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Up- and Down-regulated Expression of OsCPK25/26 Results in Increased Number of Stamens in Rice

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Abstract

Calcium-dependent protein kinases play important roles in the regulation of plant growth and development. Rice genome contains 31 CDPK genes, and the biological functions of most of these CDPK genes remain unclear. To elucidate the biological function of OsCPK25/26, we first analyzed the expression profiles for OsCPK25/26. Transcripts of OsCPK25/26 were observed in the panicles, hulls, and stamens, with significantly higher expression in stamens. Plasmid constructs of either overexpression of OsCPK25 or RNAi knockdown of OsCPK25/26 were created and introduced into the Zhonghua11 (Oryza sativa L. ssp. japonica) rice variety, and single-copy homozygous lines of the resultant transgenic plants were characterized. The expression levels of OsCPK25/26 in panicles were increased 11–15-fold in the overexpressing plants and decreased 1.5–1.7-fold in the plants expressing RNAi against OsCPK25/26 compared with that in the control plants. The numbers of stamens in some florets increased with varying degrees, ranging from six to 13 stamens in both the overexpressing and RNAi plants. Compared with the control plants, the proportion of florets with increased stamen numbers in T2 generation was 30 % in the overexpressing plants and 36 % in RNAi plants and that in T3 generation was 50 and 51 %, respectively. Our results suggest that OsCPK25/26 are involved in the regulation of stamen development, and changes (increases or decreases) of OsCPK25/26 expression levels result in an increased number of stamens. Thus, the optimal expression levels of OsCPK25/26 are a key regulating factor in maintaining normal stamen numbers in rice.

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Abbreviations

CDPK:

Calcium-dependent protein kinase

OC:

Overexpression of OsCPK25

RC:

RNA interference of OsCPK25/26

WT:

Wild type

Nc:

Negative control

RT-PCR:

Reverse transcription polymerase chain reaction

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Acknowledgments

This research was funded by the National High Technology Research and Development Program of China (863 Program), the National Program of Transgenic Variety Development of China, and the National Natural Science Foundation of China.

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Authors and Affiliations

  1. National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research, Huazhong Agricultural University, Wuhan, 430070, China

    Wei Zhang, Fei Zhou, Hao Chen, Xianghua Li & Yongjun Lin

  2. Hubei Academy of Agricultural Science, Wuhan, China

    Bingliang Wan

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  1. Wei Zhang
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Correspondence to Yongjun Lin.

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Supplementary Fig. 1

The modification for p1300S vector and constructs for OsCPK25 overexpression and OsCPK25/26 RNA interference (RNAi). a The expression component framework of target gene in p1300S vector. b The construct for OsCPK25 overexpression. c The construct for OsCPK25/26 RNAi. The rice waxy-A intron 1 sequence was used to improve the suppression efficiency of the target gene (the diagram is not to scale). (TIFF 2331 kb)

Supplementary Fig. 2

Southern blot analysis of total genomic DNA of T0 overexpression transgenic plants. DNA samples were digested with Hind III and hybridized with radioactive probes (no Hind III restriction site was present within the T-DNA region). The number of electrophoresis bands shows the copy number of inserted T-DNA. M DNA marker, NT Zhonghua11, lanes 1 to 34 transgenic plants. (TIFF 1894 kb)

Supplementary Fig. 3

The germination test of Single-copy lines of OsCPK25 OC and OsCPK25/26 RC. A The positive homozygous seeds of OsCPK25 OC. B The heterozygous seeds of OsCPK25 OC. C The negative control seeds of OsCPK25 OC. D The positive homozygous seeds of OsCPK25/26 RC. E The heterozygous seeds of OsCPK25/26 RC. F The negative control seeds of OsCPK25/26 RC. (TIFF 6174 kb)

Supplementary Fig. 4

The expression patterns for the four genes that regulate floral organ number in 35 different tissues of Minghui 63 (O. sativa L. ssp. indica). Tissues: (1) seed, 72 h after imbibition; (2) calli, 15 days after induction; (3) calli, 15 days after subculture; (4) calli, screening stage; (5) calli, 5 days after regeneration; (6) embryo and radicle after germination; (7) leaf and root at three-leaf stage; (8) root, seedling with two tillers; (9) shoot, seedling with two tillers; (10) leaf, 4–5-cm young panicle; (11) sheath, 4–5-cm young panicle; (12) panicle, 4–5-cm young panicle; (13) leaf, young panicle at the secondary-branch primordium differentiation stage; (14) sheath, young panicle at the secondary-branch primordium differentiation stage; (15) young panicle at the secondary-branch primordium differentiation stage; (16) young panicle at the pistil and stamen primordium differentiation stage; (17) young panicle at the pollen-mother cell formation stage; (18) flag leaf, 5 days before heading; (19) stem, 5 days before heading; (20) stem, heading stage; (21) panicle, heading stage; (22) flag leaf, 14 days after heading; (23) hull, 1 day before flowering; (24) stamen, 1 day before flowering; (25) spikelet, 3 days after pollination; (26) endosperm, 7 days after pollination; (27) endosperm, 14 days after pollination; (28) endosperm, 21 days after pollination; (29) plumule, 48 h after emergence, dark; (30) radicle, 48 h after emergence, dark; (31) plumule, 48 h after emergence, light; (32) radicle, 48 h after emergence, light; (33) seedling, trefoil stage, GA3; (34) seedling, trefoil stage, KT; (35) seedling, trefoil stage, NAA. The signal value represents the expression level. The error bars are standard error of the mean from two replicates. (TIFF 8518 kb)

Supplementary Fig. 5

The expression patterns for the four genes that regulate floral organ number in 35 different tissues of Shanyou 63 (O. sativa L. ssp. indica). Tissues: (1) seed, 72 h after imbibition; (2) calli, 15 days after induction; (3) calli, 15 days after subculture; (4) calli, screening stage; (5) calli, 5 days after regeneration; (6) embryo and radicle after germination; (7) leaf and root at the three-leaf stage; (8) root, seedling with two tillers; (9) shoot, seedling with two tillers; (10) leaf, 4–5-cm young panicle; (11) sheath, 4–5-cm young panicle; (12) panicle, 4–5-cm young panicle; (13) leaf, young panicle at the secondary-branch primordium differentiation stage; (14) sheath, young panicle at the secondary-branch primordium differentiation stage; (15) young panicle at the secondary-branch primordium differentiation stage; (16) young panicle at the pistil and stamen primordium differentiation stage; (17) young panicle at the pollen-mother cell formation stage; (18) flag leaf, 5 days before heading; (19) stem, 5 days before heading; (20) stem, heading stage; (21) panicle, heading stage; (22) flag leaf, 14 days after heading; (23) hull, 1 day before flowering; (24) stamen, 1 day before flowering; (25) spikelet, 3 days after pollination; (26) endosperm, 7 days after pollination; (27) endosperm, 14 days after pollination; (28) endosperm, 21 days after pollination; (29) plumule, 48 h after emergence, dark; (30) radicle, 48 h after emergence, dark; (31) plumule, 48 h after emergence, light; (32) radicle, 48 h after emergence, light; (33) seedling, trefoil stage, GA3; (34) seedling, trefoil stage, KT; (35) seedling, trefoil stage, NAA. The signal value represents the expression level. The error bars are standard error of the mean from two replicates. (TIFF 8518 kb)

Supplementary Fig. 6

The expression patterns for the four genes that regulate floral organ number in 35 different tissues of Zhenshan 97 (O. sativa L. ssp. indica). Tissues: (1) seed, 72 h after imbibition; (2) calli, 15 days after induction; (3) calli, 15 days after subculture; (4) calli, screening stage; (5) calli, 5 days after regeneration; (6) embryo and radicle after germination; (7) leaf and root at the three-leaf stage; (8) root, seedling with two tillers; (9) shoot, seedling with two tillers; (10) leaf, 4–5-cm young panicle; (11) sheath, 4–5-cm young panicle; (12) panicle, 4–5-cm young panicle; (13) leaf, young panicle at the secondary-branch primordium differentiation stage; (14) sheath, young panicle at the secondary-branch primordium differentiation stage; (15) young panicle at the secondary-branch primordium differentiation stage; (16) young panicle at the pistil and stamen primordium differentiation stage; (17) young panicle at the pollen-mother cell formation stage; (18) flag leaf, 5 days before heading; (19) stem, 5 days before heading; (20) stem, heading stage; (21) panicle, heading stage; (22) flag leaf, 14 days after heading; (23) hull, 1 day before flowering; (24) stamen, 1 day before flowering; (25) spikelet, 3 days after pollination; (26) endosperm, 7 days after pollination; (27) endosperm, 14 days after pollination; (28) endosperm, 21 days after pollination; (29) plumule, 48 h after emergence, dark; (30) radicle, 48 h after emergence, dark; (31) plumule, 48 h after emergence, light; (32) radicle, 48 h after emergence, light; (33) seedling, trefoil stage, GA3; (34) seedling, trefoil stage, KT; (35) seedling, trefoil stage, NAA. The signal value represents the expression level. The error bars are standard error of the mean from two replicates. (TIFF 8521 kb)

Supplementary Fig. 7

The expression patterns of OIP30 in 35 different tissues of Minghui 63 (O. sativa L. ssp. indica) (a), Shanyou 63 (O. sativa L. ssp. indica) (b), and Zhenshan 97 (O. sativa L. ssp. indica) (c). Tissues: (1) seed, 72 h after imbibition; (2) calli, 15 days after induction; (3) calli, 15 days after subculture; (4) calli, screening stage; (5) calli, 5 days after regeneration; (6) embryo and radicle after germination; (7) leaf and root at three-leaf stage; (8) root, seedling with two tillers; (9) shoot, seedling with two tillers; (10) leaf, 4–5-cm young panicle; (11) sheath, 4–5-cm young panicle; (12) panicle, 4–5-cm young panicle; (13) leaf, young panicle at the secondary-branch primordium differentiation stage; (14) sheath, young panicle at the secondary-branch primordium differentiation stage; (15) young panicle at the secondary-branch primordium differentiation stage; (16) young panicle at the pistil and stamen primordium differentiation stage; (17) young panicle at the pollen-mother cell formation stage; (18) flag leaf, 5 days before heading; (19) stem, 5 days before heading; (20) stem, heading stage; (21) panicle, heading stage; (22) flag leaf, 14 days after heading; (23) hull, 1 day before flowering; (24) stamen, 1 day before flowering; (25) spikelet, 3 days after pollination; (26) endosperm, 7 days after pollination; (27) endosperm, 14 days after pollination; (28) endosperm, 21 days after pollination; (29) plumule, 48 h after emergence, dark; (30) radicle, 48 h after emergence, dark; (31) plumule, 48 h after emergence, light; (32) radicle, 48 h after emergence, light; (33) seedling, trefoil stage, GA3; (34) seedling, trefoil stage, KT; (35) seedling, trefoil stage, NAA. Signal value represents the expression level. The error bars are standard error of the mean from two replicates. (TIFF 5048 kb)

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Zhang, W., Wan, B., Zhou, F. et al. Up- and Down-regulated Expression of OsCPK25/26 Results in Increased Number of Stamens in Rice. Plant Mol Biol Rep 32, 1114–1128 (2014). https://doi.org/10.1007/s11105-014-0717-1

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