Plant Physiology Preview. Published on May 15, 2015, as DOI:10.1104/pp.15.00325
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Running head:
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Strigolactone and ethylene in leaf senescence
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Corresponding author:
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Makoto Kusaba
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Graduate School of Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima,
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Hiroshima 739-8526, Japan
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Tel: +81-82-424-7490
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E-mail: [email protected]
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Research Area:
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Signaling and Response
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Copyright 2015 by the American Society of Plant Biologists
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Title: Strigolactone regulates leaf senescence in concert with ethylene in Arabidopsis
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Hiroaki Ueda and Makoto Kusaba
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Graduate School of Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima,
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Hiroshima 739-8526, Japan
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One-sentence summary:
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Prolonged dark treatment induces ethylene synthesis and consequent induction of
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strigolactone synthesis in the leaf to promote leaf senescence.
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Footnotes
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Financial source: This study was supported by Core Research for Evolutional Science
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and Technology (to M.K.) and in part by a grant from JSPS KAKENHI (No. 26292006).
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Corresponding author:
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Makoto Kusaba
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E-mail: [email protected]
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Abstract
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Leaf senescence is not a passive degenerative process; it represents a process of nutrient
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relocation, in which materials are salvaged for growth at a later stage or to produce the
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next generation. Leaf senescence is regulated by various factors, such as darkness, stress,
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aging, and phytohormones. Strigolactone is a recently identified phytohormone, and it
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has multiple functions in plant development, including repression of branching.
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Although strigolactone is implicated in the regulation of leaf senescence, little is known
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about its molecular mechanism of action. In this study, strigolactone-biosynthesis
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mutant strains of Arabidopsis thaliana showed a delayed-senescence phenotype during
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dark incubation. The strigolactone biosynthesis genes MAX3 and MAX4 were drastically
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induced during dark incubation and treatment with the senescence-promoting
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phytohormone ethylene, suggesting that strigolactone is synthesized in the leaf during
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leaf senescence. This hypothesis was confirmed by a grafting experiment using max4 as
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the stock and Col as the scion, in which the leaves from the Col scion senesced earlier
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than max4 stock leaves. Dark incubation induced the synthesis of ethylene independent
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of strigolactone. Strigolactone biosynthesis mutants showed a delayed-senescence
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phenotype during ethylene treatment in the light. Furthermore, leaf senescence was
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strongly accelerated by the application of strigolactone in the presence of ethylene, and
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not by strigolactone alone. These observations suggest that strigolactone promotes leaf
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senescence by enhancing the action of ethylene. Thus, dark-induced senescence is
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regulated by a two-step mechanism: induction of ethylene synthesis and consequent
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induction of strigolactone synthesis in the leaf.
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INTRODUCTION
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Leaf senescence is an active nutrient-salvage system that recycles materials from
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dispensable leaves to the plant (Bleecker and Patterson, 1997; Lim et al., 2007). In
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senescing leaves, high molecular-weight compounds such as proteins and lipids are
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degraded and metabolized, and the resulting low molecular-weight compounds are
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transported to younger tissues or seeds. Leaf senescence is regulated by a complex
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system. Endogenous factors such as aging and flowering, dark treatment, nutrient
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starvation, and various stresses promote leaf senescence. Several phytohormones are
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also involved in regulating leaf senescence (Lim et al., 2007; Kusaba et al., 2013);
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ethylene, abscisic acid, jasmonic acid, and salicylic acid act as promoters, while
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cytokinins cause retardation. Although leaf senescence is induced by various factors, the
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resulting phenomena are the same, collectively known as the “senescence syndrome.”
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This includes leaf yellowing due to chlorophyll degradation, degeneration of the
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chloroplast structure and concomitant lipid degradation, degradation of photosynthetic
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proteins, and reduction in photosynthetic activity (Noodén, 2004). It is possible that the
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activation of “senescence signaling,” which integrates various senescence-regulating
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pathways, causes the senescence syndrome.
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Stay-green mutants retain green leaves under senescence-inducing conditions and
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are useful in the analysis of the molecular mechanism of leaf senescence (Thomas and
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Ougham, 2014). Several stay-green mutants have been isolated, of which many are
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transcription factor mutants (Kusaba et al., 2013; Penfold and Buchanan-Wollaston,
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2014). For example, the NAC transcription factor ORE1 is regulated at the
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transcriptional and post-transcriptional level, promoting leaf senescence via
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transcriptional modulation of target genes such as BFN1 and GLK1/2 (Kim et al., 2009;
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Matallana-Ramirez et al., 2013; Rauf et al., 2013). As mentioned previously, plant
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hormones play an important role in regulating leaf senescence. Ethylene, a key plant
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hormone that promotes leaf senescence (Jing et al., 2005; Li et al., 2013), binds to
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ethylene receptors on the endoplasmic reticulum (Shakeel et al., 2013). This binding
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represses the activity of ethylene receptors, resulting in the inactivation of the
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serine/threonine kinase CTR1, a negative regulator of ethylene signaling. This
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inactivation causes the translocation of the C-terminal end of the positive regulator,
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EIN2, to the nucleus and stabilizes the transcription factors EIN3 and EIL1, which in
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turn activate the expression of ethylene target genes. Ethylene-insensitive mutants
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exhibit a delayed-senescence phenotype, while the constitutive ethylene response
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mutant ctr1 and a line over-expressing EIN3 exhibit early-senescence phenotypes (Li et
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al., 2013; Kim et al., 2014; Xu et al., 2014).
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Strigolactone is a plant hormone that regulates various phenomena (Gomez-Roldan
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et al., 2008; Umehara et al., 2008; Xie and Yoneyama, 2010; Agusti et al., 2011; Seto et
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al., 2012; Ha et al., 2014; Waldie et al., 2014). It is involved in the suppression of shoot
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branching, root development, secondary growth, and drought tolerance. Strigolactone is
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synthesized from carotenoids by the carotenoid isomerase D27; carotenoid cleavage
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dioxygenases
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cytochrome P450, MAX1 (Abe et al., 2014; Seto et al., 2014; Seto and Yamaguchi,
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2014; Zhang et al., 2014). The strigolactone receptor with hydrolase activity,
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DAD2/D14, binds to strigolactone and interacts with the F-box protein MAX2, to
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induce strigolactone responses (Hamiaux et al., 2012; Jiang et al., 2013; Nakamura et al.,
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2013; Zhou et al., 2013).
MAX3/D17/RMS5/DAD3
and
MAX4/D10/RMS1/DAD1;
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and
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Grafting experiments using pea indicated that a novel long-distance signal
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synthesized in the root repressed branching in the shoot (Beveridge et al., 2009). Later,
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this signal substance was identified as strigolactone. Grafting experiments between
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wild-type Col and strigolactone biosynthesis mutants such as max1, max3, and max4 in
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Arabidopsis thaliana confirmed these observations (Domagalska and Leyser, 2011).
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Interestingly, grafting experiments using max1 as the stock and max3 or max4 as the
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scion showed that the mobile substance could be an intermediate metabolite of
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strigolactone, downstream of MAX3 and MAX4 and upstream of MAX1 (Booker et al.,
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2005). The mobile intermediate metabolite was suggested to be carlactone, an inactive
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precursor of strigolactone that is synthesized from all-trans-β-carotene by D27, MAX3,
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and MAX4 (Alder et al., 2012; Seto and Yamaguchi, 2014; Waldie et al., 2014).
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max2 is allelic to the stay-green mutation in Arabidopsis ore9, suggesting that
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strigolactone is involved in the regulation of leaf senescence (Woo et al., 2001;
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Stirnberg et al., 2002). This hypothesis was supported by studies on petunia and rice
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(Snowden et al., 2005; Yan et al., 2007; Yamada et al., 2014). However, little is known
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about the molecular mechanism underlying the regulation of leaf senescence by
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strigolactone. In the present study, we demonstrated that strigolactone promotes leaf
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senescence on the basis of analyses of strigolactone biosynthesis mutants and
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strigolactone-feeding experiments. Furthermore, our results suggest that the efficient
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progression of dark-induced leaf senescence requires both induction of ethylene
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synthesis and the consequent induction of strigolactone synthesis.
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RESULTS
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Strigolactone is involved in promoting leaf senescence
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ore9/max2 is a stay-green mutant, suggesting that strigolactone regulates leaf
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senescence.
However,
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strigolactone-independent phenomena in A. thaliana (Nelson et al., 2011; Shen et al.,
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2012; Walters et al., 2012; Scaffidi et al., 2013; Scaffidi et al., 2014). Therefore, we
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examined
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strigolactone-biosynthesis and strigolactone-insensitive mutants. The detached, young,
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fully expanded leaf (8th visible leaf from the top) of the wild-type Columbia ecotype
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(Col) turned yellow by the 7th day of dark treatment (DDT), but not in the light (Fig.
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1A, Supplemental Fig. 1A). In contrast, the strigolactone biosynthesis mutants max1-1,
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max3-9, and max4-11 and strigolactone-insensitive mutants At d14-1 and max2-4
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showed stay-green phenotypes, although the phenotype of max3-9 appeared slightly
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weak (Fig. 1A, B).
the
MAX2
senescence
is
reportedly
phenotype
during
involved
dark
in
several
incubation
of
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During dark incubation for 7 days, the leaves of strigolactone biosynthesis mutants
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max1-1, max3-9, and max4-11 retained high Fv/Fm values, indicating that the activity of
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photosystem II was unaffected, while those of Col exhibited a significant decrease in
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Fv/Fm values (Supplemental Figure S1B). Membrane ion leakage, an indicator of the
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loss of membrane integrity, began to increase at 6 DDT in Col, while that in the
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strigolactone biosynthesis mutants remained at a low level until 8 DDT (Supplemental
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Fig. S1C). These senescence characteristics confirmed that leaf senescence was delayed
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in strigolactone biosynthesis mutants as well as the strigolactone-insensitive mutant
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ore9/max2.
Next, we examined whether exogenously applied strigolactone could restore normal
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yellowing in the strigolactone biosynthesis mutants during dark-induced senescence
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(Fig. 1B). When treated with the artificial strigolactone analogue GR24, max1-1,
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max3-9,
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strigolactone-insensitive mutants max2-4 and At d14-1 did not, suggesting that
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strigolactone is required for normal leaf senescence in the dark.
and
max4-11
leaves
turned
yellow
at
7
DDT;
however,
the
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MAX2 is involved in not only strigolactone signaling but also karrikin signaling.
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The karrikin signaling component KAI2 is not involved in strigolactone signaling, but a
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stereoisomer of GR24 (GR24ent-5DS) signals through KAI2 (Nelson et al., 2011; Scaffidi
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et al., 2014; Waters et al., 2014). In our experiments, we used a racemic mixture of
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GR24 (rac-GR24), which includes GR24ent-5DS. We examined dark-induced leaf
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senescence of kai2-3; however, a delayed-senescence phenotype was not observed,
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confirming that strigolactone specifically regulates leaf senescence (Supplemental Fig.
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S2).
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ore9/max2 exhibits a delayed natural-senescence phenotype, suggesting that
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strigolactone is involved in regulating natural leaf senescence (Woo et al., 2001;
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Stirnberg et al., 2002). We evaluated natural senescence in 70-day-old plants (28 days
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after bolting) of Col, max4-11, max2-4, and ein3-1 eil1-3 (Supplemental Fig. S3A, B).
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Like max2-4, max4-11 also exhibited delayed yellowing during natural senescence.
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Crosstalk between strigolactone and ethylene
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The ethylene-insensitive mutants ein2-5 and ein3-1 eil1-3 exhibited delayed senescence
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in the dark (Fig. 1A). Ethylene plays an important role in the progression of senescence
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in the dark; therefore, we examined ethylene production by detached leaves during dark
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treatment (Fig. 2A). To minimize the effect of wounding on ethylene production, we
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incubated the detached leaves in the light for 24 h (0 day) before dark treatment. The
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amount of ethylene released from Col leaves increased at 1 DDT and peaked at 2 DDT
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(9.9-fold increase relative to that on 0 DDT). The increased rate of ethylene release was
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maintained until 5 DDT. In contrast, there was only a low level of ethylene release in 5
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days in the light. These results suggest that dark treatment induces ethylene synthesis
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and that the synthesized ethylene promotes leaf senescence by activating ethylene
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signaling.
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Next, we examined the role of strigolactone in ethylene-mediated leaf senescence.
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There was no significant difference in ethylene release between Col and max1-1 during
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dark incubation (Fig. 2A), suggesting that strigolactone does not promote ethylene
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synthesis.
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When detached leaves were treated with ethylene, Col leaves, but not ein2-5 and
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ein3-1 eil1-3 leaves, turned yellow within 7 days in the light (Fig. 2B, Supplemental Fig.
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S4A), confirming that ethylene promotes leaf senescence. Interestingly, the
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strigolactone biosynthesis and strigolactone-insensitive mutants exhibited delayed
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yellowing during ethylene treatment. Changes in chlorophyll content in the Col and
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mutant lines over time confirmed that most of the chlorophyll was degraded in Col, but
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there was little change in chlorophyll content in ein2-5 and max1-1 at 5 days after
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ethylene treatment (Fig. 2C). However, at 8 DDT, there was a clear decrease in
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chlorophyll content in max1-1, but not in ein2-5, suggesting that max1-1 is
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hyposensitive, rather than insensitive, to ethylene. ein2-5 and ein3-1 eil1-3 were green
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during 20 days ethylene treatment, suggesting that ein2-5 and ein3-1 eil1-3 are
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completely insensitive to ethylene with respect to the promotion of leaf senescence
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(Supplemental Fig. S4B).
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Young, fully expanded Col leaves turned yellow within 7 days of incubation with
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ethylene in the light, but not with strigolactone, suggesting that strigolactone does not
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have a potent senescence-inducing activity (Fig. 2D). Nonetheless, addition of
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strigolactone enhanced the action of ethylene in promoting leaf senescence (Fig. 2D).
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Similarly, in the presence of ethylene, but not strigolactone, there was a slight decrease
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in chlorophyll content at 5 days after ethylene treatment (Fig. 2E). Application of 0.25
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μM GR24 along with ethylene significantly increased chlorophyll degradation (SPAD
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value was significantly lower than that of the “ethylene only” control at 5% level by
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Student’s t-test). These findings suggest that there is a synergistic relationship between
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ethylene and strigolactone.
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The constitutive ethylene-response mutant ctr1 exhibits an early-senescence
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phenotype (Xu et al., 2014). In the light, most of the chlorophyll in detached leaves of
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ctr1-1 degraded within 5 days, but there was little chlorophyll degradation in the leaves
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of Col and only slight degradation of chlorophyll in the leaves of max1-1 ctr1-1 (Fig.
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2F). These findings suggest that strigolactone deficiency repressed ethylene-mediated
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senescence signaling activated in ctr1-1. Taken together, these observations suggest that
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strigolactone promotes leaf senescence by activating ethylene-mediated senescence
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signaling.
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Expression of strigolactone biosynthesis genes
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The transcript levels of MAX1, MAX3, and MAX4 increased during dark incubation (Fig.
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3A), especially those of the latter two genes. Transcripts of these two genes were hardly
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detected in the pre-senescent leaves, but their levels started to increase from 1 DDT and
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continued to increase until 7 DDT. In ein2-5, induction of MAX3 and MAX4 was
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severely repressed during dark incubation (Fig. 3A). Upregulation of MAX1 expression
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was also repressed in ein2-5 at 5 and 7 DDT. These results suggest that ethylene
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signaling is involved in the upregulation of MAX1, MAX3, and MAX4 during
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dark-induced senescence. Ethylene treatment also induced MAX1, MAX3, and MAX4 in
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the light (Fig. 3B). However, according to RNA sequencing and chromatin
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immunoprecipitation sequencing, none of these three genes appear to be direct targets of
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EIN3 (Chang et al., 2013). Among the three MAX genes, only MAX4 harbors a single
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consensus binding site for EIN3 (ATGTATCT) in the promoter region (from the
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initiation codon to the stop or initiation codon of upstream adjacent gene), consistent
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with the abovementioned observation (Boutror et al., 2010).
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Induction of MAX3 and MAX4 during dark incubation was also repressed in the
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strigolactone biosynthesis mutant max1-1 (Fig. 3C). This result raised the possibility
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that MAX3 and MAX4 are under positive feedback regulation by strigolactone. To test
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this possibility, we treated Col leaves with GR24 for 7 days in the light and examined
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the transcript levels of MAX3 and MAX4. However, neither MAX3 nor MAX4 was
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induced by GR24 (Fig. 3D). To evaluate the possibility that carlactone, which may be
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accumulated in max1-1, represses MAX3 and MAX4 expression in the dark-incubated
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max1-1 leaves, we examined MAX3 expression in max4-11 and MAX4 expression in
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max3-9. Neither MAX3 nor MAX4 was induced during dark incubation in max4-11 and
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max3-9, respectively, suggesting that repression of MAX3 and MAX4 in max1-1 is not
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due to accumulation of carlactone (Supplemental Fig. S5).
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Next, we analyzed the transcript levels of strigolactone biosynthesis genes in
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naturally senescent leaves. Transcript levels of MAX1, MAX3, MAX4, and At SGR1 were
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upregulated in senescing and fully senescent leaves, suggesting that MAX1, MAX3, and
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MAX4 play roles in natural senescence (Supplemental Fig. S3C). This result is
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consistent with the fact that strigolactone biosynthesis/strigolactone-insensitive mutants
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exhibited delayed leaf senescence under natural conditions (Supplemental Fig. S3A).
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Strigolactone is synthesized in the leaf during senescence
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Prominent induction of the strigolactone biosynthesis genes MAX3 and MAX4 in the
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leaf during dark incubation suggested that strigolactone is synthesized in the senescing
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leaf. Therefore, we conducted micro-grafting experiments and generated 2-shoot grafts,
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in which the scion did not have its own root (Notaguchi et al., 2009; Fig. 4A, B). Leaves
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of the stock and scion were detached from the grafted plant at 5 weeks after grafting and
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incubated in the dark. When max4-11 was used as the stock and Col as the scion, leaves
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from max4-11 stock exhibited delayed senescence relative to that of the scion,
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suggesting that the strigolactone concentration was higher in the detached leaves of Col
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than in those of max4-11 (Fig. 4C). Because no strigolactone was synthesized in the root
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of this grafted plant, it is likely that strigolactone was synthesized in Col leaves, but not
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in max4 leaves, during dark incubation. There was no obvious difference in the
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progression of senescence between leaves from the stock and those from the scion when
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Col was used as both the stock and scion (Fig. 4C). When max4-11 was used as the
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scion and Col as the stock, leaves from max4-11 stock exhibited delayed senescence;
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however, the difference was slightly less significant than that obtained in reciprocal
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grafting, suggesting that a small amount of strigolactone from the root may contribute to
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the promotion of leaf senescence (Fig. 4C).
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Ethylene-independent senescence-promoting pathways
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Since ethylene plays an important role in dark-induced senescence, ethylene-insensitive
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mutants exhibited a strongly delayed leaf-senescence phenotype during dark incubation.
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Nonetheless, leaves of the ethylene-insensitive mutants ein2-5 and ein3-1 eil1-3
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ultimately turned yellow in the dark (Fig. 5A, B, Supplemental Fig. S6A). The
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senescence parameters Fv/Fm and membrane ion leakage indicated the advanced
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progression of leaf senescence in ein3-1 eil1-3 leaves at 16 DDT (Supplemental Fig.
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S6B, C). In addition, MAX1, MAX3, and MAX4 were upregulated in the leaves of ein3-1
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eil1-3 at 16 DDT (Supplemental Fig. S7). These results suggest that an
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ethylene-independent pathway also contributes to the induction of strigolactone
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synthesis and promotion of leaf senescence in the dark.
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At 16 DDT, ein2-5 leaves had begun to turn yellow and those of max1-1 had died,
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but max1-1 ein2-5 leaves remained green. Therefore, max1-1 ein2-5 exhibited a
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stronger delayed-senescence phenotype than ein2-5 or max1-1 in the dark (Fig. 5A, B).
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This result suggests that strigolactone is also involved in an ethylene-independent
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senescence-promoting pathway in the dark.
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Next, we treated max1-1 ein2-5 with GR24 in the dark or light (Fig. 5C). SPAD
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value declined during a long dark incubation in max1-1 ein2-5, which is accelerated by
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the addition of GR24; this suggested that strigolactone promotes leaf senescence via an
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ethylene-independent, senescence-promoting pathway in the dark. In contrast, addition
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of GR24 did not promote leaf senescence of max1-1 ein2-5 in the light, suggesting that
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strigolactone alone does not promote leaf senescence in the light.
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Nonetheless, strigolactone promoted leaf senescence to an extent, particularly in
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older (lower) leaves of Col (11th and 12th leaves; Supplemental Fig. S8A, B). However,
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this promotion was not observed in ein3-1 eil1-3. It is possible that slightly activated
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ethylene signaling in the lower leaves contributed to the promotion of leaf senescence
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by strigolactone. Li et al. (2013) reported that the transcription activation activity of
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EIN3, which implies activation of ethylene signaling, gradually increases as leaves age.
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Consistent with this, we detected higher transcript levels of some ethylene-inducible
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genes directly targeted by EIN3 in the lower leaves than in the higher leaves
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(Supplemental Fig. S8C).
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DISCUSSION
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Strigolactone has been suggested to regulate leaf senescence because ore9/max2
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exhibits
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biosynthesis/strigolactone-insensitive
323
phenotypes and that exogenously applied strigolactone reversed the delayed-senescence
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phenotype of strigolactone biosynthesis mutants confirmed that strigolactone plays a
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role in regulating leaf senescence.
a
delayed-senescence
phenotype.
mutants
Observations
also
exhibited
that
strigolactone
delayed-senescence
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Strigolactone synthesis during leaf senescence
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Strigolactone and its mobile precursor are synthesized in the root and then transported
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to the shoot (Booker et al., 2005; Beveridge et al., 2009; Kohlen et al., 2011;
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Domagalska and Leyser, 2011; Seto and Yamaguchi, 2014). We observed that the
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strigolactone biosynthesis genes MAX3 and MAX4 were strongly induced in the leaf
334
during dark incubation. In the grafting experiment, when max4 was used as the stock,
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detached leaves from the Col scion senesced earlier than those from the max4 stock.
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These observations suggest that strigolactone synthesized in the leaf during senescence
337
promotes dark-induced leaf senescence, although we do not exclude the possibility that
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a small amount of strigolactone or its precursor is transported from the root to the leaf.
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Although previous grafting experiments have shown that the signal molecule that
340
represses branching is also produced in the aerial part (Beveridge et al., 2009;
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Domagalska and Leyser, 2011), very low transcript levels of MAX3 and MAX4 in the
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pre-senescent leaves suggest that little strigolactone is produced in pre-senescent leaves.
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Recently, Ha et al. (2014) reported that MAX3 and MAX4 in the leaf are induced by
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abscisic acid, NaCl, and dehydration stresses. The results reported by Ha et al. and those
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obtained in this study suggest that strigolactone is synthesized in the leaf under certain
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stress conditions.
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MAX3 and MAX4 were drastically induced by dark and ethylene treatments. In
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the ethylene-insensitive stay-green mutants ein2 and ein3 eil1, expression of MAX3 and
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MAX4 was severely repressed until 7 DDT; however, it was ultimately upregulated at 16
350
DDT, when leaf senescence proceeded even in ein3 eil1. This suggests that an
351
ethylene-independent pathway also contributes to the induction of MAX3 and MAX4. It
352
is possible that MAX3 and MAX4 are induced by the activation of senescence signaling
353
during leaf senescence, rather than by particular senescence-inducing pathways such as
354
ethylene signaling. This idea is consistent with the observation that the expression of
355
MAX3 and MAX4 was repressed during dark incubation in the delayed-senescence
356
mutant max1.
357
358
Actions of strigolactone and ethylene in leaf senescence
359
360
Dark incubation promoted ethylene synthesis and ethylene treatment induced leaf
361
senescence in the light, suggesting that ethylene plays a key role in the progression of
362
leaf senescence. Consistent with this idea, the ethylene-insensitive mutants ein2 and
363
ein3 eil1 exhibited a strong stay-green phenotype during dark incubation. The
364
strigolactone
365
delayed-senescence phenotype not only in the dark but also in response to ethylene
biosynthesis
and
strigolactone-insensitive
mutants
23
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
exhibited
a
366
treatment, suggesting that strigolactone biosynthesis and strigolactone-insensitive
367
mutants are hyposensitive to ethylene. Strigolactone drastically promoted the
368
senescence of young, fully expanded leaves in the presence of ethylene, although
369
strigolactone did not have potent senescence-promoting activity in the light. These
370
observations suggest that strigolactone promotes leaf senescence by enhancing the
371
action of ethylene. However, max1 ein2 exhibited a stronger delayed-senescence
372
phenotype than max1 or ein2 during dark incubation, suggesting that strigolactone is
373
also involved in an ethylene-independent, senescence-promoting pathway during dark
374
treatment. Consistent with this idea, strigolactone accelerated leaf senescence of max1
375
ein2 in the dark, but not in the light.
376
max1 and Col produced similar amounts of ethylene during dark incubation. This
377
observation may have an interesting implication. Positive regulators of leaf senescence
378
are often senescence-inducible. For example, the NAC transcription factor gene ORE1
379
is upregulated during leaf senescence (Kim et al., 2009). Leaf senescence was delayed
380
but ethylene production was not reduced in max1, suggesting that ethylene production
381
was not affected by the progression of senescence but was primarily regulated by dark
382
treatment. Thus, induction of ethylene production is thought to be an upstream trigger in
383
signal transduction of dark-induced senescence. Phytochrome-interacting factors
384
promote ethylene production in the dark (Khanna et al., 2007; Song et al., 2014). These
385
observations suggest that phytochromes regulate leaf senescence in the dark, at least
386
partly, via ethylene production (Sakuraba et al., 2014; Song et al., 2014).
387
Yamada
et
al.
(2014)
observed
that
strigolactone
biosynthesis
and
388
strigolactone-insensitive mutants in rice showed delayed-senescence phenotypes during
389
dark incubation, but the phenotypes were weaker than those of Arabidopsis mutants in
24
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
390
our study. It will be interesting to examine how ethylene acts during leaf senescence in
391
rice.
392
Kapulnik et al. (2011) reported an ethylene-strigolactone crosstalk in the regulation
393
of root hair elongation. In this case, strigolactone was thought to promote root hair
394
elongation by enhancing ethylene production, in contrast with our results with leaf
395
senescence. Thus, the crosstalk between strigolactone and ethylene observed in leaf
396
senescence is thought to be unique during the development of plants.
397
398
Multi-step regulation of leaf senescence
399
400
On the basis of all the results of this study, the crosstalk between strigolactone and
401
ethylene can be summarized as follows (Fig. 6): dark treatment induces ethylene
402
synthesis and activates ethylene signaling, resulting in the initial activation of
403
senescence signaling. Activation of senescence signaling induces the transcription of
404
MAX3 and MAX4, resulting in strigolactone synthesis in the leaf. Strigolactone further
405
activates
406
ethylene-independent, senescence-promoting pathways. Finally, activated senescence
407
signaling causes the senescence syndrome in the leaf.
senescence
signaling
by
enhancing
ethylene-dependent
and
408
409
410
CONCLUSIONS
411
412
Our results suggest that ethylene synthesis alone is not sufficient for leaf senescence and
413
that strigolactone synthesis induced by senescence signaling that integrates various
25
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
414
senescence-promoting pathways is also required for the efficient progression of leaf
415
senescence. If non-critical, ethylene-producing stimuli, such as transient stresses, result
26
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
416
in leaf senescence, then this unnecessary senescence must have disadvantages in terms
417
of fitness. The multi-step activation system involving ethylene synthesis and subsequent
418
strigolactone synthesis may prevent such unnecessary and uncontrolled leaf senescence.
419
Thus, the crosstalk between ethylene and strigolactone described in this study may be
420
an important part of a system that accurately regulates leaf senescence.
421
422
423
MATERIALS AND METHODS
424
425
Plant material and growth conditions
426
427
The A. thaliana mutant lines max1-1 (CS9564; Stirnberg et al., 2001), max3-9 (CS9567;
428
Booker et al.,2004), max2-4 (SALK_028336; Umehara et al., 2008), max4-11
429
(SALK_072570), At d14-1 (CS913109; Waters et al., 2012), ein2-5 (CS16771; Alonso
430
et al., 1999), ein3-1 (CS16710; Chao et al., 1997), eil1-1 (SALK_049679; Binder et al.,
431
2007), and ctr1-1 (CS16725; Kieber et al., 1993) were obtained from the Arabidopsis
432
Biological Resource Center. kai2-3 (CS25712) carries a Ds transposon insertion in the
433
2nd exon (Supplemental Fig. S2A). max4-11 carries a T-DNA insertion in the 5th intron
434
(Supplemental Fig. S9A). MAX4 transcript was not detected in senescent leaves of
435
max4-11, suggesting that max4-11 is a null allele (Supplemental Fig. S9B). Primers used
436
for genotyping are listed in Tables S1 and S2. Typically, plants were grown in a growth
437
chamber for 4 weeks under the following conditions: 22°C, 10-h light/14-h dark
438
(short-day) photoperiod, and 80 µmol·m−2·s−1. For the natural senescence experiments,
439
plants were grown under long-day conditions (16-h light/8-h dark).
27
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
440
441
Dark and phytohormone treatments
442
443
For the dark treatment, the 8th leaf from the top of 4-week-old plants with 16 leaves
444
was detached and incubated in a box in the dark at 22°C in which high humidity was
445
maintained. For the light (control) and phytohormone treatments, leaves were incubated
446
in continuous white light (5 µmol·m−2·s−1) at 22°C. For the strigolactone treatment,
447
leaves were incubated in 3 mM 2-(N-morpholino)ethanesulfonic acid (MES, pH 5.8)
448
containing racGR24 (Chiralix; Nijmegen, The Netherlands) and solidified with 0.5%
449
(w/v) gellan gum. For the ethylene treatment, detached leaves were incubated on a wet
450
sponge in a transparent airtight container containing 500 µL/L ethylene. The hypocotyl
451
length assay of kai2-3 was performed according to the method described by Waters et al.
452
(2012).
453
454
Measurement of senescence parameters
455
456
Chlorophyll content was measured as SPAD value by using a SPAD-502 chlorophyll
457
meter (Konica-Minolta; Tokyo, Japan), and Fv/Fm values were measured using a Junior
458
PAM chlorophyll fluorometer (Walz; Effeltrich, Germany). To measure membrane ion
459
leakage, leaves were floated on 500 μL of distilled water in the dark at 22°C. Electrolyte
460
leakage was calculated from the conductivity of water, which was measured using a
461
Twin Cond B-173 conductivity meter (Horiba; Kyoto, Japan). Membrane ion leakage
462
(%) was calculated as follows: conductivity of sample/conductivity of sample boiled for
463
15 min × 100.
28
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
464
465
Measurement of ethylene concentration
466
467
Detached leaves were incubated in 16-mL sampling vials containing 4 mL of 3 mM
468
MES (pH 5.8) solidified with 1% (w/v) agar. The leaves were incubated under
469
continuous light (20 μmol·m−2·s−1) or in the dark at 22°C for 20 h. The accumulated
470
ethylene gas was collected using a syringe and measured with a gas chromatograph
471
(GC-2014, Shimazu; Kyoto, Japan) fitted with a VZ10 column and a flame ionization
472
detector.
473
474
RNA extraction and quantitative reverse-transcriptase polymerase chain reaction
475
476
Total RNA was extracted using an Isogen kit with a spin column (Nippon Gene; Tokyo,
477
Japan). First-strand cDNA was synthesized from 500 ng of total RNA by using the
478
ReverTra Ace qPCR RT Master Mix (Toyobo; Osaka, Japan). Quantitative,
479
reverse-transcriptase polymerase chain reaction (qRT-PCR) was performed using a
480
KAPA SYBR FAST qPCR kit (Nippon Genetics; Tokyo, Japan) and a Roter-Gene Q
481
2PLEX (Qiagen; Venlo, The Netherlands). The transcript level of each gene was
482
normalized to that of Actin8 (ACT8). The primers used for RT and qRT-PCR and their
483
amplifying efficiency are listed in Table S3.
484
485
Grafting experiments
486
487
Two-shoot grafting experiments were performed as described by Notaguchi et al. (2009).
29
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
488
Grafted plants were grown for 5 weeks under a 10-h light/14-h dark photoperiod before
489
senescence induction.
490
491
492
Supplemental Data
493
494
The following materials are available in the online version of this article.
495
496
Supplemental Figure S1. Quantitative analysis of senescence parameters in
497
strigolactone biosynthesis mutants.
498
Supplemental Figure S2. Phenotype of kai2 in dark-induced senescence.
499
Supplemental Figure S3. Natural senescence of strigolactone biosynthesis mutants.
500
Supplemental
501
biosynthesis/strigolactone-insensitive and ethylene-insensitive mutants in leaf
502
senescence.
503
Supplemental Figure S5. MAX3 and MAX4 expression in max4-11 and max3-9
504
during dark incubation.
505
Supplemental Figure S6. Senescence parameters in ein3-1 eil1-3 leaves during a
506
long dark incubation.
507
Supplemental Figure S7. qRT-PCR analysis of transcript levels of strigolactone
508
biosynthesis genes in senescent ein3-1 eil1-3 leaves.
509
Supplemental Figure S8. Effect of strigolactone on the senescence of lower leaves.
510
Supplemental Figure S9. Characterization of the max4-11 allele.
511
Supplemental Figure S10. Effect of strigolactone on Fv/Fm value.
Figure
S4.
Ethylene
sensitivity
of
30
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
strigolactone
512
513
Supplemental Table S1. Primers for genotyping.
514
Supplemental Table S2. Primers for dCAPS analysis.
515
Supplemental Table S3. Primers for qRT-PCR or RT-PCR.
516
517
518
ACKNOWLEDGMENTS
519
520
We thank Yumi Nagashima for technical assistance. This study was supported by Core
521
Research for Evolutional Science and Technology (to M.K.) and in part by a grant from
522
JSPS KAKENHI (No. 26292006).
523
524
525
526
527
528
FIGURE LEGENDS
529
530
Figure 1. Strigolactone is required for normal progression of leaf senescence in the dark.
531
A, Phenotype of strigolactone biosynthesis mutants during dark-induced senescence.
532
The 8th leaf from the top was incubated in the dark or light for 7 days. The lower panel
533
shows changes in chlorophyll content over time during dark treatment. B, Stay-green
534
phenotype was restored by GR24 in strigolactone biosynthesis mutants. Detached leaves
535
of strigolactone biosynthesis and strigolactone-insensitive mutants were incubated on
31
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
536
gellan gum medium containing 25 μM GR24 in the dark for 7 days. The lower panel
537
shows chlorophyll contents of leaves in the dark for 7 days with or without 25 μM
538
GR24. Full performance in promoting leaf senescence was observed for 25 µM GR24
539
without toxic effects on leaves during incubation (Figure 2E, Supplemental Figure 10).
540
Solid and open bars indicate mock and GR24-treated leaves, respectively. A, B: Bars
541
indicate standard error (n = 6).
542
543
Figure 2. Strigolactone promotes leaf senescence in concert with ethylene. A, Dark
544
treatment promotes ethylene production. Detached leaves of Col and max1-1 were
545
incubated in the light for 24 h (day 0) before dark treatment. Ethylene accumulated in
546
sampling vials over a 20-h dark incubation was measured. B, Ethylene sensitivity of leaf
547
senescence in strigolactone biosynthesis and strigolactone-insensitive mutants. The 8th
548
leaf was treated with ethylene in the light for 7 days. C, Changes in chlorophyll content
549
over time in max1-1 and At d14-1 with ethylene in the light. D, Effect of exogenous
550
strigolactone on leaf senescence. The 8th leaf of Col was treated with 25 μM GR24 with
551
or without ethylene in the light for 7 days. The right panel shows changes in chlorophyll
552
content over time in Col during this treatment. E, Strigolactone drastically promotes leaf
553
senescence in the presence of ethylene in the light. SPAD values of Col leaves treated
554
with 0.25, 1.0, 10, and 25 μM GR24, with ethylene for 5 days. F, Genetic analysis of
555
crosstalk between strigolactone and ethylene in leaf senescence. Changes in SPAD
556
values over time in detached ctr1-1 and ctr1-1 max1-1 leaves in the light. A, C, D, and
557
F; Data for the same day after treatment were statistically compared using Tukey’s
558
multiple comparison method. Data indicated with the same letter are not significantly
559
different (p < 0.05). A, C–F; Bars indicate standard error. n = 3 in A; n = 6 in C–E; n = 5
32
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
560
in F.
561
562
Figure 3. Expression of strigolactone biosynthesis genes during leaf senescence. A,
563
qRT-PCR analysis of transcript levels of strigolactone biosynthesis genes in dark-treated
564
leaves. The 8th leaf of Col and ein2-5 was incubated in the dark. Transcript levels were
565
relative to that in Col leaves at 7 DDT. Solid and open bars indicate Col and ein2-5,
566
respectively. B, qRT-PCR analysis of transcript levels of strigolactone biosynthesis
567
genes in ethylene-treated leaves. The 8th leaf of Col was incubated in the light in the
568
presence of ethylene. Transcript levels were relative to that in Col leaves 6 days after
569
ethylene treatment. Open and solid bars indicate air- and ethylene-treated Col leaves,
570
respectively. C, qRT-PCR analysis of transcript levels of strigolactone biosynthesis
571
genes in max1-1. The 8th leaf of Col and max1-1 was incubated in the dark. Transcript
572
levels were relative to that in Col leaves at 7 DDT. D, qRT-PCR analysis of transcript
573
levels of strigolactone biosynthesis genes in the presence of GR24. The 8th leaf of Col
574
was incubated for 7 days with (Light 7 days (GR24)) or without (Light 7days) 25 μM
575
GR24 in the light. For “Dark, 7 days,” the 8th leaf of Col was incubated for 7 days in
576
the dark without GR24. Transcript levels were relative to that in Col leaves at 7 DDT.
577
A–D; ACT8 was used as a reference. Bars indicate standard error (n = 4). At SGR1 and
578
SAG12 are well-characterized senescence-inducible genes (Grbic, 2003; Park et al.,
579
2008).
580
581
Figure 4. Grafting analysis of strigolactone-regulated leaf senescence. A, Diagram of
582
the grafting experiment. Stock of the grafted plant has both shoot and root; scion has
583
only the shoot. B, Procedure of the grafting experiment. Shoots of 7-day-old plants were
33
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
584
used for the grafting experiments (left panel). Arrow indicates grafting junction. Right
585
panel: grafted 5-week-old plants. Bottom panel: scion and stock separated from the
586
grafted plant. C, Dark-induced senescence of leaves from scion and stock of the grafted
587
plant. Leaves (7th to 10th) detached from stock and scion shoots of the grafted plant
588
were incubated in the dark for 4 to 6 days (indicated within parentheses). Upper panels:
589
leaves of Col/max4-11-grafted plants. Middle panels: Col/Col-grafted plants. Lower
590
panels: max4-11/Col-grafted plants.
591
592
Figure 5. Ethylene-independent pathway that promotes dark-induced leaf senescence. A,
593
Senescence of max1-1 ein2-5 leaves during a long dark incubation. At 16 DDT, Col and
594
max1-1 leaves were dead and ein2-5 leaves had turned yellow, but max1-1 ein2-5 leaves
595
stayed green. B, Change in SPAD values over time during the 16-day dark treatment.
596
Most of the chlorophyll had degraded in ein2-5 at 16 DDT. C, Effect of strigolactone on
597
max1-1 ein2-5. SPAD values are indicated for max1-1 ein2-5 leaves incubated with or
598
without 25 μM GR24 in the dark or light until 16 days after treatment. B, C; SPAD
599
values were statistically compared among data of the same day after treatment by using
600
Tukey’s multiple comparison method. Data indicated with the same letter are not
601
significantly different (p < 0.05). Bars indicate standard error. n = 8 in B. n = 5 in C.
602
603
Figure 6. Model for action of strigolactone and ethylene in dark-induced leaf
604
senescence. Dark promotes ethylene synthesis and activates ethylene signaling,
605
resulting in initial activation of senescence signaling. Activated senescence signaling
606
promotes strigolactone synthesis by inducing strigolactone biosynthesis genes in the
607
leaf. Strigolactone enhances ethylene-dependent, and in part, ethylene-independent
34
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
608
activation of senescence signaling. Activated senescence signaling causes the
609
senescence syndrome in the leaf. This model suggests that both ethylene synthesis and
610
consequent strigolactone synthesis are required for the efficient progression of leaf
611
senescence.
612
613
Supplemental Figure S1. Quantitative analysis of senescence parameters in
614
strigolactone biosynthesis mutants. A, Phenotype of strigolactone biosynthesis mutants
615
incubated in the light. The 8th leaf from the top was incubated in the light for 7 days.
616
The lower panel shows changes in SPAD values over time. B, Changes in Fv/Fm values
617
obtained from the 8th leaf incubated in the dark. Fv/Fm values of 7 days after dark
618
treatment were compared with those of Col by using Student’s t-test (*, p < 0.05; **, p
619
< 0.01). C, Changes in membrane ion leakage over time during dark incubation, for
620
which the 11th leaf was used. A–C; Bars indicate standard error (n = 6).
621
622
Supplemental Figure S2. Phenotype of kai2 in dark-induced senescence. A, Structure
623
of the kai2-3 allele. Open boxes and horizontal bars indicate exons and introns,
624
respectively. The triangle shows the Ds transposon insertion. B, Hypocotyl length of
625
Ler and kai2-3 (Ler background) germinated under dim red light for 4 days. Hypocotyl
626
length of kai2-3 is longer than that of Ler at 1% level by Student’s t-test, suggesting that
627
kai2-3 is a loss-of-function mutant. Bars indicate standard error (n = 15). C, Leaves of
628
Ler and kai2-3 were incubated in the dark for 6 days. D, Changes in SPAD values over
629
time of the 8th leaf during 7 days of dark incubation. Bars indicate standard error (n =
630
7). Student’s t-test indicated that no statistical difference was observed between Ler and
631
kai2-3 leaves in the same day during dark incubation.
35
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
632
633
Supplemental Figure S3. Natural senescence of strigolactone biosynthesis mutants. A,
634
Delayed-senescence phenotype of max4-11 in 10-week-old (28 days after bolting) plants
635
grown under long-day conditions. B, SPAD values of leaves at 28 days after bolting,
636
where the 11th leaf at bolting was used for measurement. Bars indicate standard error (n
637
= 5). C, qRT-PCR analysis of transcript levels of strigolactone biosynthesis genes in
638
naturally senescent leaves. Pre-senescent: entirely green; senescing: yellow leaf tip;
639
fully senescent: entirely yellow leaf. Leaves were collected from 10-week-old Col
640
plants (grown under short-day conditions for 4 weeks and long-day conditions for 6
641
weeks). Transcript levels are relative to that in fully senescent Col leaves. ACT8 was
642
used as a reference. Bars indicate standard error (n = 4). mRNA levels were statistically
643
compared with that of pre-senescent leaves by using Student’s t-test (*, p < 0.05).
644
645
Supplemental
Figure
646
biosynthesis/strigolactone-insensitive
647
senescence.
648
strigolactone-insensitive mutants treated with or without ethylene in the light for 7 days.
649
Solid and open bars indicate air- and ethylene-treated leaves, respectively. SPAD values
650
of ethylene-treated leaves were statistically compared by using Tukey’s multiple
651
comparison method. Data indicated with the same letter are not significantly different (p
652
< 0.05). B, Changes in chlorophyll content over time in ein2-5 and ein3-1 eil1-3 during
653
20 days of ethylene treatment in the light. No statistical difference was observed
654
between air- and ethylene-treated leaves in the same day after dark treatment by using
655
Student’s t-test. The 8th leaf was used. Bars indicate standard error. n = 6 in A and n = 8
A,
S4.
Chlorophyll
Ethylene
and
content
sensitivity
of
strigolactone
ethylene-insensitive
mutants
in
leaf
of
biosynthesis
and
strigolactone
36
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
656
in B.
657
658
Supplemental Figure S5. MAX3 and MAX4 expression in max4-11 and max3-9 during
659
dark incubation. qRT-PCR analysis of transcript levels of MAX4 in max3-9 (A) and
660
MAX3 in max4-11 (B). The 8th leaf of Col, max3-9, and max4-11 was incubated in the
661
dark for 7 days. Transcript levels are relative to that in Col leaves at 7 DDT. ACT8 was
662
used as a reference. Bars show standard error (n = 4).
663
664
Supplemental Figure S6. Senescence parameters in ein3-1 eil1-3 leaves during a long
665
dark incubation. A, Changes in SPAD values over time during dark incubation. B,
666
Changes in Fv/Fm values over time during dark incubation. C, Changes in membrane ion
667
leakage over time during dark incubation. A–C; Bars indicate standard error. n = 8 in A
668
and C. n = 6 in B. Data indicated with the same letter are not significantly different by
669
Tukey’s multiple comparison method (p < 0.05).
670
671
Supplemental Figure S7. qRT-PCR analysis of transcript levels of strigolactone
672
biosynthesis genes in senescent ein3-1 eil1-3 leaves. Transcript levels are relative to that
673
in Col leaves at 7 DDT. ACT8 was used as a reference. Bars indicate standard error (n =
674
4).
675
676
Supplemental Figure S8. Effect of strigolactone on senescence in lower leaves. A,
677
Strigolactone promotes senescence of lower leaves in Col, but not in ein3 eil1, in the
678
light. Lower (8–12th) leaves were incubated with or without 25 µM GR24 in the light
679
for 7 days. B, Chlorophyll content in the lower leaves of Col and ein3-1 eil1-3 treated
37
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
680
with or without 25 μM GR24 in the light. Solid and open bars indicate mock and
681
GR24-treated leaves, respectively. Chlorophyll contents of mock and GR24-treated
682
leaves of the same leaf position were statistically compared by using Student’s t-test. C,
683
qRT-PCR analysis of transcript levels of ethylene-inducible genes in lower leaves of Col
684
grown under the short-day condition. ERF1 and EBF2 are direct targets of EIN3.
685
Student’s t-test revealed that ERF1 and ERF2 were significantly upregulated in the 12th
686
leaf of Col but not in ein3-1 eil1-3. Transcript levels are relative to that in the 12th leaf
687
of Col. ACT8 was used as a reference. Bars indicate standard error. n = 6 in B and n = 4
688
in C. *, p < 0.05; **, p < 0.01.
689
690
Supplemental Figure S9. Characterization of the max4-11 allele. A, Structure of the
691
max4-11 allele. Open boxes and horizontal bars indicate exons and introns, respectively.
692
The triangle shows T-DNA. B, Expression of MAX4 in the max4-11 mutant. Upper
693
panel shows RT-PCR analysis of MAX4 using MAX4F (F) and MAX4R (R) as a primer
694
pair. Lower panel shows expression of ACT8 as a reference. “0” and “7” indicate 0 and
695
7 DDT.
696
697
Supplemental Figure S10. Effect of strigolactone on Fv/Fm value. The 8th leaf from the
698
top of Col plants were incubated in the light for 7 days with 25 μM GR24. No statistical
699
difference was observed between mock and GR24-treated leaves by using Student’s
700
t-test. n = 7
701
702
Supplemental Table S1. Primers used for genotyping.
703
38
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
704
Supplemental Table S2. Primers used for dCAPS analysis.
705
706
Supplemental Table S3. Primers used for qRT-PCR or RT-PCR.
707
708
39
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
A
0 day
Chlorophyll content (SPAD)
7days
30.0
Col
max1-1
max3-9
max4-11
Atd14-1
max2-4
ein2-5
ein3-1 eil1-3
25.0
20.0
15.0
10.0
5.0
0
0
2
4
6
8
Days after treatment
1.0
B
Fv/Fm
0.8
*
0.6
*
**
**
*
**
**
0day
7days
0.4
0.2
0
Membrane ion leakage (%)
C
80
Col
max1-1
max3-9
max4-11
Atd14-1
max2-4
ein2-5
ein3-1 eil1-3
70
60
50
40
30
20
10
0
0
2
4
6
8
Days after dark treatment
Supplemental Figure S1. Quantitative analysis of senescence parameters in strigolactone
biosynthesis mutants. A, Phenotype of strigolactone biosynthesis mutants incubated in the light.
The 8th leaf from the top was incubated in the light for 7 days. The lower panel shows changes in
SPAD values over time. B, Changes in Fv/Fm values obtained from the 8th leaf incubated in the dark.
Fv/Fm values of 7 days afterDownloaded
dark treatment
were
withbythose
of Col by using Student’s tfrom on June
15,compared
2017 - Published
www.plantphysiol.org
2015
Americanin
Society
of Plant Biologists.
All rightsover
reserved.
test (*, p < 0.05; **, p Copyright
< 0.01).©C,
Changes
membrane
ion leakage
time during dark
incubation, for which the 11th leaf was used. A–C; Bars indicate standard error (n = 6).
A
kai2-3
3’
5’
**
8.0
Hypolengthcotyl (mm)
B
6.0
4.0
2.0
0
Ler
C
8th
kai2-3
kai2-3
9th 10th 11th 12th
Ler
kai2-3
30.0
Chlorophyll content (SPAD)
D
25.0
Ler
Ler
kai2-3
Kai2-3
20.0
15.0
10.0
5.0
0
0
2
4
6
8
Days after dark treatment
Supplemental Figure S2. Phenotype of kai2 in dark-induced senescence. A, Structure of the kai2-3
allele. Open boxes and horizontal bars indicate exons and introns, respectively. The triangle shows
the Ds transposon insertion. B, Hypocotyl length of Ler and kai2-3 (Ler background) germinated
under dim red light for 4 days. Hypocotyl length of kai2-3 is longer than that of Ler at 1% level by
Student’s t-test, suggesting that kai2-3 is a loss-of-function mutant. Bars indicate standard error (n
= 15). C, Leaves of Ler and kai2-3 were incubated in the dark for 6 days. D, Changes in SPAD values
over time of the 8th leaf during 7 days of dark incubation. Bars indicate standard error (n = 7).
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Student’s t-test indicated that
no statistical
difference
observed
Ler and kai2-3 leaves
Copyright
© 2015 American
Societywas
of Plant
Biologists.between
All rights reserved.
in the same day during dark incubation.
B
Chlorophyll content (SPAD)
A
Col
C
max4-11
25.0
20.0
15.0
10.0
5.0
max2-4 ein3-1 eil1-3
*
1.5
0
1.5
*
MAX3
MAX4
1.0
1.0
Relative mRNA levels
*
*
0.5
0.5
*
0
1.5
1
2
3
0
1.5
MAX1
1
2
*
3
AtSGR1
1.0
1.0
0.5
0.5
0
0
Supplemental Figure S3. Natural senescence of strigolactone biosynthesis mutants. A, Delayedsenescence phenotype of max4-11 in 10-week-old (28 days after bolting) plants grown under
long-day conditions. B, SPAD values of leaves at 28 days after bolting, where the 11th leaf at
bolting was used for measurement. Bars indicate standard error (n = 5). C, qRT-PCR analysis of
transcript levels of strigolactone biosynthesis genes in naturally senescent leaves. Pre-senescent:
entirely green; senescing: yellow leaf tip; fully senescent: entirely yellow leaf. Leaves were
collected from 10-week-old Col plants (grown under short-day conditions for 4 weeks and longday conditions for 6 weeks). Transcript levels are relative to that in fully senescent Col leaves.
ACT8 was used as a reference. Bars indicate standard error (n = 4). mRNA levels were statistically
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
compared with that of pre-senescent
leaves by
usingofStudent’s
t-test
p <reserved.
0.05).
Copyright © 2015 American
Society
Plant Biologists.
All(*,
rights
Air
air
A
ethyene
Ethylene
Chlorophyll content (SPAD)
25.0
20.0
ab
ab
ab
b
15.0
10.0
c
5.0
d
0
B
a
a
ein2-5
Chlorophyll content (SPAD)
25.0
20.0
air
Air
ethylene
Ethylene
15.0
10.0
5.0
0
0
5
10
15
20
Chlorophyll content (SPAD)
Days after treatment
ein3-1 eil1-3
25.0
20.0
air
Air
ethylene
Ethylene
15.0
10.0
5.0
0
0
5
10
15
Days after treatment
20
Supplemental Figure S4. Ethylene sensitivity of strigolactone biosynthesis/strigolactone-insensitive
and ethylene-insensitive mutants in leaf senescence. A, Chlorophyll content of strigolactone
biosynthesis and strigolactone-insensitive mutants treated with or without ethylene in the light for
7 days. Solid and open bars indicate air- and ethylene-treated leaves, respectively. SPAD values of
ethylene-treated leaves were statistically compared by using Tukey’s multiple comparison method.
Data indicated with the same letter are not significantly different (p < 0.05). B, Changes in
chlorophyll content over time in ein2-5 and ein3-1 eil1-3 during 20 days of ethylene treatment in
the light. No statistical difference
was
observed
between
air- and
ethylene-treated leaves in the
Downloaded
from
on June 15,
2017 - Published
by www.plantphysiol.org
Copyrightby
© 2015
American
Society
of PlantThe
Biologists.
All rights
same day after dark treatment
using
Student’s
t-test.
8th leaf
wasreserved.
used. Bars indicate
standard error. n = 6 in A and n = 8 in B.
Relative mRNA levels
A
1.5
MAX4
1.5
1.0
1.0
0.5
0.5
AtSGR1
0
0
0
7
0
7
0
max3-9
Col
7
0
7
max3-9
Col
Relative mRNA levels
B
1.5
1.5
MAX3
1.0
1.0
0.5
0.5
AtSGR1
0
0
0
7
Col
0
7
max4-11
0
7
Col
0
7
max4-11
Supplemental Figure S5. MAX3 and MAX4 expression in max4-11 and max3-9 during dark
incubation. qRT-PCR analysis of transcript levels of MAX4 in max3-9 (A) and MAX3 in max4-11 (B).
The 8th leaf of Col, max3-9, and max4-11 was incubated in the dark for 7 days. Transcript levels are
relative to that in Col leaves at 7 DDT. ACT8 was used as a reference. Bars show standard error (n =
4).
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Chlorophyll content (SPAD)
A
30.0
25.0
a
20.0
a
15.0
a
a
a
b
5.0
a
a
a
a
b
b
0
5
10
15
Days after dark treatment
1.0
a
0.8
Fv/Fm
a
c
10.0
0
B
Col
max1-1
ein3-1 eil1-3
Col
a
a
0.6
b
0.4
max1-1
a
a
a
ein3-1 eil1-3
a
a
b
a
0.2
0
0
C
5
10
15
Days after dark treatment
Membrane ion leakage (%)
40
Col
max1-1
ein3-1 eil1-3
30
a
a
20
10
0
a
a
0
a
b
5
b
a
a
b
10
b
15
Days after dark treatment
Supplemental Figure S6. Senescence parameters in ein3-1 eil1-3 leaves during a long dark
incubation. A, Changes in SPAD values over time during dark incubation. B, Changes in Fv/Fm values
over time during dark incubation. C, Changes in membrane ion leakage over time during dark
incubation. A–C; Bars indicate standard error. n = 8 in A and C. n = 6 in B. Data indicated with the
same letter are not significantly
different by Tukey’s multiple comparison method (p < 0.05).
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
1.5
1.5
Relative mRNA levels
MAX3
MAX4
1.0
1.0
0.5
0.5
0
0
0
7
0
7
0
16
7
0
7
16
1.5
1.5
MAX1
AtSGR1
1.0
1.0
0.5
0.5
0
0
0
7
Col
0
7
16
ein3-1 eil1-3
0
7
Col
0
7
16
ein3-1 eil1-3
Days after dark treatment
Supplemental Figure S7. qRT-PCR analysis of transcript levels of strigolactone biosynthesis genes in
senescent ein3-1 eil1-3 leaves. Transcript levels are relative to that in Col leaves at 7 DDT. ACT8 was
used as a reference. Bars indicate standard error (n = 4).
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
A
Col
ein3-1 eil1-3
Atd14-1
8th 9th 10th 11th 12th
8th 9th 10th 11th 12th
8th 9th 10th 11th 12th
Mock
GR24
Chlorophyll content (SPAD）
B
Mock
GR24
25.0
*
20.0
15.0
**
10.0
**
5.0
**
0
25.0
25.0
20.0
20.0
15.0
15.0
10.0
10.0
5.0
5.0
0
0
8th 9th 10th 11th 12th
8th 9th 10th 11th 12th
8th 9th 10th 11th 12th
ein3-1 eil1-3
Atd14-1
Col
C
Relative mRNA levels
1.5
1.5
**
*
*
ERF1
1.0
1.0
0.5
0.5
0
EBF2
0
8th
12th
Col
8th
12th
ein3-1 eil1-3
8th
12th
Col
8th
12th
ein3-1 eil1-3
Supplemental Figure S8. Effect of strigolactone on senescence in lower leaves. A, Strigolactone
promotes senescence of lower leaves in Col, but not in ein3 eil1, in the light. Lower (8–12th) leaves
were incubated with or without 25 µM GR24 in the light for 7 days. B, Chlorophyll content in the
lower leaves of Col and ein3-1 eil1-3 treated with or without 25 μM GR24 in the light. Solid and
open bars indicate mock and GR24-treated leaves, respectively. Chlorophyll contents of mock and
GR24-treated leaves of the same leaf position were statistically compared by using Student’s t-test.
C, qRT-PCR analysis of transcript levels of ethylene-inducible genes in lower leaves of Col grown
under the short-day condition. ERF1 and EBF2 are direct targets of EIN3. Student’s t-test revealed
that ERF1 and ERF2 were significantly upregulated in the 12th leaf of Col but not in ein3-1 eil1-3.
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Transcript levels are relative
to that
inAmerican
the 12th
leafofofPlant
Col.
ACT8 was
used
as a reference. Bars
Copyright
© 2015
Society
Biologists.
All rights
reserved.
indicate standard error. n = 6 in B and n = 4 in C. *, p < 0.05; **, p < 0.01.
A
max4-11
B
max4-11
Col
M
0
7
0
7
MAX4
ACT8
Supplemental Figure S9. Characterization of the max4-11 allele. A, Structure of the max4-11
allele. Open boxes and horizontal bars indicate exons and introns, respectively. The triangle
shows T-DNA. B, Expression of MAX4 in the max4-11 mutant. Upper panel shows RT-PCR
analysis of MAX4 using MAX4F (F) and MAX4R (R) as a primer pair. Lower panel shows
expression of ACT8 as a reference. “0” and “7” indicate 0 and 7 DDT.
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Fv/Fm
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Mock
GR24
Supplemental Figure S10. Effect of strigolactone on Fv/Fm value. The 8th leaf from the top of
Col plants were incubated in the light for 7 days with 25 μM GR24. No statistical difference was
observed between mock and GR24-treated leaves by using Student’s t-test. n = 7
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Supplemental Table 1. Primers for genotyping
Sequence
max2-4 F
max2-4 R
max4-11 F
max4-11 R
At d14-1 F
At d14-1 R
eil1-3 F
eil1-3 R
Lba1_SALK
DsLox_T-DNA
max3-9 F
max3-9 R
kai2-3 F
kai2-3 R
CGAGGCTTACACGGTTAGAT
CACAAATCGGCTCAATTGCC
GTAACTTCCCCAATGCTCTC
CAGAGAGGAGAAGTGTCTGA
ACACCTCTCAGATCGCTGTT
CAACGCATGCTCTTGTATCC
TAAGTGTAAAGAAGGCGTCGA
ATGACTCGGGATAAAGCTCC
TGGTTCACGTAGTGGGCCATCG
CCATGTAGATTTCCCGGACA
TTAACGGTCACTTGCAACGC
GAATTCCGAATCATACTCTGTA
CCAACTATTCGAAGCCATCC
AGTATAGCACAAGCACTCTTG
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Supplemental Table 2. Primers for dCAPS analysis
Sequence
max1-1 F
max1-1 R
ein2-5 F
ein2-5 R
ein3-1 F
ein3-1 R
ctr1-1 F
ctr1-1 R
GTGATGAACACAACAAGTGTC
CCTTTCTTGTGAAGAGGAGAAGCTGAGCCT
TGTTCTTGATTGTTGATCACAG
GAAGCCAAGCGGGTATTTCTATCTTCAGTA
TTGGACCGACTCCTCATACC
TTGAGGCCACCAATCCTCTTTCCCATTAGC
CAAAAAGGCGTTAATCTGCTG
CGTGCTGGCCTTCAATCGCGAGAGACCGAA
Restriction Enzyme
BlnI
RsaI
AluI
EcoRI
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Supplemental Table 3. Primers for qRT-PCR or RT-PCR
ACT8 F
Sequence
AGCACTTTCCAGCAGATGTG
ACT8 R
GAAATGTGATCCCGTCATGG
MAX1 F
GGCCCTATTTTCAGATTTCAG
MAX1 R
GAAGGAATGCTTCTGTTTGGA
MAX3 F
AAAGACTTTTAACGGTCACTTG
MAX3 R
TTGAATTCCGAATCATACTCAC
MAX4 F
GATGCTAGGATCGGGAGATT
MAX4 R
TGCACATATCCATCGCTCTC
AtSGR1 F
GATTGTTCCCGTTGCAAGGT
AtSGR1 R
AAGTCCTAGGGAGCGTTGAA
SAG12 F
CTATTACAGGTTATGAGGATGT
SAG12 R
AAACCACCTCCTTCAATTCCA
ERF1 F
CGATCCCTAACCGAAAACAG
ERF1 R
TCCGATAGAATATTCCGGTGA
EBF2 F
TCAGATTTAGTGGTGATGAAGA
EBF2 R
GTAATATACACCGGGACAGC
RT_ACT8 F
Efficiency
1.00
0.96
0.99
0.97
0.96
0.97
0.98
1.00
CAGCATGAAGATTAAGGTCGT
-
RT_max4-11 F
GCACGAGCATGGTATGATAC
-
RT_max4-11 R
CAACCATGCAAGCCATAAGG
-
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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