李建鑫^,, 王文平, 胡璋健, 师恺^,浙江大学农业与生物技术学院,杭州 310058

Effects of Simulated Acid Rain Conditions on Plant Photosynthesis and Disease Susceptibility in Tomato and Its Alleviation of Brassinosteroid

LI JianXin^,, WANG WenPing, HU ZhangJian, SHI Kai^,College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058

通讯作者: * 师恺,E-mail： kaishi@zju.edu.cn

责任编辑: 赵伶俐
收稿日期:2020-07-4接受日期:2020-11-13网络出版日期:2021-04-16

基金资助:

浙江省自然科学基金****项目.LR19C150001 浙江省重点研发计划.2021C2040

Received:2020-07-4Accepted:2020-11-13Online:2021-04-16
作者简介 About authors
李建鑫,E-mail： 1123819253@qq.com。






摘要
【目的】在全球气候变化大背景下,酸雨沉降日益加剧,严重影响蔬菜等农作物的产量、品质和抗性。油菜素内酯（brassinosteroid,BR）是一类植物体中广泛存在的植物激素,具有广谱调控植物抗性的作用。研究外源油菜素内酯对模拟酸雨环境下蔬菜作物光合作用和病害发生的影响,明确其对酸雨条件下蔬菜危害的缓解效应,对蔬菜作物安全生产提供指导。【方法】本研究以番茄（Solanum lycopersium L.）‘合作903’为材料,在模拟酸雨（模拟酸雨1（simulated acid rain 1,SiAR1）：NH₄NO₃（1.3 g·L^-1）、MgSO₄·7H₂O（3.1 g·L^-1）、Na₂SO₄（2.5 g·L^-1）、KHCO₃（1.3 g·L^-1）、CaCl₂·2H₂O（3.1 g·L^-1）,用1 N H₂SO₄调节pH至3.0;模拟酸雨2（SiAR2）：杭州地区春季收集的雨水,pH调至3.0）和对照（喷施dH₂O）条件下研究酸雨对番茄叶片光合作用和Pseudomonas syringae pv. tomato DC3000（Pst DC3000）引发的细菌性叶斑病发生的影响,其次在模拟酸雨和对照条件下对番茄叶片外源施用BR,研究外源施用BR对番茄叶片的光合特性和细菌性叶斑病发生的缓解作用,为了研究BR缓解作用的内在机制,测定番茄叶片光合作用关键基因FBPase、SBPase、rbcS和抗病基因PR1、NPR1的表达以及抗氧化酶的活性。【结果】模拟酸雨导致番茄叶片净光合作用速率（Pn）、光系统II实际光化学效率（Φ_PSII）及光化学淬灭系数（qP）显著下降;模拟酸雨削弱了番茄对Pst DC3000的抗性,导致细菌性叶斑病发病率上升和菌落数显著增加。外源施用BR提高酸雨和对照条件下番茄叶片的光合作用,并有效增强了酸雨条件下番茄对Pst DC3000的抗性,使酸雨条件下叶片菌落数下降,光系统II实际光化学效率提高。探究BR缓解效应的内在机制发现,外源施用BR显著提高了番茄叶片光合作用关键基因FBPase、SBPase、rbcS和抗病基因PR1、NPR1等的表达,降低了膜脂质过氧化物丙二醛（MDA）的含量,并增加了愈创木酚过氧化物酶（G-POD）和过氧化氢酶（CAT）等抗氧化酶的活性,从而缓解模拟酸雨对番茄光合和抗病性的抑制作用。【结论】外源施用BR能够显著提高番茄内源光合相关基因和抗病基因的表达及抗氧化酶的活性,有效促进酸雨环境中番茄等园艺作物的生长和增强抗病性。
关键词： 番茄;酸雨;油菜素内酯;光合作用;细菌性叶斑病

Abstract
【Objective】 In the era of climate change, the acid rain deposition has become a global environmental issue, which seriously affects the yield, quality and disease incidence of vegetables and other crops. Brassinosteroids (BRs) are a group of plant hormones widely existing in plants, which regulate plant resistance to broad-spectrum environment stresses. The aim of this study was to investigate effects of BRs on plant photosynthesis and disease susceptibility in tomato under simulated acid rain conditions and its alleviation effect, so as to provide guidance for safety production of vegetable crop. 【Method】In this study, using tomato (Solanum lycopersium L.) cultivar ‘Hezuo 903’ as material, the effects of exogenous BR foliar spray on the photosynthetic characteristics and the incidence of bacterial leaf spot disease caused by Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) were studied under two levels of simulated acid rain including simulated acid rain 1 (SiAR2): NH₄NO₃ (1.3 g·L^-1),MgSO₄·7H₂O (3.1 g·L^-1),Na₂SO₄ (2.5 g·L^-1),KHCO₃ (1.3 g·L^-1),CaCl₂·2H₂O (3.1 g·L^-1), pH (3.0, adjusted by 1N H₂SO₄ ) and simulated acid rain 2 (SiAR2): rain from Hanzhou area in spring, pH (3.0, adjusted by 1N H₂SO₄), and spraying leaves with H₂O as control condition. The alleviation of exogenous BR were studied through spraying exogenous BR on tomato leaves under two levels of simulated acid rain and control conditions. To reveal the underlying mechanism of BR induced stress alleviation, the transcript abundance of photosynthesis-related genes (e.g. FBPase, SBPase, rbcS), defense-related genes (e.g. PR1 and NPR1), and the activity of antioxidant enzymes were measured. 【Result】The results showed that the phytotoxic effect of simulated acid rain on photosynthesis in tomato was mainly reflected by the decrease of net photosynthesis rate (Pn), the photosystem II photochemical efficiency (Φ_PSII), and the photochemical quenching coefficient (qP). The simulated acid rain increased tomato susceptibility to Pst DC3000, resulting in a significant increase in disease incidence and leaf bacterial population. However, the exogenous BR was able to enhance the leaf photosynthetic capacity and decrease the susceptibility of tomato to Pst DC3000 by reducing the leaf bacterial population under two levels of simulated acid rain and control conditions Furthermore, the exogenous BR treatment was able to protect plant photosynthesis and pathogen resistance from the damages caused by simulated acid rain. The BR pretreatment not only significantly increased the transcript abundance of photosynthesis-related genes (e.g. FBPase, SBPase, and rbcS) and defense-related genes (e.g. PR1 and NPR1), but also reduced the content of malondialdehyde and enhanced the activity of G-POD and CAT in tomato plants under simulated acid rain treatments. Thus, the exogenous BR alleviated the inhibition of simulated acid rain on tomato photosynthesis and disease resistance. 【Conclusion】It was concluded that exogenous BR could increase leaf photosynthesis, transcript abundance of photosynthesis, defense-related genes, and the activity of antioxidant enzymes under simulated acid rain, and could also improve the resistance of tomato plants to bacterial leaf pathogen.
Keywords：tomato;acid rain;brassinosteroid;photosynthesis;bacterial leaf spot


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本文引用格式
李建鑫, 王文平, 胡璋健, 师恺. 模拟酸雨对番茄光合作用和病害发生的影响及油菜素内酯对其缓解效应[J]. 中国农业科学, 2021, 54(8): 1728-1738 doi:10.3864/j.issn.0578-1752.2021.08.012
LI JianXin, WANG WenPing, HU ZhangJian, SHI Kai. Effects of Simulated Acid Rain Conditions on Plant Photosynthesis and Disease Susceptibility in Tomato and Its Alleviation of Brassinosteroid[J]. Scientia Acricultura Sinica, 2021, 54(8): 1728-1738 doi:10.3864/j.issn.0578-1752.2021.08.012


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0 引言

【研究意义】酸雨（Acid rain）是指pH小于5.6的各种形式大气降水^[1]。在全球气候变化大背景下,近年来我国酸雨频现,严重影响了农作物的生长发育及其健康生产^[2,3]。但是,酸雨对农作物生长和抗性的影响,尤其是对蔬菜作物光合作用和病害发生的影响机理研究甚少,更缺乏针对酸雨危害的有效缓解途径。油菜素内酯（Brassinosteroid,BR）是甾醇类植物激素,具有高效调节生长发育的生理效应,并且能够提高植物对多种逆境的抗性^[4,5],但BR对酸雨胁迫下植物光合及抗病性是否具有调控作用尚未见报道。番茄作为一种重要蔬菜栽培种类和研究模式作物,以其为研究对象,探究外源BR对酸雨胁迫下番茄光合作用和病害发生的调控作用,对蔬菜作物的高产高效安全生产具有一定的理论和现实意义。【前人研究进展】番茄是我国种植面积最大的蔬菜作物之一,南方以露地栽培为主,北方以保护地栽培为主。近年来,我国番茄年播种面积已达两千万亩,露地播种面积约九百万亩,占总播种面积的45%。2018年,我国受酸雨影响的面积达到53万km²,酸雨造成的蔬菜减产损失超22亿元,其中酸雨污染主要分布在长江以南—云贵高原以东地区^[6]。酸雨影响植物的种子萌发、生长发育和抗性形成等一系列生命进程。其中,酸雨对植物生长的最大影响体现在其能破坏植株光合作用^[7,8]。有研究表明,长时间酸雨环境会导致番茄类囊体膜的扭曲,破坏植物叶绿体结构^[9,10]。用不同酸度的酸雨喷淋油菜发现,随着酸雨强度增加,油菜的光合作用被严重抑制^[11]。另一方面,酸雨对植物的影响还体现在其对植物抗病性的抑制上。有关研究表明,酸雨能通过影响水杨酸（salicylic acid,SA）信号路径调控辣椒与细菌性叶斑病病菌（Capsicum annuum）的相互作用^[12]。酸雨还能破坏小麦植株的角质层,导致小麦对条锈病（Puccinia striiformis f. sp. tritici）抗性的减弱^[13]。然而,酸雨会抑制香蕉叶斑病致病菌壳针孢菌（Mycosphaerella eumusae）孢子的萌发和繁殖,使酸雨区香蕉叶斑病发病率比非酸雨区发病率明显降低^[14]。但酸雨对植物与病原菌互作的影响尚未确定,有待深入研究。为了减少环境污染对农作物造成的危害,目前越来越多的植物生长调节剂被应用于促进生产,减少病害^[15,16]。随着研究的不断深入,人们发现BR不仅能够提高植物的光合作用,调控光形态建成^[17],还具有广谱抗性,能够显著提高植物对多种病原菌的抗性,包括丁香假单胞菌（Pseudomonas syringae）、白粉病菌（Oidium sp.）、稻瘟病菌（Magnaporthe grisea）、水稻黄单胞菌（Xanthomonas oryzae）^[18]。但是BR是否能够有效缓解酸雨对植物生长和抗病等进程带来的不良影响尚且未知。【本研究切入点】酸雨对作物造成了严重的危害,导致光合作用的抑制和抗病性降低。作为植物内源激素,BR在植物诸多逆境响应中发挥了重要的积极作用。因此,探究外源施用BR是否能够缓解模拟酸雨下光合作用的下降以及病害加重的效应,并解析其内在机制非常必要。【拟解决的关键问题】通过对植物光合作用和抗病响应相关参数的分析,评估模拟酸雨对番茄光合和病害抗性造成的影响,及BR对酸雨胁迫的缓解作用。进一步利用qRT-PCR技术分析外源施用BR对模拟酸雨下番茄内源光合和抗病相关基因表达的变化,并分析抗氧化酶活性的变化,深入挖掘BR缓解酸雨胁迫的内在机制,为缓解酸雨对蔬菜等园艺作物危害和园艺作物高产高效安全生产提供科学依据。

1 材料与方法

试验于2016—2019年在浙江大学蔬菜研究所温室及实验室进行。

1.1 试验材料

1.1.1 供试番茄（Solanum lycopersium L.）‘合作903’品种。番茄种子于55℃浸种10 min后置于28℃催芽2 d,待种子发芽后播种于含有草炭与蛭石为2﹕1（v/v）的50孔穴盘中,播种7 d后,将两片真叶充分展开的番茄幼苗移至营养钵（外径10 cm,高8.5 cm）中继续生长,控制生长环境条件为：温度25℃/21℃（昼/夜）,光周期为12 h /12 h（昼/夜）,光照强度为200 μmol·m^-2·s^-1,相对湿度60%左右。待番茄长至4叶1心时进行试验。模拟酸雨处理：选取生长状况一致的番茄植株分别喷施：模拟酸雨1（simulated acid rain 1,SiAR1）,参照VELIKOVA等^[19]酸雨的配方：NH₄NO₃（1.3 g·L^-1）、MgSO₄·7H₂O（3.1 g·L^-1）、Na₂SO₄（2.5 g·L^-1）、KHCO₃（1.3 g·L^-1）、CaCl₂·2H₂O（3.1 g·L^-1）,用1 N H₂SO₄调节pH至3.0;模拟酸雨2（SiAR2）,为杭州地区春季收集的雨水,pH调至3.0）;喷施dH₂O作为对照。每次均匀喷施番茄叶面,以喷满整个叶面而没有水滴落下为准。每天处理一次,连续处理4 d。外源BR处理：喷施模拟酸雨前24 h,用1 μmol?L^-1 BR喷施番茄植株叶片,以ddH₂O作为对照。

1.2 试验方法

1.2.1 光合作用参数测定 用LI-6400型光合荧光测量系统（美国LI-COR公司）测定番茄净光合作用速率（Pn）、光化学淬灭（qP）。测定条件：光强为800 μmol?m^-2?s^-1,CO₂浓度为400 μL·L^-1,叶面温度为（25±1.5）℃。叶绿素荧光使用Imaging-PAM调制荧光成像系统（IMAG-MAXI;Heinz Walz,Germany）进行测定。植株暗适应0.5 h后,光化光强度和饱和光强度分别设为280 μmol?m^-2?s^-1和4 000 μmol?m^-2?s^-1,光系统Ⅱ实际光化学效率计算公式为Φ_PSII=（Fm′-F）/Fm′^[20],其中,F为任意时间实际荧光产量,Fm′为最大荧光产量。

1.2.2 病原菌的培养与接种 丁香假单胞菌番茄变种（P. syringae pv. tomato DC3000,Pst DC3000）的培养参照THILMONY等^[21]的方法,将Pst DC3000菌种在含有25 mg·L^-1利福平的KB固体培养基上于28℃光照培养箱中培养2 d。挑取单菌落于同样浓度抗生素的KB液体培养基中于28℃、200 r/min扩大培养约8—12 h后,调浓度至OD₆₀₀=0.8—1.0,4 000×g、4℃下离心5 min,得到的沉淀即为菌体。将菌体用10 mmol?L^-1 MgCl₂ 清洗两次后重新悬浮,浓度调为OD₆₀₀= 0.01,加入有机硅至终浓度为0.03%。接病处理为将病原菌溶液均匀喷施番茄叶面,对照处理则喷施含有终浓度为0.03%有机硅的10 mmol?L^-1 MgCl₂溶液。

1.2.3 番茄植株感病评估 Pst DC3000细菌数量的计算参照KOCH等^[22]的方法,取直径1.0 cm接种过病菌的番茄叶圆片,每个处理3个生物学重复,每个重复6个叶圆片。将叶圆片在70%的酒精中浸泡10 s后,用dH₂O冲洗,置于含有500 μLMgCl₂（10 mmol?L^-1）的1.5 mL磨样管中,4 000 r/min,20 s充分研磨。吸取磨好的样品用10 mmol?L^-1的MgCl₂进行梯度稀释。取每个稀释液10 μL点在含有25 mg·L^-1利福平的KB固体培养基上,28℃培养2 d,取菌落数范围在“9—90”的点计算叶片菌落数（Colony-forming unit,CFU）的数量。台盼蓝染色参照LEGRAND等^[23]的方法并有所改进。将番茄叶圆片浸入65℃预热的染色液中,煮沸10 min后用保鲜膜包严,摇床上染色5—6 h。染色后将叶圆片置于脱色液中,脱色3—4次,每次洗涤5—6 h至叶片透明。透明叶片分别置于LEICA- DM400B显微镜下拍照。

1.2.4 抗氧化物酶活性及丙二醛（Malondialdehyde,MDA）含量测定 取0.3 g植物样品,加入3 mL 50 mmol?L^-1的PBS缓冲液,冰浴中研磨成匀浆,4℃下12 000 r/min离心20 min,取上清液用于抗氧化物酶活性测定。抗氧化酶活性及丙二醛含量测定参考陈建勋等^[24]的方法。过氧化物酶（Guaiacol peroxidase,G- POD）活性采用愈创木酚法测定,过氧化氢酶（Catalase,CAT）活性采用紫外吸收法,蛋白质含量测定参照BRADFORD^[25]的考马斯亮蓝法,MDA含量采用硫代巴比妥酸（Thiobarbituric acid,TBA）法测定^[26]。

1.2.5 植物总RNA提取和qRT-PCR检测 植物总RNA的提取参照天根生化公司（TIANGEN）试剂盒操作步骤;cDNA的合成参照TOYOBO公司反转录试剂盒操作步骤。

qRT-PCR在荧光定量PCR Light Cycler 480Ⅱ平台（Roche）上进行,反应条件参见AceQ qPCR SYBR Green Master Mix荧光染料试剂盒（Vazyme公司）,qRT-PCR引物的设计采用软件Primer 5.0,特异性引物序列如表1所示。

Table 1
表1
表1基因qRT-PCR特异性引物
Table 1Gene-specific primers designed for qRT-PCR

基因 Gene	上游引物Forward primer (5′-3′)	下游引物Reverse primer (5′-3′)
Actin	CATGTTCCCTGGTATTGCTG	GCCCTTTGAAATCCACATCT
rbcS	AGCCTGGGTTCGTATTATCG	CCTTCTGGCTTGTAGGCAAT
FBPase	CAAGAGCCCTTCAGAACACA	GCCTCCTCAGACTCACCTTC
SBPase	GGAAACAATCCGTCCTTGAT	GCCTTAAGCCTTGATGAACC
PR1	TCCGAGAGGCCAAGCTATAA	GACTGAGTTGCGCCAGACTA
NPR1	CATCCTTGCTGTTGATGGAC	TACCATCAAACACCTTCCGA

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PCR反应体系设置为：10 μL AceQ qPCR SYBR Green Master Mix,1 μL cDNA模板,上、下游引物各0.4 μL,7.2 μL ddH₂O。选取番茄的看家基因Actin作为荧光定量的内参指标,相对基因表达量参照LIVAK等^[27]的2^-ΔΔCT法进行计算。

1.3 统计方法

试验结果均为3次重复的平均值,利用Microsoft Excel 2016整理数据;运用SAS 9.1 Tukey法进行差异显著性分析;运用Origin 2017作图。

2 结果

2.1 模拟酸雨对番茄叶片光合作用和抗病性的影响

SiAR1和SiAR2处理的番茄植株相较于对照植株,其叶片的Pn分别下降了16.5%与18.4%（图1-A）,Φ_PSII分别降低15.5%与12.8%（图1-B）,而qP也分别下降了13.4%与10.3%（图1-C）。上述结果表明模拟酸雨处理导致番茄光合反应速率减弱,光能利用率降低,导致光合作用受到抑制。

图1

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图1模拟酸雨处理对番茄叶片光合作用的影响

H₂O：对照组,用dH₂O喷施叶片;SiAR1：SiAR1喷施叶片;SiAR2：SiAR2喷施叶片。不同小写字母表示处理间差异显著（P<0.05）。下同
Fig. 1Effects of simulated acid rain conditions on photosynthesis in tomato leaves

H₂O: Control, spraying leaves with dH₂O; SiAR1: spraying leaves with simulated acid rain 1; SiAR2: simulated acid rain 2. Different letters indicate significant differences (P<0.05). The same as below


利用模拟酸雨体外培养Pst DC3000病原菌,发现酸雨条件下病原菌12 h时生长量仅为对照的6.8%（图2-A）,表明模拟酸雨能显著抑制病原菌Pst DC3000增殖。但是,对模拟酸雨处理过的番茄植株接种Pst DC3000病原菌,接种2 d后观察发现SiAR1和SiAR2处理的番茄植株叶片发病比对照组更严重（图2-B）。此外,模拟酸雨加剧了Pst DC3000侵染后番茄叶片中死细胞的积累,使番茄植株发病率提高了2倍（图2-C）,同时也导致番茄叶片上的病原菌菌落数增加了约38.4%（图2-D）。综上,模拟酸雨处理削弱了番茄对Pst DC3000的抗性。

图2

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图2模拟酸雨对番茄对细菌性叶斑病发生的影响

Fig. 2Effect of simulated acid rain on plant defense to Pst DC3000 in tomato

2.2 BR对模拟酸雨环境下番茄光合作用的影响

在SiAR1和SiAR2两种模拟酸雨环境下,外源施用1 μmol?L^-1 BR后,番茄植株的Pn比对照分别提高了13.7％和14.8%（图3-A）,Φ_PSII分别提高了7.8%和11.1%（图3-B）,qP分别提高了12.8%和18.1%（图3-C）。综上,外源施用BR显著提高了模拟酸雨环境下番茄的光合效率。

图3

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图3外源BR对模拟酸雨环境下番茄光合系统的影响

Fig. 3Effects of exogenou BR on tomato photosynthesis system under simulated acid rain conditions

2.3 BR对模拟酸雨环境下番茄细菌性叶斑病抗性的影响

在非酸雨处理环境下,外源BR处理组细菌菌落数与对照组相比降低了3.8%;这一效应在模拟酸雨环境下更加明显,对番茄叶片进行SiAR1、SiAR2处理后外源施用BR,其细菌菌落数与对照相比都降低了约7.7%（图4-A）。从图4-B可以看出,接种Pst DC3000并酸雨处理的叶片Φ_PSII值均降低,而在酸雨条件下外源施用BR后能明显缓解因病原菌接种导致的Φ_PSII下降,这与细菌菌落数Φ_PSII成像图（图4-C）的结果表现一致。因此,外源施用BR使番茄叶片Pst DC3000菌落生长量降低,在一定程度上抑制了菌落的生长,并使病菌对Φ_PSII的影响减小,能够显著缓解细菌性叶斑病造成的Φ_PSII降低,尤其能够缓解酸雨环境下病菌对番茄的危害。

图4

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图4外源BR对模拟酸雨环境下番茄细菌性叶斑病抗性的影响

Fig. 4Effect of exogenous BR on tomato plant defense to bacterial leaf spot under simulated acid rain conditions

2.4 BR对模拟酸雨和Pst DC3000处理条件下光合作用和抗病相关基因表达的影响

进一步测定光合作用关键酶果糖-1,6-二磷酸酶（FBPase）、景天庚酮糖-1,7-二磷酸酯酶（SBPase）、核酮糖1,5-二磷酸羧化/加氧酶（Rubisco）等编码基因的表达量情况。模拟酸雨处理4 d后,FBPase、SBPase、rbcS等基因表达均受到了不同程度的抑制（图5）,外源施用BR后,酸雨条件下FBPase、SBPase、rbcS的表达量相对于未进行BR处理的对照增加了3—7倍（图5）。表明酸雨抑制了光合作用相关基因的表达,而酸雨环境下外源BR有助于激活光合作用相关基因的转录与表达,进而增强番茄的光合作用。同样,外源施用BR后,番茄在模拟酸雨条件下响应病原菌入侵时病程相关基因NPR1比对照上调了6—8倍（图6-A）,PR1则上调了4—13倍（图6-B）,BR诱导的内源抗病基因表达上调有助于提高番茄对Pst DC3000的抗性。

图5

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图5外源BR对在模拟酸雨下番茄叶片中光合相关基因表达的影响

Fig. 5Effects of exogenous BR on the transcript abundance of photosynthetic-related genes (A, FBPase; B, rbcS; C, SBPase) under simulated acid rain conditions

图6

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图6外源BR对在模拟酸雨下番茄叶片中抗病基因表达的影响

Fig. 6Effects of exogenous BR on the transcript abundance of defense-related genes (A, NPR1; B, PR1) under simulated acid rain conditions



此外,外源施用BR使两种模拟酸雨环境下MDA含量分别降低了22.8%和21.9%（图7-A）,使G-POD活性分别提高了19.6%和22.8%（图7-B）,CAT的活性分别提高29.1%和21.6%（图7-C）。以上结果表明,酸雨与BR都能够影响番茄的氧化还原稳态,外源施用BR能够显著降低番茄的膜脂过氧化水平,并在酸雨处理的基础上进一步提高抗氧化酶的活性,进而提高番茄植株对酸雨的抗性。

图7

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图7外源BR对番茄叶片中MDA含量和抗氧化酶活性的影响

Fig. 7Effects of exogenous BR on MDA content and antioxidant enzyme activities under simulated acid rain conditions

3 讨论

叶片是植物光合作用的主要器官,酸雨会直接影响叶片的生理状况和光合机构进而影响叶片的光合作用。前人研究表明,酸雨可以影响植物的叶绿素荧光参数、气孔运动、光合色素、光合蛋白酶等来危害植物的光合作用,使植物光合作用速率降低^[28]。本研究发现,模拟酸雨处理4 d使番茄叶片的Pn显著降低,这与姚桃峰等^[29]研究酸雨使拔节期小麦Pn显著下降的结果一致。研究发现,酸雨对不同生态型和不同生育期植物的Pn均有显著影响^[30]。酸雨不仅降低番茄的光合速率,还破坏番茄的光化学系统。本研究中酸雨使番茄PSII严重受损,类似研究也有报道,YU等^[31]研究黄瓜发现经酸雨处理3 h后,Φ_PSII随酸雨的pH减小呈显著下降趋势。GABARA等^[32]用pH 1.8的酸雨喷淋番茄,发现其PSII结构发生扭曲,酸雨还能导致活性氧积累来加重PSII的损伤^[33]。

酸雨对植物-病原菌互作的影响是复杂的,其引起的酸性环境能够显著抑制病菌的萌发和繁殖能力,本研究用酸雨体外培养病菌发现酸雨显著抑制了Pst DC3000病菌繁殖。有研究报道,酸雨处理使小麦叶枯病菌（Mycosphaerella graminicola）孢子萌发率显著降低,而且雨水中亚硫酸盐浓度的升高显著增加了小麦颖枯病菌（Phaeosphaeria nodorum）的感染率^[34]。酸雨还可能通过降低寄主植物免疫能力,增强病原菌致病力来削弱植物的抗病性。本研究中模拟酸雨处理后番茄侵染Pst DC3000,发现番茄感病更加严重,抗病性明显下降。袁志文^[35]研究发现,在邻近重酸雨区的马尾松落针病（Lophodermium pinastri）和赤落叶病（Meloderma desmazierii）的病情较无酸雨区更加严重,酸性降水增加了对表皮的侵蚀,植物的免疫系统受到损害,病菌扩散更为迅速,导致死细胞数量明显增多。上述研究结果说明酸雨影响植物光合作用的同时,加剧了细菌性叶斑病的发生。

BR作为植物体内重要的激素,能够调节植物叶片的发育,并参与光形态建成,直接和间接的调节植物的光合作用^[36]。本试验中外源施用BR能显著提高模拟酸雨环境下番茄叶片的Pn、qP和Φ_PSII,增加酸雨胁迫下PSII反应中心的光能利用率,有效缓解酸雨对光合作用的损害。JIN等^[37]在高温胁迫下对榕树进行BR处理,显著提高了抗氧化酶活性并有效减轻高温对PSII反应中心的损伤。研究发现酸雨胁迫与其他逆境胁迫类似,都会诱导植物产生过量活性氧,破坏光合结构,而外源BR有助于缓解活性氧等有害物质的积累。陆晓民^[38]用1 mg·L^-1 BR处理大豆,提高了渍水胁迫下大豆幼苗的叶绿素含量、脯氨酸含量,并抑制了MDA增生,降低了细胞膜透性,提高了大豆的光合作用。同样,KUREPIN等^[39]的研究也证实外源BR有助于缓解弱光胁迫,提高植物的光合作用。另外有研究表明,FBPase、SBPase、Rubisco都是光合碳代谢和光呼吸的关键酶,可以直接影响碳固定^[40]。本研究中酸雨抑制了FBPase、SBPase、rbcS的表达,而外源施用BR显著提高了这些基因的表达。光照条件下,BR可以通过BR信号转导因子BZR1（Brassinazole resistant 1）结合于光信号转导中的转录调节因子来调控光合基因的表达^[41]。在模拟酸雨条件下,外源施用BR能够诱导其信号元件参与到光信号途径中,调控光合基因的表达,进而提高植物的光合作用,增强PSII光化学反应能力,保护植物的光合结构^[42]。

BR除了具有促进植物生长发育等作用外,其信号途径在植物免疫反应中也发挥着积极的正调节作用^[43]。BRI1（Brassinosteroid insensitive 1）是细胞膜表面受体,它能够识别BR信号。有研究报道,烟草中BR参与到乙烯介导的对Pst DC3000的抗性,外源BR与乙烯均能提高烟草的抗性,而且BRI1的缺失也导致烟草对Pst DC3000更加敏感^[44]。本试验对番茄叶片外源施用BR后,显著抑制了病原菌在植物中的生长。有研究报道外施BR可以增强烟草抵御P. syringae和Oidium sp.侵染^[43];同样喷施BR后,葡萄对霜霉病（Plasmopara viticola）和灰霉病（Botrytis cinerea）的抗性都显著增强^[45]。本研究中,外源施用BR显著提高了番茄植株响应Pst DC3000时内源抗病基因PR1和NPR1的表达,这与拟南芥中BR信号能够提高抗病基因表达,激活免疫反应的模式类似^[46]。另外,多种逆境胁迫都会导致植物积累活性氧等有害物质,而植物通过提高抗氧化酶活性来缓解逆境胁迫,酸雨胁迫也不例外。本试验中不仅酸雨使抗氧化酶活性增加,外源施用BR也使酸雨条件下多种抗氧化酶活性提高,有助于激活番茄的抗性,使番茄更能抵御酸雨的侵害。

4 结论

模拟酸雨不仅导致番茄作物光合作用下降,还加剧了番茄细菌性叶斑病的发生。外源施用BR则能显著促进番茄内源光合与抗病相关基因的表达,提高抗氧化酶活性,有效缓解模拟酸雨对光合作用的危害,同时提高对细菌性叶斑病的抗性。因此,外源施用BR有助于提高植物对酸雨胁迫的抗性,研究结果为应对酸雨危害,探索酸雨胁迫缓解途径提供了科学依据。

参考文献 View Option 原文顺序
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被引期刊影响因子

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Fruit ripening is a unique plant developmental process with direct implications for our food supply, nutrition, and health. In contrast to climacteric fruit, where ethylene is pivotal, the hormonal control of ripening in nonclimacteric fruit, such as grape (Vitis vinifera), is poorly understood. Brassinosteroids (BRs) are steroidal hormones, essential for normal plant growth and development but not previously implicated in the ripening of nonclimacteric fruit. Here we show that increases in endogenous BR levels, but not indole-3-acetic acid (IAA) or GA levels, are associated with ripening in grapes. Putative grape homologs of genes encoding BR biosynthesis enzymes (BRASSINOSTEROID-6-OXIDASE and DWARF1) and the BR receptor (BRASSINOSTEROID INSENSITIVE 1) were isolated, and the function of the grape BRASSINOSTEROID-6-OXIDASE gene was confirmed by transgenic complementation of the tomato (Lycopersicon esculentum) extreme dwarf (dx/dx) mutant. Expression analysis of these genes during berry development revealed transcript accumulation patterns that were consistent with a dramatic increase in endogenous BR levels observed at the onset of fruit ripening. Furthermore, we show that application of BRs to grape berries significantly promoted ripening, while brassinazole, an inhibitor of BR biosynthesis, significantly delayed fruit ripening. These results provide evidence that changes in endogenous BR levels influence this key developmental process. This may provide a significant insight into the mechanism controlling ripening in grapes, which has direct implications for the logistics of grape production and down-stream processing.

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The microstructure of leaves and ultrastructure of chloroplasts were examined in tomato (Lycopersicon esculentum L.) plants treated with elevated temperature. Plants were exposed to 35A degrees C for 30 d after florescence. The plants grown continuously under 25A degrees C served as controls. Compared with the controls, the net photosynthetic rate (P (N)) in stressed plants decreased significantly. Stomatal conductance, intercellular CO2 concentrations, the rate of transpiration, and the limitation of stomatal conductance showed that the decrease in P (N) was caused mainly by nonstomatal restrictions. Meanwhile, stomata density increased significantly in the stressed plants. The stomata status of opening and closing became disorganized with a prolonged 35A degrees C exposure. The damage of chloroplast membrane occurred earlier and was more serious in the plants under elevated temperature. At the same time, the thylakoids were loosely distributed with lesser grana, but the number of lipid droplets increased in chloroplasts. The number of starch grains in chloroplasts increased first and then decreased. In addition, the length of the main nerve in leaves increased and the main vein showed distortion in the plants stressed by 35A degrees C. An increase was observed in the number of cells on the abaxial side of the main vein and these cells were overly congregated. The thickness of a vertical section became thinner in the stressed leaves. The cells of the upper epidermis thinned, and the ratio of palisade tissue to spongy tissue decreased. Generally, the photosynthetic apparatus of tomato changed significantly and the changed chloroplast ultrastructure might be one of the important reasons that caused the decrease of P (N) under 35A degrees C.

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Rare earth elements (REEs) pollution and acid rain are environmental issues, and their deleterious effects on plants attract worldwide attention. These two issues exist simultaneously in many regions, especially in some rice-growing areas. However, little is known about the combined effects of REEs and acid rain on plants. Here, the combined effects of lanthanum chloride (LaCl3), one type of REE salt, and acid rain on photosynthesis in rice were investigated. We showed that the combined treatment of 81.6 mu M LaCl3 and acid rain at pH 4.5 increased net photosynthetic rate (P-n), stomatic conductance (G(s)), intercellular CO2 concentration (C-i), Hill reaction activity (HRA), apparent quantum yield (AQY) and carboxylation efficiency (CE) in rice. The combined treatment of 81.6 mu M LaCl3 and acid rain at pH 3.5 began to behave toxic effects on photosynthesis (decreasing P-n, G(s), HRA, AQY and CE, and increasing C-i), and the maximally toxic effects were observed in the combined treatment of 2449.0 mu M LaCl3 and acid rain at pH 2.5. Moreover, the combined effects of LaCl3 and acid rain on photosynthesis in rice depended on the growth stage of rice, with the maximal effects occurring at the booting stage. Furthermore, the combined treatment of high-concentration LaCl3 and low-pH acid rain had more serious effects on photosynthesis in rice than LaCl3 or acid rain treatment alone. Finally, the combined effect of LaCl3 and acid rain on P-n in rice resulted from the changes in stomatic (G(s), C-i) and non-stomatic (HRA, AQY and CE) factors. (C) 2014 Elsevier Ltd.

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Aims Our objective was to investigate the effect of sulfur acid rain (SAR), nitric acid rain (NAR) and their mixture (MAR) on the physiology, growth and yield of the crop rape (Brassica napus).
Methods Fifteen 4 m × 5 m plots were distributed stochastically for exploring SAR, NAR and MAR stress, with a roughly 0.6 m spacing between plots to avoid interference. We used three replicate treatments of pH = 7.0, 1.5, 3.1, 4.1 and 5.1 and examined growth and development at 5-day intervals from budding to the end of flowering (March 10 to April 25, 2006). On April 12 for the peak period of flowering, we measured leaf cell membrane permeability,
contents of malondialdehyde (MDA) and photosynthesis pigment in leaves, photosynthesis speed, leaf injury rate and leaf area as physiological and growth parameters. On May 10, we measured the yield index during the ripening stage.
Important findings These kinds of acid rains can inhibit physiology, growth and yield with effects differing among treatments. At pH ≤ 4.1, SAR, NAR and MAR damage the membrane of rape leaves, decreasing photosynthetic pigment content and photosynthetic speed, thereby diminishing photosynthesis of the crop. For pH ≤ 3.1, leaf area is decreased and the leaf injury rate increases greatly. The value of pH = 4.1 can be used as the threshold for impact on rape yield. Over the range of pH = 1.5–7.0, leaf membrane permeability, MDA content and leaf injury rate follow the pattern of NAR > MAR > SAR and for the photosynthetic speed, content of photosynthesis pigment, leaf area and yield the order is SAR > MAR > NAR. At pH > 4.1, the differences between
these treatments of acid rain are insignificant. For pH ≤ 3.1, the stress differences become large (p < 0.05), with greater differences at stronger acidities, following the pattern NAR > MAR > SAR. This indicates the first two
treatments have stronger inhibiting effects on the physiology, growth and yield of rape.
MAI B R, ZHENG Y F, WU R J, LIANG J, LIU X. Effects of simulated sulfur-rich, nitric-rich and mixed acid rain on the physiology, growth and yield of rape (Brassica napus)
Chinese Journal of Plant Ecology, 2010,34(4):427-437. (in Chinese)
DOI:10.3773/j.issn.1005-264x.2010.04.008URL [本文引用: 1]
Aims Our objective was to investigate the effect of sulfur acid rain (SAR), nitric acid rain (NAR) and their mixture (MAR) on the physiology, growth and yield of the crop rape (Brassica napus).
Methods Fifteen 4 m × 5 m plots were distributed stochastically for exploring SAR, NAR and MAR stress, with a roughly 0.6 m spacing between plots to avoid interference. We used three replicate treatments of pH = 7.0, 1.5, 3.1, 4.1 and 5.1 and examined growth and development at 5-day intervals from budding to the end of flowering (March 10 to April 25, 2006). On April 12 for the peak period of flowering, we measured leaf cell membrane permeability,
contents of malondialdehyde (MDA) and photosynthesis pigment in leaves, photosynthesis speed, leaf injury rate and leaf area as physiological and growth parameters. On May 10, we measured the yield index during the ripening stage.
Important findings These kinds of acid rains can inhibit physiology, growth and yield with effects differing among treatments. At pH ≤ 4.1, SAR, NAR and MAR damage the membrane of rape leaves, decreasing photosynthetic pigment content and photosynthetic speed, thereby diminishing photosynthesis of the crop. For pH ≤ 3.1, leaf area is decreased and the leaf injury rate increases greatly. The value of pH = 4.1 can be used as the threshold for impact on rape yield. Over the range of pH = 1.5–7.0, leaf membrane permeability, MDA content and leaf injury rate follow the pattern of NAR > MAR > SAR and for the photosynthetic speed, content of photosynthesis pigment, leaf area and yield the order is SAR > MAR > NAR. At pH > 4.1, the differences between
these treatments of acid rain are insignificant. For pH ≤ 3.1, the stress differences become large (p < 0.05), with greater differences at stronger acidities, following the pattern NAR > MAR > SAR. This indicates the first two
treatments have stronger inhibiting effects on the physiology, growth and yield of rape.

[12] DUTTA B, LANGSTON D B, LUO X, CARLSON S, KICHLER J, GITAITIS R. A risk assessment model for bacterial leaf spot of pepper (Capsicum annuum), caused by Xanthomonas euvesicatoria, based on concentrations of macronutrients, micronutrients, and micronutrient ratios
Phytopathology, 2017,107(11):1331-1338.
DOI:10.1094/PHYTO-05-17-0187-RURLPMID:28686086 [本文引用: 1]
The phytopathogenic bacterium Xanthomonas euvesicatoria causes bacterial leaf spot (BLS) of pepper and has a worldwide distribution. BLS is difficult to control and an integrated management strategy that incorporates crop rotation, use of clean seed and clean plants, weed control, resistant varieties, applications of bactericides, biocontrol agents, and systemic acquired resistance (SAR) inducers is generally recommended. However, even with that arsenal of weapons, BLS can still be responsible for severe losses under favorable environmental conditions. Thus, additional tools need to be added to an overall integrated management strategy to combat BLS. In this article, we developed several models from 2012 to 2014 that were based on how macronutrients, micronutrients, and micronutrient ratios affect BLS severity. Factors used to select a model for validation included highly significant P values, high adjusted R(2) values, low variance inflation factor values (<5), root mean square error, Mallow's Cp, and high Akaike's information criterion correction values. In addition, salicylic acid (SA) concentrations and relative expression of nonexpresser pathogenesis-related gene1 (NPR1) and pathogenesis-related protein 1 (PR1) in pepper tissues were also considered in model selection. A model (ECGA1) consisting of concentrations of copper, manganese, potassium, and the iron/zinc ratio as independent variables was used for validation in three different commercial pepper fields in Georgia: Colquitt County and Worth County in 2015 and Tift County in 2016. When area under the disease progress curve (AUDPC) values for two field sites (Colquitt and Worth Counties) in 2015 were pulled together and plotted against ECGA1-predicted values for both sites, the resulting relationship was highly significant (P = 0.0001) with an R(2) value of 0.92. A significant relationship between observed AUDPC versus predicted values was also observed in Tift County in 2016 (P < 0.001; adjusted R(2) = 0.98). Relative gene expression of both NPR1 and PR1 genes was significantly (P < 0.01) higher in pepper grown in predicted low-risk sites compared with pepper from high-risk sites in Colquitt, Worth, and Tift Counties. Although BLS severity will fluctuate depending on environmental conditions, the data indicate that the level of risk at a particular location may be influenced by how macronutrient and micronutrient concentrations affect plant disease resistance genes in the SAR pathway.

[13] 王树和, 孙佩璐, 周文楠, 刘婷, 马占鸿. 模拟酸雨对小麦条锈病流行学组分的影响
植物保护学报, 2018,45(1):173-180.
[本文引用: 1]

WANG S H, SUN P L, ZHOU W N, LIU T, MA Z H. Effect of simulated acid rain on disease progress of wheat yellow rust
Journal of Plant Protection, 2018,45(1):173-180. (in Chinese)
[本文引用: 1]

[14] TRUJILLO E E, KADOOKA C Y, TANIMOTO V, BERGFELD S, SHISHIDO G, KAWAKAMI G. Effective biomass reduction of the invasive weed species banana poka by septoria leaf spot
Plant Disease, 2001,85(4):357-361.
DOI:10.1094/PDIS.2001.85.4.357URLPMID:30831966 [本文引用: 1]
Inoculations of Septoria passiflorae for biological control of banana poka (Passiflora tripartita var. tripartita) at different forest sites in Hawaii, Kauai, and Maui resulted in successful establishment of the Septoria leaf spot disease at all sites during 1996. Semi-annual monitoring of sites in 1997 revealed low disease incidence and no disease spread to adjacent non-inoculated plants. Site inspections in March 1998 revealed light disease epidemics causing visible defoliation at inoculated sites on Kauai and Maui. Banana poka biomass reduction at sites with light epidemics of the disease in Kauai and Maui were estimated to be less than 10% in 1998, whereas in 1999 biomass reduction ranged from 50 to 95%. Five of 11 inoculation sites in 1996 on the island of Hawaii showed no disease. These five sites on Kaloko had frequent acid rainfall averaging 3.2 pH, which inhibited spore germination and infection. Six sites, free of acid rain, three at Hilo Forest Reserve and three at Puuwaawaa Wildlife Sanctuary, had severe disease epidemics by 1998, and vine defoliation was >90%. Widespread epidemics of the disease occurred in 1999, resulting in estimated 80 to 95% biomass reductions in more than 2,000 hectares of native forest infested with banana poka.

[15] VERMA V, RAVINDRAN P, KUMAR P P. Plant hormone-mediated regulation of stress responses
BMC Plant Biology, 2016,16(1):86.
DOI:10.1186/s12870-016-0771-yURL [本文引用: 1]

[16] HAN S Y, LIU H, YAN M, QI F Y, WANG Y Q, SUN Z Q, HUANG B Y, DONG W Z, TANG F S, ZHANG X Y, HE G H. Differential gene expression in leaf tissues between mutant and wild-type genotypes response to late leaf spot in peanut (Arachis hypogaea L.)
PLoS ONE, 2017,12(8):e0183428.
DOI:10.1371/journal.pone.0184210URLPMID:28859154 [本文引用: 1]
BACKGROUND: Depression is under-diagnosed and under-treated in most areas of the US. New York City is currently looking to close gaps in identifying and treating depression through the adoption of a screening and collaborative care model deployed throughout the city. METHODS: We examine the cost-effectiveness of universal two-stage screening with the 2- and 9-item Patient Health Questionnaires (PHQ-2 and PHQ-9) in New York City followed by collaborative care for those who screen positive. We conducted microsimulations on hypothetical adult participants between ages 20 and 70. RESULTS: The incremental cost-effectiveness of the interventions over the average lifespan of a 20-year-old adult in NYC is approximately $1,726/QALY gained (95% plausible interval: cost-saving, $10,594/QALY gained). CONCLUSIONS: Two-stage screening coupled with collaborative care for depression in the clinical setting appears to be significantly less expensive than most clinical preventive interventions, such as HIV screening in high-risk patients. However, effectiveness is dependent on the city's ability to manage scale up of collaborative care models.

[17] AHAMMED G J, CHOUDHARY S P, CHEN S, XIA X, SHI K, ZHOU Y, YU J. Role of brassinosteroids in alleviation of phenanthrene- cadmium co-contamination-induced photosynthetic inhibition and oxidative stress in tomato
Journal of Experimental Botany, 2013,64(1):199-213.
DOI:10.1093/jxb/ers323URLPMID:23201830 [本文引用: 1]
Heavy metal pollution often occurs together with organic contaminants. Brassinosteroids (BRs) induce plant tolerance to several abiotic stresses, including phenanthrene (PHE) and cadmium (Cd) stress. However, the role of BRs in PHE+Cd co-contamination-induced stress amelioration is unknown. Here, the interactive effects of PHE, Cd, and 24-epibrassinolide (EBR; a biologically active BR) were investigated in tomato plants. The application of Cd (100 microM) alone was more phytotoxic than PHE applied alone (100 microM); however, their combined application resulted in slightly improved photosynthetic activity and pigment content compared with Cd alone after a 40 d exposure. Accumulation of reactive oxygen species and membrane lipid peroxidation were induced by PHE and/or Cd; however, the differences in effect were insignificant between Cd and PHE+Cd. The foliar application of EBR (0.1 microM) to PHE- and/or Cd-stressed plants alleviated photosynthetic inhibition and oxidative stress by causing enhancement of the activity of the enzymes and related transcript levels of the antioxidant system, secondary metabolism, and the xenobiotic detoxification system. Additionally, PHE and/or Cd residues were significantly decreased in both the leaves and roots after application of EBR, more specifically in PHE+Cd-stressed plants when treated with EBR, indicating a possible improvement in detoxification of these pollutants. The findings thus suggest a potential interaction of EBR and PHE for Cd stress alleviation. These results advocate a positive role for EBR in reducing pollutant residues for food safety and also strengthening phytoremediation.

[18] YU M H, ZHAO Z Z, HE J X. Brassinosteroid signaling in plant-microbe interactions
International Journal of Molecular Sciences, 2018,19(12):4091.
DOI:10.3390/ijms19124091URL [本文引用: 1]

[19] VELIKOVA V, YORDANOV I, KURTEVA M, TSONEV T. Effects of simulated acid rain on the photosynthetic characteristics of Phaseolus vulgaris L
Photosynthetica, 1998,34(4):523-535.
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[20] SUN Y J, GENG Q W, DU Y P, YANG X H, ZHAI H. Induction of cyclic electron flow around photosystem I during heat stress in grape leaves
Plant Science, 2017,256:65-71.
DOI:10.1016/j.plantsci.2016.12.004URLPMID:28167040 [本文引用: 1]
Photosystem II (PSII) in plants is susceptible to high temperatures. The cyclic electron flow (CEF) around PSI is thought to protect both PSII and PSI from photodamage. However, the underlying physiological mechanisms of the photosynthetic electron transport process and the role of CEF in grape at high temperatures remain unclear. To investigate this issue, we examined the responses of PSII energy distribution, the P700 redox state and CEF to high temperatures in grape leaves. After exposing 'Cabernet Sauvignon' leaves to various temperatures (25, 30, 35, 40 and 45 degrees C) in the light (600mumol photons m(-2)s(-1)) for 4h, the maximum quantum yield of PSII (Fv/Fm) significantly decreased at high temperatures (40 and 45 degrees C), while the maximum photo-oxidizable P700 (Pm) was not affected. As the temperature increased, higher initial rates of increase in post-illumination Chl fluorescence were detected, which were accompanied by an increase in high energy state quenching (qE). The chloroplast NAD(P)H dehydrogenase-dependent CEF (NDH-dependent CEF) activities were different among grape cultivators. 'Gold Finger' with greater susceptibility to photoinhibition, exhibited lower NDH-dependent CEF activities under acute heat stress than a more heat tolerant 'Cabernet Sauvignon'. These results suggest that overclosure of PSII reaction centers at high temperature resulted in the photoinhibition of PSII, while the stimulation of CEF in grape played an important role in the photoprotection of PSII and PSI at high temperatures through contributing to the generation of a proton gradient.

[21] THILMONY R, UNDERWOOD W, HE S Y. Genome-wide transcriptional analysis of the Arabidopsis thaliana interaction with the plant pathogen Pseudomonas syringae pv. tomato DC3000 and the human pathogen Escherichia coli O157:H7
The Plant Journal, 2006,46(1):34-53.
DOI:10.1111/j.1365-313X.2006.02725.xURLPMID:16553894 [本文引用: 1]
Pseudomonas syringae pv. tomato DC3000 (Pst) is a virulent pathogen that causes disease on tomato and Arabidopsis. The type III secretion system (TTSS) plays a key role in pathogenesis by translocating virulence effectors from the bacteria into the plant host cell, while the phytotoxin coronatine (COR) contributes to virulence and disease symptom development. Recent studies suggest that both the TTSS and COR are involved in the suppression of host basal defenses. However, little is known about the interplay between the host gene expression changes associated with basal defenses and the virulence activities of the TTSS and COR during infection. In this study, we used the Affymetrix full genome chip to determine the Arabidopsis transcriptome associated with basal defense to Pst DC3000 hrp mutants and the human pathogenic bacterium Escherichia coli O157:H7. We then used Pst DC3000 virulence mutants to characterize Arabidopsis transcriptional responses to the action of hrp-regulated virulence factors (e.g. TTSS and COR) during bacterial infection. Additionally, we used bacterial fliC mutants to assess the role of the pathogen-associated molecular pattern flagellin in induction of basal defense-associated transcriptional responses. In total, our global gene expression analysis identified 2800 Arabidopsis genes that are reproducibly regulated in response to bacterial pathogen inoculation. Regulation of these genes provides a molecular signature for Arabidopsis basal defense to plant and human pathogenic bacteria, and illustrates both common and distinct global virulence effects of the TTSS, COR, and possibly other hrp-regulated virulence factors during Pst DC3000 infection.

[22] KOCH E, SLUSARENKO A. Arabidopsis is susceptible to infection by a downy mildew fungus
The Plant Cell, 1990,2(5):437-445.
DOI:10.1105/tpc.2.5.437URLPMID:2152169 [本文引用: 1]
A population of Arabidopsis thaliana growing locally in a suburb of Zurich called Weiningen was observed to be infected with downy mildew. Plants were collected and the progress of infection was investigated in artificial inoculations in the laboratory. The plants proved to be highly susceptible, and pronounced intercellular mycelial growth, haustoria formation, conidiophore production, and sporulation of the causal organism Peronospora parasitica were all observed. The formation of oogonia, antheridia, and oospores also occurred. In contrast, Arabidopsis strain RLD was resistant to infection and none of the above structures was formed. The fungus was localized very soon after penetration of RLD leaf cells, which responded with a typical hypersensitive reaction. The differential interaction of an isolate of P. parasitica with two strains of Arabidopsis opens up the possibility of cloning resistance determinants from a host that is very amenable to genetic and molecular analysis.

[23] LEGRAND C, BOUR J M, JACOB C, CAPIAUMONT J, MARTIAL A, MARC A, WUDTKE M, KRETZMER G, DEMANGEL C, DUVAL D, HACHE J. Lactate dehydrogenase (LDH) activity of the number of dead cells in the medium of cultured eukaryotic cells as marker
Journal of Biotechnology, 1992,25(3):231-243.
DOI:10.1016/0168-1656(92)90158-6URLPMID:1368802 [本文引用: 1]
One significant problem in monitoring a culture's evolution is to assess change in cell viability. We have demonstrated that LDH release could be a good indicator of cellular damage of many cell lines, especially during shear stress or sonication. Moreover, we have found a significant correlation between the number of dead cells, determined by Trypan Blue staining, and LDH activity measurements in the supernatant of hybridoma strains, whatever the culture conditions. We have also shown that when viability is still near 100% no LDH is released even at high cell concentrations. Therefore, LDH should serve as a potential marker of cell injury and death.

[24] 陈建勋, 王晓峰. 植物生理学实验指导(第二版). 广州: 华南理工大学出版社, 2006: 64-66.
[本文引用: 1]

CHEN J X, WANG X F. Guidance of Plant Physiological Experiment (2^nd edition)
Guangzhou: South China University of Technology Publishers, 2006: 64-66. (in Chinese)
[本文引用: 1]

[25] BRADFORD M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein- dye binding
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[26] HODGES D M, DELONG J M, FORNEY C F, PRANGE R K. Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds
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[27] LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method
Methods, 2001,25(4):402-408.
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The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.

[28] WYRWICKA A, SK?ODOWSKA M. Influence of repeated acid rain treatment on antioxidative enzyme activities and on lipid peroxidation in cucumber leaves
Environmental and Experimental Botany, 2006,56(2):198-204.
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[29] 姚桃峰, 王润元, 王鹤龄, 赵鸿. 拔节期模拟酸雨对春小麦叶片光合特性的影响
安徽农业科学, 2010,38(15):8069-8073.
[本文引用: 1]

YAO T F, WANG R Y, WANG H L, ZHAO H. Stress effects of simulated acid rain on photosynthetic characteristics of field-grown spring wheat at the jointing stage
Journal of Anhui Agricultural Sciences, 2010,38(15):8069-8073. (in Chinese)
[本文引用: 1]

[30] SUN Z G, WANG L H, CHEN M M, WANG L, LIANG C J, ZHOU Q, HUANG X H. Interactive effects of cadmium and acid rain on photosynthetic light reaction in soybean seedlings
Ecotoxicology and Environmental Safety, 2012,79:62-68.
DOI:10.1016/j.ecoenv.2011.12.004URL [本文引用: 1]
Interactive effects of cadmium (Cd2+) and acid rain on photosynthetic light reaction in soybean seedlings were investigated under hydroponic conditions. Single treatment with Cd2+ or acid rain and the combined treatment decreased the content of chlorophyll, Hill reaction rate, the activity of Mg2+-ATPase, maximal photochemical efficiency and maximal quantum yield, increased initial fluorescence and damaged the chloroplast structure in soybean seedlings. In the combined treatment, the change in the photosynthetic parameters and the damage of chloroplast structure were stronger than those of any single pollution. Meanwhile, Cd2+ and acid rain had the interactive effects on the test indices in soybean seedlings. The results indicated that the combined pollution of Cd2+ and acid rain aggravated the toxic effect of the single pollution of Cd2+ or acid rain on the photosynthetic parameters due to the serious damage to the chloroplast structure. (C) 2011 Elsevier Inc.

[31] YU J Q, YE S F, HUANG L F. Effects of simulated acid precipitation on photosynthesis, chlorophyll fluorescence, and antioxidative enzymes in Cucumis sativus L
Photosynthetica, 2002,40(3):331-335.
DOI:10.1023/A:1022658504882URL [本文引用: 1]
The effects of simulated acid rain on gas exchange, chlorophyll fluorescence, and anti-oxidative enzyme activity in cucumber seedlings (Cucumis sativus L. cv. Jingchun No. 4) were investigated. Acid rain significantly reduced net photosynthetic rate and mainly non-stomatal factors contributed to the decrease of photosynthesis during the experimental period. The reduced photosynthesis was associated with a decreased maximal photochemical efficiency (F_v/F_m) and the average quantum yield of the photosystem 2 (PS2) reaction centres (_PS2). Meanwhile, acid rain significantly increased the activities of guaiacol peroxidase (GPX) and superoxide dismutase (SOD), but decreased the activity of catalase (CAT) together with an increased content of malonyldialdehyde (MDA), Hence the changes in photosynthesis in acid rain treatment might be a secondary effect of acidity damage probably due to lipid peroxidation of lipids and proteins in thylakoid membrane rather than direct effect on PS2 reaction centre.

[32] GABARA B, SK?ODOWSKA M, WYRWICKA A, GLI?SKA S, GAPI?SKA M. Changes in the ultrastructure of chloroplasts and mitochondria and antioxidant enzyme activity in Lycopersicon esculentum Mill. leaves sprayed with acid rain
Plant Science, 2003,164(4):507-516.
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[33] JOZEF K, BO?IVOJ K, MARTIN B, FRANTI?EK S, JOSEF H. Physiological responses of root-less epiphytic plants to acid rain
Ecotoxicology, 2011,20(2):348-357.
DOI:10.1007/s10646-010-0585-xURL [本文引用: 1]
Selected physiological responses of Tillandsia albida (Bromeliaceae) and two lichens (Hypogymnia physodes and Xanthoria parietina) exposed to simulated acid rain (AR) over 3 months were studied. Pigments were depressed in all species being affected the most in Tillandsia. Amounts of hydrogen peroxide and superoxide were elevated and soluble proteins decreased only in AR-exposed Hypogymnia. Free amino acids were slightly affected among species and only glutamate sharply decreased in AR-exposed Xanthoria. Slight increase in soluble phenols but decrease in flavonoids in almost all species suggests that the latter are not essential for tolerance to AR. Almost all phenolic acids in Tillandsia leaves decreased in response to AR and activities of selected enzymes (phenylalanine ammonia-lyase, polyphenol oxidase, ascorbate- and guaiacol-peroxidase) were enhanced by AR. In lichens, considerable increase in metabolites (physodalic acid, atranorin and parietin) in response to AR was found but amount of ergosterol was unchanged. Macronutrients (K, Ca, Mg) decreased more pronouncedly in comparison with micronutrients in all species. Xanthoria showed higher tolerance in comparison with Hypogymnia, suggesting that could be useful for long-term biomonitoring.

[34] CHANDRAMOHAN P, SHAW M W. Sulphate and sulphurous acid alter the relative susceptibility of wheat to Phaeosphaeria nodorum and Mycosphaerella graminicola
Plant Pathology, 2013,62(6):1342-1349.
DOI:10.1111/ppa.12052URL [本文引用: 1]
The relative abundances of DNA of Mycosphaerella graminicola and Phaeosphaeria nodorum in archived wheat samples are closely correlated with UK anthropogenic emissions of oxidized sulphur over the last 160years. To test whether this could be a causal relationship, possible modes of action of sulphur on the two fungi were examined. Mycelial growth of the two fungi in solutions of sulphurous acid was similar. Sulphurous acid at pH 4 reduced percentage germination of P.nodorum conidia more strongly than M.graminicola conidia. In spray inoculations of wheat cv. Squarehead's Master, Cappelle Desprez and Riband with water or sulphurous acid (pH 4), the ratio of leaves infected by P.nodorum to leaves infected by M.graminicola was increased by factors of 25, 21 and 06, respectively at pH 4. The same three cultivars of wheat were grown in sand and vermiculite and fertilized with nutrient solution containing 25 or 05mm sulphate. Both pathogens infected less frequently at 25mm sulphate, by a factor of about 2. The severity of infection by M.graminicola was reduced on all three cultivars by a factor of about 45 at 25mm sulphate, but severity of P.nodorum was reduced only by a factor of about 2. Both elevated free sulphate concentrations in soil and sulphite in rainwater could therefore increase the prevalence of P.nodorum relative to M.graminicola, which is consistent with the historical changes in abundance.

[35] 袁志文. 酸雨与马尾松病害的发生调查初报
生态学杂志, 1988,7(5):50-52.
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YUAN Z W. Acid rain and the occurrence of diseases in Pinus massoniana
Chinese Journal of Ecology, 1988,7(5):50-52. (in Chinese)
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[36] YANG A J, ANJUM S A, WANG L, SONG J X, ZONG X F, LV J, ZOHAIB A, ALI I, YAN R, ZHANG Y, DONG Y F, WANG S G. Effect of foliar application of brassinolide on photosynthesis and chlorophyll fluorescence traits of Leymus chinensis under varying levels of shade
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[37] JIN S H, LI X Q, WANG G G, ZHU X T. Brassinosteroids alleviate high-temperature injury in Ficus concinna seedlings via maintaining higher antioxidant defence and glyoxalase systems
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Habitat fragmentation strongly affects the genetic diversity of plant populations, and this has always attracted much research interest. Although numerous studies have investigated the effects of habitat fragmentation on the genetic diversity of plant populations, fewer studies have compared species with contrasting breeding systems while accounting for phylogenetic distance. Here, we compare the levels of genetic diversity and differentiation within and among subpopulations in metapopulations (at fine-scale level) of two closely related Zingiber species, selfing Zingiber corallinum and outcrossing Zingiber nudicarpum. Comparisons of the genetic structure of species from unrelated taxa may be confounded by the effects of correlated ecological traits or/and phylogeny. Thus, we possibly reveal the differences in genetic diversity and spatial distribution of genetic variation within metapopulations that relate to mating systems. Compared to outcrossing Z. nudicarpum, the subpopulation genetic diversity in selfing Z. corallinum was significantly lower, but the metapopulation genetic diversity was not different. Most genetic variation resided among subpopulations in selfing Z. corallinum metapopulations, while a significant portion of variation resided either within or among subpopulations in outcrossing Z. nudicarpum, depending on whether the degree of subpopulation isolation surpasses the dispersal ability of pollen and seed. A stronger spatial genetic structure appeared within subpopulations of selfing Z. corallinum potentially due to restricted pollen flow and seed dispersal. In contrast, a weaker genetic structure was apparent in subpopulations of outcrossing Z. nudicarpum most likely caused by extensive pollen movement. Our study shows that high genetic variation can be maintained within metapopulations of selfing Zingiber species, due to increased genetic differentiation intensified primarily by the stochastic force of genetic drift among subpopulations. Therefore, maintenance of natural variability among subpopulations in fragmented areas is key to conserve the full range of genetic diversity of selfing Zingiber species. For outcrossing Zingiber species, maintenance of large populations is an important factor to enhance genetic diversity. Compared to outcrossing Z. nudicarpum, the subpopulation genetic diversity in selfing Z. corallinum was significantly lower, but the metapopulation genetic diversity did not differ. Most genetic variation resided among subpopulations in selfing Z. corallinum metapopulations, while a significant portion of variation resided either within or among subpopulations in outcrossing Z. nudicarpum, depending on whether the degree of subpopulation isolation surpasses the dispersal ability of pollen and seed. Our study shows that selfing Z. corallinum could maintain high genetic diversity through differentiation intensified primarily by the stochastic force of genetic drift among subpopulations at fine-scale level, but not local adaptation.

[38] 陆晓民, 陈勇, 贡伟, 陈运梅. 油菜素内酯对毛豆幼苗生长及其抗渍性的影响
生物学杂志, 2006,23(3):37-38.
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LU X M, CHEN Y, GONG W, CHEN Y M. Effect of brassinolide on the seedling growth and water logging resistance of soybean
Journal of Biology, 2006,23(3):37-38. (in Chinese)
[本文引用: 1]

[39] KUREPIN L V, JOO S H, KIM S K, PHARIS R P, BACK T G. Interaction of brassinosteroids with light quality and plant hormones in regulating shoot growth of young sunflower and Arabidopsis seedlings
Journal of Plant Growth Regulation, 2012,31(2):156-164.
DOI:10.1007/s00344-011-9227-7URL [本文引用: 1]
Sunflower hypocotyls elongate as light quality changes from the normal red to far-red (R/FR) ratio of sunlight to a lower R/FR ratio. This low R/FR ratio-induced elongation significantly increases endogenous concentrations of indole-3-acetic acid (IAA) and also of three gibberellins (GAs): GA(20), GA(1), and GA(8). Of these, it is likely GA(1) that drives low R/FR-induced growth. Brassinosteroids are also involved in shoot growth. Here we tested three R/FR ratios: high, normal, and low. Significant hypocotyl elongation occurred with this stepwise reduction in R/FR ratio, but endogenous castasterone concentrations in the hypocotyls remained unchanged. Brassinolide was also applied to the seedlings and significantly increased hypocotyl growth, though one that was uniform across all three R/FR ratios. Applied brassinolide increased hypocotyl elongation while significantly reducing (usually) levels of IAA, GA(20), and GA(8), but not that of GA(1), which remained constant. Given the above, we conclude that endogenous castasterone does not mediate the hypocotyl growth that is induced by enriching FR light, relative to R light. Similarly, we conclude that the hypocotyl growth that is induced by applied brassinolide does not result from an interaction of brassinolide with changes in light quality. The ability of applied brassinolide to influence IAA, GA(20), and GA(8) content, yet have no significant effect on GA(1), is hard to explain. One speculative hypothesis, though, could involve the brassinolide-induced reductions that occurred for endogenous IAA, given IAA's known ability to differentially influence the expression levels of GA20ox, GA3ox, and GA2ox, key genes in GA biosynthesis.

[40] YOSHIDA K, HISABORI T. Determining the rate-limiting step for light-responsive redox regulation in Chloroplasts
Antioxidants, 2018,7(11):153-160.
DOI:10.3390/antiox7110153URL [本文引用: 1]

[41] LUO X M, LIN W H, ZHU S W, ZHU J Y, SUN Y, FAN X Y, CHENG M L, HAO Y Q, OH E, TIAN M M, LIU L J, ZHANG M, XIE Q, CHONG K, WANG Z Y. Integration of light and brassinosteroid- signaling pathways by a GATA transcription factor in Arabidopsis
Developmental Cell, 2010,19(6):872-883.
DOI:10.1016/j.devcel.2010.10.023URL [本文引用: 1]

Summary

Light and brassinosteroid (BR) antagonistically regulate the developmental switch from etiolation in the dark to photomorphogenesis in the light in plants. Here, we identify GATA2 as a key transcriptional regulator that mediates the crosstalk between BR- and light-signaling pathways. Overexpression of GATA2 causes constitutive photomorphogenesis in the dark, whereas suppression of GATA2 reduces photomorphogenesis caused by light, BR deficiency, or the constitutive photomorphogenesis mutant cop1. Genome profiling and chromatin immunoprecipitation experiments show that GATA2 directly regulates genes that respond to both light and BR. BR represses GATA2 transcription through the BR-activated transcription factor BZR1, whereas light causes accumulation of GATA2 protein and feedback inhibition of GATA2 transcription. Dark-induced proteasomal degradation of GATA2 is dependent on the COP1 E3 ubiquitin ligase, and COP1 can ubiquitinate GATA2 invitro. This study illustrates a molecular framework for antagonistic regulation of gene expression and seedling photomorphogenesis by BR and light.

Graphical Abstract

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[42] WHITNEY S M, HOUTZ R L, ALONSO H. Advancing our understanding and capacity to engineer nature’s CO₂-sequestering enzyme, Rubisco
Plant Physiology, 2011,155(1):27-35.
DOI:10.1104/pp.110.164814URLPMID:20974895 [本文引用: 1]

[43] NAKASHITA H, YASUDA M, NITTA T, ASAMI T, FUJIOKA S, ARAI Y, SEKIMATA K, TAKATSUTO S, YAMAGUCHI I, YOSHIDA S. Brassinosteroid functions in a broad range of disease resistance in tobacco and rice
The Plant Journal, 2003,33(5):887-898.
DOI:10.1046/j.1365-313x.2003.01675.xURLPMID:12609030 [本文引用: 2]
Brassinolide (BL), considered to be the most important brassinosteroid (BR) and playing pivotal roles in the hormonal regulation of plant growth and development, was found to induce disease resistance in plants. To study the potentialities of BL activity on stress responding systems, we analyzed its ability to induce disease resistance in tobacco and rice plants. Wild-type tobacco treated with BL exhibited enhanced resistance to the viral pathogen tobacco mosaic virus (TMV), the bacterial pathogen Pseudomonas syringae pv. tabaci (Pst), and the fungal pathogen Oidium sp. The measurement of salicylic acid (SA) in wild-type plants treated with BL and the pathogen infection assays using NahG transgenic plants indicate that BL-induced resistance does not require SA biosynthesis. BL treatment did not induce either acidic or basic pathogenesis-related (PR) gene expression, suggesting that BL-induced resistance is distinct from systemic acquired resistance (SAR) and wound-inducible disease resistance. Analysis using brassinazole 2001, a specific inhibitor for BR biosynthesis, and the measurement of BRs in TMV-infected tobacco leaves indicate that steroid hormone-mediated disease resistance (BDR) plays part in defense response in tobacco. Simultaneous activation of SAR and BDR by SAR inducers and BL, respectively, exhibited additive protective effects against TMV and Pst, indicating that there is no cross-talk between SAR- and BDR-signaling pathway downstream of BL. In addition to the enhanced resistance to a broad range of diseases in tobacco, BL induced resistance in rice to rice blast and bacterial blight diseases caused by Magnaporthe grisea and Xanthomonas oryzae pv. oryzae, respectively. Our data suggest that BDR functions in the innate immunity system of higher plants including dicotyledonous and monocotyledonous species.

[44] XIONG J, HE R, YANG F, ZOU L, YI K, LIN H, ZHANG D. Brassinosteroids are involved in ethylene-induced Pst DC3000 resistance in Nicotiana benthamiana
Plant Biology, 2020,22(2):309-316.
DOI:10.1111/plb.13074URLPMID:31758615 [本文引用: 1]
Plant immunity is regulated by a huge phytohormone regulation network. Ethylene(ET) and brassinosteroids (BRs) play critical roles in plant response to biotic stress; however, the relationship between BR and ET in plant immunity is unclear. We used chemical treatments, genetic approaches and inoculation experiments to investigate the relationship between ET and BR in plant defense against Pst DC3000 in Nicotiana benthamiana. Foliar applications of ET and BR enhanced plant resistance to Pst DC3000 inoculation, while treatment with brassinazole (BRZ, a specific BR biosynthesis inhibitor) eliminated the ET induced plant resistance to Pst DC3000. Silencing of DWARF 4(DWF4, a key BR biosynthetic gene), BRASSINOSTEROID INSENSITIVE 1 (BRI1, aBR receptor) and BRASSINOSTEROID-SIGNALING KINASE 1 (BSK1, downstream of BRI1) also neutralised the ET-induced plant resistance to Pst DC3000. ET can induce callose deposition and reactive oxygen species (ROS) accumulation to resistPst DC3000, BRZ-treated and gene-silenced were completely eliminate this response. Our results suggest BR is involved in ET-induced plant resistance, the involvement of ET in plant resistance is possibly by the induction of callose deposition and ROS accumulation, in a BR-dependent manner.

[45] 刘庆. 24-表油菜素内酯诱导葡萄抵抗霜霉病和灰霉病的研究
[D]. 杨凌: 西北农林科技大学, 2016.
[本文引用: 1]

LIU Q. Effct of 24-epibrassinolide induced resistance of grapevine against downy mildew and grape against grey mould
[D]. Yangling: Northwest A&F University, 2016. (in Chinese)
[本文引用: 1]

[46] YAN H J, ZHAO Y F, SHI H, LI J, WANG Y C, TANG D Z. Brassinosteroid-signaling kinase1 phosphorylates MAPKKK5 to regulate immunity in Arabidopsis
Plant Physiology, 2018,176(4):2991-3002.
DOI:10.1104/pp.17.01757URLPMID:29440595 [本文引用: 1]
Arabidopsis (Arabidopsis thaliana) immune receptor FLAGELLIN SENSING2 (FLS2) rapidly forms a complex to activate pathogen-associated molecular pattern-triggered immunity (PTI) upon perception of the bacterial protein flagellin. The receptor-like cytoplasmic kinase BRASSINOSTEROID-SIGNALINGKINASE1 (BSK1) interacts with FLS2 and is critical for the activation of PTI. However, it is unknown how BSK1 transduces signals to activate downstream immune responses. We identified MEK Kinase5 (MAPKKK5) as a potential substrate of BSK1 by whole-genome phosphorylation analysis. In addition, we demonstrated that BSK1 interacts with and phosphorylates MAPKKK5. In the bsk1-1 mutant, the Ser-289 residue of MAPKKK5 was not phosphorylated as it was in the wild type. Similar to the bsk1 mutant, the mapkkk5 mutant displayed enhanced susceptibility to virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae pv tomato DC3000, and to the fungal powdery mildew pathogen Golovinomyces cichoracearum Phosphorylation of the Ser-289 residue is not involved in MAPKKK5-triggered cell death but is critical for MAPKKK5-mediated resistance to both bacterial and fungal pathogens. Furthermore, MAPKKK5 interacts with multiple MAPK kinases, including MKK1, MKK2, MKK4, MKK5, and MKK6. Overall, these results indicate that BSK1 regulates plant immunity by phosphorylating MAPKKK5 and suggest a direct regulatory mode of signaling from the immune complex to the MAPK cascade.