Original research
Downregulation of Rice DWARF 14 LIKE Suppress Mesocotyl Elongation via a Strigolactone Independent Pathway in the Dark

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Abstract

Strigolactones (SLs) are a class of plant hormones that control plant development in response to environmental conditions. In rice, mesocotyl elongation is regulated by SLs in the dark, while mesocotyls are longer in SL deficient or insensitive mutants. SLs are perceived by DWARF14 (D14), which is a member of a small gene family. In this study, we examined the function of another D14 family gene in rice, D14 LIKE (D14L), focusing on mesocotyl growth. The mesocotyls of D14L RNAi lines are longer than those of WT in the dark. This phenotype is enhanced when the D14L RNAi lines are combined with the d14 mutation, suggesting that D14 and D14L work independently to inhibit mesocotyl elongation. This phenotype is alleviated by the exogenous supply of GR24, a synthetic SL, suggesting that D14L is not necessary for SL signaling. D14L mRNA is predominantly expressed in vascular bundles and crown root primordia. Our results suggest that D14L and D14 confer their effects via an SL independent pathway and an SL signaling pathway respectively.

Introduction

Strigolactones (SLs) were first reported as a class of plant hormones that suppress shoot branching (Gomez-Roldan et al., 2008, Umehara et al., 2008). Since then, the elucidation of their biosynthesis and signaling pathways has rapidly progressed (Seto et al., 2012). In the biosynthesis pathway, β-carotenoid is catalyzed to carlactone, which is a precursor for SLs. The enzymes involved in this process are DWARF 27 (D27), carotenoid cleavage dioxygenase (CCD7), and CCD8 (Alder et al., 2012, Seto et al., 2014). MORE AXILLARY GROWTH 1 (MAX1) is one of the enzymes involved in converting carlactone to SLs (Scaffidi et al., 2013, Seto et al., 2014). In the signaling pathway, SLs are perceived by DWARF 14 (D14), an α/β-fold hydrolase protein (Arite et al., 2009, Hamiaux et al., 2012). Recognition of SLs by D14 induces an interaction between D14, DWARF 3 (D3) and DWARF 53 (D53). D3 and D53 are an F-box protein and a Clp ATPase protein, respectively. Then, D53 protein is ubiquitinated by D3 and degraded via the 26S proteasome pathway to start the cascade of downstream events (Jiang et al., 2013, Zhou et al., 2013).

Several additional biological functions of SLs have also been discovered. These include the regulation of root architecture (Kapulnik et al., 2011, Ruyter-Spira et al., 2011, Arite et al., 2012, Rasmussen et al., 2012), secondary growth in the cambium (Agusti et al., 2011), leaf senescence (Woo et al., 2001, Woo et al., 2004, Snowden et al., 2005, Yan et al., 2007), control of leaf shape (Stirnberg et al., 2002, Booker et al., 2004, Waters et al., 2012, Scaffidi et al., 2013), drought resistance (Bu et al., 2014, Ha et al., 2014), lamina joint inclination (Li et al., 2014), and tiller angle (Sang et al., 2014). In addition, it was shown that SLs are involved in photomorphogenesis in Arabidopsis. Treatment of wild-type (WT) Arabidopsis with SLs suppresses hypocotyl elongation in light (Scaffidi et al., 2014). On the other hand, more axillary growth 2 (max2), which is a mutant of the Arabidopsis D3 ortholog, is the only SL deficient or insensitive mutant that exhibits any abnormalities in photomorphogenesis (Stirnberg et al., 2002, Shen et al., 2007, Nelson et al., 2011, Shen et al., 2012, Waters et al., 2012). Recent findings indicated that the unique phenotype of max2 is explained by a redundancy among D14 paralogs. The D14 family genes are classified into two clades: D14 and D14L. The D14 clade is further subdivided into the core D14 and the D14 LIKE 2 (DLK2) subclades (Waters et al., 2012). MAX2 and D14L were identified as the mutated genes in the Arabidopsis karrikin insensitive 1 (kai1) and karrikin insensitive 2 (kai2) mutants, respectively (Nelson et al., 2011, Waters et al., 2012). Karrikins are chemicals found in smoke, and they can stimulate seed germination and enhance photomorphogenesis (Flematti et al., 2004, Nelson et al., 2010). kai2 mutant has longer hypocotyls than WT and shows several light-hyposensitive phenotypes as observed in max2 (Sun and Ni, 2010, Nelson et al., 2011, Waters et al., 2012). This indicated that MAX2 and D14L function in karrikin signaling.

In contrast to the situation in Arabidopsis, disruption of SL biosynthesis or signaling causes extra elongation of the mesocotyl in the dark. The mesocotyl phenotype is rescued by the treatment of GR24, a synthetic SL analog, in SL deficient mutants, but not in d14 and d3 mutants (Hu et al., 2010, Hu et al., 2014). This indicates that SL suppresses mesocotyl elongation in the dark. Interestingly, the mesocotyl phenotype in d3 mutant is severer than that in other mutants including d14, indicating a possibility that D3 controls mesocotyl development not only in the SL signaling pathway with D14 but also in an SL independent pathway.

Here, as a first step toward understanding how D14 family genes and D3 regulate skotomorphogenesis in rice, we show that D14L suppresses mesocotyl elongation in the dark via an SL independent pathway.

Section snippets

D14L suppresses mesocotyl elongation in the dark

To examine the role of D14L in mesocotyl growth, we measured mesocotyl elongation in D14L RNAi lines. We first confirmed that the expression of D14L but not D14 was significantly reduced in the seedling shoot of RNAi lines (Fig. S1A and B). Seeds of the wild-type (WT) plants, the D14L RNAi lines, and the d3-2 mutant (Yoshida et al., 2012) were germinated and grown on agar plates for 8 days. No difference was observed among WT, D14L RNAi lines and d3-2 mutant in light (Fig. 1A). In contrast, the

D14, D14L, and D3 suppress mesocotyl elongation in rice

In this study we showed that D14L suppresses mesocotyl elongation in the dark. It was previously reported that D14 and D3 also regulate mesocotyl elongation in the dark (Hu et al., 2010). However, the additive phenotype observed in d14-1 mutants expressing the D14L RNAi construct indicated that D14 and D14L function in independent pathways. In Arabidopsis two signaling pathways, the SL signaling pathway mediated by D14 and the karrikin signaling pathway mediated by KAI2, depend on the function

Plant materials and growth conditions

The d3-1 (Ishikawa et al., 2005), d3-2 (Yoshida et al., 2012), d14-1 (Arite et al., 2009), and D14L RNAi (Yoshida et al., 2012) lines were described previously. The D14L RNAi lines 8 and 43, and d3-2 are in the Nipponbare background. The d14-1, D14L RNAi d14-1, and d3-1 lines are in the Shiokari background.

To observe the mesocotyl phenotypes and to examine D14L mRNA expression levels, seeds were sterilized in sodium hypochlorite and grown on 0.7% (w/v) agar in a growth chamber at 28°C. For in

Acknowledgments

We thank Prof. Mikio Nakazono and Dr. Zhongyuan Hu for advice on plant growth and GR24 treatment conditions. This work was supported by a grant from the Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) of Japan to J.K.

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