拟南芥金属蛋白酶FtSH4通过生长素与活性氧调控叶片衰老

doi:10.11983/CBB17044

摘要/Abstract

摘要：

植物金属蛋白酶FtSH基因家族在拟南芥(Arabidopsis thaliana)中有12个成员, 目前各基因的功能还不清楚。该文利用细胞生物学和遗传学方法初步分析了拟南芥FtSH4在叶片衰老中的功能。ftsh4-4突变体叶片中H₂O₂含量及细胞死亡率增加, 叶绿素含量降低; 此外, 突变体中过氧化物酶基因表达上调, 过氧化物酶活性增加, 出现早衰表型。外源抗氧化剂AsA、内源和外源生长素能够通过降低ftsh4-4体内H₂O₂含量、过氧化物酶基因的表达及过氧化物酶活性, 恢复ftsh4-4叶片的衰老表型。ftsh4-4突变体中生长素响应因子基因ARF2和ARF7上调表达, 外源生长素和抗氧化剂能够降低ARF2和ARF7的表达, 并且ARF2突变能够降低ftsh4-4的H₂O₂含量并恢复其早衰表型。以上结果表明, FtSH4基因通过生长素与活性氧在调控植物叶片衰老中起重要作用。

关键词: FtSH4,, 拟南芥,, 活性氧,, 生长素,, 叶片衰老

Abstract:

The plant metalloproteinases FtSH gene family has 12 members in Arabidopsis, and their functions are still unclear. In the present study, we analyzed the function of FtSH4 on leaf senescence using cell biology and genetics methods. The ftsh4-4 mutant displayed a premature leaf senescence phenotype with increased H₂O₂content and cell death rate, decreased chlorophyll content, increased peroxidase gene expression and peroxidase activity. The ftsh4-4 leaf senescence phenotype could be rescued by applying the exogenous antioxidant AsA and endogenous or exogenous auxin by decreasing H₂O₂ content, peroxidase gene expression level and peroxidase activity. The expression of auxin response factor genes ARF2 and ARF7 was increased in the ftsh4-4 mutant and was reduced by exogenous auxin or AsA. Moreover, H₂O₂ content and the senescence phenotype of ftsh4-4 could be rescued by the arf2-8 mutant. These results indicate that FtSH4 gene plays an important role in the regulation of leaf senescence through auxin and reactive oxygen species.

Key words: FtSH4,, Arabidopsis,, reactive oxygen species,, auxin,, leaf senescence

张盛春, 李清明, 阳成伟. 拟南芥金属蛋白酶FtSH4通过生长素与活性氧调控叶片衰老. 植物学报, 2017, 52(4): 453-464.

Shengchun Zhang, Qingming Li, Chengwei Yang. Arabidopsis Metalloprotease FtSH4 Regulates Leaf Senescence Through Auxin and Reactive Oxygen Species. Chinese Bulletin of Botany, 2017, 52(4): 453-464.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB17044

https://www.chinbullbotany.com/CN/Y2017/V52/I4/453

图/表 7

表1 引物序列

Table 1 Primers used in this study

Primer name	Primer sequence (5ʹ-3ʹ)
PER33F	TCTTCTCCATCACTTCTTCTTA
PER33R	ATCCTCCAACACATATTCTCTA
PER37F	CGCCAACACTCTTTGACAACAAG
PER37R	ACTCATCCTTATCATTGCCTTCGC
ARF2F	AATATAGCACCTTCATCTCCT
ARF2R	ATCACACTCTACACTCTCAG
ARF7F	GCTAATGCTAATAACAGTCCTT
ARF7R	TCCACATTCTTCAGTCTCAA
SAG12F	ATGATGAGCAAGCACTGATGAAGG
SAG12R	TCCGTTAGTAGATTCGCCGTATCC
SAG13F	GCGACAACATAAGGACGAACTCTG
SAG13R	GAAGACAAAGAAATGCCACAAGCG
SAG101F	GGGATGAGAGACGATGTGAGAGAG
SAG101R	CGGGTGTTCATAAACTCGGTCAAG
SEN1F	GGACATCCGACTAGAGCCATCAAC
SEN1R	ATCGCCGTGAAGCCAGCAG
SEN4F	AACCGCCAATTTCCACACTTACTC
SEN4R	CTCTTGTTGCCCAATCGTCTGC
UBQ10F	CCGACTACAACATTCAGAAG
UBQ10R	TATCAATGGTGTCAGAACTCT

图1 FtSH4突变导致拟南芥叶片早衰^(A) ftsh4-4突变体表现出衰老表型(Bar=1 cm); (B) ftsh4-4突变体叶绿素含量降低; (C) ftsh4-4突变体相对电导率增加; (D) ftsh4-4突变体过氧化物酶活性增加。* 表示差异显著(P<0.05); ** 表示差异极显著(P<0.01) (Student’s t-test)。

Figure 1 FtSH4 mutation causes leaf senescence of Arabi- dopsis^(A) The phenotype of premature senescence observed in the ftsh4-4 mutant (Bar=1 cm); (B) The chlorophyll content decreased in ftsh4-4 mutant; (C) The relative electrolytic leakage increased in ftsh4-4 mutant; (D) The peroxidase activities increased in ftsh4-4 mutant. * indicates significant difference at P<0.05; ** indicates significant difference at P<0.01 (Student’s t-test).

图2 外源抗氧化剂AsA能恢复拟南芥ftsh4-4突变体叶片衰老表型^(A) 外源AsA处理能恢复ftsh4-4突变体早衰表型(Bar=1 cm); (B) 外源AsA处理能降低ftsh4-4突变体中H2O2含量(Bar=1 cm); (C) 外源AsA处理能减少ftsh4-4突变体中细胞死亡数目(Bar=1 cm); (D) 外源AsA处理能降低ftsh4-4突变体中过氧化物酶基因的表达。* 表示差异显著(P<0.05); ** 表示差异极显著(P<0.01) (Student’s t-test)。

Figure 2 Exogenous AsA rescued the leaf senescence phenotype of Arabidopsis ftsh4-4 mutant^(A) Exogenous AsA rescued the leaf senescence phenotype of ftsh4-4 mutant (Bar=1 cm); (B) Exogenous AsA reduced the H2O2 level of ftsh4-4 mutant (Bar=1 cm); (C) Exogenous AsA reduced the cell death of ftsh4-4 mutant (Bar=1 cm); (D) Exogenous AsA reduced the expression of peroxidase genes in ftsh4-4 mutant. * indicates significant difference at P<0.05; ** indicates significant difference at P<0.01 (Student’s t-test).

图3 外源生长素(IAA)能恢复拟南芥ftsh4-4突变体早衰表型^(A) 外源IAA处理能恢复ftsh4-4突变体早衰表型(Bar=1 cm); (B) 外源IAA处理能减少ftsh4-4突变体中细胞死亡数目

Figure 3 Exogenous IAA restored the leaf senescence phenotype of Arabidopsis ftsh4-4 mutant^(A) Exogenous IAA restored the leaf senescence phenotype of ftsh4-4 mutant (Bar=1 cm); (B) Exogenous IAA reduced the cell death of ftsh4-4 mutant

图4 增加内源生长素含量能恢复拟南芥ftsh4-4突变体早衰表型^(A) 增加内源IAA含量能恢复ftsh4-4突变体早衰表型(Bar=1 cm); (B) iaaM-ftsh4-4中细胞死亡数目减少(Bar=1 cm); (C) iaaM-ftsh4-4中叶绿素含量恢复到WT水平; (D) iaaM-ftsh4-4中相对电导率恢复到WT水平; (E) 内源激素降低ftsh4-4突变体中衰老基因SAG12、SAG13、SAG101、SEN1及SEN4的表达。* 表示差异显著(P<0.05); ** 表示差异极显著(P<0.01) (Student’s t-test)。

Figure 4 Increasing endogenous IAA restored the leaf senescence phenotype of Arabidopsis ftsh4-4 mutant^(A) Increasing endogenous IAA restored the leaf senescence phenotype of ftsh4-4 mutant (Bar=1 cm); (B) Cell death decreased in iaaM-ftsh4-4 (Bar=1 cm); (C) The chlorophyll content of iaaM-ftsh4-4 transgeneic line restored to the wild type level; (D) The relative electrolytic leakage of iaaM- ftsh4-4 transgeneic line restored to the wild type level; (E) Endogenous IAA decreased the expression of senescence-associated genes SAG12, SAG13, SAG101, SEN1 and SEN4 in ftsh4-4 mutant. * indicates significant difference at P<0.05; ** indicates significant difference at P<0.01 (Student’s t-test).

图5 ARF2参与FtSH4介导的拟南芥叶片衰老过程^(A) 生长素响应因子基因ARF2和ARF7在ftsh4-4中上调表达并受外源IAA和AsA抑制; (B) ARF2突变抑制了ftsh4-4的叶片衰老(Bar=1 cm); (C) ARF2突变使ftsh4-4中细胞死亡数目减少(Bar=1 cm); (D) ARF2突变使ftsh4-4中叶绿素含量增加; (E) ARF2突变使ftsh4-4中相对电导率降低; (F) ARF2突变使ftsh4-4中衰老相关标记基因SAG12、SAG13、SAG101、SEN1及SEN4下调表达。* 表示差异显著(P<0.05); ** 表示差异极显著(P<0.01) (Student’s t-test)。

Figure 5 ARF2 is involved in FtSH4-mediated leaf senescence of Arabidopsis^(A) The expression levels of ARF2 and ARF7 increased in the ftsh4-4 and were inhibited by the exogenous IAA and AsA; (B) ARF2 mutation rescued the leaf senescence of ftsh4-4 mutant (Bar=1 cm); (C) ARF2 mutation reduced the cell death of ftsh4-4 mutant (Bar=1 cm); (D) ARF2 mutation increased the chlorophyll content of ftsh4-4 mutant; (E) ARF2 mutation reduced the relative electrolytic leakage of ftsh4-4 mutant; (F) ARF2 mutation reduced the expression of senescence-associated genes SAG12, SAG13, SAG101, SEN1 and SEN4. * indicates significant difference at P<0.05; ** indicates significant difference at P<0.01 (Student’s t-test).

图6 生长素处理降低拟南芥ftsh4-4突变体中H2O2含量和过氧化物酶活性^(A) ARF2突变使ftsh4-4中H2O2含量减少(Bar=1 cm); (B) ARF2突变使ftsh4-4中过氧化物酶活性降低; (C)外施生长素能降低ftsh4-4突变体中H2O2含量(Bar=1 cm); (D) 增加内源生长素含量能降低ftsh4-4突变体中H2O2含量(Bar=1 cm); (E) 增加内源生长素含量能降低ftsh4-4突变体中过氧化物酶活性; (F) 外源生长素处理降低ftsh4-4突变体中过氧化物酶基因的表达。* 表示差异显著(P<0.05); ** 表示差异极显著(P<0.01) (Student’s t-test)。

Figure 6 IAA treatment reduces the H2O2 level and peroxidase activities of Arabidopsis ftsh4-4 mutant^(A) ARF2 mutation reduced the H2O2 level of ftsh4-4 mutant (Bar=1 cm); (B) ARF2 mutation reduced the peroxidase activities of ftsh4-4 mutant; (C) Exogenous IAA rescued the H2O2 level of ftsh4-4 mutant (Bar=1 cm); (D) Increasing endogenous IAA reduced the H2O2 level of ftsh4-4 mutant (Bar=1 cm); (E) Increasing endogenous IAA reduced the peroxidase activities of ftsh4-4 mutant; (F) Exogenous IAA reduced the peroxidase genes expression of ftsh4-4 mutant. * indicates significant difference at P<0.05; ** indicates significant difference at P<0.01 (Student’s t-test).

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