Utilization of genes encoding osmoprotectants in transgenic plants for enhanced abiotic stress tolerance

Global agriculture in the context of growing and expanding populations is under huge pressure to provide increased food, feed, and fiber. The recent phenomenon of climate change has further added fuel to the fire. It has been practically established now that the global temperature has been on the increase with associated fluctuations in annual rainfall regimes, and the resultant drought and flood events and increasing soil and water salinization. These challenges would be met with the introduction and utilization of new technologies coupled with conventional approaches. In recent years, transgenic technology has been proved very effective in terms of production of improved varieties of crop plants, resistant to biotic stresses. The abiotic stresses such as salt and drought are more complex traits, controlled by many genes. Transgenic plant development for these stresses has utilized many single genes. However, much emphasis has been placed on genes catalyzing the biosynthetic pathways of osmoprotectants. This review focuses on the current status of research on osmoprotectant genes and their role in abiotic stress tolerance in transgenic plants

Transcriptome Analysis Of Leaves, Flowers And Fruits Perisperm Of Coffea Arabica L. Reveals The Differential Expression Of Genes Involved In Raffinose Biosynthesis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coffea arabica L. is an important crop in several developing countries. Despite its economic importance, minimal transcriptome data are available for fruit tissues, especially during fruit development where several compounds related to coffee quality are produced. To understand the molecular aspects related to coffee fruit and grain development, we report a large-scale transcriptome analysis of leaf, flower and perisperm fruit tissue development. Illumina sequencing yielded 41,881,572 high-quality filtered reads. De novo assembly generated 65,364 unigenes with an average length of 1,264 bp. A total of 24,548 unigenes were annotated as protein coding genes, including 12,560 full-length sequences. In the annotation process, we identified nine candidate genes related to the biosynthesis of raffinose family oligossacarides (RFOs). These sugars confer osmoprotection and are accumulated during initial fruit development. Four genes from this pathway had their transcriptional pattern validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, we identified similar to 24,000 putative target sites for microRNAs (miRNAs) and 134 putative transcriptionally active transposable elements (TE) sequences in our dataset. This C. arabica transcriptomic atlas provides an important step for identifying candidate genes related to several coffee metabolic pathways, especially those related to fruit chemical composition and therefore beverage quality. Our results are the starting point for enhancing our knowledge about the coffee genes that are transcribed during the flowering and initial fruit development stages.121Brazilian Coffee Research Consortium, National Institute for Coffee Science and Technology (INCT-Cafe)Coordination for the Improvement of Higher Education Personnel (CAPES)National Council of Technological and Scientific Development (CNPq)Brazilian Innovation Agency (FINEP)Center for Computational Engineering and Sciences at Unicamp/SP-BrazilCAPESFundacao Araucaria (FA)CNPqCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Transcriptome analysis of leaves, flowers and fruits perisperm of Coffea arabica L. reveals the differential expression of genes involved in raffinose biosynthesis

Coffea arabica L. is an important crop in several developing countries. Despite its economic importance, minimal transcriptome data are available for fruit tissues, especially during fruit development where several compounds related to coffee quality are produced. To understand the molecular aspects related to coffee fruit and grain development, we report a large-scale transcriptome analysis of leaf, flower and perisperm fruit tissue development. Illumina sequencing yielded 41,881,572 high-quality filtered reads. De novo assembly generated 65,364 unigenes with an average length of 1,264 bp. A total of 24,548 unigenes were annotated as protein coding genes, including 12,560 full-length sequences. In the annotation process, we identified nine candidate genes related to the biosynthesis of raffinose family oligossacarides (RFOs). These sugars confer osmoprotection and are accumulated during initial fruit development. Four genes from this pathway had their transcriptional pattern validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, we identified ~24,000 putative target sites for microRNAs (miRNAs) and 134 putative transcriptionally active transposable elements (TE) sequences in our dataset. This C. arabica transcriptomic atlas provides an important step for identifying candidate genes related to several coffee metabolic pathways, especially those related to fruit chemical composition and therefore beverage quality. Our results are the starting point for enhancing our knowledge about the coffee genes that are transcribed during the flowering and initial fruit development stages. (Résumé d'auteur

Transcriptome Analysis of Leaves, Flowers and Fruits Perisperm of Coffea arabica L. Reveals the Differential Expression of Genes Involved in Raffinose Biosynthesis.

Coffea arabica L. is an important crop in several developing countries. Despite its economic importance, minimal transcriptome data are available for fruit tissues, especially during fruit development where several compounds related to coffee quality are produced. To understand the molecular aspects related to coffee fruit and grain development, we report a largescale transcriptome analysis of leaf, flower and perisperm fruit tissue development. Illumina sequencing yielded 41,881,572 high-quality filtered reads. De novo assembly generated 65,364 unigenes with an average length of 1,264 bp. A total of 24,548 unigenes were annotated as protein coding genes, including 12,560 full-length sequences. In the annotation process, we identified nine candidate genes related to the biosynthesis of raffinose family oligossacarides (RFOs). These sugars confer osmoprotection and are accumulated during initial fruit development. Four genes from this pathway had their transcriptional pattern validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, we identified ~24,000 putative target sites for microRNAs (miRNAs) and 134 putative transcriptionally active transposable elements (TE) sequences in our dataset. This C. arabica transcriptomic atlas provides an important step for identifying candidate genes related to several coffee metabolic pathways, especially those related to fruit chemical composition and therefore beverage quality. Our results are the starting point for enhancing our knowledge about the coffee genes that are transcribed during the flowering and initial fruit development stages

Functional Genomics for Tolerance to Abiotic Stress in Cereals

The world food grain production needs to be doubled by the year 2050 to meet the ever growing demands of the population (Tilman et al., 2002). This goal needs to be achieved despite decreased arable land, dwindling water resources, and the environmental constraints such as drought, water logging, excess heat, frost, salinity, metal toxicity and nutrient imbalances, which cause major losses in cereal grain production. Drought, salinity and cold stress alone are known to cause nearly 35% of cereal crop losses throughout the world (Quarrie et al., 1999). The effectiveness of traditional breeding approaches to deal with the problem is limited due to complex nature of stress tolerance traits and due to incompatibility barriers encountered during transfer of genes from wild species to cultivated ones. Therefore, newer strategies need to be used for developing crop plants tha

Rôle des facteurs de transcription CBF dans le contrôle du développement de l'eucalyptus en condition de stress

Eucalyptus, feuillu le plus planté dans le monde, est fortement exposé au froid en raison de l'absence de dormance. Les facteurs de transcription CBF (CRT-binding Factor) jouent un rôle important dans la réponse aux stress abiotiques, particulièrement au froid. Préalablement à mes travaux, 17 gènes CBF avaient été identifiés chez E. grandis. Une étude fonctionnelle par surexpression de gènes CBF chez un hybride d'E. urophylla x E. grandis avait été initiée avec un phénotypage des lignées transgéniques au stade microboutures. Mon travail s'est articulé autour d'une étude in silico du régulon potentiel des CBF et de la caractérisation des sur-exprimants CBF après les avoir enracinés puis acclimatés ex vitro. Les résultats obtenus mettent en évidence de profondes modifications dans la tolérance, la croissance, la morphologie et l'anatomie des transformants qui, le plus souvent, ont pu être reliées à la surexpression de gènes impliqués dans ces processus. Au-delà d'un impact sur les feuilles, ces travaux suggèrent fortement pour la première fois un contrôle très important des CBF sur la formation de la tige.Eucalyptus, the most widely planted hardwood in the world, is highly exposed to the cold due to the lack of dormancy. CBF transcription factors play an important role in the response to abiotic stress, particularly cold stress. Previously, 17 CBF genes have been identified in E. grandis and a functional study of CBF genes by overexpressing in a hybrid E. urophylla x E. grandis has been initiated through the phenotyping of transgenic lines at microcutting stage. My work involved in silico study of the potential CBF regulon and the characterization of these transformants after rooting and ex vitro acclimatization. The results show substantial changes in tolerance, growth, morphology and anatomy of transformants which most oftent could be associated to the overexpression of genes involved in the corresponding process. Beyond the impact on the leaves, this work strongly suggests for the first time a very important control of CBF on stem formation

Plants and Environment

Changing environmental condition and global population demands understanding the plant responses to hostile environment. Significant progress has been made over the past few decades through amalgamation of molecular breeding with non-conventional breeding. Understanding the cellular and molecular mechanisms to stress tolerance has received considerable scientific scrutiny because of the uniqueness of such processes to plant biology, and also its importance in the campaign "Freedom From Hunger". The main intention of this publication is to provide a state-of-the-art and up-to-date knowledge of recent developments in understanding of plant responses to major abiotic stresses, limitations and the current status of crop improvement. A better insight will help in taking a multidisciplinary approach to address the issues affecting plant development and performance under adverse conditions. I trust this book will act as a platform to excel in the field of stress biology

복숭아 품종의 저온 요구도와 내한성 비교

학위논문 (박사) -- 서울대학교 대학원 : 농업생명과학대학 식물생산과학부(원예과학전공), 2021. 2. 이희재.Deciduous fruit trees temporarily suspend their growth through dormancy and increase their cold hardiness through cold acclimation to adapt low temperature in winter. Here, physiological changes during dormancy, cold acclimation, and deacclimation were compared among peach cultivars to elucidate different responses to freezing temperatures. Dormancy completion of peach cultivars ranged from early January to late February. Chilling requirements were calculated by using Chill Hours, Utah, Dynamic, North Carolina, and Low Chilling models and ranged from 263 to 2123 chill hour, 377 to 1134 chill unit, and 21.3 to 74.8 chilling portion. Low-chill cultivars might be readily deacclimated when exposed to warm spells and sudden temperature drop during late winter or early spring which might induce freezing damage. Changes in soluble sugar content, related enzyme activity, and gene expression were monitored in the shoots of a cold-hardy Soomee (SM) and a cold-sensitive Odoroki (OD) peach cultivar during cold acclimation (CA) and deacclimation (DA). Although both cultivars had similar seasonal patterns of cold hardiness, SM was cold-hardier than OD from December to March. During this period, total soluble sugar content was higher in SM than in OD, but starch content was significantly lower in SM, along with higher total amylase activity and related gene expression. Of the detected soluble sugars, sucrose was predominant and its content was most significantly correlated with cold hardiness. Fructose and glucose contents in SM, but not in OD, increased during CA and then decreased during DA concurrently with increased acid invertase activity and its gene expression. Raffinose and stachyose were detectable only from November to April. Their contents and the expression of the genes involved in their synthesis were also significantly correlated with cold hardiness. To compare DA resistance and sugar metabolism between the cultivars, changes in sugar content and transcriptome of SM and Kanoiwa Hakuto (KH) were compared after accelerated DA at 15℃ and delayed DA at –5℃ in February. During the accelerated DA for 4 weeks, KH lost cold hardiness more rapidly than SM, and starch content decreased more in SM than KH. The most enriched pathway with differentially expressed genes (DEGs) was carbohydrate metabolism in which starch and sucrose metabolism was also most enriched with 43 DEGs encoding 20 related enzymes. Transcripts encoding catabolic enzymes, including β-galactosidase (LOC18769018 and LOC18783479) and β-glucosidase (LOC18770931, LC18779132, and LOC18779303) were up-regulated in SM relative to KH, while those encoding anabolic enzymes such as galactinol synthase (LOC18789982) and raffinose synthase (LOC18778819) were down-regulated. Changes in gene expression and enzyme activities related to soluble sugar metabolism were different among cultivars which consequently cause different cold hardiness of peach trees.온대 과수는 휴면을 통해 생장을 일시적으로 멈추고 저온 순화를 통해 내한성을 증가시켜 겨울철 저온에 적응하기 한다. 본 논문에서는 복숭아 품종별로 동해가 다르게 나타나는 원인을 구명하기 위해 휴면과 저온 순화 과정 중의 생리적 변화를 비교하였다. 복숭아 품종의 휴면은 1월과 2월 사이에 타파되었으며, Chill Hours, Utah, Dynamic 모델 등을 적용한 결과, 263-2123 chill hour, 377-1134 chill unit, 21.3-74.8 chilling portion의 저온 요구도를 나타내었다. 저온 요구도가 낮은 품종은 휴면 타파 시기가 빠르기 때문에 쉽게 탈순화되어 늦겨울과 초봄의 저온에 동해를 입을 가능성이 높은 것으로 판단되었다. 내한성이 강한 수미 품종과 약한 오도로끼 품종의 신초를 대상으로 하여 시기별로 유리당 함량의 변화를 조사하고 주요 당과 관련된 효소 활성의 변화, 관련 유전자의 발현양을 조사하였다. 두 품종은 시기별로 내한성이 유사하게 변화하였으나 12월부터 3월까지는 수미가 더 강한 내한성을 나타내었다. 이 시기의 총 유리당 함량은 수미에서 더 높았으며 관련 유전자 발현양도 더 높았다. 복숭아 신초에서는 유리당 중에서 sucrose의 함량이 가장 높은 것으로 조사되었으며 내한성과의 상관관계가 가장 높았다. 또한 fructose와 glucose는 순화 기간 동안 수미에서만 유의하게 증가하였으며 오도로끼에서는 증가하지 않았다. 이는 수미에서 활성이 높았던 acid invertase에 의해 sucrose가 fructose와 glucose로 분해되었기 때문인 것으로 보인다. Raffinose와 stachyose는 11월부터 4월 사이에서만 검출되었으며 이외의 시기에서는 검출되지 않아 내한성과 유의한 상관관계를 보였다. 품종별 탈순화 반응에 따른 대사 변화를 분석하기 위해 수미와 가납암백도 품종을 2월에 촉진 탈순화(15℃)와 지연 탈순화(–5℃) 처리하고 내한성, 전사체, 유리당과 전분 함량 변화를 분석하였다. 4주간의 촉진 탈순화 과정에서 내한성의 소실이 수미에서보다 가납암백도에서 더 빠르게 이루어졌으며 전분 함량의 감소는 수미에서 더 크게 나타났다. 전사체 분석 결과 carbohydrate metabolism에 연관된 차별 발현 유전자가 가장 많이 나타났으며, 그 중에서 starch and sucrose metabolism에 관련된 것이 가장 많았다. 43개의 차별 발현 유전자가 starch and sucrose metabolism에 연관되어 있었고, 이들은 20개의 효소를 암호화하였다. 촉진 탈순화 과정에서 β-galactosidase(LOC18769018, LOC18783479), β-glucosidase(LOC18770931, LC18779132, LOC18779303)와 같은 분해 효소 유전자의 발현이 가납암백도에서보다 수미에서 더 증가하였으며, galactinol synthase(LOC18789982)나 raffinose synthase(LOC18778819)와 같은 합성 효소 유전자의 발현은 수미에서보다 가납암백도에서 더 증가하였다. 본 실험을 통해 유리당 대사에 관련된 유전자 발현과 효소 활성의 변화가 품종별로 차이가 있다는 것을 확인하였으며 이는 품종 간에 내한성이 다르게 나타나는 데 영향을 끼침을 알 수 있었다.GENERAL INTRODUCTION 1 LITERATURE REVIEW 3 Dormancy and cold hardiness 3 Chilling requirement and models 4 CA 5 DA 6 Reacclimation 7 Cold hardiness determination 7 Mechanisms of freezing injury 8 Sugar metabolism and cold hardiness 8 Functions of soluble sugars against freezing stress 11 LITERATURE CITED 13 CHAPTER 1 24 ABSTRACT 24 INTRODUCTION 25 MATERIALS AND METHODS 28 Plant materials 28 Chilling requirements 28 Chilling accumulation over the last 100 years of climate records 32 Statistical analysis 32 RESULTS AND DISCUSSION 33 Chilling requirements of peach cultivars 33 Chilling accumulation over the last 100 years of climate records 42 LITERATURE CITED 48 CHAPTER 2 55 ABSTRACT 55 INTRODUCTION 57 MATERIALS AND METHODS 60 Plant materials 60 Cold hardiness determination 60 Determination of sugar and starch contents 61 Determination of enzyme activities 62 Determination of relative gene expressions 65 Statistical analysis 65 RESULTS AND DISCUSSION 69 Seasonal changes in cold hardiness 69 Seasonal changes in soluble sugar and starch contents 71 Seasonal changes in enzyme activities 74 Seasonal changes in relative gene expressions 78 Interconversion of soluble sugars and starch 84 Correlation of cold hardiness with carbohydrate content, enzyme activity, and gene expression 85 Changes in soluble sugar contents and related metabolism 88 LITERATURE CITED 94 CHAPTER 3 103 ABSTRACT 103 INTRODUCTION 105 MATERIALS AND METHODS 107 Plant materials and treatments 107 Cold hardiness determination 107 Determination of sugar and starch contents 109 Total RNA extraction 110 cDNA library construction, sequencing, and assembling 111 Identification and functional annotation of the DEGs 111 Determination of relative gene expressions 112 RESULTS AND DISCUSSION 115 Cold hardiness: LT50 values and tissue browning 115 Changes in soluble sugar contents 118 Transcriptome sequencing data statistics 121 Functional annotation and GO term enrichment of the DEGs 124 Transcription factor (TF) identification and classification 128 Functional annotation of the transcripts during accelerated DA 134 Comparison of DEGs between SM and KH 139 Validation of DEG data by qRT-PCR 144 LITERATURE CITED 147 CONCLUSIONS 154 ABSTRACT IN KOREAN 155Docto