‘赣南早’脐橙早熟性状回复型突变体的生理与转录组分析
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摘要
【目的】明确早熟品种‘赣南早’脐橙(wild type,WT)与其早熟性状回复型突变体(mutant type, MT)在生理和转录水平的差异,探究柑橘果实成熟的调控机制。【方法】测定MT和WT的果实品质及成熟相关生理指标,采用RNASeq分析MT和WT果实的转录差异。【结果】MT具有稳定的早熟性状回复现象。MT和WT果皮的各可溶性糖含量差异显著。MT和WT有机酸含量在果肉间及果皮间差异均显著。MT果皮的赤霉素(gibberellin,GA)、吲哚乙酸(indole acetic acid,IAA)和茉莉酸(jasmonic acid,JA)显著高于WT,脱落酸(abscisic acid,ABA)则相反。转录组测序分析表明,MT与WT果皮间DEGs数量为980个,果肉间为289个。在果皮DEGs中,有38种GO分类、6个KEGG代谢通路被显著性富集,而果肉中未见GO分类和KEGG代谢通路的显著性富集。ABA合成基因CsNCED1在MT果皮和果肉中均下调表达,而分解基因CsCYP707A1在MT果皮中上调表达。【结论】MT成熟期比WT推迟约30 d。MT果皮GA含量及GA合成基因CsCPS1、CsKAO表达量均高于WT,可能与果皮的褪绿延迟相关。MT果皮ABA积累抑制可能受合成基因CsNCED1下调表达及分解基因CsCYC707A1上调表达的影响。MT和WT果皮的生理和转录组水平差异均比果肉间差异大,说明果皮在柑橘果实成熟过程中具有重要作用。
【Objective】Selection of early or late ripening varieties is an important target of citrus breeding. The regulation mechanism of citrus fruit ripening is of great significance for breeding early or late ripening varieties. Previous studies regarding ripening mechanism of citrus fruits mainly focused on the flesh, little attention was paid on the pericarp. Herein, both flesh and pericarp of‘Gannanzao'navel orange(Wild type, WT), and its restorative mutant(Mutant type, MT) were investigatedin order to explore the regulation mechanism of the ripening of navel orange through comprehensive comparison of the physiological and transcriptional differences between the WT and the MT.【Methods】The fruit qualities and physiological properties, including the contents of soluble sugar, organic acid and phytohormones, in both flesh and pericarp of the WT and the MT were determined. Transcriptome data of both flesh and pericarp of the WT and the MT were obtained and analyzed by high-throughput sequencing.【Results】MT featured an obvious and stable late-maturing character. The soluble sugar content was significantly different only in the pericarp of the WT and the MT, while the organic acid content was significantly different in both of the flesh and the pericarp. The MT had much lower contents of both malic acid and citric acid, and higher content of quinic acid compared with those of the WT. At200 DAF, the contents of GA, IAA and JA in the pericarp of the MT were significantly higher than those of the WT, whereas the content of ABA in the pericarp of the MT was obviously lower. than that of the WT. Meanwhile, the comparison of transcriptome sequencing between the MT and the WT showed that the number of differentially expressed genes(DEGs) were 980 and 289 in the pericarp and the flesh respectively, and 94 DEGs were the common DEGs of the pericarp and the flesh. Interestingly, the significant enrichment(p ≤ 0.05) of GO terms and KEGG pathways were only found in the pericarp. A total of 38 GO terms were significantly enriched in the pericarp. Furthermore, a total of 6 KEGG pathways being involved in photosynthesis, photosynthesis-antenna proteins, peroxisome,cutin, suberine and wax biosynthesis, protein digestion and absorption, and ubiquinone and other terpenoid-quinone biosynthesis were significantly enriched(p ≤ 0.05) in the pericarp. In ABA synthesis signal transduction pathway, the key limiting gene CsNCED1 of ABA synthesis was downregulated in both of the pericarp and the flesh in the MT, and the decomposition gene CsCYP707 A1 was up-regulated in the MT pericarp.【Conclusion】The ripening date of the mutant(MT) was 30 days later than that of the WT. The delay of both chlorisis and color transition might be related to the increase of GA accumulation and the up-regulated expression of CsCPS1 and CsKAO. The down-regulated and up-regulated expression of CsNCED1 and CsCYC707 A1 in the MT pericarp might lead to the inhibition of ABA accumulation. The differences of the physiological and transcriptome levels in the pericarp between the WT and the MT were greater than those in the flesh, indicating that the pericarp may play an important role in citrus fruit ripening. It seems that the investigation of the physiology and transcriptome both in the flesh and the pericarp are essentially necessary for studying the mechanism of the citrus fruit ripening.
【Objective】Selection of early or late ripening varieties is an important target of citrus breeding. The regulation mechanism of citrus fruit ripening is of great significance for breeding early or late ripening varieties. Previous studies regarding ripening mechanism of citrus fruits mainly focused on the flesh, little attention was paid on the pericarp. Herein, both flesh and pericarp of‘Gannanzao'navel orange(Wild type, WT), and its restorative mutant(Mutant type, MT) were investigatedin order to explore the regulation mechanism of the ripening of navel orange through comprehensive comparison of the physiological and transcriptional differences between the WT and the MT.【Methods】The fruit qualities and physiological properties, including the contents of soluble sugar, organic acid and phytohormones, in both flesh and pericarp of the WT and the MT were determined. Transcriptome data of both flesh and pericarp of the WT and the MT were obtained and analyzed by high-throughput sequencing.【Results】MT featured an obvious and stable late-maturing character. The soluble sugar content was significantly different only in the pericarp of the WT and the MT, while the organic acid content was significantly different in both of the flesh and the pericarp. The MT had much lower contents of both malic acid and citric acid, and higher content of quinic acid compared with those of the WT. At200 DAF, the contents of GA, IAA and JA in the pericarp of the MT were significantly higher than those of the WT, whereas the content of ABA in the pericarp of the MT was obviously lower. than that of the WT. Meanwhile, the comparison of transcriptome sequencing between the MT and the WT showed that the number of differentially expressed genes(DEGs) were 980 and 289 in the pericarp and the flesh respectively, and 94 DEGs were the common DEGs of the pericarp and the flesh. Interestingly, the significant enrichment(p ≤ 0.05) of GO terms and KEGG pathways were only found in the pericarp. A total of 38 GO terms were significantly enriched in the pericarp. Furthermore, a total of 6 KEGG pathways being involved in photosynthesis, photosynthesis-antenna proteins, peroxisome,cutin, suberine and wax biosynthesis, protein digestion and absorption, and ubiquinone and other terpenoid-quinone biosynthesis were significantly enriched(p ≤ 0.05) in the pericarp. In ABA synthesis signal transduction pathway, the key limiting gene CsNCED1 of ABA synthesis was downregulated in both of the pericarp and the flesh in the MT, and the decomposition gene CsCYP707 A1 was up-regulated in the MT pericarp.【Conclusion】The ripening date of the mutant(MT) was 30 days later than that of the WT. The delay of both chlorisis and color transition might be related to the increase of GA accumulation and the up-regulated expression of CsCPS1 and CsKAO. The down-regulated and up-regulated expression of CsNCED1 and CsCYC707 A1 in the MT pericarp might lead to the inhibition of ABA accumulation. The differences of the physiological and transcriptome levels in the pericarp between the WT and the MT were greater than those in the flesh, indicating that the pericarp may play an important role in citrus fruit ripening. It seems that the investigation of the physiology and transcriptome both in the flesh and the pericarp are essentially necessary for studying the mechanism of the citrus fruit ripening.
引文
[1] PAN X,ZHU B,ZHU H,CHEN Y,TIAN H,LUO Y,FU D.iTRAQ protein profile analysis of tomato green-ripe mutant reveals new aspects critical for fruit ripening[J]. Journal of Proteome Research,2014,13(4):1979.
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[4] JIA H F,LU D,SUN J H,LI C L,XING Y,QIN L,SHEN Y Y.Type 2C protein phosphatase ABI1 is a negative regulator of strawberry fruit ripening[J]. Journal of Experimental Botany,2013,64(6):1677-1687.
[5] NICOLAS P,LECOURIEUX D,KAPPEL C,CLUZET S,CRAMER G R,DELROT S,LECOURIEUX F. The bZIP transcription factor VvABF2 is an important transcriptional regulator of ABA-dependent grape berry ripening processes[J]. Plant Physiology,2013,35(5):523-525.
[6] KARPPINEN K,HIRVEL?E,NEVALA T,SIPARI N,SUOKAS M,JAAKOLA L. Changes in the abscisic acid levels and related gene expression during fruit development and ripening in bilberry(Vaccinium myrtillus L.)[J]. Phytochemistry,2013,95(6):127.
[7] JIA H,LI C,CHAI Y,XING Y,SHEN Y. Sucrose promotes strawberry fruit ripening by stimulation of abscisic acid biosynthesis[J]. Pakistan Journal of Botany,2013,45(1):169-175.
[8] CONCHA C M,FIGUEROA N E,POBLETE L A,O?ATE F A,SCHWAB W,FIGUEROA C R. Methyl jasmonate treatment induces changes in fruit ripening by modifying the expression of several ripening genes in Fragaria chiloensis fruit[J]. Plant Physiology&Biochemistry,2013,70(1):433-444.
[9]蒋天梅,殷学仁,王平,孙崇德,徐昌杰,李鲜,陈昆松.乙烯调控非跃变型果实成熟衰老研究进展[J].园艺学报,2011,38(2):371-378.JIANG Tianmei,YIN Xueren,WANG Ping,SUN Chongde,XU Changjie,LI Xian,CHEN Kunsong. Research advance in regulation of ethylene during ripening and senescence of non-climacteric fruit[J]. Acta Horticulturae Sinica,2011,38(2):371-378.
[10] IRELAND H S,GUNASEELAN K,MUDDUMAGE R,TACKEN E J,PUTTERILL J,JOHNSTON J W,SCHAFFER R J. Ethylene regulates apple(Malus′domestica)fruit softening through a dose′time-dependent mechanism and through differential sensitivities and dependencies of cell wall-modifying genes[J]. Plant&Cell Physiology,2014,55(5):1005.
[11] LIU Y Z,TANG P,TAO N G,XU Q,PENG S A,DENG X X,XIANG K S,HUANG R H. Fruit coloration difference between Fengwan,a late-maturing mutant and its original cultivar Fengjie72-1 of navel orange(Citrus sinensis Osbeck)[J]. Journal of Plant Physiology&Molecular Biology,2006,32(1):31-36.
[12] ZHANG Y J,WANG X J,WU J X,CHEN S Y,CHEN H,CHAI L J,YI H L. Comparative transcriptome analyses between a spontaneous late-ripening sweet orange mutant and its wild type suggest the functions of ABA,sucrose and JA during citrus fruit ripening[J]. Plos One,2014,9(12):e116056.
[13] WU J,XU Z,ZHANG Y,CHAI L,YI H,DENG X. An integrative analysis of the transcriptome and proteome of the pulp of a spontaneous late-ripening sweet orange mutant and its wild type improves our understanding of fruit ripening in Citrus[J]. Journal of Experimental Botany,2014,65(6):1651-1671.
[14]钟八莲,赖晓桦,杨斌华,米兰芳,谢上海,黄彩英,杨文侠,张湟.纽荷尔脐橙芽变早熟品种—赣南早脐橙[J].中国南方果树,2013,42(2):48-51.ZHONG Balian,LAI Xiaohua,YANG Binhua,MI Lanfang,XIE Shanghai,HUANG Caiying,YANG Wenxia,ZHANG Huang.‘Gannanzao’,a new early-ripening navel orange cultivar of‘Newhall’[J]. South China Fruits,2013,42(2):48-51.
[15]米兰芳.橙汁加工品种综合品质分析与评价[D].武汉:华中农业大学,2009.MI Lanfang. Comprehensive analysis evaluation of quality of orange juice processing cultivars[D]. Wuhan:Huazhong Agricultural University,2009.
[16] PAN X,WELTI R,WANG X. Quantitative analysis of major plant hormones in crude plant extracts by high-performance liquid chromatography-mass spectrometry[J]. Nature Protocols,2010,5(6):986-992.
[17] LIU Y,LIU Q. Efficient isolation of RNA from fruit peel and pulp of ripening navel orange(Citrus sinensis Osbeck)[J]. Journal of Huazhong Agricultural University,2006,25(3):300-304.
[18] XU Q,CHEN L L,RUAN X,CHEN D,ZHU A,CHEN C,BERTRAND D,JIAO W B,HAO B H,LYON M P. The draft genome of sweet orange(Citrus sinensis)[J]. Nature Genetics,2013,45(1):59.
[19]中国国家标准化管理委员会.脐橙:GB/T 21488—2008[S].北京:中国标准出版社,2008.China National Standardization Management Committee. Navel orange:GB/T 21488—2008[S]. Beijing:China Standards Press,2008.
[20]续丽红,陈健美,谢丽红,钟八莲,米兰芳.赣南早脐橙果实成熟过程中主要品质指标的变化[J].中国南方果树,2016,45(2):65-68.XU Lihong,CHEN Jianmei,XIE Lihong,ZHONG Balian,MI Lanfang. Changes of main quality Indexes of‘Gannanzao’navel orange during fruit ripening[J]. South China Fruits,2016,45(2):65-68.
[21] ENRIQUETA A,MANUEL C,MARíAJESúS R,LORENZO Z A,MANUEL T. Regulation of color break in citrus fruits.Changes in pigment profiling and gene expression induced by gibberellins and nitrate,two ripening retardants[J]. Journal of Agricultural&Food Chemistry,2006,54(13):4888.
[22] WANG Y,WANG Y,JI K,DAI S,HU Y,SUN L,LI Q,CHEN P,SUN Y,DUAN C. The role of abscisic acid in regulating cucumber fruit development and ripening and its transcriptional regulation[J]. Plant Physiology&Biochemistry,2013,64(5):70.
[23] WANG X,YIN W,WU J,CHAI L,YI H. Effects of exogenous abscisic acid on the expression of citrus fruit ripening-related genes and fruit ripening[J]. Scientia Horticulturae,2016,201:175-183.
[24] MA Y,SZOSTKIEWICZ I,KORTE A,MOES D,YANG Y,CHRISTMANN A,GRILL E. Regulators of PP2C phosphatase activity function as abscisic acid sensors[J]. Science,2009,324(5930):1064.
[25] FUJII H,SHIMADA T,SUGIYAMA A,NISHIKAWA F,ENDO T,NAKANO M,IKOMA Y,SHIMIZU T,OMURA M. Profiling ethylene-responsive genes in mature mandarin fruit using a citrus 22K oligoarray[J]. Plant Science,2007,173(3):340-348.
[26] RODRIGO M J,ZACARIAS L. Effect of postharvest ethylene treatment on carotenoid accumulation and the expression of carotenoid biosynthetic genes in the flavedo of orange(Citrus sinensis L. Osbeck)fruit[J]. Postharvest Biology&Technology,2007,43(1):14-22.
[27] KOMATSU A,TAKANOKURA Y,MORIGUCHI T,OMURA M,AKIHAMA T. Differential expression of three sucrose-phosphate synthase isoforms during sucrose accumulation in citrus fruits(Citrus unshiu Marc.)[J]. Plant Science,1999,140(2):169-178.
[2] ZENG J,GAO C,DENG G,JIANG B,YI G,PENG X,ZHONG Y,ZHOU B,LIU K. Transcriptome analysis of fruit development of a Citrus late-ripening mutant by microarray[J].Scientia Horticulturae,2012,134(2):32-39.
[3] SUN L,WANG Y P,CHEN P,REN J,JI K,LI Q,LI P,DAI S J,LENG P. Transcriptional regulation of SlPYL,SlPP2C,and SlSnRK2 gene families encoding ABA signal core components during tomato fruit development and drought stress[J]. Journal of Experimental Botany,2011,62(15):5659.
[4] JIA H F,LU D,SUN J H,LI C L,XING Y,QIN L,SHEN Y Y.Type 2C protein phosphatase ABI1 is a negative regulator of strawberry fruit ripening[J]. Journal of Experimental Botany,2013,64(6):1677-1687.
[5] NICOLAS P,LECOURIEUX D,KAPPEL C,CLUZET S,CRAMER G R,DELROT S,LECOURIEUX F. The bZIP transcription factor VvABF2 is an important transcriptional regulator of ABA-dependent grape berry ripening processes[J]. Plant Physiology,2013,35(5):523-525.
[6] KARPPINEN K,HIRVEL?E,NEVALA T,SIPARI N,SUOKAS M,JAAKOLA L. Changes in the abscisic acid levels and related gene expression during fruit development and ripening in bilberry(Vaccinium myrtillus L.)[J]. Phytochemistry,2013,95(6):127.
[7] JIA H,LI C,CHAI Y,XING Y,SHEN Y. Sucrose promotes strawberry fruit ripening by stimulation of abscisic acid biosynthesis[J]. Pakistan Journal of Botany,2013,45(1):169-175.
[8] CONCHA C M,FIGUEROA N E,POBLETE L A,O?ATE F A,SCHWAB W,FIGUEROA C R. Methyl jasmonate treatment induces changes in fruit ripening by modifying the expression of several ripening genes in Fragaria chiloensis fruit[J]. Plant Physiology&Biochemistry,2013,70(1):433-444.
[9]蒋天梅,殷学仁,王平,孙崇德,徐昌杰,李鲜,陈昆松.乙烯调控非跃变型果实成熟衰老研究进展[J].园艺学报,2011,38(2):371-378.JIANG Tianmei,YIN Xueren,WANG Ping,SUN Chongde,XU Changjie,LI Xian,CHEN Kunsong. Research advance in regulation of ethylene during ripening and senescence of non-climacteric fruit[J]. Acta Horticulturae Sinica,2011,38(2):371-378.
[10] IRELAND H S,GUNASEELAN K,MUDDUMAGE R,TACKEN E J,PUTTERILL J,JOHNSTON J W,SCHAFFER R J. Ethylene regulates apple(Malus′domestica)fruit softening through a dose′time-dependent mechanism and through differential sensitivities and dependencies of cell wall-modifying genes[J]. Plant&Cell Physiology,2014,55(5):1005.
[11] LIU Y Z,TANG P,TAO N G,XU Q,PENG S A,DENG X X,XIANG K S,HUANG R H. Fruit coloration difference between Fengwan,a late-maturing mutant and its original cultivar Fengjie72-1 of navel orange(Citrus sinensis Osbeck)[J]. Journal of Plant Physiology&Molecular Biology,2006,32(1):31-36.
[12] ZHANG Y J,WANG X J,WU J X,CHEN S Y,CHEN H,CHAI L J,YI H L. Comparative transcriptome analyses between a spontaneous late-ripening sweet orange mutant and its wild type suggest the functions of ABA,sucrose and JA during citrus fruit ripening[J]. Plos One,2014,9(12):e116056.
[13] WU J,XU Z,ZHANG Y,CHAI L,YI H,DENG X. An integrative analysis of the transcriptome and proteome of the pulp of a spontaneous late-ripening sweet orange mutant and its wild type improves our understanding of fruit ripening in Citrus[J]. Journal of Experimental Botany,2014,65(6):1651-1671.
[14]钟八莲,赖晓桦,杨斌华,米兰芳,谢上海,黄彩英,杨文侠,张湟.纽荷尔脐橙芽变早熟品种—赣南早脐橙[J].中国南方果树,2013,42(2):48-51.ZHONG Balian,LAI Xiaohua,YANG Binhua,MI Lanfang,XIE Shanghai,HUANG Caiying,YANG Wenxia,ZHANG Huang.‘Gannanzao’,a new early-ripening navel orange cultivar of‘Newhall’[J]. South China Fruits,2013,42(2):48-51.
[15]米兰芳.橙汁加工品种综合品质分析与评价[D].武汉:华中农业大学,2009.MI Lanfang. Comprehensive analysis evaluation of quality of orange juice processing cultivars[D]. Wuhan:Huazhong Agricultural University,2009.
[16] PAN X,WELTI R,WANG X. Quantitative analysis of major plant hormones in crude plant extracts by high-performance liquid chromatography-mass spectrometry[J]. Nature Protocols,2010,5(6):986-992.
[17] LIU Y,LIU Q. Efficient isolation of RNA from fruit peel and pulp of ripening navel orange(Citrus sinensis Osbeck)[J]. Journal of Huazhong Agricultural University,2006,25(3):300-304.
[18] XU Q,CHEN L L,RUAN X,CHEN D,ZHU A,CHEN C,BERTRAND D,JIAO W B,HAO B H,LYON M P. The draft genome of sweet orange(Citrus sinensis)[J]. Nature Genetics,2013,45(1):59.
[19]中国国家标准化管理委员会.脐橙:GB/T 21488—2008[S].北京:中国标准出版社,2008.China National Standardization Management Committee. Navel orange:GB/T 21488—2008[S]. Beijing:China Standards Press,2008.
[20]续丽红,陈健美,谢丽红,钟八莲,米兰芳.赣南早脐橙果实成熟过程中主要品质指标的变化[J].中国南方果树,2016,45(2):65-68.XU Lihong,CHEN Jianmei,XIE Lihong,ZHONG Balian,MI Lanfang. Changes of main quality Indexes of‘Gannanzao’navel orange during fruit ripening[J]. South China Fruits,2016,45(2):65-68.
[21] ENRIQUETA A,MANUEL C,MARíAJESúS R,LORENZO Z A,MANUEL T. Regulation of color break in citrus fruits.Changes in pigment profiling and gene expression induced by gibberellins and nitrate,two ripening retardants[J]. Journal of Agricultural&Food Chemistry,2006,54(13):4888.
[22] WANG Y,WANG Y,JI K,DAI S,HU Y,SUN L,LI Q,CHEN P,SUN Y,DUAN C. The role of abscisic acid in regulating cucumber fruit development and ripening and its transcriptional regulation[J]. Plant Physiology&Biochemistry,2013,64(5):70.
[23] WANG X,YIN W,WU J,CHAI L,YI H. Effects of exogenous abscisic acid on the expression of citrus fruit ripening-related genes and fruit ripening[J]. Scientia Horticulturae,2016,201:175-183.
[24] MA Y,SZOSTKIEWICZ I,KORTE A,MOES D,YANG Y,CHRISTMANN A,GRILL E. Regulators of PP2C phosphatase activity function as abscisic acid sensors[J]. Science,2009,324(5930):1064.
[25] FUJII H,SHIMADA T,SUGIYAMA A,NISHIKAWA F,ENDO T,NAKANO M,IKOMA Y,SHIMIZU T,OMURA M. Profiling ethylene-responsive genes in mature mandarin fruit using a citrus 22K oligoarray[J]. Plant Science,2007,173(3):340-348.
[26] RODRIGO M J,ZACARIAS L. Effect of postharvest ethylene treatment on carotenoid accumulation and the expression of carotenoid biosynthetic genes in the flavedo of orange(Citrus sinensis L. Osbeck)fruit[J]. Postharvest Biology&Technology,2007,43(1):14-22.
[27] KOMATSU A,TAKANOKURA Y,MORIGUCHI T,OMURA M,AKIHAMA T. Differential expression of three sucrose-phosphate synthase isoforms during sucrose accumulation in citrus fruits(Citrus unshiu Marc.)[J]. Plant Science,1999,140(2):169-178.
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