Developmental and molecular biological dissection of a heat tolerant tomato mutant ht7

Developmental and Molecular Biological Dissection of a Heat Tolerant Tomato Mutant HT7 A Dissertation Submitted to the Graduate School of Life and Environmental Sciences, the University of Tsukuba in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Agricultural Science (Doctoral Program in Biosphere Resource Science and Technology) PHAM THI DUNG Table of Contents Abbreviation iii Abstract iv Chapter General Introduction 1.1 Introduction 1.2 Hypothesis 1.3 Objectives Chapter The Unique Phenotypes of HT7 Mutant under Long-term Heat Stress 2.1 Introduction 2.2 Plant materials and Methods 2.3 Results 11 2.4 Discussion 17 Chapter 50 Flower Morphology and Pollen Viability of Mutant Line 50 3.1 Introduction 51 3.2 Materials and Methods 52 3.3 Results 54 3.4 Discussion 58 Chapter 74 Biochemical Responses of HT Mutant to Heat Stress 74 4.1 Introduction 75 4.2 Materials and Methods 77 4.3 Results 81 4.4 Discussion 84 Chapter 96 Expression of Transcription Factor, Flower Structure and other Heat Stress -Related Genes in Mutant 96 5.1 Introduction 97 5.2 Materials and Methods 99 5.3 Results 100 5.4 Discussion 103 i Chapter 115 Whole Genome Sequencing (NGS) for Detecting the Responsible Gene(s) to Thermotolerance 115 6.1 Introduction 116 6.2 Materials and Methods 118 6.3 Results 120 Ongoing research 123 Chapter 137 General Discussions and Conclusions 137 137 7.1 General discussion 138 7.2 Conclusions 143 Acknowledgments 150 References 152 ii Abbreviation ABA : Abscisic acid CO : Control condition DAHS : Days after heat stress DAS : Days after sowing EMS : Ethyl methanesulfonate Fru : Fructose Glu : Glucose GRH : Greenhouse condition HSPs : Heat shock proteins HS : Heat stress condition HSR : Heat stress response HT : Heat tolerant line: HT HT7 : Heat-tolerant line HPLC : High Performance Liquid Chromatography MS : Murashige and Skoog PGM : Pollen germination medium PCR : Polymerase chain reaction PCD : Program cell death ROS : Reactive oxygen species RE : Restriction enzyme qRT-PCR :Reverse transcription polymerase chain reaction SA : Salicylic acid Suc : Sucrose TSS : Total soluble solids content TFs : Transcription factors NGS : Next generation sequencing WT : Wild-type Micro-Tom iii Abstract Tomato (Solanum lycopersicum) is an undeniable importance as a vegetable crop in term of the merits of fresh and industrial products It is also well known as an incredible source of rich nutrient components such as vitamin C, β-carotene and lycopene that positively impact on human health (Bergougnoux, 2014) However, the yield and quality of tomato are adversely affected as a consequence of various biotic and abiotic stresses High temperature is one of the major abiotic stresses in globe, which causes the multifarious negative effects on plant morphology, physiology, biochemistry and molecular pathways at all vegetative and reproductive stages, causing the reduction of fruit set and quality (Hasanuzzaman et al., 2013; Bita and Gerats, 2013) We isolated heat-tolerant (HT) mutants conferring improved fruit production under longterm ambient high temperature among EMS tomato mutant populations, created by National BioResource Project in Japan In this project, we focused on the HT7 mutant which produced normal fruits with seeds in heat stress (HS) condition and tried to understand the mechanism of heat tolerance through the developmental and molecular dissection Mutant and WT were evaluated in two fixed cultivated conditions, HS condition (35o/25 °C) or control condition (CO) (25 °C), 16 h/8 h light/dark, 60.0 μmol m-2 s-1, and opened greenhouse in summer 2018 The HT7 mutant expressed several unique traits under long-term exposure to elevated temperature: HT7 kept the narrow plant canopy by producing fewer lateral shoots than WT; HT7 had around 30-40 flowers per plant that was lower number than WT; rate of fruit setting (6 %) was five times higher than WT, resulting in two times higher fruit yield than WT; HT7 consistently produced normal fruits in HS condition, while WT could not get fruit set for every cultivation Typically, HT7 fruits contained seeds that could germinate, while 100 % of WT fruits did not have seeds In the opened greenhouse in summer 2018, 100 % of HT7 and WT fruits produced fruits without seeds when bud formation and anthesis were exposed to the highest temperature peak in the summer (more than 45 oC at noon) In HS condition, HT7 mutant remained the flower structure as more suitable for selfpollination HT7 had less than 20 % of abnormal flowers, while WT had more than 40 %; HT7 produced pollens within pollen sacs, and their walls dehisced for releasing pollens, while 40 % WT flowers did not produce pollens and pollen sac walls did not dehisce well at anthesis In long term HS condition, HT7 produced two times of higher viable pollens and total pollens than WT; both HT7 and WT pollens germinated very low in the pollen germination medium, rating at % iv and %, while these rates were more than 80 % in CO; HT7 and WT pollens did not elongate pollen tubes well In short term HS, HT7 produced two times higher in viable pollens than WT in -1, -3, -5 days before anthesis, however, the pollen germinate rate was not significant differences In the greenhouse, HT7 and WT flowers had more than 85 % of non-viable pollens due to very hot temperature These observations suggest that improved production of active pollens and its release from anthers due to dehiscence in HS condition are involved in stable fruit setting and development in the phenotype of increasing HT7 mutant To adapt with the elevated temperature, tomatoes have established various heat response reactions The 2-weeks-old seedlings of HT7 and WT died after 12 h constantly treated at 42 oC; the stomata diameters of HT7 were the biggest in HS and smallest in GRH, while those in WT were the biggest in CO and smallest in GRH The stomata density on leaves of HT7 was lower than in WT in three conditions; HT7 released lower at 10 days and higher at 60 days in HS in total ionic leakage than WT; fruits of HT7 and WT released similar amount of hydrogen peroxide The amount of fructose, glucose and sucrose in HT7 fruits were lower than in WT These results suggest that alteration of stomata and biochemical factors are involved in increasing heat tolerance of HT7 mutant Transcription factors, heat shock proteins and other related genes to heat stress play important roles in thermotolerance In leaves, HT7 mutant expressed SIHsfA1a, SIHsfA1d genes lower than those of WT, in contrasted, HT7 expressed SIHsfA1b1 and SIHsfA1b3 genes higher than those of WT; the relative gene expression levels of HT7 in SIHsp21 and SIHsp101 were three to four times higher than those of WT However, in the anther cone, the expression levels of SIHsfA1a, SIHsfA1b1 and SIHsp101 genes were not significant differences between HT7 and WT In the greenhouse, the expression levels of ABCDE model genes were different; HT7 expressed SlAGL6 and SlNAM2 genes lower than those of WT, while the expression levels of SlLePI, SlMC and SlTM6 genes were higher than in WT Regarding to leaf senesce genes, RbcL, SENU3 and DREB2A expressed highest at hours in HS HT7 expressed RbcL and DREB2A lower than WT at days in HS condition These results suggest that enhanced expression of heat stress-related genes in HS condition are involved in increasing heat tolerance of HT7 mutant Isolation of gene(s) responsible for heat tolerance in mutant HT7 is a final goal of this study The whole genome sequencing by NGS was carried out on the bulked DNA extracting from the leaves of F2 population which showed higher fruit setting in HS condition Total reads of v 327,921,773 (WT – like F2), 326,421,481 (HT7 – like F2), 151,100,978 (WT), and 158,203,158 (HT7) were called The mapping with bowtie2 using SL3.1 tomato genome reference (Heinz 1706) indicated that 96 % of reads were mapped concordantly or discordantly on the reference in both case and there were no contamination sequences in fastq datasets In total, there are 11,083 reads including 7,940 reads for SNP and 3,143 reads for INDEL mutants in F2 NGS sequence In addition, a single strong QTL was found at the upper arm of ch06 in HT7 and also in bulked F2 plants with HT7 phenotype These finding suggested that the QTL on ch06 is potentially associated with HT7 phenotype Therefore, we selected several mutant positions on ch06 to confirm the suggestion Sanger sequencing and restriction enzyme digestion were performed in different mutative positions on ch06, however, the strong responsible gene(s) were not detected yet and need to further analysis of the data In conclusion, HT7 mutant had more normal flowers compatibly for self-pollination, narrow plant canopy, higher viable pollens, higher fruit setting rate, and higher fruit number per plant and resulted in higher fruit yield in HS condition Particularly, HT7 produced normal fruits containing seeds and stayed green in leaves in HS condition Taken together, HT7 can be a prominent breeding material for enhancing heat tolerance In addition, HT7 mutant showed enhanced expression of heat stress-related genes in HS condition which may induce the increasedheat tolerance HT7 mutant is a useful material for elucidating a heat tolerant mechanism in tomato vi CHAPTER General Introduction 1.1 Introduction Climate change including very high temperatures is predicted to induce enormous negative effects to plant growth and development, resulting in a devastating reduction of crop productivity, and will become a novel threat causing severe spread famine on global food security (FAO-STAT, http://faostat.fao.org; Verisk Maplecroft, https://www.maplecroft.com; Bita and Gerats, 2013) According to the research of the Intergovernmental Panel on Climate Change (IPCC), the global surface temperature will increase 0.3 oC during the next decade (Jones et al., 1999; Abdrabo and Adger, 2014) High temperature is one of the major global abiotic stresses, which causes the multifarious negative effects on plant morphology, physiology, biochemistry and molecular pathways at all vegetative and reproductive stages, causing the reduction of fruit set and quality (Hasanuzzaman et al., 2013; Bita and Gerats, 2013) Plants respond to elevated temperature and ensure survival by various mechanisms at different levels such as in transcription, post-transcription, translation, post-translation, and regulation; and in calcium, phytohormone, sugar and lipid signaling; and in primary and secondary metabolisms (Bita and Gerats, 2013) Moreover, thermotolerances are dependent on a complex network of distinct and interconnected pathways to maintain protein homeostasis and minimize cellular damage (Keller et al., 2018) Tomato (Solanum lycopersicum) is an undeniable importance of a vegetable crop in term of the merits of fresh and industrial products It is also well known as an incredible source of rich nutrient components such as vitamin C, β-carotene and lycopene that positively impact on human health (Bergougnoux, 2014) However, the yield and quality of tomato are adversely affected as a consequence of various biotic and abiotic stresses The responses to heat stress of tomato, however, vary between germplasms and development stages and timing of heat-treated events These responses are not only observed in the leaves (vegetative organs) (Zhou et al., 2017) but also in gametophytes (reproductive organs) (Firon et al., 2006) Pollens are not only affected by elevated temperature in both of prior and following dehiscence There was a correlation between a decrease of pollen viability and fruit set in tomato in stressed conditions (Firon et al., 2006) In addition, the flower structure was negatively changed due to the high temperature causing the significant reduce in fruit production, such as the stigma exertion and the association with the jasmonate signaling and other plant hormone pathways (Pan et al., 2019) Heat stress transcription factors (Hsfs) play critical role for thermotolerance in tomato and these involved a complex network for heat response regulatory (Bita and Gerats, 2013; Ohama et al., 2017) There are several tolerant genes, such as HsfA2,HsfA3, induced-heat shock protein Hsps, MBF1 homologue ER24, SlAPX3 (a reactive oxygen species scavenger), and calcium dependent protein kinase (CDPK2) were isolated from the anther (Frank et al., 2009; Zinn et al., 2010) In addition, the ABCDE model genes controlling the floral structure were identified in Arabidopsis, rice and wheat, such as: the MADS-box gene AP1 (class A); PISTILLATA (PI) and AP3 (class B); AGAMOUS (AG) (class C); SHATTERPROOF1 and (SHP1 and SHP2), SEEDSTICK (STK) (class D); and SEPALLATA1 (SEP1-sepals), SEPALLATA2 (SEP2-petals), SEPALLATA3 (SEP3stamens) and SEPALLATA4 (SEP4-carpels) (class E) (Murai, 2013) Many scientists and breeders have tried to solve the problems in tomato production arising from HS Although some tomato varieties have developed improved thermotolerance, their quality and fruit yield are not sufficient (Bita and Gerats, 2013) Thus, it is necessary to create novel genetic resources for heat-tolerant (HT) tomato cultivars with high and stable fruit production Tomato is not only the largest vegetable crop among Solanaceae in over the World, but also the highest economic value in Japan among fruits and vegetables (Ministry of Agriculture, Forestry and Fisheries) The Solanaceae Genomics Network website (SGN, http://solgenomics.net/) is one of popular genomic online database where researches efficiently find out the needed information In the USA, the C M Rick Tomato Genetics Resource Center (TGRC) at the University of California, Davis (http://tgrc.ucdavis.edu/) crucially contributes the seed banks of 1,023 monogenic mutants at 625 putative genetic loci In Japan, tomato has been selected as an important bioresource among the National BioResource Project (NBRP) sponsored by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) This project had been launched and improved an online database TOMATOMA (http://tomatoma.nbrp.jp/index.jsp) (Saito et al., 2011; Shikata et al., 2016) base on 10,793 M2 mutagenized lines of Micro-Tom, created by EMS mutagenesis (4,371 lines) and gamma-ray irradiation (6,422 lines) (Watanabe et al., 2007; Matsukura et al., 2008) Acknowledgments This dissertation was written for submitting to the Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan in particular fulfillment of the requirements for the Degree of Doctor of Philosophy in Agriculture Science (Doctoral Program in Biosphere Resource Science and Technology) This is one of the most important challenges in my life I am grateful to Japanese Government (MONBUKAGAKUSHO:MEXT) scholarship for their financial aid support for three years in my doctoral program, from 2017 October to 2020 September I would like to thank to Tsukuba Plant Innovation Center, University of Tsukuba, through the National Bio-Resource Project (NBRP) of the Japan Agency for Research and Development (AMED), Japan for providing the tomato seeds (TOMJP00001), EMS mutant line for conducting the experiments in this thesis I would like to express my deeply gratitude to my supervisor Prof Hiroshi Ezura for his inspiring lectures, persistent advice and constant support that wonderfully help me during my program I would like to express my great thanks to my sub-supervisors, Dr Ken Hoshikawa and Dr Yoshihito Shinozaki for their advice, care, consistent encouragement and support on my research and my academic life in Japan I would also like to thank to academic advisor committee members, Profs Kenji Miura, Naoya Fukuda and Tohru Ariizumi for their precious advices in my projects I would like to thank to Dr Ryoichi Yano and Dr Mariko Takayama for their assistance in paperwork of Doctoral entrance and in genome sequencing experiment I gratefully thank to all sensei Prof Ryo Ohsawa, Prof Chiaki Matsukura, Dr Kyoko Hiwasa-Tanase, Dr Satoko Nonaka, Dr Yoshihito Okabe, Dr Naomichi Fujiuchi, Dr Lombardo Fabien, Dr Seung Won Kang; to our lab-mates Johan Hunziker, Kosuke Kuwabara, Eri Kuroda, Y.C Lin, Damayanti Farida, Satoshi Fujita, Kasozi Michael; to our lab technicians Yoko Fujimori, Naoko Ito, Michiko Miyamoto, Naomi Inage, Reiko Masuda, Mieko Yamaguchi, Yumiko Yoshida; to our lab secretary Momoyo Okada, as well as the pos-doctoral fellows Yu Lu, Mitalo Oscar and other members in Sosaikaki’s lab for their fruitful discussion, technical assistance, paperwork assistance and encouragement 150 I would like to thank to Prof Brad Day (Michigan State University, USA), Prof Antonio Granell (Universidad Politécnica de Valencia, Spain); Dr Pierre Baldet (French National Institute for Agriculture, Food, and Environment - INRAE) for their advices in my research I would like to thank Prof Kenji Miura, Prof Naoya Fukuda and their laboratory members for teaching and letting me use their instrumentation facilities I sincerely thank to Prof Yooichi Kainoh (University of Tsukuba) for introducing the MEXT scholarship to Vietnam National University of Agriculture I would like to thank Prof Kazuo Watanabe (University of Tsukuba) for the precious classes in the framework of Program Global Food Security I would like to thank all sensei, all staffs and members in Program Global Food Security, in Gene Research Center building; in the office F306, B602; in the Graduate school of Life and Environmental Science, who are in-charge of my doctor program and study in Japan I would also like to express my gratitude to my university Vietnam National University of Agriculture, Faculty of Biotechnology, Department of Molecular and Applied Biotechnology for the important support during my study processing I am thankful to my master supervisor Prof Dulal Borthakur, previous lab-mate Mike Honda (University of Hawaii-Manoa, USA) and all my friends who always encourage and help me a lot in both academic and daily life Above all, I would like to express my deeply gratitude to my family; my grandmother Pham Thi Nhi; my blood parents Pham Ngoc Dung, Nguyen Thi Nhien; my parents-in-law Tran Ngoc Dang, Tran Thi Mo; my husband Tran Anh Duc, my son Tran Dang Nguyen; my godmother Nguyen Thi Hoang; my uncle Pham Ngoc Cuong, my brothers Pham Ngoc Duy, Tran Hung Viet, Le Van Truong; my sisters Pham Thi Bich, Tran Thi Thu Tham, Tran Thi Thanh Nga and all other beloved family members for their unconditional love, encouragement, and other essential support, without which I would not have grown and made it possibly Thank you very much With the highest appreciation Yours sincerely, PHAM THI DUNG 151 References Abdrabo, M., Adger, W.N., 2014 Climate change 2014: impacts, adaptation, and vulnerability-IPCC WGII AR5 summary for policymakers : https://www.researchgate.net/publication/272150376 Abdul-Baki, A a., Stommel, J.R., 1995 Pollen viability and 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