Welcome to Acta Agronomica Sinica，

Acta Agronomica Sinica ›› 2019, Vol. 45 ›› Issue (9): 1327-1337.doi: 10.3724/SP.J.1006.2019.82069

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

Expression of OsUBA and its function in promoting seed germination and flowering

ZHANG Shuang-Shuang,WANG Li-Wei,YAO Nan,GUO Guang-Yan,XIA Yu-Feng,BI Cai-Li()   

  1. Life Science College, Hebei Normal University, Shijiazhuang 050024, Hebei, China
  • Received:2018-12-25 Accepted:2019-05-12 Online:2019-09-12 Published:2019-05-17
  • Contact: Cai-Li BI E-mail:beicaili@sina.com
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31101129);the Doctoral Foundation of Hebei Normal University(L2018B14)

RichHTML355

22

PDF

1215

Abstract:

Autophagy is a cellular process by which dysfunctional or unnecessary cellular components are degraded. Autophagy plays important roles in growth and development, and makes responses to nutritional deficiency and biotic/abiotic stress responsiveness in eukaryotes. NBR1 (Next to BRCA1 gene 1, NBR1) proteins were the most important autophagy cargo receptors found in plants. Up to date, only limited reports about NBR1 were available in plants, and the function of NBR1 proteins in rice remains unclear. An UBA (Ubiquitin associated, UBA) domain-containing gene was cloned from cDNA of rice (Oryza sativa L. japonica cv. Nipponbare) seedlings and was named as OsUBA. The open reading frame of OsUBA is 2538 bp in length and encodes 845 amino acids residues. Conserved domain prediction and phylogenetic analysis suggested that OsUBA belongs to NBR1 proteins. Promoter analysis suggested that many motifs related to light-, stress-, and hormone responsiveness were predicted in OsUBA promoter sequence. Gus staining of the rice plants harboring OsUBA p:Gus showed that OsUBA was highly expressed in anthers, germinating seeds, and roots, OsUBA transcripts could also be detected in stems and leaves. Expression of OsUBA was dramatically inhibited by 200 μmol L -1 ABA treatment, and slightly increased by 100 μmol L -1 GA treatment. Compared with the wild type control, the OsUBA-overexpressors germinated more quickly and flowered obviously earlier. The seed germination of the OsUBA-overexpressed rice lines was obviously inhibited by 3 μmol L -1 ABA treatment, and promoted slightly by 100 μmol L -1GA treatment. These results suggested that the expression and function of OsUBA may be related to regulations of flowering time, seed germination and biotic/abiotic stress responsiveness in rice.

Key words: rice (Oryza sativa L.), NBR1, transgenic, flowering time, seed germination

Fig. 1

PCR amplification of OsUBA gene in rice M: 5 kb DNA marker; 1: PCR product."

Fig. 2

Conserved domain prediction of OsUBA protein"

Fig. 3

Phylogenetic tree of OsUBA and its homologues in plants The star indicates OsUBA."

Fig. 4

PCR amplification of OsUBA promoter M: 5 kb DNA marker; 1: PCR product."

Table 1

Prediction of cis-elements of OsUBA promoter by PlantCARE software"

功能
Function		 元件名称(核心序列)
Element (core sequence)
ABA响应元件
cis-acting element involved in the abscisic acid responsiveness		 ABRE (ACGTG)
分生组织表达调控元件
cis-acting regulatory element related to meristem expression		 CAT-box (GCCACT)
茉莉酸甲酯响应元件
cis-acting regulatory element involved in the MeJA-responsiveness		 CGTCA-motif (CGTCA)
赤霉素响应元件
cis-acting element involved in gibberellin-responsiveness		 TATC-box (TATCCCA), P-box (CCTTTTG), GARE-motif (TCTGTTG)
水杨酸响应元件
cis-acting element involved in salicylic acid responsiveness		 TCA-element (CCATCTTTTT)
缺氧特异诱导增强子样元件
Enhancer-like element involved in anoxic specific inducibility		 GC-motif (CCCCCG)
功能
Function		 元件名称(核心序列)
Element (core sequence)
低温响应元件
cis-acting element involved in low-temperature responsiveness		 LTR (CCGAAA)
光响应相关元件
cis-acting regulatory element involved in light responsiveness		 G-box (CACGTC), Sp1 (CC(G/A)CCC)
部分光反应元件
Part of a light responsive element		 Box 4 (ATTAAT), GATA-motif (GATAGGG), I-box (GATAAGGTG), LAMP-element (CCTTATCCA), chs-CMA1a (TTACTTAA), chs-CMA2c (ATATACGTGAAGG)

Fig. 5

Identification of OsUBA p:Gus vector by double digestion M: 5 kb DNA marker; 1: plasmid of pCAMBIA1391-OsUBAp (undigested); 2: identification of pCAMBIA1391-OsUBAp plasmid by Hind III/Sal I digestion."

Fig. 6

Analysis of Gus expression in OsUBA p:Gus transgenic rice plants WT: rice cv. Nipponbare; L6 and L2: two homozygous rice lines harboring OsUBA p:Gus."

Fig. 7

Expression of OsUBA in different organs and tissues A: roots and leaf from rice seedlings grown in normal conditions for 7 d; B: the main stem node during flowering; C: the 2nd internode of the main stem during flowering; D: flower; E: seed germinated for 1 d; F: seed germinated for 3 d. The scale in A represents 1 cm, and the scale in B and C equals to 2 mm, the scale in D, E, and F means 1 mm."

Fig. 8

Expression of OsUBA under ABA and GA treatments A: untreated control, normally grown for 24 h; B: 200 μmol L-1 ABA treatment for 24 h; C: 100 μmoL L-1 GA treatment for 24 h; Bar = 1 mm."

Fig. 9

Identification of 35S:OsUBA vector by PCR and double digestion A: amplification of positive clone harboring pCAMBIA1300- OsUBA; M: 5 kb marker; 1: positive clone; B: identification of pCAMBIA1300-OsUBA positive clone by Xba I/Kpn I digestion; M: 5 kb marker; 1: plasmid of pCAMBIA1300-OsUBA (undigested); 2: Xba I/Kpn I digestion of pCAMBIA1300-OsUBA plasmid."

Fig. 10

Relative expression analysis of OsUBA in OsUBA- overexpressing rice plants"

Fig. 11

Comparison of flowering time between OsUBA-overexpressing rice plants and the wild type control"

Fig. 12

ABA and GA treatment affects seed germination of OsUBA-overexpressing rice plants and the wild type control A: The seed germination course of the OsUBA-overexpressing rice plants and the wild type control on 1/2 MS medium; B: 1/2 MS medium supplemented with 100 μmol L-1 GA; C: 1/2 MS medium supplemented with 3 μmol L-1 ABA."

[1] Yoshimoto K . Beginning to understand autophagy, an intracellular self-degradation system in plants. Plant Cell Physiol, 2012,53:1355-1365.
[2] Bassham D C . Plant autophagy: more than a starvation response. Curr Opin Plant Biol, 2007,10:587-593.
[3] Johansen T, Lamark T . Selective autophagy mediated by autophagic adapter proteins. Autophagy, 2011,7:279-296.
[4] Lamark T, Kirkin V, Dikic I, Johansen T . NBR1 and p62 as cargo receptors for selective autophagy of ubiquitinated targets. Cell Cycle, 2009,8:1986-1990.
[5] Kirkin V, Lamark T, Sou Y S, Bjørkøy G, Nunn J L, Bruun J A, Shvets E, McEwan D G, Clausen T H, Wild P, Bilusic I, Theurillat J P, Øvervatn A, Ishii T, Elazar Z, Komatsu M, Dikic I, Johansen T . A role for NBR1 in autophagosomal degradation of ubiquitinated substrates. Mol Cell, 2009,33:505-516.
[6] Katsuragi Y, Ichimura Y, Komatsu M . p62/SQSTM1 functions as a signaling hub and an autophagy adaptor. FEBS J, 2015,282:4672-4678.
[7] Waters S, Marchbank K, Solomon E, Whitehouse C, Gautel M . Interactions with LC3 and polyubiquitin chains link nbr1 to autophagic protein turnover. FEBS Lett, 2009,583:1846-1852.
[8] Svenning S, Lamark T, Krause K, Johansen T . Plant NBR1 is a selective autophagy substrate and a functional hybrid of the mammalian autophagic adapters NBR1 and p62/SQSTM1. Autophagy, 2011,7:993-1010.
[9] Zhou J, Wang J, Cheng Y, Chi Y J, Fan B, Yu J Q, Chen Z . NBR1-mediated selective autophagy targets insoluble ubiquitinated protein aggregates in plant stress responses. PLoS Genet, 2013,9:e1003196.
[10] Zientara-Rytter K, Lukomska J, Moniuszko G, Gwozdecki R, Surowiecki P, Lewandowska M, Liszewska F, Wawrzyńska A, Sirko A . Identification and functional analysis of Joka2, a tobacco member of the family of selective autophagy cargo receptors. Autophagy, 2011,7:1145-1158.
[11] Zientara-Rytter K, Sirko A . Significant role of PB1 and UBA domains in multimerization of Joka2, a selective autophagy cargo receptor from tobacco. Front Plant Sci, 2014,5:13.
[12] Dagdas Y F, Belhaj K, Maqbool A, Chaparro-Garcia A, Pandey P, Petre B, Tabassum N, Cruz-Mireles N, Hughes R K, Sklenar J, Win J, Menke F, Findlay K, Banfield M J, Kamoun S, Bozkurt T O . An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor. eLife, 2016,14:5.
[13] Li X, Zhang S, Ma J, Guo G, Zhang X, Liu X, Bi C . TaUBA, a UBA domain-containing protein in wheat (Triticum aestivum L.), is a negative regulator of salt and drought stress response in transgenic Arabidopsis. Plant Cell Rep, 2015,34:755-766.
[14] Lescot M, Déhais P, Moreau Y, De Moor B, Rouzé P, Rombauts S . PlantCARE: a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res, 2002,30:325-327.
[15] Zhou J, Jiao F, Wu Z, Li Y, Wang X, He X, Zhong W, Wu P . OsPHR2 is involved in phosphate-starvation signaling and excessive phosphate accumulation in shoots of plants. Plant Physiol, 2008, 146:1673-1686.
[16] Livak K J, Schmittgen T D . Analysis of relative gene expression data using real-time quantitative PCR and the 2 -ΔΔCt method. Methods, 2001,25:402-408.
[17] 孔德艳, 陈守俊, 周立国, 高欢, 罗利军, 刘灶长 . 水稻开花光周期调控相关基因研究进展. 遗传, 2016,38:532-542.
Kong D Y, Chen S J, Zhou L G, Gao H, Luo L J, Liu Z C . Research progress of photoperiod regulated genes on flowering time in rice. Hereditas (Beijing), 2016,38:532-542 (in Chinese with English abstract).
[18] Li L, Li X, Liu Y, Liu H . Flowering responses to light and temperature. Sci China Life Sci, 2016,59:403-408.
[19] Song Y H, Ito S, Imaizumi T . Flowering time regulation: photoperiod- and temperature-sensing in leaves. Trends Plant Sci, 2013,18:575-583.
[20] Gangappa S N, Maurya J P, Yadav V, Chattopadhyay S . The regulation of the Z- and G-box containing promoters by light signaling components, SPA1 and MYC2, in Arabidopsis. PLoS One, 2013,8:e62194.
[21] Jiao Y, Lau O S, Deng X W . Light-regulated transcriptional networks in higher plants. Nat Rev Genet, 2007,8:217-230.
[22] Harrison-Lowe N J, Olsen L J . Autophagy protein 6 (ATG6) is required for pollen germination in Arabidopsis thaliana. Autophagy, 2008,4:339-348.
[23] Kurusu T, Koyano T, Kitahata N, Kojima M, Hanamata S, Sakakibara H, Kuchitsu K . Autophagy-mediated regulation of phytohormone metabolism during rice anther development. Plant Signal Behav, 2017,12:e1365211.
[24] Kurusu T, Koyano T, Hanamata S, Kubo T, Noguchi Y, Yagi C, Nagata N, Yamamoto T, Ohnishi T, Okazaki Y, Kitahata N, Ando D, Ishikawa M, Wada S, Miyao A, Hirochika H, Shimada H, Makino A, Saito K, Ishida H, Kinoshita T, Kurata N, Kuchitsu K . OsATG7 is required for autophagy-dependent lipid metabolism in rice postmeiotic anther development. Autophagy, 2014,10:878-888.
[25] North H, Baud S, Debeaujon I, Dubos C, Dubreucq B, Grappin P, Jullien M, Lepiniec L, Marion-Poll A, Miquel M, Rajjou L, Routaboul J M, Caboche M . Arabidopsis seed secrets unravelled after a decade of genetic and omics-driven research. Plant J, 2010,61:971-981.
[26] Salem M A, Li Y, Wiszniewski A, Giavalisco P . Regulatory-associated protein of TOR (RAPTOR) alters the hormonal and metabolic composition of Arabidopsis seeds, controlling seed morphology, viability and germination potential. Plant J, 2017,92:525-545.
[27] Shu K, Liu X D, Xie Q, He Z H . Two faces of one seed: Hormonal regulation of dormancy and germination. Mol Plant, 2016,9:34-45.
[28] Steinbrecher T, Leubner-Metzger G . The biomechanics of seed germination. J Exp Bot, 2017,68:765-783.
[29] 徐恒恒, 黎妮, 刘树君, 王伟青, 王伟平, 张红, 程红焱, 宋松泉 . 种子萌发及其调控的研究进展. 作物学报, 2014,40:1141-1156.
Xu H H, Li N, Liu S J, Wang W Q, Wang W P, Zhang H, Cheng H Y, Song S Q . Research progress in seed germination and its control. Acta Agron Sin, 2014,40:1141-1156 (in Chinese with English abstract).
[30] 伍静辉, 谢楚萍, 田长恩, 周玉萍 . 脱落酸调控种子休眠和萌发的分子机制. 植物学报, 2018,53:542-555.
Wu J H, Xie C P, Tian C E, Zhou Y P . Molecular mechanism of abscisic acid regulation during seed dormancy and germination. Chin Bull Bot, 2018,53:542-555 (in Chinese with English abstract).
[31] Kucera B, Cohn M A, Leubner-Metzger G L . Plant hormone interactions during seed dormancy release and germination. Seed Sci Res, 2005,15:282-307.
[32] Honig A, Avin-Wittenberg T, Galili G . Selective autophagy in the aid of plant germination and response to nutrient starvation. Autophagy, 2012,8:838-839.
[33] Han C, Zhen S, Zhu G, Bian Y, Yan Y . Comparative metabolome analysis of wheat embryo and endosperm reveals the dynamic changes of metabolites during seed germination. Plant Physiol Biochem, 2017,115:320-327.
[34] Wasternack C, Hause B . Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. an update to the 2007 review in Annals of Botany. Ann Bot, 2013,111:1021-1058.
[35] Kumar D . Salicylic acid signaling in disease resistance. Plant Sci, 2014,228:127-134.
[36] Vishwakarma K, Upadhyay N, Kumar N, Yadav G, Singh J, Mishra R K, Kumar V, Verma R, Upadhyay R G, Pandey M, Sharma S . Abscisic acid signaling and abiotic stress tolerance in plants: a review on current knowledge and future prospects. Front Plant Sci, 2017,8:161.
[1] Ma Yi-Na, Wu Xiao-Ming-Yu, Li Ou-Qi, Wang Yuan, Chen Li, Zhang Ying-Chuan, Zhao Lun, Wen Jing, Fu Ting-Dong, Shen Jin-Xiong. Functional analysis of the Bna-miR1040-EIF3A module in regulating flowering time in rapeseed (Brassica napus) [J]. Acta Agronomica Sinica, 2026, 52(2): 349-362.
[2] HE Peng-Xu, YAO Li-Rong, CHEN Yuan-Ling, YAN Yan, ZHANG Hong, WANG Jun-Cheng, LI Bao-Chun, YANG Ke, SI Er-Jing, MENG Ya-Xiong, MA Xiao-Le, WANG Hua-Jun. Differences and correlations in physiological and molecular mechanisms of barley germination under drought stress [J]. Acta Agronomica Sinica, 2025, 51(9): 2412-2432.
[3] XU Yi-Wei, ZHANG Ying-Ying, LI Rui, YAN Yong-Liang, LIU Yun-Jun, KONG Zhao-Sheng, ZHENG Jun, WANG Yi-Ru. csp2 gene of Deinococcus gobiensis improves drought tolerance in maize [J]. Acta Agronomica Sinica, 2025, 51(8): 1981-1990.
[4] WANG Qiong, ZOU Dan-Xia, CHEN Xing-Yun, ZHANG Wei, ZHANG Hong-Mei, LIU Xiao-Qing, JIA Qian-Ru, WEI Li-Bin, CUI Xiao-Yan, CHEN Xin, WANG Xue-Jun, CHEN Hua-Tao. Genome-wide association analysis and candidate genes prediction of flowering time and maturity date traits in soybean (Glycine max L.) [J]. Acta Agronomica Sinica, 2025, 51(6): 1558-1568.
[5] LI Wen-Jia, LIAO Yong-Jun, HUANG Lu, LU Qing, LI Shao-Xiong, CHEN Xiao-Ping, JIN Jing-Wei, WANG Run-Feng. Genome-wide associate analysis of flowering traits and identification of candidate genes in peanut [J]. Acta Agronomica Sinica, 2025, 51(5): 1400-1408.
[6] LI Jia-Xin, HUANG Ying-Ying, WU Lu-Mei, ZHAO Lun, YI Bin, MA Chao-Zhi, TU Jin-Xing, SHEN Jin-Xiong, FU Ting-Dong, WEN Jing. Phylogenetic and functional analysis of the BnaSLY1 genes in Brassica napus L. [J]. Acta Agronomica Sinica, 2025, 51(1): 44-57.
[7] LIU Bo, CHI Ming, CAO Meng-Qi, TANG Da, YANG Heng-Zhao, ZHANG Wei-Hua, XUE Cong. Impact of potato StuPPO9 gene overexpression on drought resistance in Nicotiana benthamiana [J]. Acta Agronomica Sinica, 2024, 50(9): 2237-2247.
[8] QIAO Zhi-Xin, ZHANG Jie-Dao, WANG Yu, GUO Qi-Fang, LIU Yan-Jing, CHEN Rui, HU Wen-Hao, SUN Ai-Qing. Difference in germination characteristics of different winter wheat cultivars under drought stress [J]. Acta Agronomica Sinica, 2024, 50(6): 1568-1583.
[9] LIU Wei, WANG Yu-Bin, LI Wei, ZHANG Li-Feng, XU Ran, WANG Cai-Jie, ZHANG Yan-Wei. Overexpression of soybean isopropyl malate dehydrogenase gene GmIPMDH promotes flowering and growth [J]. Acta Agronomica Sinica, 2024, 50(3): 613-622.
[10] WU Yu, LIU Lei, CUI Ke-Hui, QI Xiao-Li, HUANG Jian-Liang, PENG Shao-Bing. Changes of root characteristics of super hybrid rice variety contributing to high nitrogen accumulation under low nitrogen application at seedling stage [J]. Acta Agronomica Sinica, 2024, 50(2): 414-424.
[11] LI Yan, FANG Yu-Hui, WANG Yong-Xia, PENG Chao-Jun, HUA Xia, QI Xue-Li, HU Lin, XU Wei-Gang. Transcriptomics profile of transgenic OsPHR2 wheat under different phosphorus stress [J]. Acta Agronomica Sinica, 2024, 50(2): 340-353.
[12] ZHENG Guang-Jie, YE Chang, XU Chun-Mei, CHEN Song, CHU Guang, CHEN Li-Peng, ZHANG Xiu-Fu, WANG Dan-Ying. Effect of low-temperature stress on glucose and water status in rice germ and its relationship with seedling emergence [J]. Acta Agronomica Sinica, 2024, 50(12): 3055-3068.
[13] WANG Zi-Ran, LU Yi-Wei, YANG Jing-Yi, WANG Cheng-Long, SONG Ya-Ping, MA Jin-Hu. Effects of exogenous SA on physiological characteristics and stress-resistant gene expression of soybean under Cd stress [J]. Acta Agronomica Sinica, 2024, 50(11): 2883-2895.
[14] XIAO Sheng-Hua, LU Yan, LI An-Zi, QIN Yao-Bin, LIAO Ming-Jing, BI Zhao-Fu, ZHUO Gan-Feng, ZHU Yong-Hong, ZHU Long-Fu. Function analysis of an AP2/ERF transcription factor GhTINY2 in cotton negatively regulating salt tolerance [J]. Acta Agronomica Sinica, 2024, 50(1): 126-137.
[15] YUE Run-Qing, LI Wen-Lan, MENG Zhao-Dong. Acquisition and resistance analysis of transgenic Maize Inbred Line LG11 with insect and herbicide resistance [J]. Acta Agronomica Sinica, 2024, 50(1): 89-99.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
Add: No. 80 Xueyuan South Rd, Beijing 100081, P. R. China E-mail: zwxb301@caas.cn
Supported by Beijing Magtech Co.Ltd