[Autophagy 5:4, 546-547; 16 May 2009]; ©2009 Landes Bioscience
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Autophagy regulates progression of programmed cell death during petal senescence in Japanese morning glory
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Article Addendum
Kenichi Shibuya,1,* Tetsuya Yamada2 and Kazuo Ichimura1
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Key words: autophagy, flower, InPSR26, morning glory, petal, programmed cell death, senescence
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1National Institute of Floricultural Science; National Agriculture and Food Research Organization (NARO); Tsukuba, Japan; 2Faculty of Agriculture; Tokyo University of Agriculture and Technology (TUAT); Tokyo, Japan
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expression during petal senescence in Japanese morning glory.5 In a recent functional study,7 we produced transgenic plants with reduced expression of PSRs. Transgenic plants with reduced InPSR26 expression (PRS26r lines) showed accelerated visible petal senescence. InPSR26 encodes a putative membrane protein with unknown function that is dominantly expressed in petal limbs. The transcript level of InPSR26 increases after flower opening and reaches a maximum a few hours prior to visible petal senescence. In the PSR26r plants, the collapse of the epidermal cells in the petals occurred in concert with petal wilting, which was earlier than in wild-type (WT) plants. Furthermore, ion and anthocyanin leakage was higher in the petal limbs of PSR26r transgenic plants than in those of WT plants at 8 h after flower opening, suggesting that there are more dead cells in the petals of transgenic plants. Hastened PCD in the petals of PSR26r transgenic plants is also reflected in the acceleration of DNA degradation. These results indicate that reduction of InPSR26 expression results in the acceleration of PCD during petal senescence and that InPSR26 plays a role in the delay of PCD in petal senescence.7 As autophagy is one of the main mechanisms responsible for the degradation and remobilization of macromolecules, it is thought to play an important role in PCD in senescing tissue.8 In the vacuoles of senescing morning glory petals, numerous vesicles and cytoplasmic components have been observed, indicating that autophagy occurs during petal senescence.9 We isolated three autophagy-related genes, InATG4, InATG8, and InPI3K, from Japanese morning glory and found that their transcript levels increased prior to visible senescence in petals. We visualized autophagy using monodansylcadaverine (MDC) staining, which has been shown to specifically stain autophagic structures in Arabidopsis (Arabidopsis thaliana).10 The percentage of protoplasts containing MDC-stained structures was greater in the petal limbs of opened flowers compared to buds, and it increased with the progression of petal senescence. These results suggest that autophagic activity is upregulated during flower opening and petal senescence, although the specificity of MDC staining in this cell type should be confirmed in another way. In the petal limbs of PSR26r lines with reduced expression of InPSR26, transcript levels of InATG8 and InATG4 and the percentage of protoplasts containing MDC-stained structures were significantly lower than in WT plants. These data suggest that the autophagic process is suppressed in these transgenic plants and that InPSR26 plays a role in the regulation of autophagy in PCD during petal senescence.7
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Petal senescence is a type of programmed cell death (PCD) that is tightly regulated by multiple genes. We recently reported that a putative membrane protein, InPSR26, regulates progression of PCD during petal senescence in Japanese morning glory (Ipomoea nil). Reduced InPSR26 expression in transgenic plants (PSR26r lines) resulted in accelerated petal senescence with hastened development of PCD symptoms, and transcript levels of autophagy-related genes were reduced in the petals. Autophagy visualized by monodansylcadaverine staining indicated reduced autophagic activity in the PSR26r plants. The results from our recent studies suggest that InPSR26 acts to delay the progression of PCD during petal senescence, possibly through regulation of the autophagic process. In this addendum, we discuss the role of autophagy in petal senescence as it relates to these findings.
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Petals in flowering plants senesce and are eventually removed after pollination or a certain period after flower opening, regardless of the pollination event.1 The time to petal senescence is species-specific and is thought to be regulated by programmed cell death (PCD).2-4 Japanese morning glory (Ipomoea nil) has ephemeral flowers that open in the morning and generally show visible petal senescence symptoms within 12 hours after flower opening. We previously showed that treatment with cycloheximide and actinomycin D, which inhibit protein and RNA synthesis, respectively, dramatically delays petal senescence, and also that DNA degradation and chromatin condensation occur during petals senesce in Japanese morning glory,5,6 indicating that petal senescence of this plant is regulated by PCD. To identify the genes regulating PCD in petals (PSRs: petal senescence-related genes), we isolated genes showing changes in
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*Correspondence to: Kenichi Shibuya; National Institute of Floricultural Science; National Agriculture and Food Research Organization (NARO); 2-1 Fujimoto; Tsukuba 305-8519 Japan; Tel.: +81.29.838.6821; Fax: +81.29.838.6841; Email:
[email protected] Submitted: 01/21/09; Revised: 02/18/09; Accepted: 02/27/09 Previously published online as an Autophagy E-publication: http://www.landesbioscience.com/journals/autophagy/article/8310 Addendum to: Shibuya K, Yamada T, Suzuki T, Shimizu K, Ichimura K. InPSR26, a putative membrane protein, regulates programmed cell death during petal senescence in Japanese morning glory. Plant Physiol 2009; 149:816–24; PMID: 19036837; DOI: 10.1104/pp.108.127415.
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Autophagy in petal senescence
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13. Hanaoka H, Noda T, Shirano Y, Kato T, Hayashi H, Shibata D, et al. Leaf senescence and starvation-induced chlorosis are accelerated by the disruption of an Arabidopsis autophagy gene. Plant Physiol 2002; 129:1181-93. 14. Thompson AR, Doelling JH, Suttangkakul A, Vierstra RD. Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways. Plant Physiol 2005; 138:2097-110. 15. Xiong Y, Contento AL, Bassham DC. AtATG18a is required for the formation of autophagosomes during nutrient stress and senescence in Arabidopsis thaliana. Plant J 2005; 42:535-46. 16. Bassham DC, Laporte M, Marty F, Moriyasu Y, Ohsumi Y, Olsen LJ, Yoshimoto K. Autophagy in development and stress responses of plants. Autophagy 2006; 2:2-11.
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Transgenic plants showing a reduction of InPSR26 expression showed suppressed autophagy, and PCD symptoms were accelerated. We found that treatment with an autophagy inhibitor, 3-methyladenine, hastens PCD in petals of Japanese morning glory.11 Taken together, these data suggest that autophagy is a mechanism that delays PCD during petal senescence. Suppression of autophagy results in acceleration of leaf senescence under nutrient-deficient conditions in Arabidopsis loss-of-function mutants for ATG genes,12-16 suggesting that autophagy plays a role in cell survival and delays cell death in leaf senescence. Here, in petal senescence, what is the role of autophagy and why does it delay cell death in petals which will eventually be discarded? One interpretation is that autophagy in plants is essential for the recovery and translocation of nutrients from dying tissues to growing tissues, such as developing seeds, in that it delays precocious death in order for this transfer to be accomplished (Fig. 1). Indeed, PSR26r transgenic plants produced fewer seeds per flower compared to WT plants.7 Petals may play a role as sinks of nutrients in addition to attracting pollinators in some plants, and autophagy is likely to play an important role in nutrient recycling and relocation to developing seeds.
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Figure 1. A PCD model of petal senescence. Autophagy is induced in PCD during petal senescence and apparently delays cell death. If autophagic processes are suppressed, precocious cell death occurs, resulting in an acceleration of petal senescence. InPSR26 is involved in the regulation of autophagy in PCD during petal senescence.
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References
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