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The role of ANAC072 in the regulation of chlorophyll degradation during age- and dark-induced leaf senescence

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Abstract

Key message

ANAC072 positively regulates both age- and dark-induced leaf senescence through activating the transcription of NYE1.

Abstract

Leaf senescence is integral to plant development, which is age-dependent and strictly regulated by internal and environmental signals. Although a number of senescence-related mutants and senescence-associated genes (SAGs) have been identified and characterized in the past decades, the general regulatory network of leaf senescence is still far from being elucidated. Here, we report the role of ANAC072, an SAG identified through bioinformatics analysis, in the regulation of chlorophyll degradation during natural and dark-induced leaf senescence. The expression of ANAC072 was increased with advancing leaf senescence in Arabidopsis. Leaf degreening was significantly delayed under normal or dark-induced conditions in anac072-1, a knockout mutant of ANAC072, with a higher chlorophyll level detected. In contrast, an overexpression mutant, anac072-2, with ANAC072 transcription markedly upregulated, showed an early leaf-yellowing phenotype. Consistently, senescent leaves of the loss-of-function mutant anac072-1 exhibited delays in the decrease of photosynthesis efficiency of photosystem II (F v/F m ratio) and the increase of plasma membrane ion leakage rate as compared with corresponding leaves of wild-type Col-0 plants, whereas the overexpression mutant anac072-2 showed opposite changes. Our data suggest that ANAC072 plays a positive role during natural and dark-induced leaf senescence. In addition, the transcript level of NYE1, a key regulatory gene in chlorophyll degradation, relied on the function of ANAC072. Combining these analyses with electrophoretic mobility shift assay and chromatin immunoprecipitation, we demonstrated that ANAC072 directly bound to the NYE1 promoter in vitro and in vivo, so ANAC072 may promote chlorophyll degradation by directly upregulating the expression of NYE1.

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Acknowledgments

This work was supported by National Natural Science Foundation of China (31170218), Science and Technology Commission of Shanghai Municipality (13JC400900), National Key Project of Transgenic Variety Development of China (2014ZX08003-004), Fudan University, State Key Laboratory of Genetic Engineering.

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    1. State Key Laboratory of Genetic Engineering, Institute of Plant Biology, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai, 200433, China

      Shou Li, Jiong Gao, Lingya Yao, Guodong Ren, Xiaoyu Zhu, Shan Gao, Kai Qiu, Xin Zhou & Benke Kuai

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    1. Shou Li
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    Communicated by F. Sato.

    S. Li and J. Gao contributed equally to this work.

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    299_2016_1991_MOESM1_ESM.pdf

    Supplementary material 1 Fig. S1 ANAC072 gene overexpressed in anac072-2 gain of function mutant. qRT-PCR of ANAC072 transcript levels. ACT2 gene was an internal control for normalizing the variation in cDNA amounts used. Data are mean ± SD from three biological replicates. a Expression of ANAC072 during dark-induced senescence. The fourth leaves of Col-0 and anac072-2 were detached from 4-week-old plants and incubated at darkness for the indicated days. The expression of ANAC072 in Col-0 at 0 day was set to 1. b Expression of ANAC072 during natural senescence. The fourth leaves of Col-0 and anac072-2 were detached from plants at the indicated times. ACT2 gene was an internal control. The expression of ANAC072 in Col-0 at 4 weeks old was set to 1 (PDF 8 kb)

    299_2016_1991_MOESM2_ESM.pdf

    Supplementary material 2 Fig. S2 Expression of NYE1 in Col-0, anac072-1, and anac072-2 during natural senescence. The fourth leaves of Col-0, anac072-1, and anac072-2 were detached at the indicated times. qRT-PCR of ANAC072 transcript levels. ACT2 gene was an internal control. The expression of ANAC072 in Col-0 at 4 weeks old was set to 1. Data are mean ± SD from three biological replicates (PDF 6 kb)

    299_2016_1991_MOESM3_ESM.pdf

    Supplementary material 3 Fig. S3 Promoter sequence analysis of NYE1. 914-bp upstream of ATG was analyzed. Putative ANAC072 binding core motif was marked in bold and underlined (PDF 80 kb)

    299_2016_1991_MOESM4_ESM.pdf

    Supplementary material 4 Fig. S4 ANAC072 expression in ANAC072 genomic DNA complemented lines. The fourth rosette leaves of Col-0 and genomic DNA fragment containing ANAC072 complemented lines were detached from plants at 4 weeks old. After 5-day incubation in darkness, total RNA was extracted, and transcript levels of ANAC072 were determined by qRT-PCR. ACT2 gene was an internal control. The expression of ANAC072 in Col-0 was set to 1. Data are mean ± SD from three biological replicates. *P < 0.05, **P < 0.01 (t-test) (PDF 81 kb)

    299_2016_1991_MOESM5_ESM.pdf

    Supplementary material 5 Fig. S5 Expression of ORE1 and NAP in Co-0, anac072-1 and anac072-2 during natural and dark-induced senescence. For natural senescence, the fourth leaves of different lines were detached at the indicated times and used for transcript detection. For dark-induced senescence, the fourth leaves of different lines were detached from 4-week-old plants and incubated in darkness for the indicated days. qRT-PCR analysis of transcript levels. ACT2 gene was an internal control. Data are mean ± SD from three biological replicates. a Expression of ORE1 during natural senescence. The expression of ORE1 in Col-0 at 4 weeks old was set to 1. b Expression of ORE1 during dark-induced senescence. The expression of ORE1 in Col-0 at 0 day was set to 1. c Expression of NAP during natural senescence. The expression of NAP in Col-0 at 4 weeks old was set to 1. d Expression of NAP during dark-induced senescence. The expression of NAP in Col-0 at 0 day was set to 1 (PDF 15 kb)

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    Li, S., Gao, J., Yao, L. et al. The role of ANAC072 in the regulation of chlorophyll degradation during age- and dark-induced leaf senescence. Plant Cell Rep 35, 1729–1741 (2016). https://doi.org/10.1007/s00299-016-1991-1

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