1 School of Landscape Architecture, Zhejiang A&F University, Hangzhou 311300, China;
2 Zhejiang Provincial Key Laboratory of Germplasm Innovation and Utilization for Garden Plants, Zhejiang Agriculture & Forestry University, Hangzhou 311300, China;
3 Key Laboratory of National Forestry and Grassland Administration on Germplasm Innovation and Utilization for Southern Garden Plants, Zhejiang Agriculture & Forestry University, Hangzhou 311300, China
Abstract：Ambient temperature is significantly affected flower bud development and flower opening of sweet osmanthus (Osmanthus fragrans). To further understand SQUAMOSA promoter-binding protein-like (SPL) genes and miR156 (microRNA156) on how to respond to ambient temperature in regulating flower bud development of sweet osmanthus, the variety of Osmanthus fragrans 'Yanhonggui' were selected, and 10 OfSPLs (OfSPL1A: MT756841; OfSPL1B: MT756842; OfSPL5: MT756843; OfSPL6: MT756844; OfSPL7: MT756845; OfSPL8: MT756849; OfSPL10: MT756846; OfSPL11: MT756847; OfSPL12: MT756848; OfSPL13: MT756850) were cloned by PCR. Meanwhile, the expression of miR156 and OfSPLs were identified by real-time quantitative PCR (qRT-PCR) to further analyze their temporal and spatial expression patterns. The results of OfSPL sequences analysis revealed that the cloned OfSPLs cDNA length between 1 004~3 824 bp, the sizes of OfSPL proteins ranged from 232 amino acid (aa) to 1 006 aa, and all of OfSPLs included a conserved SBP domain. Furthermore, all of OfSPL genes showed high homology with OlSPL genes from the species of Olea europaea by amino acid sequence alignment and evolutionary analysis. To investigate the intracellular localization of the proteins coded by the OfSPL genes, the bioinformatics method was used for predicting. The predicted results showed that all of the OfSPL proteins located in the nucleus. miR156 plays its role through regulating SPL gene expression by translational repression or degradation of mRNA in a sequence-specific manner. Therefore, the binding site of SPL genes was further analyzed. The expression of OfPL6, OfPL10, OfPL11, and OfSPL12 might be affected by miR156 through targeting their binding sites. Under different temperature treatments, the expression of OfSPL1A, OfSPL5, OfSPL6, OfSPL10, and OfSPL13 significantly increased at 19 ℃ comparing with the control 25 ℃. In contrast, the expression of miR156-1 and miR156-2 under 19 ℃ treatment were lower than the control 25 ℃. The results indicated that the expression of miR156-1 and miR156-2 may be inhibited by low ambient temperature, and resulted in the genes of OfSPL6 and OfSPL10 upregulation and then promoted the flower bud differentiation. Additionally, OfSPL1A, OfSPL5, and OfSPL13 may also involve in the flower bud development of O. fragrans at low ambient temperature, but they were not directly regulated by miR156-1 and miR156-2. This study could provide a reference for further research on the regulation of flower bud differentiation and flowering time by ambient temperature in O. fragrans.
[1] 代法国,胡宗利,陈国平,等.2010.植物特有的SBP-box基因家族的研究进展[J].生命科学,22(2):155-160.
(Dai F G,Hu Z L,Chen G P,et al.Progress in the plant specific SBP-box gene family[J].Chinese Bulletin of Life Sciences,22(2):155-160.)
[2] 冯凯,侯静,戴晓港,等.2017.簸箕柳SPL基因家族分析[J].南京林业大学学报(自然科学版),2(41) 55-62.
(Feng K,Hou J,Dai X G,et al.Analyzing the SPL gene family in Salix suchowensis[J].Journal of Nanjing Forestry University (Natural Sciences Edition),2(41) 55-62.)
[3] 宋长年,钱剑林,房经贵,等.2010.枳SPL9和SPL13全长cDNA克隆,亚细胞定位和表达分析[J].中国农业科学,43(10):2105-2114.
(Song C N,Qian J L,Fang J G,et al.Cloning,subcellular localization and expression analysis of SPL9 and SPL13 genes from Poncirus trifoliata[J].Scientia Agricultura Sinica,43(10):2105-2114.)
[4] 王英,张超,付建新,等.2016.桂花花芽分化和花开放研究进展[J].浙江农林大学学报,33(2):340-347.
(Wang Y,Zhang C,Fu J X,et al.Progresses on flower bud differentiation and flower opening in Osmanthus fragrans[J].Journal of Zhejiang A&F University,33(2):340-347.)
[5] 臧德奎,向其柏,刘玉莲,等.2003.中国桂花的研究历史,现状与桂花品种国际登录[J].植物资源与环境学报,12(4):49-53.
(Zang D K,Xiang Q B,Liu Y L,et al.The studying history and the application to International Cultivar Registration Authority of sweet osmanthus(Osmanthus fragrans Lour.)[J].Journal of Plant Resources and Environment,12(4):49-53.)
[6] 朱益民,王千千,董彬,等.2019.桂花OfSVP响应环境低温对花芽分化的影响[J].园艺学报,46(6):1134-1144.
(Zhu Y M,Wang Q Q,Dong B,et al.2019.Effect of OfSVP on flower bud differentiation in response to ambient temperature in Osmanthus fragrans[J].Acta Horticulturae Sinica,46(6):1134-1144.)
[7] 朱云美,张玲,高用顺,等.2016.温度对植物开花时间调控的研究进展[J].亚热带农业研究,12(2):130-135.
(Zhu Y M,Zhang L,Gao Y S,et al.2016.Recent research progress on plant flowering time regulation by temperature[J].Subtropical Agriculture Research,12(2):130-135.)
[8] An F M,Chan M T.2012.Transcriptome-wide characterization of miRNA-directed and non-miRNA-directed endonucleolytic cleavage using Degradome analysis under low ambient temperature in Phalaenopsis aphrodite subsp.formosana[J].Plant and Cell Physiology,53(10):1737-1750.
[9] Bergonzi S,Albani M C,Van Themaat E V L,et al.2013.Mechanisms of age-dependent response to winter temperature in perennial flowering of Arabis alpina[J].Science,340(6136):1094-1097.
[10] Cardon G H,Höhmann S,Nettesheim K,et al.1997.Functional analysis of the Arabidopsis thaliana SBP‐box gene SPL3:A novel gene involved in the floral transition[J].The Plant Journal,12(2):367-377.
[11] Capovilla G,Schmid M,Posé D.2015.Control of flowering by ambient temperature[J].Journal of Experimental Botany,66(1):59-69.
[12] Chen X,Zhang Z,Liu D,et al.2010.SQUAMOSA promoter‐binding protein‐like transcription factors:Star players for plant growth and development[J].Journal of Integrative Plant Biology,52(11):946-951.
[13] Han H,Liu G,Zhang J,et al.2016.Four SQUAMOSA PROMOTER BINDING PROTEIN-LIKE homologs from a basal eudicot tree (Platanus acerifolia) show diverse expression pattern and ability of inducing early flowering in Arabidopsis[J].Trees,30(4):1417-1428.
[14] Kim J J,Lee J H,Kim W,et al.2012.The microRNA156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 module regulates ambient temperature-responsive flowering via FLOWERING LOCUS T in Arabidopsis[J].Plant Physiology,159(1):461-478.
[15] Klein J,Saedler H,Huijser P.1996.A new family of DNA binding proteins includes putative transcriptional regulators of the Antirrhinum majus floral meristem identity gene SQUAMOSA[J].Molecular and General Genetics,250(1):7-16.
[16] Lännenpää M,Jänönen I,Hölttä‐Vuori M,et al.2004.A new SBP‐box gene BpSPL1 in silver birch (Betula pendula)[J].Physiologia Plantarum,120(3):491-500.
[17] Lei K J,Lin Y M,Ren J,et al.2015.Modulation of the phosphate-deficient responses by microRNA156 and its targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 in Arabidopsis[J].Plant and Cell Physiology,57(1):192-203.
[18] Liu Q,Harberd N P,Fu X,et al.2016.SQUAMOSA promoter binding protein-like transcription factors:targets for improving cereal grain yield[J].Molecular Plant,9(6):765-767.
[19] Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method[J].Methods,25(4):402-408.
[20] May P,Liao W,Wu Y,et al.2013.The effects of carbon dioxide and temperature on microRNA expression in Arabidopsis development[J].Nature Communications,4(1):1-11.
[21] Preston J C,Hileman L.2013.Functional evolution in the plant SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) gene family[J].Frontiers in Plant Science,4(80):1-13.
[22] Quint M,Delker C,Franklin K A,et al.2016.Molecular and genetic control of plant thermomorphogenesis[J].Nature Plants,2(1):1-9.
[23] Schwarz S,Grande A V,Bujdoso N,et al.2008.The microRNA regulated SBP-box genes SPL9 and SPL15 control shoot maturation in Arabidopsis[J].Plant Molecular Biology,67(1-2):183-195.
[24] UsamiT,HoriguchiG,Yano S,et al.2009.The more and smaller cells mutants of Arabidopsis thaliana identify novel roles for SQUAMOSA PROMOTER BINDING PROTEIN-LIKE genes in the control of heteroblasty[J].Development,136(6):955-964.
[25] Wang J W,Czech B,Weigel D.2009.miR156-regulated SPL transcription factors define an endogenous flowering pathway in Arabidopsis thaliana[J].Cell,138(4):738-749.
[26] Wang J W,Park M Y,Wang L J,et al.2011.miRNA control of vegetative phase change in trees[J].PLOS Genetics,7(2):e1002012.
[27] Xie K,Wu C,Xiong L.2006.Genomic organization,differential expression,and interaction of SQUAMOSA promoter-binding-like transcription factors and microRNA156 in rice[J].Plant Physiology,142(1):280-293.
[28] Xing S,Quodt V,Chandler J,et al.2013.SPL8 acts together with the brassinosteroid-signaling component BIM1 in controlling Arabidopsis thaliana male fertility[J].Plants,2(3):416-428.
[29] Xu M,Hu T,Zhao J,et al.2016.Developmental functions of miR156-regulated SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes in Arabidopsis thaliana[J].PLOS Genetics,12(8):e1006263.
[30] Zhang T,Wang J,Zhou C.2015.The role of miR156 in developmental transitions in Nicotiana tabacum[J].Science China Life Sciences,58(3):253-260.
[31] Zhang Y,Schwarz S,Saedler H,et al.2007.SPL8,a local regulator in a subset of gibberellin-mediated developmental processes in Arabidopsis[J].Plant Molecular Biology,63(3):429-439.
