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. 2015 May 15;169(1):138–147. doi: 10.1104/pp.15.00325

Strigolactone Regulates Leaf Senescence in Concert with Ethylene in Arabidopsis1

Hiroaki Ueda 1, Makoto Kusaba 1,*
PMCID: PMC4577378  PMID: 25979917

Prolonged dark treatment induces ethylene synthesis and consequent induction of strigolactone synthesis in the leaf to promote leaf senescence.

Abstract

Leaf senescence is not a passive degenerative process; it represents a process of nutrient relocation, in which materials are salvaged for growth at a later stage or to produce the next generation. Leaf senescence is regulated by various factors, such as darkness, stress, aging, and phytohormones. Strigolactone is a recently identified phytohormone, and it has multiple functions in plant development, including repression of branching. Although strigolactone is implicated in the regulation of leaf senescence, little is known about its molecular mechanism of action. In this study, strigolactone biosynthesis mutant strains of Arabidopsis (Arabidopsis thaliana) showed a delayed senescence phenotype during dark incubation. The strigolactone biosynthesis genes MORE AXIALLY GROWTH3 (MAX3) and MAX4 were drastically induced during dark incubation and treatment with the senescence-promoting phytohormone ethylene, suggesting that strigolactone is synthesized in the leaf during leaf senescence. This hypothesis was confirmed by a grafting experiment using max4 as the stock and Columbia-0 as the scion, in which the leaves from the Columbia-0 scion senesced earlier than max4 stock leaves. Dark incubation induced the synthesis of ethylene independent of strigolactone. Strigolactone biosynthesis mutants showed a delayed senescence phenotype during ethylene treatment in the light. Furthermore, leaf senescence was strongly accelerated by the application of strigolactone in the presence of ethylene and not by strigolactone alone. These observations suggest that strigolactone promotes leaf senescence by enhancing the action of ethylene. Thus, dark-induced senescence is regulated by a two-step mechanism: induction of ethylene synthesis and consequent induction of strigolactone synthesis in the leaf.

Leaf senescence is an active nutrient-salvage system that recycles materials from dispensable leaves to the plant (Bleecker and Patterson, 1997; Lim et al., 2007). In senescing leaves, high-molecular weight compounds, such as proteins and lipids, are degraded and metabolized, and the resulting low-molecular weight compounds are transported to younger tissues or seeds. Leaf senescence is regulated by a complex system. Endogenous factors, such as aging and flowering, dark treatment, nutrient starvation, and various stresses, promote leaf senescence. Several phytohormones are also involved in regulating leaf senescence (Lim et al., 2007; Kusaba et al., 2013); ethylene, abscisic acid, jasmonic acid, and salicylic acid act as promoters, whereas cytokinins cause retardation. Although leaf senescence is induced by various factors, the resulting phenomena are the same and collectively known as the senescence syndrome. This includes leaf yellowing caused by chlorophyll degradation, degeneration of the chloroplast structure and concomitant lipid degradation, degradation of photosynthetic proteins, and reduction in photosynthetic activity (Noodén, 2004). It is possible that the activation of senescence signaling, which integrates various senescence-regulating pathways, causes the senescence syndrome.

Stay-green mutants retain green leaves under senescence-inducing conditions and are useful in the analysis of the molecular mechanism of leaf senescence (Thomas and Ougham, 2014). Several stay-green mutants have been isolated, of which many are transcription factor mutants (Kusaba et al., 2013; Penfold and Buchanan-Wollaston, 2014). For example, the NAC (for no apical meristem [NAM], Arabidopsis transcription activation factor [ATAF1-2], and cup-shaped cotyledon [CUC2]) transcription factor ORESARA1 (ORE1) is regulated at the transcriptional and posttranscriptional levels and promotes leaf senescence through transcriptional modulation of target genes, such as BIFUNCTIONAL NUCLEASE1 and GOLDEN2-LIKE1/2 (Kim et al., 2009; Matallana-Ramirez et al., 2013; Rauf et al., 2013). As mentioned previously, plant hormones play an important role in regulating leaf senescence. Ethylene, a key plant hormone that promotes leaf senescence (Jing et al., 2005; Li et al., 2013), binds to ethylene receptors on the endoplasmic reticulum (Shakeel et al., 2013). This binding represses the activity of ethylene receptors, resulting in the inactivation of the Ser/Thr kinase CONSTITUTIVE TRIPLE RESPONSE1 (CTR1), a negative regulator of ethylene signaling. This inactivation causes the translocation of the C-terminal end of the positive regulator, ETHYLENE INSENSITIVE2 (EIN2), to the nucleus and stabilizes transcription factors EIN3 and ETHYLENE INSENSITIVE3-LIKE1 (EIL1), which in turn, activate the expression of ethylene target genes. Ethylene-insensitive mutants exhibit a delayed senescence phenotype, whereas the constitutive ethylene response mutant ctr1 and a line overexpressing EIN3 exhibit early senescence phenotypes (Li et al., 2013; Kim et al., 2014; Xu et al., 2014).

Strigolactone is a plant hormone that regulates various phenomena (Gomez-Roldan et al., 2008; Umehara et al., 2008; Xie et al., 2010; Agusti et al., 2011; Seto et al., 2012; Ha et al., 2014; Waldie et al., 2014). It is involved in the suppression of shoot branching, root development, secondary growth, and drought tolerance. Strigolactone is synthesized from carotenoids by the carotenoid isomerase D27, carotenoid cleavage dioxygenases MORE AXIALLY GROWTH3 (MAX3)/DWARF17 (D17)/RAMOSUS5 (RMS5)/DECREASED APICAL DOMINANCE3 (DAD3) and MAX4/D10/RMS1/DAD1, and cytochrome P450 MAX1 (Abe et al., 2014; Seto et al., 2014; Seto and Yamaguchi, 2014; Zhang et al., 2014). The strigolactone receptor with hydrolase activity, DAD2/D14, binds to strigolactone and interacts with the F-box protein MAX2 to induce strigolactone responses (Hamiaux et al., 2012; Jiang et al., 2013; Nakamura et al., 2013; Zhou et al., 2013).

Grafting experiments using pea (Pisum sativum) indicated that a long-distance signal synthesized in the root repressed branching in the shoot (Beveridge et al., 2009). Later, this signal substance was identified as strigolactone. Grafting experiments between wild-type Columbia-0 (Col-0) and strigolactone biosynthesis mutants, such as max1, max3, and max4, in Arabidopsis (Arabidopsis thaliana) confirmed these observations (Domagalska and Leyser, 2011). Interestingly, grafting experiments using max1 as the stock and max3 or max4 as the scion showed that the mobile substance could be an intermediate metabolite of strigolactone: downstream of MAX3 and MAX4 and upstream of MAX1 (Booker et al., 2005). The mobile intermediate metabolite was suggested to be carlactone, an inactive precursor of strigolactone that is synthesized from all trans-β-carotene by D27, MAX3, and MAX4 (Alder et al., 2012; Seto and Yamaguchi, 2014; Waldie et al., 2014).

max2 is allelic to the stay-green mutation in Arabidopsis ore9, suggesting that strigolactone is involved in the regulation of leaf senescence (Woo et al., 2001; Stirnberg et al., 2002). This hypothesis was supported by studies on petunia (Petunia hybrida) and rice (Oryza sativa; Snowden et al., 2005; Yan et al., 2007; Yamada et al., 2014). However, little is known about the molecular mechanism underlying the regulation of leaf senescence by strigolactone. In this study, we showed that strigolactone promotes leaf senescence on the basis of analyses of strigolactone biosynthesis mutants and strigolactone-feeding experiments. Furthermore, our results suggest that the efficient progression of dark-induced leaf senescence requires both induction of ethylene synthesis and the consequent induction of strigolactone synthesis.

RESULTS

Strigolactone Is Involved in Promoting Leaf Senescence

ore9/max2 is a stay-green mutant, suggesting that strigolactone regulates leaf senescence. However, MAX2 is reportedly involved in several strigolactone-independent phenomena in Arabidopsis (Nelson et al., 2011; Shen et al., 2012; Waters et al., 2012; Scaffidi et al., 2013, 2014). Therefore, we examined the senescence phenotype during dark incubation of strigolactone biosynthesis and strigolactone-insensitive mutants. The detached, young, fully expanded leaf (eighth visible leaf from the top) of the wild-type Col-0 turned yellow by the seventh day of dark treatment (DDT) but not in the light (Fig. 1A; Supplemental Fig. S1A). In contrast, the strigolactone biosynthesis mutants max1-1, max3-9, and max4-11 and strigolactone-insensitive mutants Atd14-1 and max2-4 showed stay-green phenotypes, although the phenotype of max3-9 appeared slightly weak (Fig. 1).

Figure 1.

Figure 1.

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Strigolactone is required for normal progression of leaf senescence in the dark. A, Phenotype of strigolactone biosynthesis mutants during dark-induced senescence. The eighth leaf from the top was incubated in the dark or light for 7 d. The graph shows changes in chlorophyll content over time during dark treatment. B, Stay-green phenotype was restored by GR24 in strigolactone biosynthesis mutants. Detached leaves of strigolactone biosynthesis and strigolactone-insensitive mutants were incubated on gellan gum medium containing 25 μm GR24 in the dark for 7 d. The graph shows chlorophyll contents of leaves in the dark for 7 d with or without 25 μm GR24. Full performance in promoting leaf senescence was observed for 25 µm GR24 without toxic effects on leaves during incubation (Fig. 2E; Supplemental Fig. S10). Black and white bars indicate mock and GR24-treated leaves, respectively. Bars indicate se (n = 6).

During dark incubation for 7 d, the leaves of strigolactone biosynthesis mutants max1-1, max3-9, and max4-11 retained high variable PSII fluorescence in the dark-adapted state (Fv)/maximum PSII fluorescence in the dark-adapted state (Fm) values, indicating that the activity of PSII was unaffected, whereas those of Col-0 exhibited a significant decrease in Fv/Fm values (Supplemental Fig. S1B). Membrane ion leakage, an indicator of the loss of membrane integrity, began to increase at 6 DDT in Col-0, whereas that in the strigolactone biosynthesis mutants remained at a low level until 8 DDT (Supplemental Fig. S1C). These senescence characteristics confirmed that leaf senescence was delayed in strigolactone biosynthesis mutants as well as the strigolactone-insensitive mutant ore9/max2.

Next, we examined whether exogenously applied strigolactone could restore normal yellowing in the strigolactone biosynthesis mutants during dark-induced senescence (Fig. 1B). When treated with the artificial strigolactone analog GR24, max1-1, max3-9, and max4-11 leaves turned yellow at 7 DDT; however, the leaves of strigolactone-insensitive mutants max2-4 and Atd14-1 did not, suggesting that strigolactone is required for normal leaf senescence in the dark.

MAX2 is involved in not only strigolactone signaling but also, karrikin signaling. The karrikin signaling component KAI2 is not involved in strigolactone signaling, but a stereoisomer of GR24 (GR24ent-5DS) signals through KARRIKIN INSENSITIVE2 (KAI2; Nelson et al., 2011; Scaffidi et al., 2014; Waters et al., 2014). In our experiments, we used a racemic mixture of GR24 (racGR24), which includes GR24ent-5DS. We examined dark-induced leaf senescence of kai2-3; however, a delayed senescence phenotype was not observed, confirming that strigolactone specifically regulates leaf senescence (Supplemental Fig. S2).

ore9/max2 exhibits a delayed natural senescence phenotype, suggesting that strigolactone is involved in regulating natural leaf senescence (Woo et al., 2001; Stirnberg et al., 2002). We evaluated natural senescence in 70-d-old plants (28 d after bolting) of Col-0, max4-11, max2-4, and ein3-1 eil1-3 (Supplemental Fig. S3, A and B). Like max2-4, max4-11 also exhibited delayed yellowing during natural senescence.

Cross Talk between Strigolactone and Ethylene

The ethylene-insensitive mutants ein2-5 and ein3-1 eil1-3 exhibited delayed senescence in the dark (Fig. 1A). Ethylene plays an important role in the progression of senescence in the dark; therefore, we examined ethylene production by detached leaves during dark treatment (Fig. 2A). To minimize the effect of wounding on ethylene production, we incubated the detached leaves in the light for 24 h (0 d) before dark treatment. The amount of ethylene released from Col-0 leaves increased at 1 DDT and peaked at 2 DDT (9.9-fold increase relative to that on 0 DDT). The increased rate of ethylene release was maintained until 5 DDT. In contrast, there was only a low level of ethylene release in 5 d in the light. These results suggest that dark treatment induces ethylene synthesis and that synthesized ethylene promotes leaf senescence by activating ethylene signaling.

Figure 2.

Figure 2.

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Strigolactone promotes leaf senescence in concert with ethylene. A, Dark treatment promotes ethylene production. Detached leaves of Col-0 and max1-1 were incubated in the light for 24 h (day 0) before dark treatment. Ethylene accumulated in sampling vials over a 20-h dark incubation was measured. FW, Fresh weight. B, Ethylene sensitivity of leaf senescence in strigolactone biosynthesis and strigolactone-insensitive mutants. The eighth leaf was treated with ethylene in the light for 7 d. C, Changes in chlorophyll content over time in max1-1 and Atd14-1 with ethylene in the light. D, Effect of exogenous strigolactone on leaf senescence. The eighth leaf of Col-0 was treated with 25 μm GR24 with or without ethylene in the light for 7 d. The graph shows changes in chlorophyll content over time in Col-0 during this treatment. E, Strigolactone drastically promotes leaf senescence in the presence of ethylene in the light. SPAD values of Col-0 leaves treated with 0.25, 1.0, 10, or 25 μm GR24 with ethylene for 5 d. F, Genetic analysis of cross talk between strigolactone and ethylene in leaf senescence. Changes in SPAD values over time in detached ctr1-1 and ctr1-1 max1-1 leaves in the light. A, C, D, and F, Data for the same day after treatment were statistically compared using Tukey’s multiple comparison method. Data indicated with the same letter are not significantly different (P < 0.05). Bars indicate se (n = 3 in A, n = 6 in C–E , and n = 5 in F).

Next, we examined the role of strigolactone in ethylene-mediated leaf senescence. There was no significant difference in ethylene release between Col-0 and max1-1 during dark incubation (Fig. 2A), suggesting that strigolactone does not promote ethylene synthesis.

When detached leaves were treated with ethylene, Col-0 leaves but not ein2-5 and ein3-1 eil1-3 leaves turned yellow within 7 d in the light (Fig. 2B; Supplemental Fig. S4A), confirming that ethylene promotes leaf senescence. Interestingly, the strigolactone biosynthesis and strigolactone-insensitive mutants exhibited delayed yellowing during ethylene treatment. Changes in chlorophyll content in the Col-0 and mutant lines over time confirmed that most of the chlorophyll was degraded in Col-0 but that there was little change in chlorophyll content in ein2-5 and max1-1 at 5 d after ethylene treatment (Fig. 2C). However, at 8 DDT, there was a clear decrease in chlorophyll content in max1-1 but not in ein2-5, suggesting that max1-1 is hyposensitive rather than insensitive to ethylene. ein2-5 and ein3-1 eil1-3 were green during 20 d of ethylene treatment, suggesting that ein2-5 and ein3-1 eil1-3 are completely insensitive to ethylene with respect to the promotion of leaf senescence (Supplemental Fig. S4B).

Young, fully expanded Col-0 leaves turned yellow within 7 d of incubation with ethylene in the light but not with strigolactone, suggesting that strigolactone does not have a potent senescence-inducing activity (Fig. 2D). Nonetheless, addition of strigolactone enhanced the action of ethylene in promoting leaf senescence (Fig. 2D). Similarly, in the presence of ethylene but not strigolactone, there was a slight decrease in chlorophyll content at 5 d after ethylene treatment (Fig. 2E). Application of 0.25 μm GR24 along with ethylene significantly increased chlorophyll degradation (SPAD value was significantly lower than that of the ethylene-only control at the 5% level by Student’s t test). These findings suggest that there is a synergistic relationship between ethylene and strigolactone.

The constitutive ethylene response mutant ctr1 exhibits an early senescence phenotype (Xu et al., 2014). In the light, most of the chlorophyll in detached leaves of ctr1-1 degraded within 5 d, but there was little chlorophyll degradation in the leaves of Col-0 and only slight degradation of chlorophyll in the leaves of max1-1 ctr1-1 (Fig. 2F). These findings suggest that strigolactone deficiency repressed ethylene-mediated senescence signaling activated in ctr1-1. Taken together, these observations suggest that strigolactone promotes leaf senescence by activating ethylene-mediated senescence signaling.

Expression of Strigolactone Biosynthesis Genes

The transcript levels of MAX1, MAX3, and MAX4 increased during dark incubation (Fig. 3A), especially those of the latter two genes. Transcripts of these two genes were hardly detected in the presenescent leaves, but their levels started to increase from 1 DDT and continued to increase until 7 DDT. In ein2-5, induction of MAX3 and MAX4 was severely repressed during dark incubation (Fig. 3A). Up-regulation of MAX1 expression was also repressed in ein2-5 at 5 and 7 DDT. These results suggest that ethylene signaling is involved in the up-regulation of MAX1, MAX3, and MAX4 during dark-induced senescence. Ethylene treatment also induced MAX1, MAX3, and MAX4 in the light (Fig. 3B). However, according to RNA sequencing and chromatin immunoprecipitation sequencing, none of these three genes seem to be direct targets of EIN3 (Chang et al., 2013). Among three MAX genes, only MAX4 harbors a single consensus binding site for EIN3 (ATGTATCT) in the promoter region (from the initiation codon to the stop or initiation codon of upstream adjacent gene), consistent with the abovementioned observation (Boutrot et al., 2010).

Figure 3.

Figure 3.

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Expression of strigolactone biosynthesis genes during leaf senescence. A, qRT-PCR analysis of transcript levels of strigolactone biosynthesis genes in dark-treated leaves. The eighth leaf of Col-0 and ein2-5 was incubated in the dark. Transcript levels were relative to those in Col-0 leaves at 7 DDT. Black and white bars indicate Col-0 and ein2-5, respectively. B, qRT-PCR analysis of transcript levels of strigolactone biosynthesis genes in ethylene-treated leaves. The eighth leaf of Col-0 was incubated in the light in the presence of ethylene. Transcript levels were relative to those in Col-0 leaves 6 d after ethylene treatment. Black and white bars indicate air- and ethylene-treated Col-0 leaves, respectively. C, qRT-PCR analysis of transcript levels of strigolactone biosynthesis genes in max1-1. The eighth leaf of Col-0 and max1-1 was incubated in the dark. Transcript levels were relative to those in Col-0 leaves at 7 DDT. D, qRT-PCR analysis of transcript levels of strigolactone biosynthesis genes in the presence of GR24. The eighth leaf of Col-0 was incubated for 7 d with (light 7 d [GR24]) or without (light 7 d) 25 μm GR24 in the light. For dark 7 d, the eighth leaf of Col-0 was incubated for 7 d in the dark without GR24. Transcript levels were relative to those in Col-0 leaves at 7 DDT. ACT8 was used as a reference. Bars indicate se (n = 4). AtSGR1 and SENESCENCE-ASSOCIATED GENE12 (SAG12) are well-characterized senescence-inducible genes (Grbic, 2003; Park et al., 2007).

Induction of MAX3 and MAX4 during dark incubation was also repressed in the strigolactone biosynthesis mutant max1-1 (Fig. 3C). This result raised the possibility that MAX3 and MAX4 are under positive feedback regulation by strigolactone. To test this possibility, we treated Col-0 leaves with GR24 for 7 d in the light and examined the transcript levels of MAX3 and MAX4. However, neither MAX3 nor MAX4 were induced by GR24 (Fig. 3D). To evaluate the possibility that carlactone, which may be accumulated in max1-1, represses MAX3 and MAX4 expression in dark-incubated max1-1 leaves, we examined MAX3 expression in max4-11 and MAX4 expression in max3-9. Neither MAX3 nor MAX4 were induced during dark incubation in max4-11 and max3-9, respectively, suggesting that repression of MAX3 and MAX4 in max1-1 is not caused by accumulation of carlactone (Supplemental Fig. S5).

Next, we analyzed the transcript levels of strigolactone biosynthesis genes in naturally senescent leaves. Transcript levels of MAX1, MAX3, MAX4, and Arabidopsis STAY-GREEN1 (AtSGR1) were up-regulated in senescing and fully senescent leaves, suggesting that MAX1, MAX3, and MAX4 play roles in natural senescence (Supplemental Fig. S3C). This result is consistent with the fact that strigolactone biosynthesis/strigolactone-insensitive mutants exhibited delayed leaf senescence under natural conditions (Supplemental Fig. S3A).

Strigolactone Is Synthesized in the Leaf during Senescence

Prominent induction of the strigolactone biosynthesis genes MAX3 and MAX4 in the leaf during dark incubation suggested that strigolactone is synthesized in the senescing leaf. Therefore, we conducted micrografting experiments and generated two-shoot grafts, in which the scion did not have its own root (Notaguchi et al., 2009; Fig. 4, A and B). Leaves of the stock and scion were detached from the grafted plant at 5 weeks after grafting and incubated in the dark. When max4-11 was used as the stock and Col-0 was used as the scion, leaves from max4-11 stock exhibited delayed senescence relative to that of the scion, suggesting that the strigolactone concentration was higher in the detached leaves of Col-0 than in those of max4-11 (Fig. 4C). Because no strigolactone was synthesized in the root of this grafted plant, it is likely that strigolactone was synthesized in Col-0 leaves but not max4 leaves during dark incubation. There was no obvious difference in the progression of senescence between leaves from the stock and those from the scion when Col-0 was used as both the stock and the scion (Fig. 4C). When max4-11 was used as the scion and Col-0 was used as the stock, leaves from max4-11 stock exhibited delayed senescence; however, the difference was slightly less significant than that obtained in reciprocal grafting, suggesting that a small amount of strigolactone from the root may contribute to the promotion of leaf senescence (Fig. 4C).

Figure 4.

Figure 4.

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Grafting analysis of strigolactone-regulated leaf senescence. A, Diagram of the grafting experiment. Stock of the grafted plant has both shoot and root; scion has only the shoot. B, Procedure of the grafting experiment. Shoots of 7-d-old plants were used for the grafting experiments (upper left). Arrow indicates the grafting junction. Upper right, Grafted 5-week-old plants. Lower, Scion and stock separated from the grafted plant. C, Dark-induced senescence of leaves from scion and stock of the grafted plant. Leaves (7th–10th) detached from stock and scion shoots of the grafted plant were incubated in the dark for 4 to 6 d as indicated. Top, Leaves of Col-0/max4-11-grafted plants. Middle, Col-0/Col-0-grafted plants. Bottom, max4-11/Col-0-grafted plants.

Ethylene-Independent, Senescence-Promoting Pathways

Because ethylene plays an important role in dark-induced senescence, ethylene-insensitive mutants exhibited a strongly delayed leaf senescence phenotype during dark incubation. Nonetheless, leaves of the ethylene-insensitive mutants ein2-5 and ein3-1 eil1-3 ultimately turned yellow in the dark (Fig. 5, A and B; Supplemental Fig. S6A). The senescence parameters Fv/Fm and membrane ion leakage indicated the advanced progression of leaf senescence in ein3-1 eil1-3 leaves at 16 DDT (Supplemental Fig. S6, B and C). In addition, MAX1, MAX3, and MAX4 were up-regulated in the leaves of ein3-1 eil1-3 at 16 DDT (Supplemental Fig. S7). These results suggest that an ethylene-independent pathway also contributes to the induction of strigolactone synthesis and promotion of leaf senescence in the dark.

Figure 5.

Figure 5.

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Ethylene-independent pathway that promotes dark-induced leaf senescence. A, Senescence of max1-1 ein2-5 leaves during dark incubation. At 16 DDT, Col-0 and max1-1 leaves were dead, and ein2-5 leaves had turned yellow; however, max1-1 ein2-5 leaves stayed green. B, Change in SPAD values over time during the 16-d dark treatment. Most of the chlorophyll had degraded in ein2-5 at 16 DDT. C, Effect of strigolactone on max1-1 ein2-5. SPAD values are indicated for max1-1 ein2-5 leaves incubated with or without 25 μm GR24 in the dark or light until 16 d after treatment. B and C, SPAD values were statistically compared among data of the same day after treatment by using Tukey’s multiple comparison method. Data indicated with the same letter are not significantly different (P < 0.05). Bars indicate se (n = 8 in B and n = 5 in C).

At 16 DDT, ein2-5 leaves had begun to turn yellow, and those of max1-1 had died; however, max1-1 ein2-5 leaves remained green. Therefore, max1-1 ein2-5 exhibited a stronger delayed senescence phenotype than ein2-5 or max1-1 in the dark (Fig. 5, A and B). This result suggests that strigolactone is also involved in an ethylene-independent, senescence-promoting pathway in the dark.

Next, we treated max1-1 ein2-5 with GR24 in the dark or light (Fig. 5C). SPAD value declined during a long and dark incubation in max1-1 ein2-5, which is accelerated by the addition of GR24; this suggested that strigolactone promotes leaf senescence through an ethylene-independent, senescence-promoting pathway in the dark. In contrast, addition of GR24 did not promote leaf senescence of max1-1 ein2-5 in the light, suggesting that strigolactone alone does not promote leaf senescence in the light.

Nonetheless, strigolactone promoted leaf senescence to an extent, particularly in older (lower) leaves of Col-0 (11th and 12th leaves; Supplemental Fig. S8, A and B). However, this promotion was not observed in ein3-1 eil1-3. It is possible that slightly activated ethylene signaling in the lower leaves contributed to the promotion of leaf senescence by strigolactone. Li et al. (2013) reported that the transcription activation activity of EIN3, which implies activation of ethylene signaling, gradually increases as leaves age. Consistent with this, we detected higher transcript levels of some ethylene-inducible genes directly targeted by EIN3 in the lower leaves than in the higher leaves (Supplemental Fig. S8C).

DISCUSSION

Strigolactone has been suggested to regulate leaf senescence, because ore9/max2 exhibits a delayed senescence phenotype. Observations that strigolactone biosynthesis/strigolactone-insensitive mutants also exhibited delayed senescence phenotypes and that exogenously applied strigolactone reversed the delayed senescence phenotype of strigolactone biosynthesis mutants confirmed that strigolactone plays a role in regulating leaf senescence.

Strigolactone Synthesis during Leaf Senescence

Strigolactone and its mobile precursor are synthesized in the root and then transported to the shoot (Booker et al., 2005; Beveridge et al., 2009; Domagalska and Leyser, 2011; Kohlen et al., 2011; Seto and Yamaguchi, 2014). We observed that the strigolactone biosynthesis genes MAX3 and MAX4 were strongly induced in the leaf during dark incubation. In the grafting experiment, when max4 was used as the stock, detached leaves from the Col-0 scion senesced earlier than those from the max4 stock. These observations suggest that strigolactone synthesized in the leaf during senescence promotes dark-induced leaf senescence, although we do not exclude the possibility that a small amount of strigolactone or its precursor is transported from the root to the leaf. Although previous grafting experiments have shown that the signal molecule that represses branching is also produced in the aerial part (Beveridge et al., 2009; Domagalska and Leyser, 2011), very low transcript levels of MAX3 and MAX4 in the presenescent leaves suggest that little strigolactone is produced in presenescent leaves. Recently, Ha et al. (2014) reported that MAX3 and MAX4 in the leaf are induced by abscisic acid, NaCl, and dehydration stresses. The results reported by Ha et al. (2014) and those obtained in this study suggest that strigolactone is synthesized in the leaf under certain stress conditions.

MAX3 and MAX4 were drastically induced by dark and ethylene treatments. In the ethylene-insensitive stay-green mutants ein2 and ein3 eil1, expression of MAX3 and MAX4 was severely repressed until 7 DDT; however, it was ultimately up-regulated at 16 DDT, when leaf senescence proceeded, even in ein3 eil1. This suggests that an ethylene-independent pathway also contributes to the induction of MAX3 and MAX4. It is possible that MAX3 and MAX4 are induced by the activation of senescence signaling during leaf senescence rather than particular senescence-inducing pathways, such as ethylene signaling. This idea is consistent with the observation that the expression of MAX3 and MAX4 was repressed during dark incubation in the delayed senescence mutant max1.

Actions of Strigolactone and Ethylene in Leaf Senescence

Dark incubation promoted ethylene synthesis, and ethylene treatment induced leaf senescence in the light, suggesting that ethylene plays a key role in the progression of leaf senescence. Consistent with this idea, the ethylene-insensitive mutants ein2 and ein3 eil1 exhibited a strong stay-green phenotype during dark incubation. The strigolactone biosynthesis and strigolactone-insensitive mutants exhibited a delayed senescence phenotype not only in the dark but also, in response to ethylene treatment, suggesting that strigolactone biosynthesis and strigolactone-insensitive mutants are hyposensitive to ethylene. Strigolactone drastically promoted the senescence of young, fully expanded leaves in the presence of ethylene, although strigolactone did not have potent senescence-promoting activity in the light. These observations suggest that strigolactone promotes leaf senescence by enhancing the action of ethylene. However, max1 ein2 exhibited a stronger delayed senescence phenotype than max1 or ein2 during dark incubation, suggesting that strigolactone is also involved in an ethylene-independent, senescence-promoting pathway during dark treatment. Consistent with this idea, strigolactone accelerated leaf senescence of max1 ein2 in the dark but not in the light.

max1 and Col-0 produced similar amounts of ethylene during dark incubation. This observation may have an interesting implication. Positive regulators of leaf senescence are often senescence inducible. For example, the NAC transcription factor gene ORE1 is up-regulated during leaf senescence (Kim et al., 2009). Leaf senescence was delayed, but ethylene production was not reduced in max1, suggesting that ethylene production was not affected by the progression of senescence but was primarily regulated by dark treatment. Thus, induction of ethylene production is thought to be an upstream trigger in signal transduction of dark-induced senescence. Phytochrome-interacting factors promote ethylene production in the dark (Khanna et al., 2007; Song et al., 2014). These observations suggest that phytochromes regulate leaf senescence in the dark at least partly through ethylene production (Sakuraba et al., 2014; Song et al., 2014).

Yamada et al. (2014) observed that strigolactone biosynthesis and strigolactone-insensitive mutants in rice showed delayed senescence phenotypes during dark incubation but that the phenotypes were weaker than those of Arabidopsis mutants in our study. It will be interesting to examine how ethylene acts during leaf senescence in rice.

Kapulnik et al. (2011) reported an ethylene-strigolactone cross talk in the regulation of root hair elongation. In this case, strigolactone was thought to promote root hair elongation by enhancing ethylene production, which is in contrast with our results with leaf senescence. Thus, the cross talk between strigolactone and ethylene observed in leaf senescence is thought to be unique during the development of plants.

Multistep Regulation of Leaf Senescence

On the basis of all of the results of this study, the cross talk between strigolactone and ethylene can be summarized as follows (Fig. 6). Dark treatment induces ethylene synthesis and activates ethylene signaling, resulting in the initial activation of senescence signaling. Activation of senescence signaling induces the transcription of MAX3 and MAX4, resulting in strigolactone synthesis in the leaf. Strigolactone further activates senescence signaling by enhancing ethylene-dependent and -independent, senescence-promoting pathways. Finally, activated senescence signaling causes the senescence syndrome in the leaf.

Figure 6.

Figure 6.

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Model for action of strigolactone and ethylene in dark-induced leaf senescence. Dark promotes ethylene synthesis and activates ethylene signaling, resulting in initial activation of senescence signaling. Activated senescence signaling promotes strigolactone synthesis by inducing strigolactone biosynthesis genes in the leaf. Strigolactone enhances ethylene-dependent and in part, ethylene-independent activation of senescence signaling. Activated senescence signaling causes the senescence syndrome in the leaf. This model suggests that both ethylene synthesis and consequent strigolactone synthesis are required for the efficient progression of leaf senescence.

CONCLUSION

Our results suggest that ethylene synthesis alone is not sufficient for leaf senescence and that strigolactone synthesis induced by senescence signaling that integrates various senescence-promoting pathways is also required for the efficient progression of leaf senescence. If noncritical, ethylene-producing stimuli, such as transient stresses, result in leaf senescence, then this unnecessary senescence must have disadvantages in terms of fitness. The multistep activation system involving ethylene synthesis and subsequent strigolactone synthesis may prevent such unnecessary and uncontrolled leaf senescence. Thus, the cross talk between ethylene and strigolactone described in this study may be an important part of a system that accurately regulates leaf senescence.

MATERIALS AND METHODS

Plant Material and Growth Conditions

The Arabidopsis (Arabidopsis thaliana) mutant lines max1-1 (CS9564; Stirnberg et al., 2002), max3-9 (CS9567; Booker et al., 2004), max2-4 (SALK_028336; Umehara et al., 2008), max4-11 (SALK_072570), Atd14-1 (CS913109; Waters et al., 2012), ein2-5 (CS16771; Alonso et al., 1999), ein3-1 (CS16710; Chao et al., 1997), eil1-1 (SALK_049679; Binder et al., 2007), and ctr1-1 (CS16725; Kieber et al., 1993) were obtained from the Arabidopsis Biological Resource Center. kai2-3 (CS25712) carries a Dissociator transposon insertion in the second exon (Supplemental Fig. S2A). max4-11 carries a transfer DNA insertion in the fifth intron (Supplemental Fig. S9A). MAX4 transcript was not detected in senescent leaves of max4-11, suggesting that max4-11 is a null allele (Supplemental Fig. S9B). Primers used for genotyping are listed in Supplemental Tables S1 and S2. Typically, plants were grown in a growth chamber for 4 weeks under the following conditions: 22°C, 10-h-light/14-h-dark (short-day) photoperiod, and 80 µmol m−2 s−1. For the natural senescence experiments, plants were grown under long-day conditions (16-h-light/8-h-dark cycle).

Dark and Phytohormone Treatments

For the dark treatment, the eighth leaf from the top of 4-week-old plants with 16 leaves was detached and incubated in a box in which high humidity was maintained in the dark at 22°C. For the light (control) and phytohormone treatments, leaves were incubated in continuous white light (5 µmol m−2 s−1) at 22°C. For the strigolactone treatment, leaves were incubated in 3 mm MES (pH 5.8) containing racGR24 (Chiralix) and solidified with 0.5% (w/v) gellan gum. For the ethylene treatment, detached leaves were incubated on a wet sponge in a transparent airtight container containing 500 µL L−1 ethylene. The hypocotyl length assay of kai2-3 was performed according to the method described by Waters et al. (2012).

Measurement of Senescence Parameters

Chlorophyll content was measured as SPAD value by using an SPAD-502 Chlorophyll Meter (Konica-Minolta), and Fv/Fm values were measured using a Junior PAM Chlorophyll Fluorometer (Walz). To measure membrane ion leakage, leaves were floated on 500 μL of distilled water in the dark at 22°C. Electrolyte leakage was calculated from the conductivity of water, which was measured using a Twin Cond B-173 Conductivity Meter (Horiba). Membrane ion leakage was calculated as follows: conductivity of sample/conductivity of sample boiled for 15 min × 100.

Measurement of Ethylene Concentration

Detached leaves were incubated in 16-mL sampling vials containing 4 mL of 3 mm MES (pH 5.8) solidified with 1% (w/v) agar. The leaves were incubated under continuous light (20 μmol m−2 s−1) or in the dark at 22°C for 20 h. The accumulated ethylene gas was collected using a syringe and measured with a gas chromatograph (GC-2014; Shimazu) fitted with a VZ10 column and a flame ionization detector.

RNA Extraction and Quantitative Reverse Transcription PCR

Total RNA was extracted using an Isogen Kit with a spin column (Nippon Gene). First-strand complementary DNA was synthesized from 500 ng of total RNA by using the ReverTra Ace qPCR RT Master Mix (Toyobo). Quantitative reverse transcription (qRT)-PCR was performed using a KAPA SYBR FAST qPCR Kit (Nippon Genetics) and a Roter-Gene Q 2PLEX (Qiagen). The transcript level of each gene was normalized to that of Actin8 (ACT8). The primers used for reverse transcription PCR and qRT-PCR and their amplifying efficiencies are listed in Supplemental Table S3.

Grafting Experiments

Two-shoot grafting experiments were performed as described by Notaguchi et al. (2009). Grafted plants were grown for 5 weeks under a 10-h-light/14-h-dark photoperiod before senescence induction.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers MAX1 (AT2G26170), MAX3 (AT2G44990), MAX4 (AT4G32810), MAX2 (AT2G42620), AtD14 (AT3G03990), EIN2 (AT5G03280), EIN3 (AT3G20770), EIL1 (AT2G2750), CTR1 (AT5G03730), KAI2 (AT4G37470), ACT8 (AT1G49240), ERF1 (AT3G23240), EBF2 (AT5G25350 ), AtSGR1 (AT4G22920), and SAG12 (AT5G45890).

Supplemental Data

The following supplemental materials are available.

Supplementary Material

Supplemental Data
supp_169_1_138__index.html (973B, html)

Acknowledgments

We thank Yumi Nagashima for technical assistance.

Glossary

Col-0

Columbia-0

DDT

day of dark treatment

Fm

maximum PSII fluorescence in the dark-adapted state

Fv

variable PSII fluorescence in the dark-adapted state

qRT

quantitative reverse transcription

Footnotes

1

This work was supported by Core Research for Evolutional Science and Technology (to M.K.) and, in part, by the Japan Society for Scientific Research (KAKENHI grant no. 26292006).

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Associated Data

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Supplementary Materials

Supplemental Data
supp_169_1_138__index.html (973B, html)
supp_pp.15.00325_PP2015-00325R2_Supplemental_Tables.pdf (18.1KB, pdf)
supp_pp.15.00325_00325revsupp.pptx (1.6MB, pptx)

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