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Identification and characterization of dwarf 62, a loss-of-function mutation in DLT/OsGRAS-32 affecting gibberellin metabolism in rice

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Abstract

A dwarf mutant, dwarf 62 (d62), was isolated from rice cultivar 93-11 by mutagenesis with γ-rays. Under normal growth conditions, the mutant had multiple abnormal phenotypes, such as dwarfism, wide and dark-green leaf blades, reduced tiller numbers, late and asynchronous heading, short roots, partial male sterility, etc. Genetic analysis indicated that the abnormal phenotypes were controlled by the recessive mutation of a single nuclear gene. Using molecular markers, the D62 gene was fine mapped in 131-kb region at the short arm of chromosome 6. Positional cloning of D62 gene revealed that it was the same locus as DLT/OsGRAS-32, which encodes a member of the GRAS family. In previous studies, the DLT/OsGRAS-32 is confirmed to play positive roles in brassinosteroid (BR) signaling. Sequence analysis showed that the d62 carried a 2-bp deletion in ORF region of D62 gene which led to a loss-of-function mutation. The function of D62 gene was confirmed by complementation experiment. RT-PCR analysis and promoter activity analysis showed that the D62 gene expressed in all tested tissues including roots, stems, leaves and panicles of rice plant. The d62 mutant exhibited decreased activity of α-amylase in endosperm and reduced content of endogenous GA1. The expression levels of gibberellin (GA) biosynthetic genes including OsCPS1, OsKS1, OsKO1, OsKAO, OsGA20ox2/SD1 and OsGA2ox3 were significantly increased in d62 mutant. Briefly, these results demonstrated that the D62 (DLT/OsGRAS-32) not only participated in the regulation of BR signaling, but also influenced GA metabolism in rice.

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Abbreviations

bp:

Base pair

BR:

Brassinosteroid

GA:

Gibberellin

GRAS:

GAI-RGA-SCR

GUS:

β-Glucuronidase

InDel:

Insertion/Deletion

ORF:

Open reading frame

SSR:

Simple sequence repeat

UTR:

Untranslated region

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Acknowledgments

This work was supported by National Natural Science Foundation of China (no. 31071394), Zhejiang Provincial Natural Science Foundation of China (no. Z3100089), National Key Project of GMO Breeding of China (no. 2008ZX08001-006), the Science and Technology Office of Zhejiang Province (no. 2007C12902), and the 151 Foundation for the Talents of Zhejiang Province. We thank Wanli Guo for providing the binary vector pCAMBIA1301. We also appreciate the efforts taken by Alfred Quampah and Mahmood Ul Hassan in revising the English of the manuscript.

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Correspondence to Chunhai Shi.

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W. Li and J. Wu contributed equally to this paper.

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Supplementary material 1 (DOC 63 kb)

Suppl. Fig. 1

cDNA and deduced amino acid sequence of D62 from cultivar 93-11 (wild type). The full-length cDNA consisted of 3,084 nucleotides containing an ORF of 1854-bp. The 5′-UTR was 770-bp long, and the 3′-UTR was 460-bp long. The ORF was predicted to encode a polypeptide of 617 amino acids. The 2-bp GC deletion of d62 allele is indicated 199 × 196 mm (300 × 300 DPI) (JPEG 2.05 MB)

Suppl. Fig. 2

Identification of transgenic plants by GUS reporter gene. The constructs consisted of GUS reporter gene driven by the CaMV 35S promoter. GUS staining of leaf blades from regenerated plants containing the empty vector pCAMBIA1301 (+), untransformed d62 mutant plants (−) and independent transgenic lines of d62 mutant plants containing pCT6.1 vector (1–6) are shown 199 × 89 mm (300 × 300 DPI) (JPEG 1.34 MB)

Suppl. Fig. 3

Alignment of D62 and rice DELLA-like proteins. Full-length amino acid sequences of D62, SLRL1 and SLRL2 from rice were aligned using Clustal X program. Black and gray boxes indicate identical and similar amino acids, respectively. The lines above the alignment indicate the locations of the conserved regions in the GRAS proteins as defined by Pysh et al. (1999) 199 × 103 mm (300 × 300 DPI) (JPEG 1.76 MB)

Suppl. Fig. 4

Alignment of D62 and SCR proteins. Full-length amino acid sequences of D62, OsSCR, ZmSCR and AtSCR were aligned using Clustal X program. Black and gray boxes indicate identical and similar amino acids, respectively. The lines above the alignment indicate the locations of the conserved regions in the GRAS proteins as defined by Pysh et al. (1999) 199 × 143 mm (300 × 300 DPI) (JPEG 2.91 MB)

Suppl. Fig. 5

Elongation of the second leaf sheath (a) and the seedling height (b) in response to GA3 treatment. The length of the second leaf sheaths and the seedling height were measured after 7 days when the wild-type and d62 seeds were germinated on 1/2 MS medium containing various concentrations of GA3. mean ± SD, n = 12, 150 × 71 mm (300 × 300 DPI) (JPEG 362 kb)

Suppl. Fig. 6

Expression analysis of the GA biosynthetic genes by RT-PCR. Total RNA was isolated from wild-type (WT) and d62 mutant plants treated with 10−4 M GA3 solution (+) or control solution (−). RT-PCR was performed using primers specific for each gene. The rice Actin1 gene was used as a control 99 × 103 mm (300 × 300 DPI) (JPEG 383 kb)

Suppl. Fig. 7

Expression analysis of the OsGA3ox2/D18 by real-time qPCR159 × 83 mm (200 × 200 DPI) (JPEG 152 kb)

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Li, W., Wu, J., Weng, S. et al. Identification and characterization of dwarf 62, a loss-of-function mutation in DLT/OsGRAS-32 affecting gibberellin metabolism in rice. Planta 232, 1383–1396 (2010). https://doi.org/10.1007/s00425-010-1263-1

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