宋凝曦^,1,2, 李霞^,1,2,3,*, 王净^1,2, 吴博晗^1,4, 曹悦^1,5, 杨杰^1,3, 谢寅峰²¹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, 江苏南京 210014
²南京林业大学生物与环境学院, 江苏南京 210037
³江苏省粮食作物现代产业技术协同创新中心, 江苏扬州 225009
⁴江苏大学环境与安全工程学院, 江苏镇江 212013
⁵南京农业大学生命科学学院, 江苏南京 210095

Effects on drought tolerance by pladienolide B and rice with high expression of C₄-PEPC

SONG Ni-Xi^,1,2, LI Xia^,1,2,3,*, WANG Jin^1,2, WU Bo-Han^1,4, CAO Yue^1,5, YANG Jie^1,3, XIE Yin-Feng²¹Institute 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, Jiangsu, China
²College of Biology and Environment, Nanjing Forestry University, Nanjing 210037, Jiangsu, China
³Collaborative Innovation Center for Modern Production Technology of Grain Crops of Jiangsu Province, Yangzhou 225009, Jiangsu, China
⁴School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang 212013, Jiangsu, China
⁵School of Life Sciences, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China

通讯作者: *李霞, E-mail:jspplx@jaas.ac.cn

收稿日期:2020-10-25接受日期:2021-03-19网络出版日期:2021-04-08

基金资助:

国家自然科学基金项目.31571585 国家重点研发计划项目.2016YFD0300501-03

Corresponding authors: *E-mail:jspplx@jaas.ac.cn
Received:2020-10-25Accepted:2021-03-19Published online:2021-04-08

Fund supported:

National Natural Science Foundation of China.31571585 National Key Research and Development Program of China.2016YFD0300501-03

作者简介 About authors
E-mail:362600124@qq.com



摘要
为揭示可变剪接机制参与植物耐旱性的内在机制, 以高表达转玉米C₄型磷酸烯醇式丙酮酸羧化酶(phosphoenolpyruvate carboxylase, PEPC)基因(C₄-PEPC)水稻(PC)和受体“Kitaake” (WT)为材料, 通过盆栽和水培试验, 研究外施可变剪接抑制剂大环内酯类(pladienolide B, PB)联合干旱处理下, 功能叶片的光合参数、总可溶性糖及其组分、主要抗氧化酶活性以及抗氧化物质含量、Ca²⁺、NO、H₂O₂、ABA含量、与蔗糖非发酵1 (sucrose nonfermenting-1, SNF1)相关蛋白激酶(SNF1-related protein kinase 3s, SnRK3s)以及剪接因子基因表达的变化。结果表明: 在盆栽试验中, 与单独自然干旱处理(drought stress, DS)相比, 在孕穗期外施0.5 µmol L^-1 PB联合干旱处理(DS+PB), 显著下降了供试水稻的产量及其构成因子的数值, 其中, 在DS+PB处理下, PC的株高、穗数、每穗实粒重和单株产量均显著高于WT。在水培试验中, 与10% PEG-6000模拟干旱处理(PEG)相比, 外施0.5 µmol L ^-1 PB和10% PEG-6000模拟干旱处理(PEG+PB), 均显著降低了供试水稻功能叶片的净光合速率(P_n)、相对含水量、总可溶性糖含量、脯氨酸含量、SOD酶活性、POD酶活性、CAT酶活性、PEPC酶活性、糖组分(蔗糖、葡萄糖、果糖含量)、Ca²⁺、NO和H₂O₂的含量, 其中, PC中3个糖组分以及钙离子含量始终高于WT。与PEG处理相比, PEG+PB处理也导致水稻叶片内糖信号SnRK2s基因(SAPK8和SAPK9)和3个SnRK1基因表达量下降, 其中PC的SAPK8、SAPK10、OsK1a和OsK24表达均高于WT; 进一步10 mmol L^-1 EGTA钙离子螯合剂引入实验证明, 钙离子通过调节剪接因子相关基因的表达参与水稻干旱响应。相关性分析也表明, PC中钙离子含量分别与叶片可溶性蛋白含量、ABA含量以及剪接因子RS33的表达水平呈显著或极显著相关。综上, 可变剪接参与水稻干旱响应, 水稻可通过叶内糖信号SnRK1s和SnRK2s基因以及钙离子, 参与调节剪接因子相关基因的表达, 对水稻耐旱起积极的作用。与WT相比, PC的增益效果更强, 这与其内源高钙离子和糖组分含量密切相关。
关键词： 水稻;可变剪接;磷酸烯醇式丙酮酸羧化酶;大环内酯;干旱

Abstract
To investigate the intrinsic mechanism of alternative splicing (AS) participated in drought tolerance in plants, the effects of macrolides pladienolide B (PB, one of the AS inhibitors) were studied using the phosphoenolpyruvate carboxylase (C₄-PEPC) rice (PC) and “Kitaake” (WT) rice lines in pot experiments and hydroponics experiments, respectively. The changes of photosynthetic parameters, total soluble sugar and sugar components contents, some main antioxidant enzyme activities and antioxidant contents, Ca²⁺, NO, H₂O₂, ABA contents, transcript levels of sucrose nonfermenting-1 (SNF1)-related protein kinases (SnRKs), arginine/serine-rich proteins (SR proteins), and PEPC both in C₄ and C₃ type of the functional leaves in rice lines were measured. Agronomic traits of the WT and PC were recorded in the mature period. In pot experiment, compared with the natural drought treatment alone, the treatment of 0.5 µmol L^-1 PB with drought at booting stage had a significant decline on agronomic traits of the tested rice. Among them, plant height, panicle number per plant, filled grain number per panicle, and grain yield per plant in PC were significantly higher than those of WT. In the hydroponics experiment with 0.5 µmol L ^-1 PB combined with 10% mmol L^-1 polyethylene glycol 6000 (PEG-6000) to simulate drought stress, compared with PEG treatment, net photosynthetic rate, relative water content, total soluble sugar content, proline content, SOD activity, POD activity, CAT activity, and PEPC activity were significantly increased in PC than those of WT. Similarly, the contents of sucrose, glucose, fructose, and Ca²⁺ of leaves in PC lines were significantly higher than those of WT. It was noteworthy that the relative gene expression levels of C₄-PEPE, Osppc2a, SnRK2s (SAPK8 and SAPK9), SnRK1s (OsK1a, OsK24, and OsK35), and SR proteins (SR33, SR40, RS29, RS2Z21, and RS2Z38) under PEG+PB treatment were significantly lower than those under PEG treatment. It was further verified by using 10 mmol L^-1 EGTA experiments [Ethylene glycolbis (aminoethylether)-tetra-acetic acid, a chelate solution of calcium ions] that Ca²⁺ were involved in the drought response by regulating the transcript levels of SR proteins genes in rice. In addition, the Ca²⁺ content in PC lines was significantly correlated with soluble protein content, ABA content, and RS33 transcript level, respectively. In conclusion, AS participated in drought response by regulating the expression of SR related genes through sugar signal both SnRK1s and SnRK2s, and calcium ion as well, which played a positive role in drought tolerance in rice. Compared with WT, PC had a stronger positive effect, which was closely related to its high endogenous Ca ²⁺ content and the contents of sucrose, glucose, fructose.
Keywords：rice;alternative splicing;phosphate phosphoenolpyruvate carboxylase;macrolides pladienolide B;drought


PDF (2605KB)元数据多维度评价相关文章导出EndNote|Ris|Bibtex收藏本文
本文引用格式
宋凝曦, 李霞, 王净, 吴博晗, 曹悦, 杨杰, 谢寅峰. 大环内酯类和高表达玉米C₄-PEPC基因对水稻耐旱性的影响[J]. 作物学报, 2021, 47(10): 1927-1940 DOI:10.3724/SP.J.1006.2021.03064
SONG Ni-Xi, LI Xia, WANG Jin, WU Bo-Han, CAO Yue, YANG Jie, XIE Yin-Feng. Effects on drought tolerance by pladienolide B and rice with high expression of C₄-PEPC[J]. Acta Agronomica Sinica, 2021, 47(10): 1927-1940 DOI:10.3724/SP.J.1006.2021.03064


水稻作为世界上最重要的粮食作物之一, 是全世界一半以上人口的主粮^[1]。近年来高温和干旱等极端天气频发, 水稻受到不利影响而减产^[2]。在非生物胁迫中, 干旱是影响全球农业最重要的环境因素^[3]。然而, 耐旱性是一个复杂的性状, 涉及一系列生理、形态、细胞和分子适应途径, 导致世界范围内抗旱性遗传改良的进展缓慢^[4,5]。在低浓度CO₂、高温、强光以及干旱等逆境条件下, C₄植物具有较高的水分、氮素利用和光合效率以及较高的生物学产量^[6]。并已通过基因工程将C₄光合基因如磷酸烯醇式丙酮酸羧化酶(phosphoenolpyruvate carboxylase, PEPC)基因(C₄-PEPC)引入C₃植物, 获得了高表达玉米C₄型PEPC转基因作物, 提高了其光合效率和产量^[7,8,9,10], 耐旱性也得到改善^{[11,12,13,14,15]}。与未转基因的野生型水稻(以下简称WT)相比, 高表达玉米C₄-PEPC转基因水稻(以下简称PC)在干旱条件下表现更强的光合优势^{[11,12,13,14,15]}、更多的有效穗数、千粒重以及产量^[15,17-18], 因此, 通过增强C₃植物的C₄光合特性作为一项重要的提高作物耐旱性改良技术途径, 已被广泛重视^[19]。

磷酸烯醇式丙酮酸羧化酶(PEPC)是重要的胞质代谢酶, 也是C₄植物和CAM植物光合作用的关键酶, 并在非光合组织中发挥重要的作用^[20]。已有研究表明, 磷脂酸^[21]、H₂O₂^[17]、NO^[22]、钙离子^[15,18]以及ATP^[23]等第二信使分子, 均参与调节了PC水稻C₄-PEPC的表达和酶活性的发挥, 通过调节气孔运动, 参与干旱响应, 而且这些调节过程与其内源糖水平的差异密切相关^[24]。与WT相比, PC的内源糖代谢活跃^[25]。通过葡萄糖激活糖信号-蔗糖非发酵1 (sucrosenonfermenting-1, SNF1)相关蛋白激酶(sucrosenonfermenting-1-related protein kinase,SnRKs)家族中SnRK3s家族基因, 并与B类钙调磷酸酶(calcineurin-B-like, CBL)作用诱导NO参与叶片气孔调节, 从而增强叶片的保水能力^[26]。PC还通过蔗糖参与SnRK2糖信号调节, 增强花青素调节因子和合成相关基因的表达水平, 增加了花青素代谢以快速响应干旱^[27], 综上, PC存在多种对干旱逆境响应和适应的策略, 但其内在的生理调节机制还需要深入研究。

表观遗传机制调控植物应答外界胁迫是近年来发现的重要机制之一, 它是DNA序列不改变的前提下, 通过基因转录、基因转录后以及蛋白翻译水平上参与植物逆境响应^[28], 其中可变剪接(alternative splicing, AS)是一种生物体基因转录后调控的表观遗传方式^[29]。在植物中的许多基因均被发现存在AS, 并广泛参与对非生物胁迫逆境的转录调控, 为植物提供了一条迅速应对环境变化的有效调控方案^[30]。磷酸化蛋白组学研究已鉴定了糖信号SnRK2的底物蛋白包括RNA剪接相关的重要蛋白质^[31]。可变剪接的抑制剂大环内酯类(pladienolide B, PB)可以模拟非生物胁迫信号, 包括盐、干旱和脱落酸(abscisic acid, ABA), 激活拟南芥中的非生物胁迫和ABA响应基因RD29A和MAPK18, 并诱导了SnRK2s相关酶基因的表达水平, 通过ABA参与了对拟南芥气孔孔径的调节, 抑制拟南芥的可变剪接过程^[32]。PC水稻在响应干旱逆境时C₄-PEPC的启动子有去甲基化现象发生, 增强了C₄-PEPC的表达^[18]。已有证据表明DNA甲基化可调节可变剪接^[33], 那么可变剪接是否参与PC水稻的干旱响应, 这是值得深入研究的一个科学问题。

本研究通过引用可变剪接的抑制剂PB, 联合自然干旱和PEG模拟干旱处理, 分别通过盆栽和水培试验, 在水稻水分敏感时期——孕穗期至齐穗期, 进行自然干旱和PB联合处理, 在收获期考种, 分析可变剪接机制对其产量的影响; 同时, 通过水培实验, 在四叶一心期, 通过PEG-6000模拟干旱和PB联合, 测定其倒二叶的光合参数, 并分析叶片中相对含水量、总可溶性糖及其组分、Ca²⁺、NO、H₂O₂、ABA含量、SnRKs亚家族基因、剪接因子基因表达、抗氧化酶以及非酶促的抗氧化分子的变化, 探究可变剪接机制在水稻应对干旱响应的作用。本研究旨在丰富可变剪接机制参与水稻耐旱的内在生理机制, 并为通过表观遗传机制改良水稻耐旱性提供新依据。

1 材料与方法

1.1 试验材料

所用水稻(Oryza sativa)为高表达玉米C₄-PEPC转基因水稻(以下简称PC), 其由Ku等^[7]以日本粳稻Kitaake为受体, 将完整的玉米C₄-PEPC基因导入水稻创制; 未转基因野生型水稻Kitaake (以下简称WT)为对照。选择连续种植20代的同年收获的大小一致、颗粒饱满的供试种子, 经过75%乙醇消毒15 min, 用清水冲洗5次。然后将种子放入黑暗30°C培养箱中, 浸种催芽3 d后, 待幼苗长到二叶期时, 转移至国际水稻研究所标准水稻培养营养液^[34]中, 置人工气候箱以30℃/25℃ (昼/夜)、14 h/10 h (光/暗)培养。待秧苗长到四叶一心时, 选择株型、长势和叶片均一的植株, 于晴天的傍晚进行处理, 并统一测定生理指标。

1.2 试验设计

1.2.1 水培试验处理 参照张金飞等^[26]方法, 将植株分为4组, 在晴天的傍晚叶面喷施0.5 µmol L^-1 PB溶液、0.5 µmol L^-1 PB +10 mmol L^-1 EGTA (钙离子螯合剂)溶液, 以清水喷施作为对照, 处理后转移到30℃/25℃ (昼/夜)人工气候箱中先暗处理10 h, 再光照处理2 h, 随后测定植株处理前倒二叶的光合参数。之后将植株转移到含有10% PEG-6000培养液中(PEG模拟干旱胁迫, 简称PEG处理; 单独PEG处理, 简称PEG; PB喷施联合PEG处理, 简称PEG+PB; PB和EGTA喷施并联合PEG处理, 简称PEG+PB+EGTA), 在光照处理2 h后, 再测定植株处理后倒二叶的光合参数。之后取下不同处理的倒二叶, 迅速在-80℃液氮中保存, 用于测定其他生理指标。同时测定处理前后叶片的相对含水量(relative water content, RWC), 在苗期以RWC 作为耐旱性指标。

1.2.2 盆栽试验处理 于2019年4月25日至9月10日在江苏省农业科学院专用网室中进行。盆栽用土为稻田黏壤土, 盆钵随机区组排放, 进行常规水肥和病虫管理。在孕穗期, 待盆钵土中的水自然落干后, 对水稻叶片喷施0.5 µmol L^-1 PB溶液, 以喷清水为对照。一组为正常灌溉处理(对照, CK), 另一组自然干旱处理(drought stress, DS)。外源PB处理是在水稻开花前5 d开始处理, 再将如上每个处理随机分成2组, 水稻植株每5 d喷施0.5 µmol L^-1 PB溶液1次, 共处理5次, 每株共喷施50 mL溶液(CK+PB和DS+PB处理), 另外2组水稻植株喷施同体积的清水(CK和DS处理)。5次处理结束后, 所有处理均恢复正常灌溉, 开花后50 d收获, 考察产量及其构成因子, 以单株产量作为耐旱性指标。

1.3 测定方法

1.3.1 光合参数 参照Li等^[21]方法, 使用LI-6400 (Li-Cor, Lincoln, NE, USA)便携式光合作用测定系统, 采用红蓝光源叶室(LI-6400-40), 设置光量子通量密度(photosynthetic photon quanta flux density, PPFD) 1000 μmol m^-2 s^-1, 流速500 μmol s^-1。室外温度28~30℃, 相对湿度68%~79%, CO₂浓度为(390.0±10.5) μmol mol^-1, 上午9:00—10:00, 选取水稻倒二叶测定净光合速率(net photosynthesis rate, P_n)、气孔导度(stomatal conductance, G_s)和胞间CO₂浓度(intercellular CO₂ concentration, C_i)等, 通过CE=P_n/C_i, 计算出羧化效率(carboxylation efficiency, CE)。每个处理测定5张叶片, 室外进行, 每个处理5次重复。

1.3.2 相对含水量的测定 参照Smart等^[35]的方法测定。

1.3.3 可溶性总糖、糖组分以及可溶性蛋白 参考蒽酮比色法^[36]测定植株叶片可溶性总糖; 参照文献中方法^[37]测定蔗糖、葡萄糖和果糖; 参照考马斯亮蓝G-250法^[38]测定可溶性蛋白。

1.3.4 抗氧化酶活性及其抗氧化物质 总SOD活性测定根据Giannopolitis等^[39]方法测定; POD活性根据Smith等^[40]方法测定; CAT活性根据Havir等^[41]方法测定; 脯氨酸含量参考Walter等^[42]方法测定; 谷胱甘肽参照Wang等^[43]方法测定; 抗坏血酸参照Doulis等^[44]方法测定。

1.3.5 Ca²⁺、NO、H₂O₂和ABA含量测定 叶片总Ca²⁺含量参照Yang等^[45]方法测定; NO参照Murphy与Noack^[46]方法测定; H₂O₂参照Ren等^[17]方法测定; ABA含量的测定参照Xiong等^[47]方法测定。

1.3.6 总RNA提取和qRT-PCR 总RNA的提取和实时荧光定量聚合酶链式反应(quantitative real-time PCR, qRT-PCR)参照Jung等^[48]方法制备, 参考Chen等^[22]方法反转录, Real-time PCR采用TB Green Premix Ex Taq TliRNaseH Plus试剂盒进行。qRT-PCR反应条件为95℃ 10 min; 94℃ 30 s, 58℃ 40 s和68℃各1 min, 共32次循环, 重复3次。采用Primer 3设计引物, 以水稻组成性表达的Actin基因作为内部参照, 引物序列见表1, 所有引物均由生工生物工程(上海)有限公司合成。

Table 1
表1
表1qRT-PCR的基因和引物
Table 1Genes and primers for qRT-PCR

基因名称 Gene name	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)	基因登录号 Gene accession number
Act	CCCTCAAACATCGGTATGGA	TTGATCTTCATGCTGCTTGG	Os01g4349863
OsK1a	AACCAGAGGTAACAGGCAGG	AACCAGAGGTAACAGGCAGG	Os05g4433941
OsK24	CGTGTTGGCTTCAGTGAAT	CCTTCTCTATCTAAGGGCCG	Os08g4349863
OsK35	TTGTGTTGGCTTCAGTGAAA	CCTTCGCTGTCTAAGGACTG	Os03g4332495
C4-PEPC	CCCACTATCCTTCGCAAGAC	CTAGCCAGTGTTCTGCATGCCGG	E17154
Osppc2a	CTGGTTGAGATGGTTTTCGC	GGTGTGAATTCAGGCACTTC	Os01g55350
SAPK8	ATAGATGATAATGTCCAGCG	GTTCCTACAGTGGATTTTGG	Os03g0764800
SAPK9	CACAGCAACGCCGTCTCC	CACACTTCCACCGCTACCAA	Os12g0586100
SAPK10	TGCTGATGTGTGGTCGTGTG	TGCTGGTATGGTCGCCTCT	Os03g0610900
RS29	GGGATCTTTGATTTGCTGCGA	CAATTTATGTGTTCCACGCCG	Os04g02870
RS33	ACTCCCGGTAAGCACATGAC	GGGGCTAGCATGTTTCACTG	Os02g03040
SR40	CAATCTGGGGACTGCTTTC	TCCTGCTTGGGCTTTTACT	Os12g38430
SR33	ATATTGCCTGCTACCCGAAAG	CAGAGCAGCACCCAGTTTATTAC	Os07g47630
SCL25	AAAGTGCACTCTGCGAACTCTCT	GCGGTTCACTGAAAAGGACAA	Os07g43950
SCL26	GAAGAGAAAATGAACCAGACCG	AAACCCTAGCGAAAAGGAAATC	Os03g24890
SCL30	CACGCTGATATGTGGGTCTCAT	CCTCCCAACGGAAATCTCTAAC	Os12g38430
SCL57	TCCCACACCGATTTATCTTTTT	CATGTTCTTTAGCCTGTCCTGA	Os11g47830
RSZ21	ATGGTCATTGACAAGTGTGCG	CGATAGATCTAATCCCGCAAGG	Os06g08840
RS2Z36	TGTCAAAACAGCCCAAGAAATC	CCATCCATCGTACACCAATAGC	Os05g02880

Act: 肌动蛋白; SnRK: 蔗糖非发酵相关蛋白激酶; OsK1a OsK24 OsK35: SnRK1s亚家族基因; SAPK8-10: SnRK2s亚家族基因; C₄-PEPC: C₄型磷酸烯醇式丙酮酸羧化酶基因; Osppc2a: C₃型磷酸烯醇式丙酮酸羧化酶基因; RS SR SCL RSZ: 丝氨酸/精氨酸富集蛋白基因。
Act: actin; SnRK: sucrose nonfermenting-1-related protein kinase; OsK1a OsK24 OsK35: gene of SnRK1s; SAPK8-10: gene of SnRK2s; C₄-PEPC: the gene of C₄ phosphoenolpyruvate carboxylase; Osppc2a: the gene of C₃ phosphoenolpyruvate carboxylase; RS SR SCL RSZ: genes of serine/arginine-rich protein.

新窗口打开|下载CSV

1.4 统计与分析方法

使用SPSS 25.0软件对数据进行统计分析, 采用TB tool软件对数据进行作图及相关性分析, 使用2^-ΔΔCt方法分析qRT-PCR数据。

2 结果与分析

2.1 孕穗期干旱胁迫下喷施PB对水稻产量及其构成因子的影响

如表2所示, 与CK相比, 单独PB处理, 对水稻产量构成因子没有产生显著影响; 但与CK相比, 在单独自然干旱(DS)处理下, 供试材料的产量构成因子均降低, 但PC的株高(79.03 cm/90.14%)、穗长(11.04 cm/82.70%)、每穗实粒数(24.58个/73.42%)、千粒重(23.38 g/90.83%)以及单株产量(2.83 g/ 68.52%)的下降程度均小于WT (株高: 72.35 cm/82.80%; 穗长: 10.75 cm/92.12%; 每穗实粒数: 21.38个/68.88%; 千粒重: 20.74 g/81.89%; 单株产量: 2.06 g/53.93%)。而与DS处理相比, DS+PB处理使供试材料产量构成因子的降低得更多, PC主要产量构成因子的下降程度小于WT, 如PC的千粒重 (19.95 g/77.51%)、干物质重(叶: 2.45 g/65.16%和穗: 2.47 g/36.48%)以及单株产量(2.54 g/61.50%)均显著高WT的(千粒重: 17.87 g/70.63%; 叶重: 2.03 g/57.02%; 穗重: 2.14 g/34.57%; 单株产量: 1.71 g/44.76%), 提示增强可变剪接水平可显著缓解孕穗期干旱胁迫对水稻产量造成的损失, 而且对PC的效果更显著。

Table 2
表2
表2孕穗期外施PB联合干旱处理对水稻产量及其构成因子的影响
Table 2Effects of spraying PB at booting stage combined with drought treatment on agronomic traits in rice lines

性状 Trait	转玉米C4-PEPC水稻 C4-PEPC rice (PC)				Kitaake (WT)
	CK	CK+PB	DS	DS+PB	CK	CK+PB	DS	DS+PB
株高 Plant height (cm)	87.67 a 100.00%	84.32 a 96.18%	79.03 b 90.14%	67.83 c 77.37%	87.38 a 100.00%	85.33 a 97.65%	72.35 bc 82.80%	52.62 d 60.22%
最大分蘖数 The largest tiller number per plant	10.73 b 100.00%	9.79 b 91.24%	8.66 c 80.71%	8.29 c 77.26%	13.97 a 100.00%	12.22 a 87.47%	8.23 c 58.91%	7.19 d 51.47%
每株总穗数 Panicle number per plant	9.33 b 100.00%	9.18 b 98.39%	6.33 c 67.85%	5.74 d 61.52%	11.04 a 100.00%	10.57 ab 95.74%	6.29 c 56.97%	5.33 d 48.28%
穗长 Panicle length (cm)	13.35 a 100.00%	12.47 a 93.41%	11.04 b 82.70%	8.63 d 64.64%	11.67 ab 100.00%	11.40 ab 97.69%	10.75 c 92.12%	7.67 d 65.72%
每穗总粒数 Total grain number per panicle	37.61 a 100.00%	38.47 a 102.29%	33.63 b 89.42%	28.58 c 75.99%	37.33 a 100.00%	35.87 a 96.09%	28.83 c 77.23%	27.33 c 73.21%
每穗实粒数 Filled grain number per panicle	33.48 ab 100.00%	32.87 ab 98.18%	24.58 c 73.42%	19.33 d 57.74%	31.04 b 100.00%	30.12 b 97.04%	21.38 d 68.88%	18.65 d 60.08%
结实率 Gain filling ratio (%)	89.34 a 100.00%	85.44 a 95.63%	80.24 c 89.81%	69.14 e 77.39%	83.24 bc 100.00%	83.97 b 100.88%	74.01 d 88.91%	68.32 e 82.08%
千粒重 1000-grain weight (g)	25.74 a 100.00%	24.87 a 96.62%	23.38 b 90.83%	19.95 c 77.51%	25.30 a 100.00%	23.84 ab 94.23%	20.74 c 81.98%	17.87 d 70.63%
每株叶干重 Leaf weight per plant (g)	3.76 a 100.00%	3.87 a 102.93%	2.53 c 67.29%	2.45 c 65.16%	3.56 b 100.00%	3.47 b 97.47%	2.29 c 64.33%	2.03 d 57.02%
每株茎干重 Stem weight per plant (g)	2.93 a 100.00%	2.85 a 97.27%	1.23 b 41.98%	0.73 c 24.91%	2.59 a 100.00%	2.44 a 94.21%	1.51 b 58.30%	0.81 c 31.27%
每株穗干重 Panicle weight per plant (g)	6.77 a 100.00%	6.59 a 97.34%	4.95 c 73.12%	2.47 e 36.48%	6.19 b 100.00%	5.93 b 95.80%	4.12 d 66.56%	2.14 f 34.57%
总干重 Total dry weight per plant (g)	13.46 a 100.00%	13.31 a 98.89%	8.71 c 64.71%	5.65 d 41.98%	12.34 b 100.00%	11.84 b 95.95%	7.52 c 60.94%	4.98 e 40.36%
单株产量 Yield per plant (g)	4.13 a 100.00%	4.05 a 98.06%	2.83 c 68.52%	2.54 d 61.50%	3.82 b 100.00%	3.71 b 97.12%	2.06 e 53.93%	1.71 f 44.76%

CK: 正常灌溉和叶面喷施清水; CK+PB: 正常灌溉和叶面喷施0.5 µmol L^-1 PB溶液; DS: 干旱胁迫+叶面喷施清水; DS+PB: 干旱胁迫+叶面喷施0.5 µmol L^-1 PB溶液。不同的小写字母表示在5%水平差异显著, 每个参数下面的百分比是同一个株系处理数据和清水对照(CK)比值的百分数。
CK: irrigation and leaf spray application of water; CK+PB: irrigation and leaf spray application of 0.5 µmol L^-1 PB; DS: drought stress and leaf spray application of water; DS+PB: drought stress and leaf spray application of 0.5 µmol L^-1 PB. Values followed by different lowercase letters are significantly different at the 5% probability level. Values below each parameter are the percentage of the ratio between the treated data and that of the water control (CK) in the same line in rice.

新窗口打开|下载CSV

2.2 喷施PB联合10%PEG-6000模拟干旱胁迫对水稻叶片光合参数的影响

如表3所示, 与CK相比, 在PEG模拟干旱处理下, PC和WT的净光合速率(P_n)、气孔导度(G_s)、胞间CO₂浓度(C_i)、蒸腾速率(T_r)和羧化效率(CE)均降低, 但该处理下PC的P_n (16.73 μmol CO₂ m^-2 s^-1/83.03%)、G_s (0.45 mol m^-2 s^-1/61.64%)、C_i (345.18 μmol mol^-1/94.26%)和CE (0.048 mol m^-2 s^-1/76.19%)始终高于WT (P_n: 13.46 μmol CO₂ m^-2 s^-1/73.87%; G_s: 0.33 mol m^-2 s^-1/44.00%; C_i: 343.04 μmol mol^-1/92.97%; CE: 0.039 mol m^-2 s^-1/76.47%); 与PEG处理相比, PEG+PB处理进一步加重了干旱对水稻的P_n (PC: 15.60 μmol CO₂ m^-2 s^-1/77.46%; WT: 10.68 μmol CO₂ m^-2 s^-1/58.62%)的抑制, 但是G_s、C_i和T_r则差异不显著, WT的CE (0.032 mol m^-2 s^-1/62.75%)与其P_n的变化类似, 显著低于其PEG处理的, 而PC (CE: 0.046 mol m^-2 s^-1/73.01%)的则与其PEG处理的没有显著差异。值得关注的是, 在PEG+PB处理下, PC的P_n、G_s和CE始终高于同样处理下WT, 说明外施PB显著地加重了干旱胁迫导致的水稻光合能力下降, 反证增强可变剪接水平可以缓解干旱胁迫导致的水稻光合能力下降, 尤其对PC有益。

Table 3
表3
表3喷施PB联合10% PEG-6000模拟干旱处理对水稻叶片光合参数的变化
Table 3Changes of photosynthetic parameters in rice leaves when spraying PB combined with 10% PEG-6000 treatment

光合参数 Photosynthetic parameters	转玉米C4-PEPC水稻 C4-PEPC rice (PC)			Kitaake (WT)
	CK	PEG	PEG+PB	CK	PEG	PEG+PB
净光合速率 Net photosynthetic rate Pn (μmoL CO2 m-2 s-1)	20.14 a 100.00%	16.73 c 83.07%	15.60 d 77.46%	18.22 b 100.00%	13.46 c 73.87%	10.68 f 58.62%
气孔导度 Stomatal conductance Gs (mol m-2 s-1)	0.73 a 100.00%	0.45 b 61.64%	0.46 b 63.01%	0.75 a 100.00%	0.33 c 44.00%	0.31 c 41.33%
胞间CO2浓度 Intercellular CO2 concentration Ci (μmol mol-1)	366.57 a 100.00%	345.18 ab 94.16%	337.18 b 91.98%	368.96 a 100.00%	343.04 b 92.97%	354.09 ab 95.97%
蒸腾速率 Transpiration rate Tr (mol H2O m-2 s-1)	9.06 a 100.00%	6.31 c 69.65%	6.24 c 68.87%	9.46 a 100.00%	7.14 b 75.48%	7.34 b 77.59%
羧化效率 Carboxylation efficiency CE (mol m-2 s-1)	0.063 a 100.00%	0.048 c 76.19%	0.046 c 73.02%	0.051 b 100.00%	0.039 d 76.47%	0.032 e 62.75%

CK: 培养液和叶面喷施清水; PEG: 10% PEG培养液和叶面喷施清水; PEG+PB: 10% PEG水稻培养液和叶面喷施0.5 µmol L^-1 PB溶液。不同的小写字母表示在5%水平差异显著。每个参数下面的百分比与表2的一致。
CK: rice culture solution and leaf spray application of water; PEG: 10% PEG rice culture solution and leaf spray application of water; PEG+PB: 10% PEG rice culture solution and leaf spray application of 0.5 µmol L^-1 PB. Values labeled with different lowercase letters are significantly different at the 5% probability level. The percentages below each parameter are consistent with those in Table 2.

新窗口打开|下载CSV

2.3 喷施PB联合10% PEG-6000模拟干旱胁迫对水稻叶片相对含水量、渗透调节物质含量、抗氧化酶活性以及PEPC酶活性的影响

由表4看出, 与CK相比, PEG处理显著降低了PC和WT叶片的相对含水量和可溶性蛋白, 而渗透调节物质可溶性总糖和脯氨酸的含量则诱导增加; 而与PEG处理相比, PEG+PB的4个参数均显著下降, 并且在PEG和PEG+PB处理下, PC的相对含水量、可溶性蛋白、可溶性总糖以及脯氨酸含量都显著高于WT。不同处理下水稻叶片超氧化物歧化酶(superoxide dismutase, SOD)、过氧化物酶(peroxidase, POD)和过氧化氢酶(catalase, CAT)活性也发生了变化。同样地, PEG处理下PC和WT叶片SOD和POD活性均上升; PEG+PB处理下PC和WT的SOD、POD和CAT酶活性均下降; 另外, PEG处理下PC和WT的抗氧化物质抗坏血酸(ascorbic acid, AsA)含量均上升; 而PEG+PB处理则下降了AsA和谷胱甘肽(glutathione, GSH)含量下降, 同步地, PEPC酶活性也显示了与可溶性总糖等渗透调节物质类似的变化, 说明PC因C₄-PEPC的导入使其耐旱性更强, 表现为渗透调节物质积累和抗氧化防护能力的增强, 而且与可变剪接水平有关。

Table 4
表4
表4不同处理对水稻叶片相对含水量、渗透调节物质、抗氧化酶活性以及PEPC酶活性的影响
Table 4Effects of different treatments on the content of relative water content, osmotic regulation components, antioxidant enzyme activity, and PEPC activities in rice

生理指标 Physiological indexes	转玉米C4-PEPC水稻 C4-PEPC rice (PC)			Kitaake (WT)
	CK	PEG	PEG+PB	CK	PEG		PEG+PB
相对含水量 Relative water content (%)	88.43 a 100.00%	78.43 b 88.69%	73.47 c 83.08%	88.96 a 100.00%	69.17 d 77.75%	61.42 e 69.04%
总的可溶性蛋白含量 Total soluble protein content (mg g-1)	14.25 a 100.00%	12.92 b 90.67%	9.92 d 69.61%	14.01 a 100.00%	11.72 c 83.65%	8.02 e 57.24%
总的可溶性糖含量 Total soluble sugar content (mg g-1)	21.23 c 100.00%	28.33 a 133.44%	12.41 e 58.46%	19.02 d 100.00%	23.04 b 121.14%	9.59 f 50.42%
脯氨酸含量 Proline content (mg g-1)	63.38 c 100.00%	75.49 a 119.11%	41.85 d 66.03%	64.15 c 100.00%	68.04 b 106.06%	38.03 e 59.28%
超氧化物歧化酶活性 Superoxide dismutase activity (U g-1)	56.78 b 100.00%	84.54 a 148.89%	33.56 d 59.11%	51.74 c 100.00%	56.53 b 109.26%	29.78 e 57.56%
过氧化物酶活性 Peroxidase activity (U g-1)	65.46 c 100.00%	89.44 a 136.63%	32.73 e 50.01%	59.07 d 100.00%	76.64 b 129.74%	29.53 f 49.99%
过氧化氢酶活性 Catalase activity (U g-1)	176.27 a 100.00%	137.32 c 77.90%	126.27 d 71.63%	160.34 b 100.00%	130.56 d 81.42%	96.11 e 59.94%
抗坏血酸含量 Ascorbic acid content (µg g-1)	11.38 c 100.00%	16.19 a 142.27%	7.07 d 62.13%	12.43 c 100.00%	14.88 b 119.71%	6.72 d 54.06%
谷胱甘肽含量 Glutathione content (µmol L-1)	22.31 a 100.00%	15.52 c 69.57%	11.09 e 49.71%	20.31 b 100.00%	13.21 d 65.04%	10.52 e 51.80%
磷酸烯醇式丙酮酸羧化酶活性 PEPC activity (U mg-1 prot)	60.54 c 100.00%	90.97 a 150.26%	68.32 b 112.85%	20.03 e 100.00%	29.44 d 146.98%	21.14 e 105.54%

CK、PEG和PEG+PB处理与表3的相同。不同小写字母表示在5%水平差异显著, 每个参数下面的百分比与表2的一致。
CK, PEG, and PEG+PB treatments are the same as those in Table 3. Different lowercase letters indicate significant differences at the 5% probability level. The percentages below each parameter are consistent with those in Table 2.

新窗口打开|下载CSV

2.4 喷施PB联合10% PEG-6000模拟干旱胁迫对水稻叶片可溶性糖组分以及Ca²⁺、NO、H₂O₂含量的影响

由表5可知, 与CK相比, 单独PEG处理显著提高了PC的蔗糖和葡萄糖含量, 而PEG+PB则显著下降了2个供试材料的蔗糖和葡萄糖含量; 而果糖含量则不论是PEG处理还是PEG+PB处理均导致其含量下降。值得注意的是, PEG+PB处理下, PC的3个糖组分含量始终高于WT; 与CK相比, 单独PEG处理上调了水稻叶片内的钙离子浓度水平, 而PEG+PB处理则下调了其含量, 且PC始终高于WT, 它的变化趋势与糖组分含量的变化一致; 单独PEG处理则下调2个供试材料的NO含量, 而且PEG+PB处理则进一步下调, 但是2个供试材料的变化则没有差异; 与CK相比, PEG处理只上调了WT的H₂O₂含量, 而对PC则不影响。与PEG处理相比, PEG+PB处理也同时下调了H₂O₂含量, 但是2个材料则无显著差异。看来, 可变剪接机制参与了水稻干旱响应, 其中PC和WT的差异可能与蔗糖、葡萄糖和钙离子含量等信号关系更大。

Table 5
表5
表5不同处理对水稻叶片蔗糖、葡萄糖、果糖、钙离子、NO和H₂O₂含量的影响
Table 5Effects of different treatments on the contents of sucrose, glucose, fructose, Ca²⁺, NO, and H₂O₂ in rice leaves

生理指标 Physiological indexes	转玉米C4-PEPC水稻 C4-PEPC rice (PC)			Kitaake (WT)
	CK	PEG	PEG+PB	CK	PEG	PEG+PB
蔗糖含量 Sucrose content (mg g-1)	9.67 c 100.00%	11.92 a 123.27%	5.15 d 53.26%	10.19 b 100.00%	10.09 c 99.02%	4.41 e 43.28%
葡萄糖含量 Glucose content (mg g-1)	8.71 b 100.00%	9.86 a 113.20%	4.34 c 49.83%	9.75 a 100.00%	8.47 b 86.87%	2.69 d 27.59%
果糖含量 Fructose content (mg g-1)	3.21 b 100.00%	2.93 d 91.28%	2.21 e 68.85%	3.61 a 100.00%	3.17 c 87.81%	1.79 f 49.58%
钙离子含量 Ca2+content (mmol g-1)	1.67 c 100.00%	2.07 a 123.95%	0.82 d 49.10%	1.29 b 100.00%	1.69 c 131.01%	0.43 e 33.33%
一氧化氮含量 NO content (µmol g-1)	35.61 a 100.00%	22.05 b 61.92%	18.04 d 50.66%	36.47 a 100.00%	20.34 c 55.77%	17.92 d 49.14%
过氧化氢含量 H2O2 content (µmol g-1)	103.07 b 100.00%	107.47 b 104.27%	82.48 c 80.02%	102.15 b 100.00%	128.96 a 126.25%	84.11 c 82.34%

CK、PEG和PEG+PB处理与表3的相同。不同小写字母表示在5%水平上差异显著, 每个参数下面的百分比与表2的一致。
CK, DS, and DS+PB treatments are the same as those in Table 3. Values labeled with different letters are significantly different at the 5% probability level, the percentages below each parameter are consistent with those in Table 2.

新窗口打开|下载CSV

2.5 喷施PB和EGTA联合10% PEG-6000模拟干旱胁迫对水稻叶片相对含水量、PEPC、SnRK2s和SnRK1s相关基因表达的影响

由于PB施用, 我们观察到了水稻叶片内钙离子的差异, 推测钙离子作为信号在可变剪接机制响应水稻干旱中可能起到积极的作用。为了验证这一假设, 本研究选用了钙离子的鏊合剂EGTA, 由表6可知, 叶片相对含水量在PEG、PEG+PB、PEG+PB+EGTA处理后逐渐降低, 其中PC相对含水量始终高于WT, 可知钙离子的确对水稻干旱条件下的保水能力起正效应, 但PC的效果仍大于WT。与此同时, PEG+PB处理后, 供试材料ABA含量降低, 但在PEG+PB+EGTA处理后, 与PEG+PB处理相比, 2个供试材料的ABA含量又升高, 且PC的高于WT, 推测, 钙离子受抑后, PC内ABA的诱导增强可能对其耐旱性有益。PC既包括内源的C₃型PEPC基因(Osppc2a), 也包括外源导入的玉米C₄-PEPC基因。与CK相比, PEG处理上调PC中C₄-PEPC表达量, 而PEG+PB下调PC中C₄-PEPC表达量, 而PEG+PB+EGTA处理下PC中C₄-PEPC表达水平与PEG+PB处理下无差异; 与CK相比, PEG处理上调了水稻内源Osppc2a表达水平; PEG+PB处理下调Osppc2a表达量; PEG+PB+ EGTA处理上调了供试材料Osppc2a表达水平, 表明钙离子正调节可变剪接机制响应水稻干旱, 影响外源导入C₄-PEPC表达量; 而当水稻内源的钙离子被鏊合后, ABA含量显著增加, 有利于诱导水稻内源Osppc2a表达水平的增加, 且PC的诱导程度显著大于WT。

Table 6
表6
表6不同处理对水稻叶片ABA含量、相对含水量、PEPC以及SnRKs相关基因表达量的影响
Table 6Changes of different treatments on ABA contents, relative water content, and the relative expression level of PEPC and SnRKs related genes in rice leaves

测定指标 Measurement index	转玉米C4-PEPC水稻 C4-PEPC rice (PC)				Kitaake (WT)
	CK	PEG	PEG+PB	PEG+PB+EGTA	CK	PEG	PEG+PB	PEG+PB+EGTA
ABA 含量 ABA content (μg g-1)	8.16 d	12.95 a	5.63 e	9.78 b	9.43 c	6.86 b	4.92 f	8.26 d
相对含水量 Relative water content (%)	87.43 a	76.43 b	73.47 c	59.38 e	89.56 a	68.17 d	62.42 e	51.39 f
C4-PEPC相对表达量 Relative expression of C4-PEPC	2.27 d	6.44 a	3.27 b	2.53 c
Osppc2a相对表达量 Relative expression of Osppc2a	0.96 de	1.26 c	0.55 g	1.45 b	1.04 d	1.57 a	0.78 f	1.038 d
SAPK8相对表达量 Relative expression of SAPK8	1.98 e	10.53 a	1.05 c	0.88 d	1.03 g	5.39 b	2.03 f	0.41 h
SAPK9相对表达量 Relative expression of SAPK9	3.09 a	2.84 b	1.54 d	0.68 f	1.04 e	1.87 c	0.97 e	1.77 c
SAPK10相对表达量 Relative expression of SAPK10	0.82 d	3.09 b	2.94 b	0.75 e	1.02 c	5.81 a	0.74 e	0.66 f
OsK1a相对表达量 Relative expression of OsK1a	1.75 d	14.12 a	10.05 b	1.54 e	1.03 g	10.04 b	5.43 c	1.15 f
OsK24相对表达量 Relative expression of OsK24	0.97 e	16.99 c	14.41 d	0.74 g	1.01 e	12.89 a	9.06 b	0.25 f
OsK35相对表达量 Relative expression of OsK35	2.12 g	13.05 a	3.76 f	5.88 d	1.04 h	7.41 b	3.99 e	6.28 c

CK、PEG和PEG+PB处理与表3的相同。PEG+PB+EGTA: 10% PEG水稻培养液, 并同时叶面喷施0.5 µmol L^-1 PB和10 mmol L^-1 EGTA溶液。WT中没有外源导入的玉米C₄-PEPC的表达。不同小写字母表示在5%水平差异显著.
CK, PEG, and PEG+PB treatments are the same as those in Table 3. DS+PB+EGTA: 10% PEG rice culture solution treatment with leaf spraying both 0.5 µmol L^-1 PB and 10 mmol L^-1 EGTA solutions. There is no exogenous expression of C₄-PEPC in WT. Different lowercase letters indicate significant differences at the 5% probability level.

新窗口打开|下载CSV

进一步研究结果观察到, 与CK相比, PEG处理下PC和WT的SAPK8和SAPK10表达量升高; PEG+PB处理下均导致3个水稻的SnRK2表达量下降, 可见, 可变剪接对水稻的干旱响应起正效应, 其中PC的SAPK8、SAPK9和SAPK10表达量显著高于WT; PEG+PB+EGTA处理进一步降低了供试水稻的3个基因表达量, 但是2个供试材料的基因表达水平变化不一致, 其中PC中SAPK8和SAPK10仍高于WT, 但是SAPK9则相反; 与CK相比, PEG处理下PC和WT的OsK1a、OsK24和OsK35表达量升高; 与CK相比, PEG+PB处理下则均导致其基因表达量下降, 其中PC的OsK1a和OsK24表达量均高于WT。而PEG+PB+EGTA处理后水稻的OsK1a和OsK24表达量均低于PEG+PB处理, 表明钙离子也在可变剪接机制中通过能量代谢参与水稻干旱响应调节, 其中PC的作用仍高于WT, 可见, 钙离子可通过糖信号SnRK1和SnRK2家族相关基因的表达水平, 参与水稻干旱响应中的可变剪接过程, 其中PC相关表达基因的表达水平与WT存在差异。

2.6 喷施PB和EGTA联合10% PEG-6000模拟干旱胁迫对水稻叶片剪接因子相关基因表达的影响

表7所示, 与对照CK相比, 单独PEG处理下PC和WT的剪切因子相关基因SR33、SR40、RS29、SCL25、SCL26和RSZ21均表达增加, 且PC高于WT。与对照CK相比, PEG+PB处理下PC和WT的SR33、SR40、RS29、RS2Z21和RS2Z38表达量升高, 且PC高于WT。与对照CK相比, PEG+ PB+ EGTA处理下RS29、SCL25、RS2Z21表达水平降低, 且PC下降程度小于WT。PB抑制了部分剪接因子基因的表达量, 而诱导了另外一部分剪接因子基因表达量的增加。值得关注的是, 进一步加入钙离子螯合剂(EGTA), 即PEG+PB+EGTA处理则诱导了水稻SR33、SCL26和RS2Z36表达水平的增加, 这些基因的表达水平在PEG+PB处理下则是被抑制的, 说明钙离子也参与了干旱胁迫下剪接因子基因表达水平的调节。

Table 7
表7
表7不同处理对水稻叶片剪接因子相关基因表达的影响
Table 7Effect of different treatments on gene expression of splicing factor in rice leaves

相对基因的表达 Relative expression level of genes	转玉米C4-PEPC水稻 C4-PEPC rice (PC)				Kitaake (WT)
	CK	PEG	PEG+PB	PEG+PB+ EGTA	CK	PEG	PEG+PB	PEG+PB+EGTA
SR33	2.12 c	2.47 b	3.69 a	1.36 d	1.04 e	0.97 e	2.43 b	0.79 f
SR40	1.65 e	4.57 a	4.15 b	1.17 f	1.03 g	3.36 c	2.11 d	0.86 h
RS29	0.97 e	6.13 a	5.97 a	1.08 d	1.02 d	3.14 b	2.31 c	0.23 f
RS33	1.24 c	0.57 g	0.68 f	2.72 a	1.04 d	0.72 e	0.42 h	1.35 b
SCL25	3.12 b	4.74 a	2.07 d	1.45 e	1.03 g	2.42 c	1.23 f	0.35 h
SCL26	0.76 g	5.13 a	0.95 f	2.41 c	1.01 f	4.56 b	1.24 e	1.38 d
SCL30	0.96 e	0.69 f	2.31 c	2.76 b	1.04 e	1.21 d	4.32 a	4.14 a
SCL57	0.95 e	4.39 a	1.71 c	1.89 c	1.03 d	2.38 b	2.52 b	0.83 f
RS2Z21	5.49 b	10.74 a	9.85 a	1.72 e	1.02 e	5.16 c	3.14 d	0.51 f
RS2Z36	1.89 e	2.19 d	1.97 e	4.27 a	1.01 f	2.37 c	0.96 f	3.87 b
RS2Z38	0.86 e	4.12 a	3.93 b	0.36 f	1.04 d	3.82 b	1.55 c	1.48 c

CK、PEG、PEG+PB和PEG+PB+EGTA处理与表6的相同。不同字母表示在5%水平差异显著。
CK, PEG, PEG+PB, and DS+PB+EGTA treatments are the same as those in Table 6. Different lowercase letters indicate significant differences at the 5% probability level.

新窗口打开|下载CSV

2.7 喷施PB联合干旱胁迫下PC和WT的各个指标的相关性

由图1-A可知PC各指标存在相关性, 可溶性蛋白分别与Ca²⁺含量呈极显著相关, 与RS2Z38基因表达呈显著相关, Glu与SCL26显著相关, Ca²⁺分别与ABA和RS33显著相关, ABA与OsK1a显著相关, C₄-PEPC与SCL25显著相关, SAPK8与SR33显著相关, OsK35与SR40极显著相关。在图1B中, WT的Su与RS33显著相关, Glu与SAPK8显著相关, Osppc2a与OsK35显著相关, OsK35与SCL25显著相关。

图1

新窗口打开|下载原图ZIP|生成PPT
图1PC和WT各指标相关性

A: PC各指标相关性; B: WT各指标相关性。^*P < 0.05; ^** P < 0.01。RWC: 相对含水量; Su: 可溶性糖; Sp: 可溶性蛋白; Suc: 蔗糖; Glu: 葡萄糖; Fru: 果糖。
Fig. 1Correlation of PC and WT indicators

A: relevance in PC; B: relevance in WT. ^*P < 0.05; ^** P < 0.01. RWC: relative water content; Su: soluble sugar content; Sp: soluble protein content; Suc: sucrose content; Glu: glucose content; Fru: fructose content.

3 讨论

在干旱胁迫下, 与C₃植物相比, C₄植物表现出较高的净光合速率和产量^[49]。PC水稻不仅光合效率提高, 而且具有多重非生物胁迫的耐性^[50], 尤其是耐旱性显著改善^{[11,12,13,14,15]}。本文通过施用可变剪接的抑制剂PB观察到, 可变剪接过程在水稻干旱逆境响应和适应机制中发挥了积极的作用, 其中PC的效应大于WT, 这与PC株系内源较高的钙离子和糖组分含量密切相关, 即PC水稻通过钙离子调节部分剪接因子基因的表达量, 增强植株叶片的保水能力、光合能力和氧化防护能力, 维持产量稳定, 从而表现更强的耐旱性。

糖作为信号分子参与了植物对非生物胁迫的响应, 其中SnRK作为联系糖和非生物逆境的枢纽而发挥重要的作用^[51]。植物SnRK家族基因根据结构不同, 可以分为3个亚家族, 分别为SnRK1、SnRK2和SnRK3^[52]。拟南芥剪接因子功能丧失突变体sr45-1含有更高的能量感应水平, 通过自身的AS, 调节SnRK1感受糖信号; 与此同时, SR45还可调节拟南芥一个肌醇多磷酸-5-磷酸酶(myoinositolpolyphosphate 5-phoshatase, 5PTase13)基因(5PTase13)的AS, 通过与SnRK1相互作用以稳定SnRK1的蛋白, 从而增强了SR45的葡萄糖超敏性, 建立了SR45与糖信号感受器 / 能量传感器SnRK1降解调节之间的机制联系^[53]。本研究也观察到在干旱胁迫下, 水稻叶片内葡萄糖含量增加, 伴随SnRK1基因表达量增加, 而且PC的诱导能力大于WT。ABA与其受体PYR/PLY/RCAR结合, 再与PP2C作用形成复合物, 可解除PP2C对SnRK2激酶活性的抑制作用, 活性形式的SnRK2磷酸化AREB/ABF、离子通道蛋白和NADPH氧化酶等下游底物, 进而诱导ABA响应基因的表达^[54]。PB处理就是通过抑制PP2C磷酸酶的剪接来激活ABA信号转导途径, 同时仍然在一定程度上维持ABA激活的SnRK2激酶活性^[32]。本研究观察到PB影响了干旱处理下水稻SnRK1s和SnRK2s基因的表达, 如SnRK1s相关基因OsK24和SnRK2s相关基因SAPK8表达降低, 且对PC的作用更明显。可见, 水稻糖信号SnRKs基因的表达差异, 可能是可变剪接过程对其干旱逆境的响应表现差异的内在因素。

在植物中, Ca²⁺是第二信使, 广泛参与胁迫反应, 快速反应不断变化的环境来灵活适应植物的生长发育, 为固着生物提供了一条不可缺少的快速反应和适应机制^[55]。胞外Ca²⁺通过钙感受体(calcium-sensing receptor, CAS)信号通路, 诱导保卫细胞H₂O₂和NO积累, 激活胞内Ca²⁺瞬时增加, 最终引起气孔关闭^[31]。与未转基因水稻相比, PC水稻Ca²⁺缓冲能力强, PC含有比野生型水稻更多的游离Ca^{2+ [21]}。外源施入钙离子试验和抑制剂试验均表明内源钙离子通过调节PEPC酶活性和基因表达而参与PC干旱逆境响应^[56,18]。并且Ca²⁺参与了NO调控PEPC酶活性和基因表达量, 促进PC光合作用的提高^[22]。本研究的PB联合干旱处理, 显著降低剪接因子基因RS29、SCL25和RS2Z21表达量, 仍未消除2个供试水稻材料内源钙离子含量的差异, 其中PC仍具有较高的钙离子含量, 且部分剪接因子的基因表达水平也较高。钙离子螯合剂的药理实验进一步验证, PC的内源钙离子含量的降低的确下调了部分剪接因子基因的表达, 但是同时诱导了内源ABA含量的增加。通常干旱胁迫是通过Ca²⁺诱导ABA生物合成^[57], 值得关注的是, 本研究观察到PC则在抑制了钙离子之后诱导了ABA的含量增加。推测, ABA在钙离子被抑制后作为补偿机制, 参与PC对干旱的响应, 相关假设尚需要进一步研究验证。

植物可通过剪接因子或者转录因子发生AS响应干旱胁迫, 获得了对环境胁迫的耐受性^[58], 为植物提供了一条迅速应对环境变化的有效调控方案^[59]。其中剪接因子是一种反式作用因子, 它们能识别或结合一些在内含子或外显子上的剪接增强子或剪接抑制子顺式作用元件, 导致选择不同的剪接位点, 以产生不同的剪接模式^[60]。精氨酸/丝氨酸丰富蛋白家族(arginine/serine-rich proteins, SR proteins)是真核生物中的一类剪接因子, 在前体mRNA的组成性和选择性剪接中起作用^[61]。根据其结构特点分成6个不同亚家族, 即: SR、RSZ、SC、SCL、RS2Z及RS家族^[62]。其中SCL和RS2Z亚家族相关基因已观察到在植物响应多种非生物胁迫条件下表达水平及剪接方式发生明显变化^[63]。研究发现剪接因子的上游区域均有ABA的顺式响应元件, 对ABA有显著反应, 并明确了剪接因子SR34、SR34b、SCL30a、SCL28、SCL33、RS40、SR45和SR45a, 均是参与ABA介导应激反应的潜在候选者^[64]。本研究观察到可变剪接抑制剂也显著降低了与ABA相关的SnRK2基因表达, 当水稻叶片中游离钙离子减少可诱导叶内ABA含量部分恢复, 其中PC的含量显著高于WT, 并伴随糖信号基因表达的恢复, 帮助PC维持其剪接因子相关基因的表达水平, 参与ABA介导应激反应。在水稻对干旱的响应过程中, PC通过内源钙离子以及ABA的补偿机制参与调节可变剪接过程的内在生理机制还需深入研究。

磷酸烯醇丙酮酸羧化酶的磷酸化在控制高等植物的中枢代谢中起重要作用, 它参与了干旱胁迫下C₄植物保持高光合作用、氮素和水分利用效率的机制^[65]。磷酸烯醇丙酮酸羧化酶激酶(phosphoenolpyruvate carboxylase kinases, PPCK)在PEPC磷酸化过程中起关键作用。在番茄中鉴定了2种PPCK基因, 即LePPCK1和LePPCK2, 其中LePPCK2是由于可变剪接保留了第二内含子的转录本, 并只在果实成熟期表达显著增加, 而在成熟后子房和种子仅包含正确剪接的LePPCK1, 从而参与了PEPC酶的磷酸化过程, 而且这种组织特异性调节也在马铃薯、茄子和烟草中观察到, 而且剪接的内含子序列在所报告的4个物种间高度保守^[67]。前期研究已经观察到, 在干旱条件下, PC中的PPCK酶活性及其基因(PPCK1和PPCK2)均诱导增强, 而且与信号分子钙离子、NO和H₂O₂的调节有关^[18]。本研究观察到PB联合干旱处理, 降低了PC内源Osppc2a和外源导入C₄-PEPC的表达水平, 钙离子鏊合剂联合PB和PEG处理, 加剧了PC的C₄-PEPC表达水平和酶活性的抑制, 而诱导了其内源Osppc2a表达水平的提高, 提示外源导入C₄-PEPC, 增加PC中PEPC基因的多样性, 有利于Ca²⁺调节PEPC基因和酶活性, 保持叶片水分含量, 维持光合能力稳定, 增强氧化防护能力, 表现耐旱。是否不同类型的PEPC基因在水稻响应干旱胁迫中能够发生自身的可变剪接, 还需深入研究。

综上, 可变剪接机制参与水稻干旱耐性, 并起积极的作用。外施PB联合干旱处理实验表明: 水稻可通过糖信号SnRK1s、SnRK2s基因以及钙离子, 参与调节剪接因子相关基因的表达水平而响应干旱。与WT相比, PC的增益效果更强, 且与PC内源高钙和糖含量密切相关。本研究的结果将为今后进一步从可变剪接机制研究PC的耐旱生理机制提供新思路, 从而丰富表观遗传机制参与水稻干旱耐性调节机理的信息。未来, 通过调节可变剪接的水平将可作为改良水稻耐旱性的一条新策略。

4 结论

可变剪接参与水稻干旱响应, 水稻可通过糖信号SnRK2s和SnRK3s基因以及钙离子, 参与调节剪接因子相关基因的表达水平, 对水稻耐旱起积极的作用。与WT相比, PC的增益效果更强, 与其内源高钙离子含量和糖信号的差异表现密切相关。

参考文献 View Option 原文顺序
文献年度倒序
文中引用次数倒序
被引期刊影响因子

[1] Khush G S. What it will take to feed 5.0 billion rice consumers in 2030
Plant Mol Biol, 2005, 59:1-6.
DOIURL [本文引用: 1]

[2] Todaka D, Shinozaki K, Yamaguchi-Shinozaki K. Recent advances in the dissection of drought-stress regulatory networks and strategies for development of drought-tolerant transgenic rice plants
Front Plant Sci, 2015, 6:84.
[本文引用: 1]

[3] Yang S, Vanderbeld B, Wan J, Huang Y. Narrowing down the targets: towards successful genetic engineering of drought- tolerant crops
Mol Plant, 2010, 3:469-490.
DOIURL [本文引用: 1]

[4] Li J J, Li Y, Yin Z G, Jiang J H, Zhang M H, Guo X, Ye Z J, Zhao Y, Xiong H Y, Zhang Z Y, Shao Y J, Jiang C H, Zhang H L, An G, Paek N, Ali J, Li Z C. OsASR5 enhances drought tolerance through a stomatal closure pathway associated with ABA and H₂O₂ signaling in rice
Plant Biotechnol J, 2017, 15:183-196.
DOIURL [本文引用: 1]

[5] Valliyodan B, Nguyen H T. Understanding regulatory networks and engineering for enhanced drought tolerance in plants
Curr Opin Plant Biol, 2006, 9:189-195.
PMID [本文引用: 1]
Drought stress is one of the major limitations to crop productivity. To develop crop plants with enhanced tolerance of drought stress, a basic understanding of physiological, biochemical and gene regulatory networks is essential. Various functional genomics tools have helped to advance our understanding of stress signal perception and transduction, and of the associated molecular regulatory network. These tools have revealed several stress-inducible genes and various transcription factors that regulate the drought-stress-inducible systems. Translational genomics of these candidate genes using model plants provided encouraging results, but the field testing of transgenic crop plants for better performance and yield is still minimal. Better understanding of the specific roles of various metabolites in crop stress tolerance will give rise to a strategy for the metabolic engineering of crop tolerance of drought.

[6] Zhu X G, Long S P, Ort D R. Improving photosynthetic efficiency for greater yield
Annu Rev Plant Biol, 2010, 61:235-261.
DOIURL [本文引用: 1]

[7] Ku M S B, Agarie S, Nomura M, Fukayama H, Tsuchida H, Ono K, Hirose S, Toki S, Miyao M, Matsuoka M. High-level expression of maize phosphoenolpyruvate carboxylase in transgenic rice plants
Nat Biotechnol, 1999, 17:76-80.
PMID [本文引用: 2]
Using an Agrobacterium-mediated transformation system, we have introduced the intact gene of maize phosphoenolpyruvate carboxylase (PEPC), which catalyzes the initial fixation of atmospheric CO2 in C4 plants into the C3 crop rice. Most transgenic rice plants showed high-level expression of the maize gene; the activities of PEPC in leaves of some transgenic plants were two- to threefold higher than those in maize, and the enzyme accounted for up to 12% of the total leaf soluble protein. RNA gel blot and Southern blot analyses showed that the level of expression of the maize PEPC in transgenic rice plants correlated with the amount of transcript and the copy number of the inserted maize gene. Physiologically, the transgenic plants exhibited reduced O2 inhibition of photosynthesis and photosynthetic rates comparable to those of untransformed plants. The results demonstrate a successful strategy for installing the key biochemical component of the C4 pathway of photosynthesis in C3 plants.

[8] 吴琼, 许为钢, 李艳, 齐学礼, 胡琳, 张磊, 韩琳琳. 田间条件下转玉米C₄型PEPC基因小麦的光合生理特性
作物学报, 2010, 37:2046-2052.
[本文引用: 1]

Wu Q, Xu W G, Li Y, Qi X L, Hu L, Zhang L, Han L L. Physiological characteristics of photosynthesis in transgenic wheat with maize C₄-PEPC gene under field conditions
Acta Agron Sin, 2010, 37:2046-2052 (in Chinese with English abstract).
[本文引用: 1]

[9] Lian L, Wang X, Zhu Y, He W, Cai Q H, Xie H A, Zhang M Q, Zhang J F. Physiological and photosynthetic characteristics of indica Hang2 expressing the sugarcane PEPC gene
Mol Biol Rep, 2014, 41:2189-2197.
DOIURL [本文引用: 1]

[10] Bandyopadhyay A, Datta K, Zhang J, Yang W, Raychaudhuri S, Miyao M, Datta S K. Enhanced photosynthesis rate in genetically engineered indica rice expressing pepc gene cloned from maize
Plant Sci, 2007, 172:1204-1209.
DOIURL [本文引用: 1]

[11] 方立锋, 丁在松, 赵明. 转ppc基因水稻苗期抗旱特性研究
作物学报, 2008, 34:1220-1226.
URL [本文引用: 3]

Fang L F, Ding Z S, Zhao M. Characteristics of drought tolerance in ppc overexpressed rice seedlings
Acta Agron Sin, 2008, 34:1220-1226 (in Chinese with English abstract).
URL [本文引用: 3]

[12] 周宝元, 丁在松, 赵明. PEPC过表达可以减轻干旱胁迫对水稻光合的抑制作用
作物学报, 2011, 37:112-118
URL [本文引用: 3]

Zhou B Y, Ding Z S, Zhao M. Alleviation of drought stress inhibition on photosynthesis by overexpression of PEPC gene in rice
Acta Agron Sin, 2011, 37:112-118 (in Chinese with English abstract).
URL [本文引用: 3]

[13] 丁在松, 周宝元, 孙雪芳, 赵明. 干旱胁迫下PEPC过表达增强水稻的耐强光能力
作物学报, 2012, 38:285-292.
URL [本文引用: 3]

Ding Z S, Zhou B Y, Sun X F, Zhao M. High light tolerance is enhanced by overexpressed PEPC in rice under drought stress
Acta Agron Sin, 2012, 38:285-292 (in Chinese with English abstract).
URL [本文引用: 3]

[14] Shen W J, Chen G X, Xu J G, Jiang Y, Liu L, Gao Z P, Ma J, Chen X, Chen T H, Lyu C G. Overexpression of maize phosphoenolpyruvate carboxylase improves drought tolerance in rice by stabilization the function and structure of thylakoid membrane
Photosynthetica, 2015, 53:436-446.
DOIURL [本文引用: 3]

[15] Qian B, Li X, Liu X L, Wang M. Improved oxidative tolerance in suspension cultured cells of C₄-pepc transgenic rice by H₂O₂ and Ca²⁺ under PEG-6000
J Integr Plant Biol, 2015, 57:534-549.
DOIURL [本文引用: 5]

[16] 李霞, 焦德茂, 戴传超. 转PEPC基因水稻对光氧化逆境的响应
作物学报, 2005, 27:408-413.


Li X, Jiao D M, Dai C C. Photosynthetic characteristics for rice hybrids with transgenic PEPC parent HPTER-01
Acta Agron Sin, 2005, 27:408-413 (in Chinese with English abstract).


[17] Ren C G, Li X, Liu X L, Wei X D, Dai C C. Hydrogen peroxide regulated photosynthesis in C₄-pepc transgenic rice
Plant Physiol Biochem, 2014, 74:218-229.
DOIURL [本文引用: 3]

[18] Liu X L, Li X, Zhang C, Dai C C, Zhou J Y, Ren C G, Zhang J F. Phosphoenolpyruvate carboxylase regulation in C₄-PEPC expressing transgenic rice during early responses to drought stress
Physiol Plant, 2017, 159:178-200.
DOIURL [本文引用: 5]

[19] Orta D R, Merchant S S, Alric J, Barkan A, Blankenship R E, Bock R, Moore T A. Redesigning photosynthesis to sustainably meet global food and bioenergy demand
Proc Natl Acad Sci USA, 2015, 112:8529-8536.
DOIURL [本文引用: 1]

[20] O’Leary B, Park J, Plaxton W C. The remarkable diversity of plant PEPC recent insights into the physiological function and post-translational controls of non-photosynthetic PEPCs
Biochem J, 2011, 436:15-34.
DOIURL [本文引用: 1]

[21] Li X, Wang C, Ren C G. Effects of butanol neomycin and calcium on the photosynthetic characteristics of pepc transgenic rice
Afr J Biotechnol, 2011, 10:17466-17476.
[本文引用: 3]

[22] Chen P, Li X, Huo K, Wei X D, Dai C C. Promotion of photosynthesis in transgenic rice over-expressing of maize C₄ phosphoenolpyruvate carboxylase gene by nitric oxide donors
J Plant Physiol, 2014, 171:458-466.
DOIURL [本文引用: 3]

[23] Huo K, Li X, He Y F, Wei X D, Wang C L, Zhao C F. Exogenous ATP enhance signal response of suspension cells of transgenic rice (Oryza sativa L.) expressing maize C₄-pepc encoded phosphoenolpyruvate carboxylase under PEG treatment
Plant Growth Regul, 2017, 82:55-67.
DOIURL [本文引用: 1]

[24] Li X, Wang C. Physiological and metabolic changes of transgenic rice plant with increased activity of phosphonolpyruvate carboxylase during flowering stage
Physiol Plant, 2013, 35:1503-1512.
[本文引用: 1]

[25] Zhang C, Li X, He Y F, Zhang J F, Yan T, Liu X L. Physiological investigation of C₄ phosphoenolpyruvate carboxylase introduced rice line shows that sucrose metabolism is involved in the improved drought tolerance
Plant Physiol Biochem, 2017, 115:328-342.
DOIURL [本文引用: 1]

[26] 张金飞, 李霞, 何亚飞, 谢寅峰. 外源葡萄糖增强高表达转玉米C₄型PEPC水稻耐旱性的生理机制
作物学报, 2018, 44:82-94.
URL [本文引用: 2]

Zhang J F, Li X, He Y F, Xie Y F. Physiological mechanism on drought tolerance enhanced by exogenous glucose in C₄-PEPC rice
Acta Agron Sin, 2018, 44:82-94 (in Chinese with English abstract).
URL [本文引用: 2]

[27] He Y F, Xie Y F, Li X, Yang J. Drought tolerance of transgenic rice overexpressing maize C₄-PEPC gene related to increased anthocyanin synthesis regulated by sucrose and calcium
Biol Plant, 2020, 64:136-149.
DOIURL [本文引用: 1]

[28] 杜康兮, 沈文辉, 董爱武. 表观遗传调控植物响应非生物胁迫的研究进展
植物学报, 2018, 53:581-593.
URL [本文引用: 1]

Du K X, Shen W H, Dong A W. Advances in epigenetic regulation of abiotic stress response in plants
Chin Bull Bot, 2018, 53:581-593 (in Chinese with English abstract).
URL [本文引用: 1]

[29] Syed N H, Kalyna M, Marquez Y, Barta A, Brown J W. Alternative splicing in plants coming of age
Trends Plant Sci, 2012, 17:616-623.
DOIURL [本文引用: 1]

[30] 宋凝曦, 张晓敬, 陆佳岚, 李霞, 谢寅峰. 可变剪接在植物响应胁迫中的作用
植物生理学报, 2020, 56:1201-1211.
[本文引用: 1]

Song N X, Zhang X J, Lu J L, Li X, Xie Y F. Function of alternative splicing in plant response to biotic and abiotic stress
Plant Physiol J, 2020, 56:1201-1211 (in Chinese with English abstract).
URL [本文引用: 1]

[31] Wang P, Xue L, Batelli G, Lee S, Hou Y J, Van Oosten M J, Zhang H, Tao W A, Zhu J K. Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action
Proc Natl Acad Sci USA, 2013, 110:11205-11210.
DOIURL [本文引用: 2]

[32] Ling Y, Alshareef S, Butt H, Lozano J, Li L, Galal A A, Moustafa A, Momin A A, Tashkandi M, Richardson D N, Fujii H, Arold S, Rodriguez P L, Duque P, Mahfouz M M. Pre mRNA splicing repression triggers abiotic stress signaling in plants
Plant J, 2017, 89:291-309.
DOIURL [本文引用: 2]

[33] Galit L M, Ahuvi Y, Gil A. The alternative role of DNA methylation in splicing regulation
Trends Genet, 2015, 31:274-280.
DOIPMID [本文引用: 1]
Although DNA methylation was originally thought to only affect transcription, emerging evidence shows that it also regulates alternative splicing. Exons, and especially splice sites, have higher levels of DNA methylation than flanking introns, and the splicing of about 22% of alternative exons is regulated by DNA methylation. Two different mechanisms convey DNA methylation information into the regulation of alternative splicing. The first involves modulation of the elongation rate of RNA polymerase II (Pol II) by CCCTC-binding factor (CTCF) and methyl-CpG binding protein 2 (MeCP2); the second involves the formation of a protein bridge by heterochromatin protein 1 (HP1) that recruits splicing factors onto transcribed alternative exons. These two mechanisms, however, regulate only a fraction of such events, implying that more underlying mechanisms remain to be found. Copyright © 2015 Elsevier Ltd. All rights reserved.

[34] Yoshida S, Forno D A, Cock J H. Laboratory Manual for Physiological Studies of Rice
Philippines: International Rice Research Institute, 1976. pp 61-64.
[本文引用: 1]

[35] Smart R E, Bingham G E. Rapid estimates of relative water content
Plant Physiol, 1974, 53:258-260.
PMID [本文引用: 1]
Relative water content may be accurately estimated using the ratio of tissue fresh weight to tissue turgid weight, termed here relative tissue weight. That relative water content and relative tissue weight are linearly related is demonstrated algebraically. The mean value of r(2) for grapevine (Vitis vinifera L. cv. Shiraz) leaf tissue over eight separate sampling occasions was 0.993. Similarly high values were obtained for maize (Zea mays cv. Cornell M-3) (0.998) and apple (Malus sylvestris cv. Northern Spy) (0.997) using a range of leaf ages. The proposal by Downey and Miller (1971. Rapid measurements of relative turgidity in maize (Zea mays L.). New Phytol. 70: 555-560) that relative water content in maize may be estimated from water uptake was also investigated for grapevine leaves; this was found to be a less reliable estimate than that obtained with relative tissue weight. With either method, there is a need for calibration, although this could be achieved for relative tissue weight at least with only a few subsamples.

[36] Somani B L, Khanade J, Sinha R. A modified anthrone sulfuric acid method for the determination of fructose in the presence of certain proteins
Anal Biochem, 1987, 167:327-330.
PMID [本文引用: 1]
Two milliliters of a reagent consisting of anthrone and tryptophan each at a 0.01% concentration in 75% sulfuric acid, when added to 0.9 ml of solution containing D-fructose, produced on heating (55 degrees C, 90 min) a pink color (lambdamax 520 nm) with an absorbance of 0.009 A/nmol. The absorbance is about three times higher than that of the standard anthrone-sulfuric acid reagent. Glucose yields a color only about 1% as intense as that yielded by fructose. The method is useful for the estimation of fructose in the presence of proteins. Synthetic Amadori compounds and glycosylated proteins containing ketoamine-linked fructose, however, were unreactive.

[37] Ambavaram M M, Basu S, Krishnan A, Ramegowda V, Batlang U, Rahman L, Pereira A. Coordinated regulation of photosynthesis in rice increases yield and tolerance to environmental stress
Nat Commun, 2014, 5:93.
[本文引用: 1]

[38] Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding
Anal Biochem, 1976, 72:248-254.
PMID [本文引用: 1]

[39] Giannopolitis C N, Ries S K. Superoxide dismutases: I. Occurrence in higher plants
Plant Physiol, 1997, 59:309-314.
DOIURL [本文引用: 1]

[40] Smith F G, Robinson W B, Stotz E. A colorimetric method for the determination of peroxidase in plant material
J Biol Chem, 1949, 79:881-889.
[本文引用: 1]

[41] Havir E A, McHale N A. Biochemical and developmental characterization of multiple forms of catalase in tobacco leaves
Plant Physiol, 1987, 84:450-455.
PMID [本文引用: 1]
Leaf extracts of both Nicotiana tabacum and Nicotiana sylvestris contain multiple forms of catalase (H(2)O(2):H(2)O(2) oxidoreductase, EC 1.11.1.6) which are separable at different pH values by chromatofocusing columns. Marked changes in distribution of these catalases occur during seedling development and leaf maturation. The form of catalase eluting first (peak 1) was predominant during early seedling growth and present at all stages of development. Two more acidic forms (peaks 2 and 3) appeared later and comprised 29% of the total activity by 11 days postgermination. Mature leaves of N. tabacum contained peak 1 catalase, but peaks 2 and 3 represented 62% of the total activity. No interconversion of peaks 1, 2, and 3 was detected. The three forms of catalase differed in thermal stability with peak 1 > peak 2 >> peak 3. For N. sylvestris, t((1/2)) at 55 degrees C was 31.5 and 3.0 min for peaks 1 and 3, respectively, and for N. tabacum, t((1/2)) was 41.5 and 3.2 min, respectively. All forms of catalase in tobacco show peroxidatic (measured as ethanol to acetaldehyde conversion) as well as catalatic activities. However, for both Nicotiana species the ratio peroxidatic/catalatic activity is at least 30-fold higher in peak 3 than in peaks 1 and 2. Chromatofocusing of extracts from spinach leaves separated at least four peaks of catalase activity, one of which had a 10-fold higher ratio of peroxidatic/catalatic activity than the others. Short-term growth (5 days) of tobacco seedlings under atmospheric conditions suppressing photorespiration (1% CO(2)/21% O(2)) reduced total catalase activity and caused a decline in peak 1 catalase and a substantial increase in the activity of peaks 2 and 3 relative to air-grown seedlings at the same stage.

[42] Walter T, John L. A photometric methods for the determination of proline
J Biol Chem, 1955, 215:655-660.
DOIURL [本文引用: 1]

[43] Wang X, Cai J, Jiang D. Pre-anthesis high-temperature acclimation alleviates damage to the flag leaf caused by post-anthesis heat stress in wheat
J Plant Physiol, 2011, 168:585-593.
DOIURL [本文引用: 1]

[44] Doulis A G, Debian N, Kingston-Smith A H, Foyer C H. Differential localization of antioxidants in maize leaves
Plant Physiol, 1997, 114:1031-1037.
PMID [本文引用: 1]
The aim of this work was to determine the compartmentation of antioxidants between the bundle-sheath and mesophyll cells of maize (Zea mays L.) leaves. Rapid fractionation of the mesophyll compartment was used to minimize modifications in the antioxidant status and composition due to extraction procedures. The purity of the mesophyll isolates was assessed via the distribution of enzyme and metabolite markers. Ribulose-1,5 bisphosphate and ribulose-1,5-bisphosphate carboxylase/oxygenase were used as bundle-sheath markers and phosphoenolpyruvate carboxylase was used as the mesophyll marker enzyme. Glutathione reductase and dehydroascorbate reductase were almost exclusively localized in the mesophyll tissue, whereas ascorbate, ascorbate peroxidase, and superoxide dismutase were largely absent from the mesophyll fraction. Catalase, reduced glutathione, and monodehydroascorbate reductase were found to be approximately equally distributed between the two cell types. It is interesting that, whereas H2O2 levels were relatively high in maize leaves, this oxidant was largely restricted to the mesophyll compartment. We conclude that the antioxidants in maize leaves are partitioned between the two cell types according to the availability of reducing power and NADPH and that oxidized glutathione and dehydroascorbate produced in the bundle-sheat tissues have to be transported to the mesophyll for re-reduction to their reduced forms.

[45] Yang C Q, Liu W N, Zhao Z H, Wu H Y. Determination of the content of serum calcium with methylthymol blue as chromogenic reagent
Spectrosc Spectr Anal, 1998, 18:485-487.
[本文引用: 1]

[46] Murphy M E, Noack E. Nitic oxide assay using hemoglobin method
Methods Enzymol, 1994, 233:240-250.
[本文引用: 1]

[47] Xiong H, Li J, Liu P, Duan J, Zhao Y, Guo X, Li Y, Zhang H L, Ali J, Li Z C. Overexpression of OsMYB48-1, a novel MYB-related transcription factor, enhances drought and salinity tolerance in rice
PLoS One, 2014, 9:e92913.
DOIURL [本文引用: 1]

[48] Jung H, Kim J K, Ha S W. Use of animal viral IRES sequence makes multiple truncated transcripts without mediating polycistronic expression in rice
J Korean Soc Biol Chem, 2011, 54:678-684.
[本文引用: 1]

[49] Ermakova M, Danila F R, Furbank R T, Caemmerer S. On the road to C₄ rice: advances and perspectives
Plant J, 2020, 101:940-950.
DOI [本文引用: 1]
The international C-4 rice consortium aims to introduce into rice a high capacity photosynthetic mechanism, the C-4 pathway, to increase yield. The C-4 pathway is characterised by a complex combination of biochemical and anatomical specialisation that ensures high CO2 partial pressure at RuBisCO sites in bundle sheath (BS) cells. Here we report an update of the progress of the C-4 rice project. Since its inception in 2008 there has been an exponential growth in synthetic biology and molecular tools. Golden Gate cloning and synthetic promoter systems have facilitated gene building block approaches allowing multiple enzymes and metabolite transporters to be assembled and expressed from single gene constructs. Photosynthetic functionalisation of the BS in rice remains an important step and there has been some success overexpressing transcription factors in the cytokinin signalling network which influence chloroplast volume. The C-4 rice project has rejuvenated the research interest in C-4 photosynthesis. Comparative anatomical studies now point to critical features essential for the design. So far little attention has been paid to the energetics. C-4 photosynthesis has a greater ATP requirement, which is met by increased cyclic electron transport in BS cells. We hypothesise that changes in energy statues may drive this increased capacity for cyclic electron flow without the need for further modification. Although increasing vein density will ultimately be necessary for high efficiency C-4 rice, our modelling shows that small amounts of C-4 photosynthesis introduced around existing veins could already provide benefits of increased photosynthesis on the road to C-4 rice.

[50] 严婷, 李佳馨, 李霞, 谢寅峰. 转C₄型PEPC基因水稻非生物胁迫耐受性研究进展
淮阴工学院学报, 2019, 28:62-68.
[本文引用: 1]

Yan T, Li J X, Li X, Xie Y F. Research progress on abiotic stress tolerance of transgenic rice with C₄-PEPC gene
J Huaiyin Inst Technol, 2019, 28:62-68 (in Chinese with English abstract).
[本文引用: 1]

[51] 何亚飞, 李霞, 谢寅峰. 植物中糖信号及其对逆境调控的研究进展
植物生理学报, 2016, 52:241-249.
[本文引用: 1]

He Y F, Li X, Xie Y F. Research progress in sugar signal and its regulation of stress in plants
Plant Physiol J, 2016, 52:241-249 (in Chinese with English abstract).
[本文引用: 1]

[52] 张金飞, 李霞, 谢寅峰. 植物SnRKs家族在胁迫信号通路中的调节作用
植物学报, 2017, 52:346-357.
DOIURL [本文引用: 1]
蔗糖非发酵1 (SNF1)相关蛋白激酶家族(SnRKs)是植物胁迫响应过程中的一类关键蛋白激酶。在响应生物胁迫时, SnRKs可通过参与活性氧和水杨酸介导的信号转导途径, 增强植物对生物侵害的耐受性。在响应非生物胁迫时, SnRKs通过脱落酸(ABA)介导的信号通路, 增强植株对干旱、盐碱和高温等的耐受性; 且通过独立于ABA的信号通路, SnRKs可调控胞内能量状态, 维持离子平衡。该文总结了SnRKs蛋白激酶作为胁迫信号通路中的主要调节因子的最新研究进展, 并展望了未来的研究方向。
Zhang J F, Li X, Xie Y F. The function of sucrose nonfermenting-1 related protein kinases in stress signaling
Chin Bull Bot, 2017, 52:346-357 (in Chinese with English abstract).
URL [本文引用: 1]

[53] Carvalho R F, Szakonyi D, Simpson C G, Barbosa I C R, Browm J W S, Baena-González E, Duque P. The Arabidopsis SR45 splicing factor, a negative regulator of sugar signaling, modulates SNF1-related protein kinase 1 stability
Plant Cell, 2016, 28:1910-1925.
DOIURL [本文引用: 1]

[54] Sah S K, Reddy K R, Li J X. Abscisic acid and abiotic stress tolerance in crop plant
Front Plant Sci, 2016, 7:571.
[本文引用: 1]

[55] 刘小龙, 李霞, 钱宝云, 唐玉婷. 植物体内钙信号及其在调节干旱胁迫中的作用
西北植物学报, 2014, 34:1927-1936.
[本文引用: 1]

Liu X L, Li X, Qian B Y, Tang Y T. Ca²⁺ signal transduction and its regulation role under drought stress in plant
Bot Boreali-Occident Sin, 2014, 34:1927-1936 (in Chinese with English abstract).
URL [本文引用: 1]

[56] 刘小龙, 李霞, 钱宝云. 外源Ca²⁺对PEG处理下转C₄型PEPC基因水稻光合生理的调节
植物学报, 2015, 50:206-216.
URL [本文引用: 1]

Liu X L, Li X, Qian B Y. Photosynthetic and physiological regulation of C₄ phosphoenolpyruvate carboxylase transgenic rice (Oryza sativa) by exogenous Ca²⁺ under polyethylene glycol stress
Chin Bull Bot, 2015, 50:206-216 (in Chinese with English abstract).
URL [本文引用: 1]

[57] Xiong L M, Schumaker K S, Zhu J K. Cell signaling during cold, drought, and salt stress
Plant Cell, 2002, 14:165-183.
DOIURL [本文引用: 1]

[58] Palusa S G, Ali G S, Reddy A S. Alternative splicing of pre-mRNAs of Arabidopsis serine/arginine rich proteins: regulation by hormones and stresses
Plant J, 2007, 49:1091-1107.
DOIURL [本文引用: 1]

[59] Wang Y, Zhou D, Wang S, Yang L. Large scale detection and application of expressed sequence tag single nucleotide polymorphisms in Nicotiana
Genet Mol Res, 2015, 14:7793-7800.
DOIPMID [本文引用: 1]
Single nucleotide polymorphisms (SNPs) are widespread in the Nicotiana genome. Using an alignment and variation detection method, we developed 20,607,973 SNPs, based on the expressed sequence tag sequences of 10 Nicotiana species. The replacement rate was much higher than the transversion rate in the SNPs, and SNPs widely exist in the Nicotiana. In vitro verification indicated that all of the SNPs were high quality and accurate. Evolutionary relationships between 15 varieties were investigated by polymerase chain reaction with a special primer; the specific 302 locus of these sequence results clearly indicated the origin of Zhongyan 100. A database of Nicotiana SNPs (NSNP) was developed to store and search for SNPs in Nicotiana. NSNP is a tool for researchers to develop SNP markers of sequence data.

[60] Golisz A, Sikorski P J, Kruszka K, Kufel J. Arabidopsis thaliana LSM proteins function in mRNA splicing and degradation
Nucleic Acids Res, 2013, 41:6232-6249.
DOIPMID [本文引用: 1]
Sm-like (Lsm) proteins have been identified in all organisms and are related to RNA metabolism. Here, we report that Arabidopsis nuclear AtLSM8 protein, as well as AtLSM5, which localizes to both the cytoplasm and nucleus, function in pre-mRNA splicing, while AtLSM5 and the exclusively cytoplasmic AtLSM1 contribute to 5'-3' mRNA decay. In lsm8 and sad1/lsm5 mutants, U6 small nuclear RNA (snRNA) was reduced and unspliced mRNA precursors accumulated, whereas mRNA stability was mainly affected in plants lacking AtLSM1 and AtLSM5. Some of the mRNAs affected in lsm1a lsm1b and sad1/lsm5 plants were also substrates of the cytoplasmic 5'-3' exonuclease AtXRN4 and of the decapping enzyme AtDCP2. Surprisingly, a subset of substrates was also stabilized in the mutant lacking AtLSM8, which supports the notion that plant mRNAs are actively degraded in the nucleus. Localization of LSM components, purification of LSM-interacting proteins as well as functional analyses strongly suggest that at least two LSM complexes with conserved activities in RNA metabolism, AtLSM1-7 and AtLSM2-8, exist also in plants.

[61] Zhang P, Deng H, Xiao F, Liu Y S. Alterations of alternative splicing patterns of Ser/Arg-Rich (SR) genes in response to hormones and stresses treatments in different ecotypes of rice
J Integr Agric, 2013, 12:737-748.
DOIURL [本文引用: 1]

[62] Cruz T M, Carvalho R F, Richardson D N, Duque P. Abscisic acid (ABA) regulation of Arabidopsis SR protein gene expression
Int J Mol Sci, 2014, 15:17541-17564.
DOIURL [本文引用: 1]

[63] Barta A, Kalyna M, Reddy A S. Implementing a rational and consistent nomenclature for serine/arginine-rich protein splicing factors (SR proteins) in plants
Plant Cell, 2010, 22:2926-2929.
DOIURL [本文引用: 1]

[64] Jeanneau M, Gerentes D, Foueillassar X, Zivy M, Vidal J, Toppan A, Perez P. Improvement of drought tolerance in maize: towards the functional validation of the Zm-Asr1 gene and increase of water use efficiency by over expressing C₄-PEPC
Biochimie, 2002, 84:1127-1135.
PMID [本文引用: 1]
Water availability is one of the major limiting factors for plant growth. Maize is particularly sensitive to water stress at reproductive stages with a strong impairment of photosynthesis and grain filling. Here, we describe the use of genetic transformation first to assess the role of a candidate gene Asr1-a putative transcription factor-as an explanation for genetically linked drought tolerance Quantitative Trait Loci (QTLs), and second to modify CO(2) fixation rates in leaves through changes of C(4) phosphoenolpyruvate carboxylase (C(4)-PEPC) activity. Transgenic Asr1 over-expressing lines show an increase in foliar senescence under drought conditions. The highest C(4)-PEPC overexpressing line exhibited an increase (+30%) in intrinsic water use efficiency (WUE) accompanied by a dry weight increase (+20%) under moderate drought conditions. Opposite effects were observed for transgenic plants under-expressing the corresponding proteins. The data presented here indicate the feasibility to increase the level of endogenous biochemical activities related to water economy and/or drought tolerance, and opens a way to develop maize varieties more tolerant to dry growing conditions.

[65] Marsh J T, Sullivan S, Hartwell J, Nimmo H G. Structure and expression of phosphoenolpyruvate carboxylase kinase genes in Solanaceae. A novel gene exhibits alternative splicing
Plant Physiol, 2003, 133:2021-2028.
DOIURL [本文引用: 1]