2038-2043.
[8] AUNG K,LIN S I,WU C C,et al.pho2,a phosphate over accumulator,is caused by a nonsense mutation in a microRNA399 target gene[J].Plant Physiol,2006,141(3):1000-1011.
[9] BARI R,DATT PANT B,STITT M,et al.PHO2,microRNA399,and PHR1 define a phosphate-signaling pathway in plants[J].Plant Physiol,2006,141(3):988-999.
[10] CHIOU T J,AUNG K,LIN S I,et al.Regulation of phosphate homeostasis by microRNA in Arabidopsis[J].Plant Cell,2006,18(2):412-421.
[11] PARK B S,SEO J S,ChUA N H.NITROGEN LIMITATION ADAPTATION recruits PHOSPHATE2 to target the phosphate transporter PT2 for degradation during the regulation of Arabidopsis phosphate homeostasis[J].Plant Cell,2014,26(1):454-464.
[12] HSIEH L C,LIN S I,SHIH A C,et al.Uncovering small RNA-mediated responses to phosphate deficiency in Arabidopsis by deep sequencing[J].Plant Physiol,2009,151(4):2120-2132.
[13] LEI K J,LIN Y M,REN J,et al.Modulation of the phosphate-deficient responses by the microRNA156 and its targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 in Arabidopsis[J].Plant Cell Physiol,2016,57(1):192-203.
[14] WANG J W,CZECH B,WEIGEL D.miR156-regulated SPL transcription factors define an endogenous flowering pathway in Arabidopsis thaliana[J].Cell,2009,138:738-749.
[15] BAEK D,KIM M C,ChUN H J,et al.Regulation of miR399f transcription by AtMYB2 affects phosphate starvation responses in Arabidopsis[J].Plant Physiol,2013,161(1):362-373.
[16] KIM J,YI H,CHOI G,et al.Functional characterization of phytochrome interacting factor 3 in phytochrome-mediated light signal transduction[J].Plant Cell,2003,15:2399-2407.
[17] SANTI S,SCHMIDT W.Dissecting iron deficiency-induced proton extrusion in Arabidopsis roots[J].New Phytol,2009,183:1072-1084.
[18] AMES B N.Assay of inorganic phosphate,total phosphate and phosphatase[J].Methods Enzymol,1966,8:115-118.
[19] GOU J Y,FELIPPES F,LIU C J,et al.Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPL transcription factor[J].Plant Cell,2011,23:1512-1522.
[20] SUNKAR R,ZHU J K.Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis[J].Plant Cell,2004,16:2001-2019.
[21] YANG L,CONWAY S R,POETHIG R S.Vegetative phase change is mediated by a leaf-derived signal that represses the transcription of miR156[J].Development,2011,138(2):245-249.
[22] SHIKATA M,KOYAMA T,MITSUDA N,et al.Arabidopsis SBP-Box genes SPL10,SPL11 and SPL2 control morphological change in association with shoot maturation in the reproductive phase[J].Plant Cell Physiol,2009,50:2133-2145.
[23] NAVARRO L,DUN OYER P,JAY F,et al.A plant miRNA contributes to antibacterial resistance by repressing auxin signaling[J].Science,2006,312:436-439.
[24] CHIOU T J.The role of microRNAs in sensing nutrient stress[J].Plant Cell Environ,2007,30:323-332.
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