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

62 63

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 5 Downloaded from www.plantphysiol.org on May 27, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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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 9 Downloaded from www.plantphysiol.org on May 27, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

159 160

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 11 Downloaded from www.plantphysiol.org on May 27, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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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 12 Downloaded from www.plantphysiol.org on May 27, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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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 13 Downloaded from www.plantphysiol.org on May 27, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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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 14 Downloaded from www.plantphysiol.org on May 27, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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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 15 Downloaded from www.plantphysiol.org on May 27, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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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). 16 Downloaded from www.plantphysiol.org on May 27, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

262 263

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

271

from max4-11 stock exhibited delayed senescence relative to that of the scion,

272

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

278

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).

282 283

Ethylene-independent senescence-promoting pathways

284 285

Since ethylene plays an important role in dark-induced senescence, ethylene-insensitive 17 Downloaded from www.plantphysiol.org on May 27, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

286

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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288

ultimately turned yellow in the dark (Fig. 5A, B, Supplemental Fig. S6A). The

289

senescence parameters Fv/Fm and membrane ion leakage indicated the advanced

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290

progression of leaf senescence in ein3-1 eil1-3 leaves at 16 DDT (Supplemental Fig.

291

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,

296

but max1-1 ein2-5 leaves remained green. Therefore, max1-1 ein2-5 exhibited a

297

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.

300

Next, we treated max1-1 ein2-5 with GR24 in the dark or light (Fig. 5C). SPAD

301

value declined during a long dark incubation in max1-1 ein2-5, which is accelerated by

302

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

304

of GR24 did not promote leaf senescence of max1-1 ein2-5 in the light, suggesting that

305

strigolactone alone does not promote leaf senescence in the light.

306

Nonetheless, strigolactone promoted leaf senescence to an extent, particularly in

307

older (lower) leaves of Col (11th and 12th leaves; Supplemental Fig. S8A, B). However,

308

this promotion was not observed in ein3-1 eil1-3. It is possible that slightly activated

309

ethylene signaling in the lower leaves contributed to the promotion of leaf senescence

310

by strigolactone. Li et al. (2013) reported that the transcription activation activity of

311

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

313

genes directly targeted by EIN3 in the lower leaves than in the higher leaves 20 Downloaded from www.plantphysiol.org on May 27, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

314

(Supplemental Fig. S8C).

315 316 317

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318

DISCUSSION

319 320

Strigolactone has been suggested to regulate leaf senescence because ore9/max2

321

exhibits

322

biosynthesis/strigolactone-insensitive

323

phenotypes and that exogenously applied strigolactone reversed the delayed-senescence

324

phenotype of strigolactone biosynthesis mutants confirmed that strigolactone plays a

325

role in regulating leaf senescence.

a

delayed-senescence

phenotype. mutants

Observations also

exhibited

that

strigolactone

delayed-senescence

326 327 328

Strigolactone synthesis during leaf senescence

329 330

Strigolactone and its mobile precursor are synthesized in the root and then transported

331

to the shoot (Booker et al., 2005; Beveridge et al., 2009; Kohlen et al., 2011;

332

Domagalska and Leyser, 2011; Seto and Yamaguchi, 2014). We observed that the

333

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,

335

detached leaves from the Col scion senesced earlier than those from the max4 stock.

336

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

338

a small amount of strigolactone or its precursor is transported from the root to the leaf.

339

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;

341

Domagalska and Leyser, 2011), very low transcript levels of MAX3 and MAX4 in the 22 Downloaded from www.plantphysiol.org on May 27, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

342

pre-senescent leaves suggest that little strigolactone is produced in pre-senescent leaves.

343

Recently, Ha et al. (2014) reported that MAX3 and MAX4 in the leaf are induced by

344

abscisic acid, NaCl, and dehydration stresses. The results reported by Ha et al. and those

345

obtained in this study suggest that strigolactone is synthesized in the leaf under certain

346

stress conditions.

347

MAX3 and MAX4 were drastically induced by dark and ethylene treatments. In

348

the ethylene-insensitive stay-green mutants ein2 and ein3 eil1, expression of MAX3 and

349

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

exhibited

23 Downloaded from www.plantphysiol.org on May 27, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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 Downloaded from www.plantphysiol.org on May 27, 2015 - Published by www.plant.org 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 Downloaded from www.plantphysiol.org on May 27, 2015 - Published by www.plant.org 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 Downloaded from www.plantphysiol.org on May 27, 2015 - Published by www.plant.org 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 Downloaded from www.plantphysiol.org on May 27, 2015 - Published by www.plant.org 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 Downloaded from www.plantphysiol.org on May 27, 2015 - Published by www.plant.org 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 Downloaded from www.plantphysiol.org on May 27, 2015 - Published by www.plant.org 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

strigolactone

30 Downloaded from www.plantphysiol.org on May 27, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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 www.plantphysiol.org on May 27, 2015 - Published by www.plant.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 www.plantphysiol.org on May 27, 2015 - Published by www.plant.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 www.plantphysiol.org on May 27, 2015 - Published by www.plant.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 www.plantphysiol.org on May 27, 2015 - Published by www.plant.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 Downloaded from www.plantphysiol.org on May 27, 2015 - Published by www.plant.org 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 Downloaded from www.plantphysiol.org on May 27, 2015 - Published by www.plant.org 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 Downloaded from www.plantphysiol.org on May 27, 2015 - Published by www.plant.org 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 www.plantphysiol.org on May 27, 2015 - Published by www.plant.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 Downloaded from www.plantphysiol.org on May 27, 2015 - Published by www.plant.org 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 daysDownloaded after dark treatment were compared with of Col by using Student’s tfrom www.plantphysiol.org on May 27, 2015those - Published by www.plant.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 www.plantphysiol.org on May 27, 2015 - Published by www.plant.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 www.plantphysiol.org on May 27, 2015 - Published by www.plant.org compared with that of pre-senescent leaves by usingofStudent’s t-test p

Strigolactone Regulates Leaf Senescence in Concert with Ethylene in Arabidopsis.

Leaf senescence is not a passive degenerative process; it represents a process of nutrient relocation, in which materials are salvaged for growth at a...
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