Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (5): 786-800.doi: 10.3864/j.issn.0578-1752.2019.05.002

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

Enrichment Profile of GA4 is an Important Regulatory Factor Triggering Rice Dwarf

HUANG ShengCai1,WANG Bing1,XIE GuoQiang2,LIU ZhongLai2,ZHANG MeiJuan1,ZHANG ShuQing1(),CHENG XianGuo1()   

  1. 1 Intitute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100081
    2 Jiu Jiang Academy of Agricultural Sciences, Jiujiang 332101, Jiangxi
  • Received:2018-11-06 Accepted:2018-12-09 Online:2019-03-01 Published:2019-03-12
  • Contact: ShuQing ZHANG,XianGuo CHENG E-mail:zhangshuqing@caas.cn;chengxianguo@caas.cn

Abstract:

【Objective】A dwarf rice mutant was generated by culturing rice embryo tissues and characterized to elucidate the reasons for leading to an occurence of semi-dwarf rice with more tiller number. It is expected that this study will provide a theoretical basis for scientifically cultivating rice varieties of lodging-resistant and high-yielding in overcoming the dwarf obstacle factors.【Method】In this study, both the rice dwarf mutant and wild type were phenotypically profiled, and the structural characteristics of flower and cell appearance of leaves were investigated by stereomicroscope and light microscopy; The differential gene expression profiles were analyzed by both the transcriptomics and qRT-PCR, and the sensitivity of the mutant to exogenous gibberellin was detected by spraying exogenous GA3; The enrichment profiles of gibberellin in the mutant were detected by a high performance liquid chromatography and a mass spectrometry. 【Result】Data showed that the mutant demonstrated a decrease of 56.59% in the average plant height and an increase of 47.44% in the effective tiller number compared with the wild type, respectively (P <0.01). Observation showed that the mutant led to disappearance of the epidermis and revealed a smaller stamen accompanying a delayed development in the flower organs. Although the mutant has a higher effective tiller number, but significantly lowers the seed setting rate, which only accounts for 12.62% of that in the wild type. The length and the width of grains also are significantly reduced (P <0.01). Stem longitudinal sections reveal that the mutant decreased the cell length of 23% compared with the wild type (P <0.01). However, when the mutant was exposed to exogenous gibberellin, the plant height, effective tiller number, seed setting rate, seed size, epidermis and the stem cell length were obviously restored, indicating that the dwarf mutant possibly results from the shortage of GA’s synthesis in plant. Transcriptome sequencing showed that the mutant significantly up-regulated the OsGA13ox gene, and exhibited an identical result with the qRT-PCR analyses. Since the OsGA13ox controls the conversion of the GA12 to the GA53, both of which are converted to the GA4 and the GA1, respectively. Particularly, the GA4 exhibits a higher activity than the GA1, suggesting that rice dwarf might be triggered by the reduction of GA4 enrichment in plant. Measurement confirmed that the accumulation of the GA4 in the mutant was decreased by 94.9% compared with the wild type. Additionally, as a specific receptor for SL (Strigolactone), the D14 gene is involved in the SL signaling transduction in plant, and thus inhibits branching or tillering. The qRT-PCR showed that the mutant significantly down-regulated the D14 gene compared to the wild type, however, both the wild type and the mutant significantly up-regulated after spraying GA3. The data suggested that the up-regulation of the D14 gene might lower the effective tiller number, while the down-regulation of the D14 gene possibly increase the effective tiller number. Statistical analyses demonstrates that the mutant significantly increased the effective tiller number, but both the wild type and mutant decreased the effective tiller number after spraying GA3, indicating that the expression profiles of the D14 gene in rice might be modulated by GA, and thereby exert on the tiller number.【Conclusion】Semi-dwarfed rice mutant is likely caused by a decrease of GA4 enrichment because of abnormal expression of the OsGA13ox gene, and GA might indirectly regulate rice tiller by affecting the expression of the D14 gene.

Key words: rice, semi-dwarf mutant, OsGA13ox, GA4, D14, effective tiller

Table 1

Fluorescent quantitative PCR primers"

基因 Gene 引物序列 Primer sequence (5′-3′)
β-Actin F: CTGACAGGATGAGCAAGGAG
R: GGCAATCCACATCTGCTGGA
OsGA13ox F: AGAAGTGGAGAAAAGACTACGG
R: CAATGATCTTTCTCTGGTGTGC
D14 F: AGAAAGAGAGAGAAGAAGCGAG
R: CGCGCTCCCCTTTTATATACTA

Fig. 1

Phenotypic differences between the WT and the sd13 A: Morphological characteristics at booting stage; B: Spikelet in the WT (Scale bar, 2 cm); C: Spikelet in the sd13 (Scale bar, 2 cm); D: Mature seed size"

Fig. 2

Microscopic phenotypes of the WT and the sd13 A-D: The spikelet morphology of the WT and the sd13 before and after the application of GA3 (Scale bar, 0.4 mm); E-H: The flower characteristic of the WT and the sd13 before and after the application of GA3(Scale bar, 0.4 mm)"

Fig. 3

The apical internode cell size of the WT and the sd13 before and after spraying GA (Scale bar, 25 μm)"

Fig. 4

Statistical analyses of phenotype and stem cell lengths of the WT and the sd13 ** indicates a extremely significant difference, P <0.01; * indicates a significant difference, P <0.05. The same as below"

Fig. 5

Phenotypic changes of the sd13 after GA treatment A: Phenotypic changes after application of GA by soil culture mutants; B: Mutant phenotypic changes after adding different concentrations of GA in nutrient solution, the GA①, GA② and GA③ represents the concentration of GA3 is 1.5×10-4, 3.0×10-5 and 6.0×10-6 mol·L-1, respectively"

Fig. 6

Transcriptome sequencing results A: Differential gene clustering. Each row represents a gene and each column represents a sample. The color is from red to blue, indicating that the expression level is from large to small, and the expression patterns of genes in similar clusters are similar, indicating that these genes may have similar functions or participate in regulation of the same metabolic pathway; B: Differential gene volcano map. Genes with significant differential expression are indicated by red dot (up-regulation) and green dot (down-regulation), and genes with insignificant differential expression are represented by blue dots; C: Differential gene GO enrichment map. Green represents biological processes and orange represents molecular function; D: Differential gene KEGG enrichment scatter plot. The size of the dot indicates the number of differentially expressed genes in the pathway, and the color of the dot corresponds to a different Qvalue. The smaller the value, the more significant the enrichment"

Table 2

Expressing opposite genes before and under GA application in the sd13 "

基因 Gene 功能 Function
Os01g0115600 蛋白激酶功能,结合ATP
Kinase function, binding with ATP
Os01g0781700 ATP结合功能ATP binding
Os10g0463800 功能未知Unknown function
Os03g0332100 GA13ox,催化GA12转化为GA53,决定GA4和GA1的比例[24]
GA13ox, Converts GA12 into GA53, determines the ratio of GA4 and GA1[24]
Os04g0223500 黄素腺嘌呤二核苷酸,单加氧酶活性
FAD, monooxygenase activity
Os04g0606000 酰基转移酶活性
Transferase activity, transferring acyl groups
Os06g0364500 多糖结合功能Polysaccharide binding
Os12g0231700 推测是转位子蛋白Transposon protein, putative

Fig. 7

Relative expression of the OsGA13ox gene in flowers, stem and leaf of the sd13 "

Fig. 8

GA test results in the WT and the sd13 A: The GA4 peak map of WT; B: The GA4 peak map of the sd13 ; C: GA1 content in the WT and the sd13 ; D: GA4 content in the WT and the sd13 "

Table 3

A total of 12 genes responding to GA in both WT and sd13 "

基因 Gene 功能 Function
Novel00727 Lnc RNA,可能参与调控植物的表型Lnc RNA, probably participate in regulating the phenotype of plants
Os01g0191200 磷酸激酶活性 Acid phosphatase activity
Os01g0198900 功能未知 Unknow function
Os01g0368900 谷胱甘肽-二硫化物氧化还原酶活性 Has a glutathione-disulfide oxidoreductase activity
Os01g0501800 参与光合系统Ⅱ的组装和稳定 Involved in photosystem II assembly and stabilization
Os01g0600900 光受体,捕捉光信号 The light-harvesting complex functions as a light receptor
Os01g0948200 转录因子 DNA-binding transcription factor activity
Os03g0159900 功能未知 Unknow function
Os03g0196250 功能未知 Unknow function
Os03g0203200 D14蛋白参与独脚金内酯(SL)信号通路;与赤霉素竞争DELLA结合位点[27]
Involved in strigolactone signaling pathway; Competition with GA for DELLA binding sites[27]
Os03g0251350 转录因子,调控长日照开花[28] Probable transcription factor involved in the regulation of flowering time under long day[28]
Os03g0856500 与核糖体小亚基结合,负调控蛋白翻译 Ribosomal small subunit binding, negative regulation of translational elongation

Fig. 9

Venn diagram of differential genes and relative expression levels of D14 in the stems of the WT and the sd13 "

Fig. 10

Gibberellin synthesis pathway in plants[31]"

Table 4

The predicted results of the motif in the OsGA13ox gene promoter"

基因 Gene 基序 Motif 家族 Family 功能 Function
Os03g0224200 CAGTATCTTT ARR-B 参与细胞分裂素信号的转导[39]
Participation in the transduction of cytokinin signaling[39]
Os06g0130600 CACTCACATTCTCTCTGCACA BBR-BPC 调控大麦BKn3 的表达[40]
Regulate the expression of BKn3 gene in barley[40]
Os10g0419200 TTTTTTTTTTTG C2H2 促进植物营养生长到生殖生长的转变[41]
Promote the transformation of plant vegetative growth to reproductive growth[41]
Os07g0236700 GTATCTTTTTTTTTTTGCGCC Dof[42] 可能参与碳水化合物代谢基因的光调节,植物防御机制
Possible involvement in light regulation of carbohydrate metabolism genes, plant defense mechanisms
Os03g0821200 AACAAAGCTAAAACATAAGGG Dof[42] 参与种子萌发,发芽后糊粉中的赤霉素反应等基因的调控
Involving in seed germination, regulation of gibberellin reactions in aleurone after germination
Os03g0324300 GTCTAGATTCATTAC EIL 乙烯信号响应的正向调控因子[43]
Positive regulator of ethylene signal response[43]
Os03g0324200 GTCTAGATTCATTAC EIL 在植物受伤时,参与乙烯信号的转导[44]
Participation in the transduction of ethylene signals when plants are injured[44]
Os09g0490200 TAATGAATCTA EIL 参与乙烯信号转导 Participation in ethylene signal transduction
Os11g0681200 GAAACACGCGCGCGC FAR1 参与抵抗疾病[45] Enhance the resistance of disease[45]
Os02g0680700 TAGATATTA MYB_related[46] 功能未知 Unknown function
Os08g0157600 TAGATATTA MYB_related[46] 功能未知 Unknown function
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