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The maize d2003, a novel allele of VP8, is required for maize internode elongation

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Abstract

The d2003 is a natural dwarf mutant from maize inbred line K36 and has less than one-third of K36 plant height with severely shortened internodes. In this study, we reported the cloning of d2003 gene using positional cloning. The results showed that there was a single-base insertion in the coding region of Viviparous8 (VP8) in d2003 mutant, which resulted in a premature stop codon. Further genetic allelism tests confirmed that d2003 mutation is a novel allele of VP8. VP8 is mainly expressed in the stem apex, young leaves, and developing vascular tissues, and its expression levels in nodes are significantly higher than that in internodes at 12-leaf stage. Subcellular localization demonstrated that the VP8 protein is localized to the endoplasmic reticulum and the N-terminal 26 amino acids (aa) of VP8 protein are essential to its localization in ER. Further transgenic experiments showed that lack of the 26 aa leads to loss of VP8 function in Arabidopsis amp1 phenotype rescue. These results strongly suggested that the N-terminal 26 aa is critical for VP8 protein localization, and the correct protein localization of VP8 in ER is necessary for its function.

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Acknowledgments

This work was supported by the National Basic Research Program of China (2009CB118404).

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Authors and Affiliations

  1. Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Zhongguancun South Street 12, Beijing, 100081, China

    Hongkun Lv, Jun Zheng, Tianyu Wang, Junjie Fu, Xiang Zhang, Yunsu Shi & Guoying Wang

  2. College of Agriculture and Biotechnology, China Agricultural University, Yuanmingyuan West Road 2, Beijing, 100193, China

    Junling Huai, Haowei Min & Baohua Tian

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  1. Hongkun Lv
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  2. Jun Zheng
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  3. Tianyu Wang
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Correspondence to Yunsu Shi or Guoying Wang.

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Hongkun Lv and Jun Zheng have equally contributed to this work.

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11103_2013_129_MOESM1_ESM.tif

Supplemental figure 1. Subcellular localization of VP8-YFP in T3 transgenic seedlings. (A) Subcellular localization of the VP8-YFP fusion protein in T3 transgenic seedling roots. The fluorescence signal was primarily detected in the endoplasmic reticulum. (B) Subcellular localization of the YFP (pEarleyGate 104) protein in T3 transgenic seedling roots. The fluorescence signal was detected in the whole cells. (TIFF 4275 kb)

11103_2013_129_MOESM2_ESM.tif

Supplemental figure 2. Schematic representation of the signal peptide and transmembrane regions using the Interproscan program. (TIFF 517 kb)

11103_2013_129_MOESM3_ESM.tif

Supplemental figure 3. ABA content in 3-day-old K36 and d2003 seedlings. The error bars indicate the mean deviation of three replication experiments. (TIFF 1968 kb)

11103_2013_129_MOESM4_ESM.tif

Supplemental figure 4. The internode length and VP8 expression level in corresponding internodes and nodes. (A) The length of internodes 1-6. The first is the lowest and the sixth is the uppermost nodes. (B) The VP8 expression level in the internodes and corresponding nodes. (TIFF 2290 kb)

11103_2013_129_MOESM5_ESM.tif

Supplemental figure 5. Real-time RT-PCR analysis of the transcript levels of the KO and GA20ox genes in the GA synthesis pathway. The expression was normalized to that of tubulin. The transcript levels in the K36 were set to 1. (TIFF 1344 kb)

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Lv, H., Zheng, J., Wang, T. et al. The maize d2003, a novel allele of VP8, is required for maize internode elongation. Plant Mol Biol 84, 243–257 (2014). https://doi.org/10.1007/s11103-013-0129-x

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