Cloning and Expression Analysis of Phosphate Starvation Responsive Genes, SgPHR1 and SgPHR2, in Stylosanthes guianensis

doi:10.3969/j.issn.1000-2561.2021.08.006

Abstract

Abstract:

Low phosphorus (P) stress is one of the constraints limiting crop growth and yield. PHR (phosphate starvation response) is the key factor in P signaling network, regulating plant phosphate (Pi) homeostasis. In this study, two transcription factors, SgPHR1 and SgPHR2, were cloned in stylo (Stylosanthes guianensis). The full length of SgPHR1 and SgPHR2 was 1413 bp and 849 bp, encoding 470 and 282 amino acid residues, respectively. The protein molecular weight of SgPHR1 and SgPHR2 was 51.4 kD and 30.9 kD, respectively. Both SgPHR1 and SgPHR2 belonged to the SANT family and contained MYB protein domain and CC protein domain. Both SgPHR1 and SgPHR2 included a set of potential phosphorylation sites. Subcellular localization prediction indicated that both SgPHR1 and SgPHR2 localized to the nucleus. Real-time quantitative PCR results showed that the transcript of SgPHR1 in leaves and roots was higher than that in stems, while SgPHR2 exhibited the highest expression in leaves. Furthermore, the expression of SgPHR1 and SgPHR2 was up-regulated by both -P and nitrogen (-N) deficient treatments in stylo roots. The transcription level of SgPHR1 and SgPHR2 was found to be increased during -P treatments, suggesting that SgPHR1 and SgPHR2 involved in stylo response to Pi starvation. This study would provide candidate genes for investigating the mechanism of stylo response to P deficiency.

Key words: low phosphorus stress, Stylosanthes, phosphate starvation response, gene expression, transcription factor

CLC Number:

Q945.78
Q78

HUANG Jie, SONG Jianling, AN Na, ZOU Xiaoyan, LI Jifu, LIU Guodao, CHEN Zhijian. Cloning and Expression Analysis of Phosphate Starvation Responsive Genes, SgPHR1 and SgPHR2, in Stylosanthes guianensis[J]. Chinese Journal of Tropical Crops, 2021, 42(8): 2158-2166.

Figures/Tables 10

Tab. 1 Primers for SgPHR1 and SgPHR2 cloning and qRT-PCR analysis

引物名称 Primer name	引物序列（5′-3′） Primer sequence (5°-3°)
SgPHR1-ORF-F	GAGCTCAAAACAATGAAAGCACACCC
SgPHR1-ORF-R	ACGCGTGCCTCACTCATCTGTCCTTG
SgPHR1-RT-F	GCACACCCTGCAAAGCAACTCA
SgPHR1-RT-R	AACATAGGCCCAACTACTCCAC
SgPHR2-ORF-F	GAGCTCGATCACCATATGGATTGTGTTGC
SgPHR2-ORF-R	ACGCGTCTTGTCACTTCTTGCAGGTATA
SgPHR2-RT-F	CTCAGCATGTACCGTTGACACCTG
SgPHR2-RT-R	GCTTGACCTCACTATCTGATGCGG
SgEF1a-RT-F	CACTTCAGGACGTGTACAAGATC
SgEF1a-RT-R	CTTGGAGAGCTTCATGGTGCA

Fig. 1 Plant dry weight (A) and P content (B) in stylo at different P treatments *** indicates significant difference between different P treatments at P<0.001 level.

Fig. 2 Full length cloning of SgPHR1 and SgPHR2 M: DL2000 bp DNA maker; A: PCR product of SgPHR1; B: PCR product of SgPHR2.

Tab. 2 Analysis of phosphorylation sites of SgPHR1 and SgPHR2

蛋白名称 Protein name	预测磷酸化位点 Predicted phosphorylation sites
蛋白名称 Protein name	丝氨酸（S）	苏氨酸（T）	酪氨酸（Y）
SgPHR1	14	4	4
SgPHR2	11	6	1

Fig. 3 Phosphorylation sites of SgPHR1 (A) and SgPHR2 (B) proteins

Fig. 4 Analysis of conserved domains of SgPHR1 (A) and SgPHR2 (B)

Fig. 5 Muitiple alignment analysis of SgPHR1 and SgPHR2 with PHRs homologues in other plants Proteins included PHRs from Stylosanthes guianensis, Arabidopsis thaliana (AtPHR1, NP_194590), Zea mays (ZmPHR1, JF831533.1), Oryza sativa (OsPHR1, AK063486.1), Fragaria vesca (FvPHR1, XP_004289982.1), Brassica naus (BnPHR1, JN806156.1) and Glycine max (GmPHR1, HQ007311); Red and blue boxes indicate MYB and CC domains, respectively.

Fig. 6 Phylogenetic analysis of SgPHR1 and SgPHR2 with other PHRs proteins Genebank No. included Arabidopsis thaliana (AtPHR1,NP_194590), Zea mays (ZmPHR1, JF831533.1), Glycine max (GmPHR25, NP_001350613.1), Phaseolus vulgaris (PvPHR1, ACD13206.1), Oryza sativa (OsPHR1, AK063486.1; OsPHR2, AK100065.1; OsPHR3, A2X0Q0.1; OsPHR4, XP_015644151.1), Triticum aestivum (TaPHR1, AGH13375.1), Fragaria vesca (FvPHR1, XP_004289982.1), Brassica naus (BnPHR1, JN806156.1), Prunus mume (PmPHR1, XP_016650539.1), Prunus persica (PpPHR1, XP_020425868.1), Prunus avium (PaPHR1, XP_021831311.1), Malus domestica (MdPHR1, XP_008372382.1), Pyrus bretschneideri (PbPHR1, XP_018506029.1), Medicago truncatula (MtPHR1, XP_003625354.1), Heobroma cacao (HcPHR1, XP_007050189.2), Hevea brasiliensis (HbPHR1, XP_021670552.1), Vitis vinifera (VvPHR1, XP_002270511.1), Raphanus sativus (RsPHR1, XP_018469653.1) and Glycine max (GmPHR1, HQ007311). represents PHRs proteins from stylo.

Fig. 7 Expressions of SgPHR1 (A) and SgPHR2 (B) in different tissues Different lowercase letters indicate significant difference in various tissues at P<0.05 level.

Fig. 8 Expression analysis of SgPHR1 and SgPHR2 in different treatments A: Expressions of SgPHR1 and SgPHR2 at different nutrition deficient treatments; B: Expressions of SgPHR1 and SgPHR2 at different duration of P deficiency. Different lowercase letters indicate significant difference among various treatments at P<0.05 level.

References 35

[1]	Chen S S, Luo Y, Ding G D, et al. Comparative analysis of Brassica napus plasma membrane proteins under phosphorus deficiency using label--free and MaxQuant-based proteomics approaches[J]. Journal of Proteomics, 2016, 133:144-152. DOI URL
[2]	Liu Y, Xie Y R, Wang H, et al. Light and ethylene coordinately regulate the phosphate starvation response through transcriptional regulation of phosphate starvation response1[J]. The Plant Cell, 2017, 29(9):2269-2284. DOI URL
[3]	Murakawa M, Ohta H, Shimojima M, et al. Lipid remodeling under acidic conditions and its interplay with low Pi stress in Arabidopsis[J]. Plant Molecular Biology Reporter, 2019, 101(1/2):81-93.
[4]	Yang Z B, Rao I M, Horst W J, et al. Interaction of aluminium and drought stress on root growth and crop yield on acid soils[J]. Plant and Soil, 2013, 372(1-2):3-25. DOI URL
[5]	Qu X J, Zhou J Q, Masabni J, et al. Phosphorus relieves aluminum toxicity in oil tea seedlings by regulating the metabolic profiling in the roots[J]. Plant Physiology and Biochemistry, 2020, 152:12-22. DOI URL
[6]	Koopmans G F, Chardon W J, Willigen P, et al. Phosphorus desorption dynamics in soil and the link to a dynamic concept of bioavailability[J]. Journal of Environmental Quality, 2004, 33(4):1393-1402. PMID
[7]	Tania Galindo‐Castaeda, Kathleen M Brown, Jonathan P Lynch, et al. Reduced root cortical burden improves growth and grain yield under low phosphorus availability in maize[J]. Plant Cell and Environment, 2018, 41(7):1579-1592. DOI URL
[8]	Güsewell Sabine. Regulation of dauciform root formation and root phosphatase activities of sedges (Carex) by nitrogen and phosphorus[J]. Plant & Soil, 2017, 415(1-2):57-72.
[9]	马若囡, 刘庆, 李欢, 等. 缺磷胁迫对甘薯前期根系发育及养分吸收的影响[J]. 华北农学报, 2017, 32(5):171-176.
[10]	Li C C, Li C F, Zhang H Y, et al. The purple acid phosphatase GmPAP21 enhances internal phosphorus utilization and possibly plays a role in symbiosis with rhizobia in soybean[J]. Physiologia Plantarum, 2017, 159(2):215-227. DOI URL
[11]	Mehdi Y H, Ali I D, Ali M M, et al. Agrobacterium rhizogenes transformed soybeans with AtPAP18 gene show enhanced phosphorus uptake and biomass production[J]. Biotechnology and Biotechnological Equipment, 2018, 32(4):865-873. DOI URL
[12]	Chen Z C, Liao H. Organic acid anions: An effective defensive weapon for plants against aluminum toxicity and phosphorus deficiency in acidic soils[J]. Journal of Genetics and Genomics, 2016, 43(11):631-638. DOI URL
[13]	Chen G H, Yan W, Yang S P, et al. Overexpression of rice phosphate transporter gene OsPT2 enhances tolerance to low phosphorus stress in soybean[J]. Journal of Agricultural Science and Technology, 2015, 17(2):469-482.
[14]	Amit Sharma, Alice Mühlroth, Juliette Jouhet, et al. The Myb-like transcription factor Phosphorus Starvation Response (PtPSR) controls conditional P acquisition and remodeling in marine microalgae[J]. New Phytologist, 2020, 225(6):2380-2395. DOI PMID
[15]	Xuan L T H, Du N H N, Nguyen B A T, et al. Transcription factors and their roles in signal transduction in plants under abiotic stresses[J]. Current Genomics, 2017, 18(6):483-497.
[16]	Stanislas Thiriet-Rupert, Gregory Carrier, Benoît Chénais, et al. Transcription factors in microalgae: genome-wide prediction and comparative analysis[J]. BMC Genomics, 2016, 17(1):1-16.
[17]	Wykoff D D, Grossman A R, Weeks D P, et al. Psr1, a nuclear localized protein that regulates phosphorus metabolism in Chlamydomonas[J]. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96(26):15336-15341. PMID
[18]	Rubio V, Linhares F, Solano R, et al. A conserved MYB transcription factor involved in phosphate starvation signaling both in vascular plants and in unicellular algae[J]. Genes and Development, 2001, 15(16):2122-2133. PMID
[19]	Nilsson L, Müller R, Nielsen T H, et al. Increased expression of the MYB-related transcription factor, PHR1, leads to enhanced phosphate uptake in Arabidopsis thaliana[J]. Plant Cell and Environment, 2007, 30(12):1499-1512. PMID
[20]	Guo M, Ruan W Y, Li C Y, et al. Integrative comparison of the role of the Phosphate Response1 subfamily in phosphate signaling and homeostasis in rice[J]. Plant Physiology, 2015, 168(4):1762-1776. DOI URL
[21]	Wang X H, Bai J R, Liu H M, et al. Overexpression of a maize transcription factor ZmPHR1 improves shoot inorganic phosphate content and growth of Arabidopsis under low-phosphate conditions[J]. Plant Molecular Biology Reporter, 2013, 31(3):665-677. DOI URL
[22]	Xue Y B, Xiao B X, Zhu S N, et al. GmPHR25, a GmPHR member up-regulated by phosphate starvation, controls phosphate homeostasis in soybean[J]. Journal of Experimental Botany, 2017, 68(17):4951-4967. DOI URL
[23]	Valdés L O, Arenas H C, Ramírez M, et al. Essential role of MYB transcription factor: PvPHR1 and microRNA: PvmiR399 in phosphorus-deficiency signalling in common bean roots[J]. Plant Cell and Environment, 2008, 31(12):1834-1843. DOI URL
[24]	严琳玲, 张瑜, 白昌军. 20份柱花草营养成分分析与评价[J]. 湖北农业科学, 2016(1):128-133.
[25]	孙丽莉, 陈志坚, 刘攀道, 等. 柱花草磷转运蛋白SgPT1的克隆和表达分析[J]. 草业学报, 2013, 22(4):187-196.
[26]	刘攀道, 董荣书, 丁西朋, 等. 不同磷效率柱花草基因型对外源DNA活化利用能力的比较分析[J]. 分子植物育种, 2018, 16(4):1085-1091.
[27]	孙丽莉, 田江, 陈志坚, 等. 柱花草SgSPX1基因的克隆与表达分析[J]. 热带作物学报, 2012, 33(10):1794-1799.
[28]	Ren F, Guo Q Q, Chang L L, et al. Brassica napus PHR1 gene encoding a MYB-like protein functions in response to phosphate starvation[J]. PloS One, 2012, 7(8):e44005. DOI URL
[29]	Jiang M Q, Sun L F, Isupov M N, et al. Structural basis for the target DNA recognition and binding by the MYB domain of phosphate starvation response 1[J]. The Febs Journal, 2019, 286(14):2809-2821.
[30]	Wang Y, Zhang F, Cui W X, et al. The FvPHR1 transcription factor control phosphate homeostasis by transcriptionally regulating miR399a in woodland strawberry[J]. Plant Science, 2019, 280:258-268. DOI PMID
[31]	Ruan W Y, Guo M N, Wu P, et al. Phosphate starvation induced OsPHR4 mediates Pi-signaling and homeostasis in rice[J]. Plant Molecular Biology Reporter, 2017, 93(3):327-340.
[32]	Tiwari J K, Buckseth T, Zinta R, et al. Transcriptome analysis of potato shoots, roots and stolons under nitrogen stress[J]. Scientific Reports, 2020, 10(1):921-941. DOI URL
[33]	Du X Q, Wang F L, Li H, et al. The transcription factor MYB59 regulates K/NO translocation in the Arabidopsis response to low K stress[J]. The Plant Cell, 2019, 31(3):699-714. DOI URL
[34]	Zhang X Q, Jiang H, Wang H, et al. Transcriptome analysis of rice seedling roots in response to potassium deficiency[J]. Scientific Reports, 2017, 7(1):5523. DOI URL
[35]	Zhao X M, Liu Y, Liu X, et al. Comparative transcriptome profiling of two tomato genotypes in response to potassium-deficiency stress[J]. International Journal of Molecular Sciences, 2018, 19(8):2402-2426. DOI URL