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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (8): 1934-1947.doi: 10.3724/SP.J.1006.2024.32046

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

OsRPTA18 participated in the regulation of leaf inclination in rice

HE Dan-Dan1,2(), SHU Ya-Zhou1,2(), ZHOU Hai-Lian1,2, WU Song-Guo1,2, WEI Xiao-Shuang1,2, YANG Ming-Chong1,2, LI Bo1,2, WU Zheng-Dan1,2, HAN Shi-Jian2, YANG Juan2,3, WANG Ji-Bin1,2,4,*(), WANG Ling-Qiang1,2,3,*()   

  1. 1Guangxi University / State Key Laboratory of Conservation and Utilization of Agric-Biological Resources, Nanning 530004, Guangxi, China
    2College of Agriculture, Guangxi University, Nanning 530004, Guangxi, China
    3National Demonstration Center for Experimental Plant Science Education / Guangxi University, Nanning 530004, Guangxi, China
    4Moutai Institute, Renhuai 564501, Guizhou, China
  • Received:2023-12-01 Accepted:2024-04-01 Online:2024-08-12 Published:2024-04-18
  • Contact: * E-mail: wangjibin@mtxy.edu.cn;E-mail: lqwang@gxu.edu.cn
  • About author:** Contributed equally to this work
  • Supported by:
    General Project of Guangxi Natural Science Foundation(2022GXNSFAA035479);Key Project of Guangxi Natural Science Foundation(2020GXN9)

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Abstract:

The regulatory particle triple-A ATPase (RPTA) gene family is closely related to plant growth and development, hormone regulation and stress response. In this study, a total of 33 members of the gene family were identified in rice and their gene locations, gene structures, motifs composition, and cis-elements in the promoter regions were revealed. Then, the relative expression patterns of the gene members in OsRPTA family were investigated with the data downloaded from rice CREP database. Most of the OsRPTA genes expressed highly in panicle, endosperm and callus tissues. β-D-glucuronidase (GUS) staining further indicated that one of the members, OsRPTA18, was expressed in collar, root, leaf, leaf sheath, stem node, vascular bundles of glume and lemma. Subcellular localization indicated that OsRPTA18 protein was in nucleus. Two knock-out mutants (osrpta18-1 and osrpta18-2) were generated by CRISPR/Cas9 editing technology and both exhibited reduced plant height, leaf inclination, accompanied by the decreased grain width and 1000-grain weight compared to the wild type Zhonghua 11. Transverse section staining of the lamina joints of the plants indicated the changes in the cell wall thickness and vascular bundle size, causing the obvious proliferation of thick-walled cells at adaxial side, thus decreasing asymmetric development of the lamina joints and the leaf angel in the osrpta18 mutants. These results of this study are valuable for further functional analysis of RPTA genes and utilization of the OsRPTA18 gene for the genetic improvement of rice varieties with ideal plant architecture.

Key words: rice, RPTA gene family, collar, leaf inclination

Table 1

Primers used in this study"

引物名称
Primer name		 正向引物
Forward primer (5'-3')		 反向引物
Reverse primer (5'-3')
OsRPTA18-CRISPR-Target TAGAGCTAGAAATAGCAAGTTAAAAT TAGAGCTAGAAATAGCAAGTTAAAAT
OsRPTA18-CRISPR-Identify CCGGAAGAGGAGACCCATCTCGACA CACTCCTATCTCACTGGCTGGTA
OsRPTA18-GUS TATGACCATGATTACGAATTCGAATGTGCAGAACACTGTGAGGA TGGCTGCAGGTCGACGGATCCGGTAGACAAAAGATACCCTAACCAAGA
OsRPTA18-GUS-Identify CAGTGAGCGCAACGCAATTA CCCAGTCACGACGTTGTAAAACG

Table 2

Information of RPTA family in rice"

基因名称
Gene name		 基因LOC号
Gene LOC number		 染色体Chr. 核酸长度Nucleotide length
(bp)		 氨基酸数目
Number of amino acids (aa)		 分子量
Molecular weight
(kD)		 等电点
pI		 不稳定
系数Instability index		 脂肪族
指数Aliphatic index		 平均疏水性
Grand average of hydropathicity
OsRPTA1 LOC_Os01g04814.4 1 1260 400 44.17 6.98 47.96 73.91 -0.453
OsRPTA2 LOC_Os01g12660.1 1 2928 841 93.42 8.23 48.06 87.81 -0.378
OsRPTA3.1 LOC_Os01g43480.1 1 1281 813 89.85 5.54 38.12 89.95 -0.341
OsRPTA3.2 LOC_Os01g43480.2 1 1290 754 84.23 5.76 40.71 87.17 -0.415
OsRPTA3.3 LOC_Os01g43480.3 1 1230 676 75.82 5.38 37.24 86.53 -0.480
OsRPTA4 LOC_Os01g48270.1 1 1353 411 45.57 5.42 51.96 86.85 -0.378
OsRPTA5.1 LOC_Os01g55260.1 1 3333 387 43.47 6.02 47.87 87.75 -0.428
OsRPTA5.2 LOC_Os01g55260.2 1 1074 307 34.56 6.23 51.23 97.25 -0.286
OsRPTA6 LOC_Os02g10640.1 2 1731 401 44.58 7.03 28.84 93.40 -0.333
OsRPTA7 LOC_Os02g11050.1 2 1737 425 47.22 8.90 40.91 93.58 -0.342
OsRPTA8 LOC_Os02g21970.1 2 1176 420 46.45 5.74 37.35 88.95 -0.328
OsRPTA9 LOC_Os02g51400.1 2 2568 976 109.51 5.41 53.23 81.72 -0.553
OsRPTA10 LOC_Os02g54340.1 2 2484 427 47.68 6.03 33.77 83.94 -0.406
OsRPTA11.1 LOC_Os02g56000.1 2 2097 430 47.79 4.94 41.09 92.10 -0.427
OsRPTA11.2 LOC_Os02g56000.2 2 2313 410 45.48 4.88 41.48 93.25 -0.375
OsRPTA12 LOC_Os03g18690.1 3 1464 451 49.71 5.91 45.46 90.98 -0.388
OsRPTA13 LOC_Os03g22420.1 3 1290 1111 121.68 6.13 43.49 82.72 -0.373
OsRPTA14 LOC_Os04g21660.1 4 1281 358 39.50 5.32 35.59 93.45 -0.239
OsRPTA15 LOC_Os04g39190.1 4 2922 577 62.28 9.84 50.51 92.25 -0.148
OsRPTA16 LOC_Os04g42110.1 4 1272 579 62.80 7.24 44.39 89.67 -0.231
OsRPTA17 LOC_Os05g31220.1 5 1206 392 42.30 6.73 44.03 94.65 -0.151
OsRPTA18.1 LOC_Os05g50750.1 5 1536 856 94.86 5.58 46.24 83.87 -0.485
OsRPTA18.2 LOC_Os05g50750.2 5 1446 828 91.67 5.52 47.76 84.93 -0.475
OsRPTA18.3 LOC_Os05g50750.3 5 1182 699 77.05 5.38 45.08 80.42 -0.597
OsRPTA19 LOC_Os06g01980.1 6 3246 771 84.78 7.31 55.90 78.48 -0.486
OsRPTA20 LOC_Os06g03940.1 6 1347 488 107.10 9.43 45.81 84.78 -0.381
OsRPTA21 LOC_Os06g07630.1 6 3279 430 47.77 4.94 38.87 92.77 -0.412
OsRPTA22 LOC_Os06g09290.1 6 1347 427 47.68 6.03 33.77 83.94 -0.406
OsRPTA23 LOC_Os06g12160.1 6 588 974 108.20 5.72 53.80 85.51 -0.496
OsRPTA24 LOC_Os06g39870.1 6 3609 424 46.82 9.03 39.28 93.33 -0.289
OsRPTA25 LOC_Os06g40560.1 6 1260 402 44.58 8.48 29.86 93.72 -0.315
OsRPTA26.1 LOC_Os06g45820.3 6 2928 512 55.06 8.55 33.69 87.79 -0.167
OsRPTA26.2 LOC_Os06g45820.4 6 1281 482 51.45 7.79 30.88 94.07 -0.084
OsRPTA27 LOC_Os06g50050.1 6 1290 394 43.69 7.65 63.65 92.34 -0.254
OsRPTA28 LOC_Os07g47530.1 7 1230 1082 1184.00 8.40 42.38 81.67 -0.385
OsRPTA29 LOC_Os07g49150.1 7 1353 449 49.58 5.91 44.33 89.87 -0.414
OsRPTA30 LOC_Os08g44240.1 8 3333 1093 119.51 5.91 47.33 95.12 -0.135
OsRPTA31 LOC_Os09g38730.1 9 1074 449 48.69 4.90 37.33 91.94 -0.263
OsRPTA32 LOC_Os10g30580.2 10 1731 196 21.81 4.88 57.44 75.28 -0.559
OsRPTA33 LOC_Os11g43970.1 11 1737 1203 131.21 6.81 48.55 80.52 -0.478

Fig. 1

Chromosomal distribution, protein clustering, and collinearity analysis of RPTA gene family in rice A: Chromosomal distribution of OsRPTA gene family; B: Unrooted phylogenetic tree of OsRPTA proteins; C: Collinearity analysis of OsRPTA gene family, where red and gray lines indicate the collinear relationship among RPTA gene members and among all genes in rice genome, respectively."

Fig. 2

Motif composition, conserved domains, and gene structure of RPTA gene family in rice A: motif composition of RPTA proteins; B: conserved domains of RPTA proteins; C: gene structures of OsRPTA family, where green blocks indicate 5' and 3' untranslated regions (UTR), yellow blocks indicate the exons in coding region (CDS), and black lines indicate introns."

Fig. 3

Cis-elements in the promoter regions of RPTA gene family in rice The number of cis-elements is indicated numerically. Gray box means no corresponding cis-element."

Fig. 4

Co-expression profiling of RPTA in rice The color scale representing the relative signal values is shown above (green refers to low expression; black refers to medium expression and red refers to high expression). N1, N7, N20: endosperm 14, 17, 21 days after pollination; N2, N18: flag leaves; N3-N6, N17, N24, N29: Calli; N8: Spikelet; N9, N30: young leaves; N10, N31: sheaths; N11, N22: young panicles; N12-N14: plumules; N15, N16: radicles; N19, N23: stems; N21: seed imbibition; N25: Hull; N26: stamen; N27, N28, N32: young panicles at heading stages 3, 4, and 5, respectively; N33: root; N34: shoot; N35, N36: leaf and root; N37-N39: seedlings at trefoil stage treated with GA3, KT, and NAA, respectively."

Fig. 5

β-Glucuronidase (GUS) staining of various tissues and organs from OsRPTA18pro-GUS transgenic lines A: germinating seeds; B: root, leaf, stem, and collar collected from the seedlings at three-leaf stage; C: root, leaf, stem internode, collar, cross section of a collar, cross section of a sheath, stem node, floret, cross section of a hull collected from the plants at third heading stage; D: root, leaf, stem internode, collar, cross section of a collar, cross section of a sheath, stem node, cross section of a node collected from the plants at the fifth heading stage, and a developing seed at early maturity stage."

Fig. 6

Subcellular localization of rice RPTA18 protein in Nicotona benthamiana 35S::GFP was empty vector; 35S:OSRPSTA18-GFP was constructed from OSRPSTA18 and vector pD1301S; 35S::H2B-mCherry was nuclear marker vector. Bar: 20 μm."

Fig. 7

Generating the CRISPR/Cas9 editing lines osrpta18-1 and osrpta18-2 A: gRNA target sequence and Cas9/gRNA recombination; B: the sequence comparison between mutants (osrpta18-1 and osrpta18-2) and Zhonghua 11 (ZH11) wild type (WT) plants, where the blue letters, deletion lines, lowercase letters in red represent target sequences, missing bases, and inserted bases, respectively. - represents missing bases, + represents inserted bases."

Fig. 8

Phenotypes of the CRISPR/Cas9 edited lines osrpta18-1 and osrpta18-1 A: leaf inclination of the seedling 10 days after germination (DAG) (Bar: 1 cm) (n = 26); B: plant height of the 100-DAG plants (Bar: 1 cm) (n = 60); C: leaf inclination of the 100-DAG plants (n = 45); D: grain length (n = 66), grain width (n = 34) and 1000-grain weight (n = 5) of plants. One-way ANOVA (and nonparametric or mixed). *: P < 0.05; **: P < 0.001."

Fig. 9

Alternation of cell proliferation cell wall thickness and vascular bundle in lamina joint of CRISPR/Cas9 edited lines osrpta18-1 and osrpta18-2 A: the comparison of the lamina joints; B: the cross sections of the collar, the red boxes indicate the adaxial side and the abaxial side of the collar, bar: 500 μm; C: zoomed-in view of the abaxial side of the cross section of collar, Bar: 40 μm; D: zoomed-in view of the adaxial side of the cross section of collar, bar: 40 μm."

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