Cloning and function analysis of MsPPR1 in alfalfa under drought stress

doi:10.11686/cyxb2022425

Abstract

Abstract:

Drought is an important environmental factor affecting plant growth， development and yield. pentatricopeptide repeats （PPR） family proteins play important roles in plant growth， development， stress response and other physiological processes. In this study， an MsPPR1 gene was cloned from alfalfa （Medicago sativa） cv. Zhongmu No.1， and the drought resistance function of MsPPR1 was investigated through decreasing its expression in alfalfa using a virus induced gene silencing method and heterologous overexpression in tobacco （Nicotiana benthamiana）. It was found that the open reading frame contains 3213 bp， encoding 1070 amino acids， and the relative molecular weight of the encoded protein is 121.65 kDa. MsPPR1 is a typical member of the PPR protein family， containing multiple PPR repeat domains and locating in the cytoplasm. MsPPR1 was expressed most in leaves， followed by stems and roots， and least in flowers and induced by drought， mannitol and abscisic acid treatments. The expression of MsPPR1 was decreased by the virus induced gene silencing technology in alfalfa， and decrease of MsPPR1 expression significantly reduced the drought resistance of the plants. The MsPPR1-silenced plants were more wilted， the relative water content was significantly reduced， and the relative electrolyte permeability was significantly increased. The heterologous overexpression of MsPPR1 in tobacco enhanced the drought resistance of transgenic tobacco， while the malondialdehyde content significantly decreased and the proline content increased. This study indicates that MsPPR1 is a positive regulatory factor of alfalfa drought resistance and provides a candidate gene for molecular breeding of alfalfa for drought resistance.

Key words: alfalfa, drought resistance, MsPPR1, virus-induced gene silencing

Shao-peng WANG, Jia LIU, Jun HONG, Ji-zhen LIN, Yi ZHANG, Kun SHI, Zan WANG. Cloning and function analysis of MsPPR1 in alfalfa under drought stress[J]. Acta Prataculturae Sinica, 2023, 32(7): 49-60.

Figures/Tables 9

Table 1 Primers used in the study

引物名称Primers	引物序列Primer sequence （5'-3'）
MsPPR1-F	ATGTTGTTCTCCTCAACAAAACCC
MsPPR1-R	TCAGTTCATAGACTCTGTCATGTCTGA
MsPPR1-GFP-F	TTAATTAAATGTTGTTCTCCTCAACAAAA
MsPPR1-GFP-R	GGCGCGCCGTTCATAGACTCTGTC
pBI121-MsPPR1-F	GAGAACACGGGGGACTCTAGAATGTTGTTCTCCTCAAC
pBI121-MsPPR1-R	AAGGGACTGACCACCCGGGGAGTTCATAGACTCTGT
pTRV2-MsPPR1-F	TGAGTAAGGTTACCGAATTCTGGGTTTGTGGGTATGTTGA
pTRV2-MsPPR1-R	GTGAGCTCGGTACCGGATCCTGAGAACTCATCCGCCTCCT
MsPPR1-qPCR-F	GTTTCTAGTTCCGTGGTTTCGGCGT
MsPPR1-qPCR-R	AAACTTCATCAACCCTCCCCAACTT
MsActin-qPCR-F	CAAAAGATGGCAGATGCTGAGGAT
MsActin-qPCR-R	CATGACACCAGTATGACGAGGTCG

Fig.1 Bioinformatic analysis of the deduced MsPPR1

Fig.2 Phylogenetic tree analysis of PPR family proteins in multiple species

Fig.3 Subcellular localization of MsPPR1

Fig.4 Analysis of relative expression of MsPPR1 in alfalfa

Fig.5 Expression level of MsPPR1 and phenotype under drought stress in alfalfa by virus-induced gene silencing

Fig.6 Relative expression and phenotype of MsPPR1 in different tobacco strains

Fig.7 Malondialdehyde （MDA） and proline （Pro） content of transgenic tobacco under drought stress

Fig.8 Photosynthesis parameters of transgenic tobacco under drought stress

References 31

1	Wang W Z， Ren Y J， Miao Y. Roles of PPR proteins in plant growth and development. Journal of Tropical and Subtropical Botany， 2019， 27（2）： 225-234.
	王婉珍，任育军，缪颖. PPR蛋白在植物生长发育中的作用. 热带亚热带植物学报， 2019， 27（2）： 225-234.
2	Jiang S C， Mei C， Wang X F， et al. Expression analysis of Arabidopsis SOAR1 in response to ABA and osmotic stress. Journal of Henan Agricultural Sciences， 2016， 45（2）： 29-34， 48.
	姜上川，梅超，王小芳，等. 拟南芥SOAR1基因响应ABA与渗透胁迫的表达分析. 河南农业科学， 2016， 45（2）： 29-34， 48.
3	Emami H， Kempken F. PRECOCIOUS1 （POCO1）， a mitochondrial pentatricopeptide repeat protein affects flowering time in Arabidopsis thaliana. The Plant Journal， 2019， 100（2）： 265-278.
4	Su H G， Li B， Song X Y， et al. Genome-wide analysis of the DYW subgroup PPR gene family and identification of GmPPR4 responses to drought stress. International Journal of Molecular Sciences， 2019， 20（22）： 5667.
5	Luo Z， Xiong J， Xia H， et al. Pentatricopeptide repeat gene-mediated mitochondrial RNA editing impacts on rice drought tolerance. Frontiers in Plant Science， 2022， 13： 926285.
6	Wei K， Han P. Pentatricopeptide repeat proteins in maize. Molecular Breeding， 2016， 36（12）： 170.
7	Liu Y， Jiang D， Yan J， et al. ABA-insensitivity of alfalfa （Medicago sativa L.） during seed germination associated with plant drought tolerance. Environmental and Experimental Botany， 2022， 203： 105069.
8	Holsters M， de Waele D， Depicker A， et al. Transfection and transformation of Agrobacterium tumefaciens. Molecular and General Genetics， 1978， 163（2）： 181-187.
9	Yang Y， Li R， Qi M. In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves. The Plant Journal， 2000， 22（6）： 543-551.
10	Kenneth J， Thomas D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^- ΔΔ ^CTmethod. Methods， 2001， 25（4）： 402-408.
11	Richard E， Gail E. Rapid estimates of relative water content. Plant Physiology， 1974， 53（2）： 258-260.
12	Blum A， Ebercon A. Cell membrane stability as a measure of drought and heat tolerance in wheat. Crop Science， 1981， 21（1）： 43-47.
13	Li Y. Cloning and analysis of an inducement-responsive promoter MsZPP of Medicago sativa L. Beijing： China Academy of Agricultural Sciences， 2012.
	李燕. 紫花苜蓿诱导表达启动子MsZPP的克隆及功能分析. 北京：中国农业科学院， 2012.
14	Liu J J， Zhang D Z， Zhang Y B， et al. Dynamic and comparative transcriptome analyses reveal key factors contributing to cadmium tolerance in broomcorn millet. International Journal of Molecular Science， 2022， 23（11）： 6148.
15	Li P， Kong W， Wu X， et al. Volatile organic compounds of the plant growth-promoting rhizobacteria JZ-GX1 enhanced the tolerance of Robinia pseudoacacia to salt stress. Frontiers in Plant Science， 2021， 12： 753332.
16	Liang H， Wei B， Chen J， et al. Evaluation of the drought resistance in alfalfa germplasm by chlorophyll fluorescence parameters at seedling stage. Acta Agrestia Sinica， 2020， 28（1）： 45-55.
	梁欢，韦宝，陈静，等. 基于叶绿素荧光参数的紫花苜蓿种质苗期抗旱性评价. 草地学报， 2020， 28（1）： 45-55.
17	Wang Z N， Li L H， Xu R H， et al. Advances in mitochondrial proteins responding to stresses in plants. Plant Physiology Journal， 2018， 54（2）： 221-231.
	王忠妮，李鲁华，徐如宏，等. 响应植物逆境胁迫的线粒体蛋白研究进展. 植物生理学报， 2018， 54（2）： 221-231.
18	Yuan H， Liu D. Functional disruption of the pentatricopeptide protein SLG1 affects mitochondrial RNA editing， plant development， and responses to abiotic stresses in Arabidopsis. The Plant Journal， 2012， 70（3）： 432-444.
19	Liu J M， Zhao J Y， Lu P P， et al. The E-subgroup pentatricopeptide repeat protein family in Arabidopsis thaliana and confirmation of the responsiveness PPR96 to abiotic stresses. Frontiers in Plant Science， 2016， 7： 1825.
20	Meierhoff K， Felder S， Nakamura T， et al. HCF152， an Arabidopsis RNA binding pentatricopeptide repeat protein involved in the processing of chloroplast psbB-psbT-psbH-petB-petD RNAs. The Plant Cell， 2003， 15（6）： 1480-1495.
21	Okuda K， Myouga F， Motohashi R， et al. Conserved domain structure of pentatricopeptide repeat proteins involved in chloroplast RNA editing. Proceedings of the National Academy of Sciences， 2007， 104： 8178-8183.
22	Yamazaki H， Tasaka M， Shikanai T. PPR motifs of the nucleus-encoded factor， PGR3， function in the selective and distinct steps of chloroplast gene expression in Arabidopsis. The Plant Journal， 2004， 38（1）： 152-163.
23	Chen H， Zeng Y， Yang Y， et al. Allele-aware chromosome-level genome assembly and efficient transgene-free genome editing for the autotetraploid cultivated alfalfa. Nature Communications， 2020， 11（1）： 2494.
24	Hao Y Y， Zhao X Q， Huang F D， et al. The role of PPR proteins in posttranscriptional regulation of organelle components in plants. Hereditas （Beijing）， 2021， 43（11）： 1050-1065.
	郝媛媛，赵向前，黄福灯，等. PPR蛋白在植物细胞器组分转录后调控中的作用机制. 遗传， 2021， 43（11）： 1050-1065.
25	Fisk D G， Walker M B， Barkan A. Molecular cloning of the maize gene crp1 reveals similarity between regulators of mitochondrial and chloroplast gene expression. The EMBO Journal， 1999， 18（9）： 2621-2630.
26	Gothandam K M， Kim E， Cho H， et al. OsPPR1， a pentatricopeptide repeat protein of rice is essential for the chloroplast biogenesis. Plant Molecular Biology， 2005， 58（3）： 421-433.
27	Bentolila S， Alfonso A A， Hanson M R. A pentatricopeptide repeat-containing gene restores fertility to cytoplasmic male-sterile plants. Proceedings of the National Academy of Sciences， 2002， 99（16）： 10887-10892.
28	Yao C J， Guo S M， Ma Y C， et al. Effect of drought stress on characteristics of photosynthesis and chlorophyll fluorescence of four species of Cassia. Pratacultural Science， 2017， 34（9）： 1880-1888.
	姚春娟，郭圣茂，马英超，等. 干旱胁迫对4种决明属植物光合作用和叶绿素荧光特性的影响. 草业科学， 2017， 34（9）： 1880-1888.
29	Qin D D， Dong J， Xu F C， et al. Identification of barley germplasms with high photosynthesis efficiency and correlation analysis between photosynthesis and yield. Modern Agricultural Science and Technology， 2017（23）： 38-40， 44.
	秦丹丹，董静，许甫超，等. 大麦高光效种质资源的筛选及光合特性与产量的相关性分析. 现代农业科技， 2017（23）： 38-40， 44.
30	Guo Y， Wang Z P， Lv Y C， et al. Evaluation of drought resistance and excellent germplasm screening in different Triticeae crops. Journal of Triticeae Crops， 2022， 42（10）： 1208-1219.
	郭莹，王振平，吕迎春，等. 不同麦类种质抗旱性评价及优异种质筛选. 麦类作物学报， 2022， 42（10）： 1208-1219.
31	Song Q Q， Zhu P J， He J， et al. Effect of low temperature on photosynthetic physiological characteristics of different Artocarpus heterophyllus Lam. germplasm resources. Agricultural Research and Application， 2021， 34（6）： 7-13.
	宋奇琦，朱鹏锦，何江，等. 低温对不同菠萝蜜种质资源光合生理特性的影响. 农业研究与应用， 2021， 34（6）： 7-13.

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