HTD2

Introduction

HTD2,, which is also called D88 or D14, encoding a esterase, inhibit rice branch, negative control rice tiller number.D14 may be one-horned gold lactone (Strigolactones, SLs) signal pathway of a component, in its downstream.China's 2 group and Japan's 1 team reported in the same year that cloning of the gene.

Function

Tiller number is highly regulated by controlling the formation of tiller bud and its subsequent outgrowth in response to endogenous and environmental signals.identified a rice mutant htd2 from one of the 15,000 transgenic rice lines, which is characterized by a high tillering and dwarf phenotype. Phenotypic analysis of the mutant showed that the mutation did not affect formation of tiller bud, but promoted the subsequent outgrowth of tiller bud. To isolate the htd2 gene, a map-based cloning strategy was employed and 17 new insertions-deletions (InDels) markers were developed. A high-resolution physical map of the chromosomal region around the htd2 gene was made using the F2 and F3 population. Finally, the gene was mapped in 12.8 kb region between marker HT41 and marker HT52 within the BAC clone OSJNBa0009J13. Cloning and sequencing of the target region from the mutant showed that the T-DNA insertion caused a 463 bp deletion between the promoter and first exon of an esterase/lipase/thioesterase family gene in the 12.8 kb region. Furthermore, transgenic rice with reduced expression level of the gene exhibited an enhanced tillering and dwarf phenotype. Accordingly, the esterase/lipase/thioesterase family gene (TIGR locus Os03g10620) was identified as the HTD2 gene. HTD2 transcripts were expressed mainly in leaf. Loss of function of HTD2 resulted in a significantly increased expression of HTD1, D10 and D3, which were involved in the strigolactone biosynthetic pathway. The results suggest that the HTD2 gene could negatively regulate tiller bud outgrowth by the strigolactone pathway.

mutation

The rice htd2 mutant was found in one of 15,000 T1 rice transgenic lines. The mutant was characterized with significantly increased tiller number and reduced plant height . Genetic analysis indicated that the mutation was controlled by a single recessive gene using T1 progeny and F2 population (data not shown). To investigate whether excessive tiller resulted from the formation of tiller bud or bud outgrowth, we examined axils at different nodes in detail. We found that each node produced only one bud in all nodes of the htd2plants, so there was no difference in tiller bud number at each node between the htd2 plants and wild-type plants. However, tiller buds of the htd2 plants showed less suppression. Tiller buds at the first node were dormant in wild-type plants, while those buds in the htd2 plants were able to develop tillers. Higher order tiller buds also showed higher activity in the htd2 plants than in wild-type plants. Thus, the increased tiller number in the htd2 plants was due to an increase in outgrowth of tiller buds, but not an increase in the formation of tiller bud in each node. In addition to excessive tiller, the htd2 mutant shows a reduced plant stature. At the ripening stage, the height of the mutant reached approximately 56% of the wild-type height (Table 3). We compared different length of internodes between htd2 and wild-type plants. As shown in Fig. 2, the htd2 gene significantly inhibits the length of all internodes, with progressively larger reductions in lower internodes. The htd2 mutant also reveals a significant reduction in blade length, blade width, culm diameter and panicle size (Fig. 1b–d).

Expression

D88 was expressed in leaves, culms and roots, but at a very low level in flowers. The expression levels of D88 were similar between the d88 mutant and the wild-type Lansheng, indicating that the base substitution affected only protein function. For the d14 mutant, however, RT-PCR analysis revealed a dramatic reduction in the abundance of transcripts of 957-bp in comparison to the Shiokari wild-type (Supplementary Fig. 2a). Sequencing the 524-bp fragment indicated that it was a non-specific product (Data not shown). Real time PCR confirmed the expression of D88 was reduced by more than 20 times in culms of d14 in comparison to the Shiokari wild-type (Supplementary Table 2). To further study the expression of D88, a binary vector containing the GUS gene driven by the D88 promoter (D88:GUS) was constructed and used for rice transformation. Despite differences in the intensity of GUS activity, histochemical GUS staining of all 10 independently transgenic lines showed a common pattern of GUS distribution. As shown in Fig. 6m, n, consistent with the results of RT-PCR analysis, GUS staining was nearly undetectable in pistils or anthers despite weak staining on the glume ridges. Analyses with a longitudinal section and a transversal section of the root indicated that the GUS activity was localized mainly in the parenchyma cells in the root stele and lateral roots (Fig. 6c, e–g). A relatively high level of GUS expression was observed in the vascular tissues of vein and leaf sheath, ligule base, auricle base and stem base, especially in tiller buds (Fig. 6a, b, d, h–l).

Knowledge extension

Recent studies using highly branched mutants of pea, Arabidopsis and rice have demonstrated that strigolactones, a group of terpenoid lactones, act as a new hormone class, or its biosynthetic precursors, in inhibiting shoot branching. Here, we provide evidence that DWARF14 (D14) inhibits rice tillering and may act as a new compo-nent of the strigolactone-dependent branching inhibition pathway. The d14 mutant exhibits increased shoot branch-ing with reduced plant height like the previously characterized strigolactone-deficient and -insensitive mutants d10 and d3, respectively. The d10-1 d14-1 double mutant is phenotypically indistinguishable from the d10-1 and d14-1 single mutants, consistent with the idea that D10 and D14 function in the same pathway. However, unlike with d10, the d14 branching phenotype could not be rescued by exogenous strigolactones. In addition, the d14 mutant contained a higher level of 2'-epi-5-deoxystrigol than the wild type. Positional cloning revealed that D14 encodes a protein of the α/β-fold hydrolase superfamily, some members of which play a role in metabolism or signaling of plant hormones. We propose that D14 functions downstream of strigolactone synthesis, as a component of hormone signaling or as an enzyme that participates in the conversion of strigolactones to the bioactive form.

Laps working on this gene

(1)State Key Laboratory of Rice Biology, China National Rice Research Institute, 359 Tiyuchang Road, 310006 Hangzhou, Zhejiang, China (2)Biotechnology Research Center, China Three Gorges University, 443002 Yichang, Hubei, China (3)National Center for Gene Research/Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 500 Caobao Road, 200233 Shanghai, China (4)State Key Laboratory of Rice Biology, China National Rice Research Institute, Chinese Academy of Agricultural Sciences, 310006 Hangzhou, China

Reference

1. Zhenyu Gao;Qian Qian;Xiaohui Liu;Meixian Yan;Qi Feng;Guojun Dong;Jian Liu;Bin Han

 Dwarf 88, a novel putative esterase gene affecting architecture of rice plant
 Plant Molecular Biology, 2009, 71(3): 265-276.

2.Tomotsugu Arite;Mikihisa Umehara;Shinji Ishikawa;Atsushi Hanada;Masahiko Maekawa;Shinjiro Yamaguchi;Junko Kyozuka d14, a Strigolactone-Insensitive Mutant of Rice, Shows an Accelerated Outgrowth of Tillers Plant and Cell Physiology, 2009, 50(8): 1416-1424 3. Wenzhen Liu;Chao Wu;Yaping Fu;Guocheng Hu;Huamin Si;Li Zhu;Weijiang Luan;Zhengquan He;Zongxiu Sun Identification and characterization of HTD2: a novel gene negatively regulating tiller bud outgrowth in rice Planta, 2009, 230(4): 649-658