何亚飞^{1, 2,},
许梦洁^{1, 3},
李霞^1,,
1.江苏省农业科学院粮食作物研究所/江苏省优质水稻工程技术研究中心/国家水稻改良中心南京分中心 南京 210014
2.南京林业大学生物与环境学院 南京 210037
3.南京晓庄学院 南京 211171
基金项目: 国家自然科学基金项目31571585
江苏省农业科学院基本科研业务专项ZX[16]2002
江苏省农业科学院粮食作物研究所基金LZS17-9

详细信息

作者简介:何亚飞, 主要研究方向为作物逆境生理。E-mail:1368267588@qq.com

通讯作者:李霞, 主要研究方向为植物光合生理。E-mail:jspplx@jaas.ac.cn

中图分类号:S311;S338

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出版历程

收稿日期:2017-07-17
录用日期:2017-08-22
刊出日期:2018-03-01

Effects of DCMU on anthocyanin synthesis genes and its related signals in C₄-pepc gene overexpressed rice under drought conditions

HE Yafei^{1, 2,},
XU Mengjie^{1, 3},
LI Xia^1,,
1. Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Jiangsu High Quality Rice Engineering Technology Research Center/Nanjing Branch of National Center for Rice Improvement, Nanjing 210014, China
2. College of Biology and Environment, Nanjing Forestry University, Nanjing 210037, China
3. Nanjing Xiaozhuang University, Nanjing 211171, China
Funds: the National Natural Science Foundation of China31571585
the Special Project for Basic Research of Jiangsu Academy of Agricultural SciencesZX[16]2002
the Grant from the Institute of Food Crops of Jiangsu Academy of Agriculture SciencesLZS17-9

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Corresponding author:LI Xia, E-mail:jspplx@jaas.ac.cn

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摘要
摘要:为了揭示高表达转玉米C₄-磷酸烯醇式丙酮酸羧化酶（PEPC，EC 4.1.1.31）基因水稻（PC）在耐旱中光合与花青素调节途径的内在联系，本研究以PC和未转基因野生型原种（WT）的水培苗为试验材料，在4~5叶期，通过50 μmol·L-1光合抑制剂DCMU[3-（3'，4'-dich-lorophenyl）-1，1-dimethyl-urea]预处理1 h，观察其在12% PEG-6000模拟干旱处理下的表现。结果表明，在模拟干旱条件下，DCMU预处理使两种供试材料相对含水量显著下降，且PC相对含量显著高于WT；干旱处理下，两种材料的花青素含量显著升高，DCMU和干旱处理使两种材料的花青素含量下调，且PC水稻中始终伴随着较高的花青素含量。光合数据表明，与单独12% PEG-6000处理相比，DCMU联合12% PEG-6000处理显著抑制了两种水稻材料的净光合速率、气孔导度、胞间CO₂含量及羧化效率，但PC的各指标显著高于WT。同时，DCMU联合12% PEG-6000处理显著下调两种供试材料的内源蔗糖含量，但PC中蔗糖含量显著高于WT。进一步研究发现PC中更高的蔗糖含量与花青素合成有关转录因子bHLH（OsB1，OsB2）、R2R3-MYB（OsC1）、COP1（constitutively photomorphogenic 1）、HY5（elongated hypocotyl 5）更高的转录水平同步，下游花青素合成相关基因OsPAL、OsCHI、OsCHS、OsF3H、OsF3'H、OsDFR、OsANS的表达量增加。PC水稻可能通过诱导NO和Ca²⁺感受干旱信号，参与转录因子的调节，进而参与花青素合成基因的调控，合成较多的花青素，增强PC水稻对干旱逆境的响应，增强保水能力，最终表现耐旱。
Abstract:Anthocyanins are important antioxidant materials that protects plant from damage by reactive oxygen species (ROS). Especially under adverse conditions, the regulation of sucrose in plants depends on its ability to induce anthocyanin accumulation. To determine the intrinsic relationship between photosynthetic and anthocyanin regulated pathways for C₄-phosphoenolpyruvate carboxylate (PEPC, EC 4.1.1.31) gene overexpressed rice (PC) in drought conditions, PC and untransformed wild-type (WT) were treated with 50 μmol·L-1 photosynthetic inhibitor DCMU for 1 h and the performance of the rice seedlings at 4-5 leaf stage observed under 12% PEG-6000 simulated drought. The results showed that DCMU pretreatment significantly reduced relative water contents of WT and PC under simulated 12% PEG-6000 drought condition, and relative water content of PC was significantly higher than that of WT. The anthocyanin content was higher in PC than in WT under 12% PEG-6000 simulated drought or drought plus DCMU pretreatment. 12% PEG-6000 simulated drought decreased anthocyanin contents of PC and WT, while DCMU pretreatment alleviated this effect. Compared with 12% PEG-6000, DCMU plus 12% PEG-6000 significantly inhibited net photosynthetic rate, stomatal conductance, intercellular CO₂ and carboxylation efficiency of the two rice lines, but these parameters of PC lines were significantly higher than those of WT lines. Then DCMU plus 12% PEG-6000 down-regulated endogenous sucrose content of the two materials, but sucrose content of PC lines was significantly higher than that of WT lines. Further studies showed that higher sucrose level in PC was associated with higher expression levels of transcriptional factors of bHLH (OsB1, OsB2), R2R3-MYB (OsC1), COP1 (constitutively photomorphogenic 1), HY5 (elongated hypocotyl 5), OsPAL, OsCHI, OsCHS, OsF3H, OsF3'H, OsDFR and OsANS, which resulted in synthesizing more anthocyanin to improve water retention capacity. In addition, PC rice sensed drought signals through NO and Ca²⁺, which participated in the regulation of transcription factors, regulation of anthocyanin synthesis gene, synthesis of more anthocyanin and thereby enhanced PC rice response to drought stress. This enhanced water retention capacity, stabilized photosynthetic capacity and resisted drought. Therefore, it was beneficial in molecular breeding of "C₄ Rice" to study the symphony between high yield and plant resistance.

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图1DCMU预处理对模拟干旱胁迫下高表达转C₄-pepc基因水稻(PC)和野生型水稻(WT)叶片相对含水量(A)和PEPC活性(B)的影响
CK:正常水培培养; PEG: PEG单独施加模拟干旱处理; DCMU+PEG: DCMU预处理之后联合模拟干旱胁迫处理。不同小写字母表示WT和PC的不同处理间差异显著(P＜0.05)。
Figure1.Effects of DCMU pretreatment on leaves relative water content (A) and PEPC activity (B) of C₄-pepc gene overexpressed rice (PC) and untransformed wild-type rice (WT) under simulated drought stress
CK: normal hydroponic culture; PEG: PEG simulated drought stress; DCMU+PEG: DCMU pretreatment plus simulated drought stress. Different lowercase letters indicate significant differences among different treatments of WT and PC at P < 0.05.


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图2DCMU预处理对模拟干旱胁迫下高表达转C₄-pepc基因水稻(PC)和野生型水稻(WT)叶片光合参数(A:净光合速率; B:气孔导度; C:胞间CO₂浓度; D:羧化效率)的影响
CK:正常水培培养; PEG: PEG单独施加模拟干旱处理; DCMU+PEG: DCMU预处理之后联合模拟干旱胁迫处理。不同小写字母表示WT和PC的不同处理间差异显著(P＜0.05, LSD test)。
Figure2.Effects of DCMU pretreatment on photosynthetic parameters (A: net photosynthetic rate; B: stomatal conductance; C: intercellular CO₂ concentration; D: carboxylation efficiency) of C₄-pepc gene overexpressed rice (PC) and untransformed wild-type rice (WT) leaves under simulated drought stress
CK: normal hydroponic culture; PEG: PEG simulated drought stress; DCMU+PEG: DCMU pretreatment plus simulated drought stress. Different lowercase letters indicate significant differences among different treatments of WT and PC at P < 0.05.


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图3DCMU预处理对模拟干旱胁迫下高表达转C₄-pepc基因水稻(PC)和野生型水稻(WT)叶片NO (A)、H₂O₂ (B)和Ca²⁺ (C)含量的影响
CK:正常水培培养; PEG: PEG单独施加模拟干旱处理; DCMU+PEG: DCMU预处理之后联合模拟干旱胁迫处理。不同小写字母表示WT和PC的不同处理间差异显著(P＜0.05, LSD test)。
Figure3.Effects of DCMU pretreatment on contents of NO (A), H₂O₂ (B) and Ca²⁺ (C) of C₄-pepc gene overexpressed rice (PC) and untransformed wild-type rice (WT) leaves under simulated drought stress
CK: normal hydroponic culture; PEG: PEG simulated drought stress; DCMU+PEG: DCMU pretreatment plus simulated drought stress. Different lowercase letters indicate significant differences among different treatments of WT and PC at P < 0.05.


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图4DCMU预处理对模拟干旱胁迫下高表达转C₄-pepc基因水稻(PC)和野生型水稻(WT)叶片可溶性糖(A)、蔗糖(B)、葡萄糖(C)和果糖(D)含量的影响
CK:正常水培培养; PEG: PEG单独施加模拟干旱处理; DCMU+PEG: DCMU预处理之后联合模拟干旱胁迫处理。不同小写字母表示WT和PC的不同处理间差异显著(P＜0.05)。
Figure4.Effects of DCMU pretreatment on contents of soluble sugar (A), sucrose (B), glucose (C) and fructose (D) of C₄-pepc gene overexpressed rice (PC) and untransformed wild-type rice (WT) leaves under simulated drought stresses
CK: normal hydroponic culture; PEG: PEG simulated drought stress; DCMU+PEG: DCMU pretreatment plus simulated drought stress. Different lowercase letters indicate significant differences among different treatments of WT and PC at P < 0.05.


下载: 全尺寸图片幻灯片


图5DCMU预处理对模拟干旱胁迫下高表达转C₄-pepc基因水稻(PC)和野生型水稻(WT)叶片花青素含量影响
CK:正常水培培养; PEG: PEG单独施加模拟干旱处理; DCMU+PEG: DCMU预处理之后联合模拟干旱胁迫处理。不同小写字母表示WT和PC的不同处理间差异显著(P＜0.05)。
Figure5.Effect of DCMU pretreatment on anthocyanin content of C₄-pepc gene overexpressed rice (PC) and untransformed wild-type rice (WT) leaves under simulated drought stress
CK: normal hydroponic culture; PEG: PEG simulated drought stress; DCMU+PEG: DCMU pretreatment plus simulated drought stress. Different lowercase letters indicate significant differences among different treatments of WT and PC at P < 0.05.


下载: 全尺寸图片幻灯片


图6DCMU预处理对模拟干旱胁迫下高表达转C₄-pepc基因水稻(PC)和野生型水稻(WT)叶片花青素合成酶基因表达的影响
CK:正常水培培养; PEG: PEG单独施加模拟干旱处理; DCMU+PEG: DCMU预处理之后联合模拟干旱胁迫处理。不同小写字母表示WT和PC的不同处理间差异显著(P＜0.05, LSD test)。
Figure6.Effects of DCMU pretreatment on expression of anthocyanin synthase genes of C₄-pepc gene overexpressed rice (PC) and untransformed wild-type rice (WT) leaves under simulated drought stress
CK: normal hydroponic culture; PEG: PEG simulated drought stress; DCMU+PEG: DCMU pretreatment plus simulated drought stress. Different lowercase letters indicate significant differences among different treatments of WT and PC at P < 0.05.


下载: 全尺寸图片幻灯片


图7DCMU预处理对模拟干旱胁迫下高表达转C₄-pepc基因水稻(PC)和野生型水稻(WT)叶片花青素调节因子的影响
CK:正常水培培养; PEG: PEG单独施加模拟干旱处理; DCMU+PEG: DCMU预处理之后联合模拟干旱胁迫处理。不同小写字母表示WT和PC的不同处理间差异显著(P＜0.05, LSD test)。
Figure7.Effect of DCMU pretreatment on regulators of anthocyanin of C₄-pepc gene overexpressed rice (PC) and untransformed wild-type rice (WT) leaves under simulated drought stress
CK: normal hydroponic culture; PEG: PEG simulated drought stress; DCMU+PEG: DCMU pretreatment plus simulated drought stress. Different lowercase letters indicate significant differences among different treatments of WT and PC at P < 0.05.


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表1QRT-PCR的基因和引物
Table1.Genes and primers for QRT-PCR

基因?Gene			引物序列?Primers sequence
基因描述 Gene description	引物名称 Primers name		正向引物 Forward primer (F)	反向引物 Reverse primer (R)
bHLH	ACTIN		CCCTCTTTCATCGGTATGGA	TTGATCTTCATGCTGCTTGG
	B1		GGATGGTCTCCTTGGACTGA	GGGTGGCAGATTCATCACTT
	B2		GTGGCAATAACGACGACGACTCC	CGTACGGTGTTGACGAGGTA
MYB	C1		CGGGTTCTTCTTCCACGAC	CCCGCAACTGCACTTAAAAT
Anthocyanin synthase	PAL		CAAGCTCATGACCTCCACCTA	GTTCATGGTGAGCACCTTCTT
gene	CHS		TCATGTATGGGTGGTTTGGTT	GCCAGGCATCTCTTACACAGG
	CHI		CGAGCAGTACTCGGACAAGG	TGAAGGCCTCCTTGAACTTG
	F3H		GAGCAATGGGAGGTTCAAGA	CTTCGATTTTCGACGGAAGA
	F3’H		CCGCTACAGTACCAGCCTTC	TGCCACCATTTCTAGAGTTCC
Transcription factor	DFR		CGGATGGATGTACTTCGTGTC	CATCCCGTTGCTGATGAAG
	ANS		CTCCTCCAGCTCAAGATCAAC	GTTGTGGAGGATGAAGGAGAG
	COP1		ATAATCCTGGGTCGAGCCAC	TATGGTGATCAGCAGAACCCAC
	HY5		GGCGGGTGCCGGAGATGG	CGCCGTCGTGTTCTTCTTGAGTATCTGG

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