Arabidopsis Metalloprotease FtSH4 Regulates Leaf Senescence Through Auxin and Reactive Oxygen Species

doi:10.11983/CBB17044

Abstract

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

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

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB17044

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

Figures/Tables 7

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

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).

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).

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

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).

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).

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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