NAD⁺accumulation as a metabolic off switch for orthodox pollen

Terrestrial plant pollen is classified into two categories based on its metabolic status: pollen with low-metabolism are termed “orthodox” and pollen with high-metabolism are termed “recalcitrant.” Nicotinamide adenine dinucleotide (NAD) is crucial for a number of metabolisms in all extant organisms. It has recently been shown that NAD homeostasis plays an important role in a broad range of developmental processes and responses to environment. Recently, a reverse genetic approach shed light on the significance of NAD biosynthesis on pollen fate. In orthodox Arabidopsis pollen, NAD(+) that was accumulated in excess at dispersal dramatically decreased on rehydration. The lack of a key gene that is involved in NAD biosynthesis compromised the excess accumulation. Moreover, absence of the excess accumulation phenocopied the so-called recalcitrant pollen, as demonstrated by the germination inside anthers and the loss of desiccation tolerance. Upon rehydration, NAD(+)-consuming inhibitors impaired tube germination. Taken together, our results suggest that accumulation of NAD(+) functions as a physiochemical molecular switch for suspended metabolism and that the decrease of NAD(+) plays a very important role during transitions in metabolic states. Shifting of the redox state to an oxidizing environment may efficiently control the comprehensive metabolic network underlying the onset of pollen germination

Molecular insights into plant proteins involved in NAD biosynthesis and signalling

L'importanza delle vitamine del gruppo B per la vita è stata accertata da più di un secolo. La forma biologicamente attiva della vitamina B3, il coenzima NAD+, svolge un ruolo centrale come scambiatore di elettroni, substrato enzimatico e molecola di segnalazione. Il metabolismo del NAD+ nelle piante si è rapidamente rivelato una rete complessa, che ha un impatto non solo sui principali percorsi biochimici intracellulari delle piante (fotosintesi, respirazione,...), ma anche sui processi di sviluppo a livello sistemico come la germinazione dei semi, la riproduzione e l'adattamento a diversi tipi di stress, con un ruolo particolare nell'immunità delle piante. Il lavoro di questa tesi mira a far luce sul ruolo del NAD+ nelle piante, sia a livello intracellulare che extracellulare. L'attività di ricerca si è concentrata su due proteine di A. thaliana, un modello universalmente accettato per le piante. Nella prima parte di questa tesi di dottorato, descritta nel capitolo III, forniamo una caratterizzazione completa di un enzima chiave della biosintesi del NAD+, la NaMN/NMN adenililtransferasi, che catalizza una reazione comune sia alle vie de novo che a quelle di salvataggio, unificando così tutte le vie biosintetiche del NAD+ e influenzando l'intero metabolismo della pianta. Nella seconda parte della tesi, riportata nel capitolo IV, si descrive la produzione di una forma ricombinante del dominio extracellulare del recettore lectin receptor kinase 1.8 di A. thaliana. Questo proteina transmembrana è altamente affine e specifico per NAD+. La sua interazione con il NAD+ extracellulare promuove la trascrizione di numerosi effettori immunitari.The importance of the vitamins of the group B for life, has been established since more than a century ago. The biologically active form of the B3 vitamin, i.e., the coenzyme NAD+, plays a central role electron exchanger, enzymatic substrate and signalling molecule. NAD+ metabolism in plants quickly turned out to be a complex network, impacting not only the core intracellular plant biochemistry pathways (photosynthesis, respiration,…), but also developmental processes at a systemic level such as seed germination, reproduction, and the adaptation to several types of stresses, with a particular role in plant immunity. The work of this thesis aims to shed light on the role of NAD+ in plants, both intracellularly and in the extracellular space. The activity research focused on two proteins from A. thaliana, an universally accepted model for plants. In the first part of this PhD dissertation, described in chapter III, we provide a complete characterization of a key enzyme of NAD+ biosynthesis, NaMN/NMN adenylyltransferase, which catalyses a reaction common to both de novo and salvage pathways, thus unifying all NAD+ biosynthetic routes and influencing the whole plant metabolism. In the second part of the thesis, reported in chapter IV, we describe the production of a recombinant form of the extracellular domain of the receptor of A. thaliana lectin receptor kinase 1.8. This transmembrane protein is highly affine and specific for NAD+. Its interaction with extracellular NAD+ promotes transcription of several immune effectors

The mitochondrial NAD+ transporter (NDT1) plays important roles in cellular NAD+ homeostasis in \u3ci\u3eArabidopsis thaliana\u3c/i\u3e

Nicotinamide adenine dinucleotide (NAD+) is an essential coenzyme required for all living organisms. In eukaryotic cells, the final step of NAD+ biosynthesis is exclusively cytosolic. Hence, NAD+ must be imported into organelles to support their metabolic functions. Three NAD+ transporters belonging to the mitochondrial carrier family (MCF) have been biochemically characterized in plants. AtNDT1 (At2g47490), focus of the current study, AtNDT2 (At1g25380), targeted to the inner mitochondrial membrane, and AtPXN (At2g39970), located in the peroxisomal membrane. Although AtNDT1 was presumed to reside in the chloroplast membrane, subcellular localization experiments with green fluorescent protein (GFP) fusions revealed that AtNDT1 locates exclusively in the mitochondrial membrane in stably transformed Arabidopsis plants. To understand the biological function of AtNDT1 in Arabidopsis, three transgenic lines containing an antisense construct of AtNDT1 under the control of the 35S promoter alongside a T-DNA insertional line were evaluated. Plants with reduced AtNDT1 expression displayed lower pollen viability, silique length, and higher rate of seed abortion. Furthermore, these plants also exhibited an increased leaf number and leaf area concomitant with higher photosynthetic rates and higher levels of sucrose and starch. Therefore, lower expression of AtNDT1 was associated with enhanced vegetative growth but severe impairment of the reproductive stage. These results are discussed in the context of the mitochondrial localization of AtNDT1 and its important role in the cellular NAD+ homeostasis for both metabolic and developmental processes in plants

代謝工学による環境ストレス耐性植物の分子育種

プロジェクト番号 : A10-101一般研究 : 外部資金獲得促進研究textapplication/pdfresearch repor

実績報告書（論文リスト） 環境生態学部門 ２００９－２０１０（平成２１-２２）年度

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Thriving in a salty future:morpho-anatomical, physiological and molecular adaptations to salt stress in alfalfa (Medicago sativa L.) and other crops

BackgroundWith soil salinity levels rising at an alarming rate, accelerated by climate change and human interventions, there is a growing need for crop varieties that can grow on saline soils. Alfalfa (Medicago sativa) is a cool-season perennial leguminous crop, commonly grown as forage, biofuel feedstock, and soil conditioner. It demonstrates significant potential for agricultural circularity and sustainability, for example by fixing nitrogen, sequestering carbon, and improving soil structures. Although alfalfa is traditionally regarded as moderately salt-tolerant species, modern alfalfa varieties display specific salt-tolerance mechanisms, which could be used to pave alfalfa’s role as a leading crop able to grow on saline soils.ScopeAlfalfa’s salt tolerance underlies a large variety of cascading biochemical and physiological mechanisms. These are partly enabled by alfalfa’s complex genome structure and out-crossing nature, which on the other hand entail impediments for molecular and genetic studies. This review first summarizes the general effects of salinity on plants and the broad-ranging mechanisms for dealing with salt-induced osmotic stress, ion toxicity, and secondary stress. Secondly, we address defensive and adaptive strategies that have been described for alfalfa, such as the plasticity of alfalfa’s root system, hormonal crosstalk for maintaining ion homeostasis, spatiotemporal specialized metabolite profiles, and the protection of alfalfa-rhizobia associations. Finally, bottlenecks for research of the physiological and molecular salt-stress responses as well as biotechnology-driven improvements of salt tolerance are identified and discussed.ConclusionUnderstanding morpho-anatomical, physiological, and molecular responses to salinity is essential for the improvement of alfalfa and other crops in saline land reclamation. This review identifies potential breeding targets for enhancing alfalfa performance stability and general crop robustness for rising salt levels as well as to promote alfalfa applications in saline land management

애기장대의 발달 과정 및 배추의 아종 내 형태학적 다양성에 대한 후성유전학적 조절 연구

학위논문(박사) -- 서울대학교대학원 : 농업생명과학대학 식물생산과학부, 2022. 8. 허진회.Epigenetic mechanisms play crucial roles in diverse biological processes such as cell differentiation and responses to developmental and environmental cues. Epigenetic modifications including DNA methylation and histone modifications can alter gene expression through modulating chromatin structure without DNA sequence changes. Plants can perceive and respond to internal or external signals, where gene expression may be epigenetically regulated. Moreover, some epigenetic variations may be stably inherited and conceivably contribute to important phenotypic traits. However, the detailed mechanisms of epigenetic factor-mediated developmental and environmental plasticity and their evolutionary significance associated with traits remain to be explored in plants. In this study, I investigated environmental and developmental responses regulated by DNA demethylases in Arabidopsis. DNA methylation can be actively removed by the DEMETER (DME) family of DNA glycosylases, together with REPRESSOR OF SILENCING 1 (ROS1), DEMETER-LIKE 2 (DML2) and DML3. Two ros1 mutant lines were hypersensitive to abscisic acid (ABA). Downregulation of ABA-inducible genes was accompanied by DNA hypermethylation at their promoter regions in ros1, indicating ROS1-dependent DNA demethylation is required for transcriptional activation in ABA-mediated drought and osmotic stress responses. I further extended this research to examine the combined function of DNA demethylases in reproductive development. I observed several developmental defects such as delayed growth and flowering, aberrant floral development and short unfertilized siliques in the dme ros1 dml2 dml3 (drdd) quadruple homozygous mutant. Gene expression is significantly reduced in drdd compared to WT, rather than in dme and rdd, accompanied with an increase in DNA methylation levels. These findings suggest that DNA demethylases act redundantly for the proper regulation of genes during reproductive development. In addition, I analyzed the morphological divergence between the two subspecies Chinese cabbage (Brassica rapa subsp. pekinensis) and turnip (B. rapa subsp. rapa) despite high genetic similarity. Comprehensive analysis of transcriptome and epigenome revealed that accessible chromatin regions (ACRs) were associated with the expression dynamics, histone H3 lysine 27 acetylation enrichment and the depletion of DNA methylation. The distant ACRs of the two subspecies were highly conserved but displayed divergent chromatin accessibility with differential enrichment of transcription factor motifs. These results indicate that subspecies-specific divergence of distal enhancers might be responsible for morphotype diversification. Taken together, this study will broaden the understanding of regulatory mechanisms of DNA demethylation in response to environmental and developmental cues and provide insights into distal enhancer-derived subspecies diversification during evolution.후성유전학적 기작은 세포 분화, 내부 발달 신호 및 외부 환경 요인에 대한 반응 등 다양한 생물학적 반응을 조절하는 데 중요한 역할을 한다. DNA 메틸화, 히스톤 변형 등의 후성유전학적 변형은 DNA 염기서열의 변화 없이 염색질 구조 변화를 통해 유전자 발현을 변화시킨다. 식물은 내부 및 외부 자극을 인지하고 반응하여 후성유전학적으로 유전자 발현을 조절한다. 그리고 일부 후성유전학적 변이는 안정적으로 후대에 전달됨으로써 주요 형질에 기여하는 것으로 생각된다. 그러나 세부적인 후성유전학적 조절 기작 및 형질과 연관된 진화적 중요성은 아직 식물에서 많이 밝혀지지 않았다. DNA 메틸화 수준은 DNA 메틸화와 탈메틸화 기작에 의해 조절된다. 애기장대에서 DNA 메틸화는 DNA 탈메틸화 효소에 의해 제거되는데, DEMTER (DME), REPRESSOR OF SILENCING 1 (ROS1), DEMETER-LIKE 2 (DML2) 및 DML3가 이를 담당한다. DME는 암배우체의 중심세포에서 주로 발현되는 반면에, ROS1, DML2 및 DML3는 영양조직에서 발현된다. 본 논문에서는 애기장대에서 DNA 탈메틸화 효소에 의한 환경 및 발달 반응 조절을 연구하였다. ros1 돌연변이체는 앱시스산 처리 시 야생형에 비해 유묘 및 뿌리 발달이 저해되었다. ros1 돌연변이체에서 앱시스산 유도성 유전자들의 발현이 감소하였으며, 프로모터 지역에서 DNA 메틸화 수준이 증가하였다. 따라서 ROS1에 의한 DNA 탈메틸화는 앱시스산이 매개하는 가뭄 및 수분 스트레스 반응에 필요한 유전자들의 발현을 활성화하는 데 중요한 역할을 하는 것으로 생각된다. 더 나아가 생식 생장에 있어 DNA 탈메틸화 효소의 역할을 밝히고자 하였다. 애기장대 dme ros1 dml2 dml3 (drdd) 사중 돌연변이체는 성장 지연, 개화 시기 지연, 비정상적 화기 구조 및 꼬투리 발달 등의 표현형을 보였다. 사중 돌연변이체에서는 dme 또는 rdd 돌연변이체 비해 유전자 발현 감소와 DNA 메틸화 증가가 두드러졌다. 이는 여러DNA 탈메틸화 효소가 생식 생장에 필요한 유전자의 발현을 중복적으로 조절함을 시사한다. 또한 본 논문에서는 배추(Brassica rapa subsp. pekinensis)와 순무(B. rapa subsp. rapa)의 형태학적 다양성에 대한 연구를 수행하였다. 배추와 순무는 같은 종으로 유사한 유전체를 가지고 있지만, 상이한 표현형을 보인다. 전사체 및 후성유전체 분석을 수행하여 배추와 순무의 열린 염색질 지역은 유전자 발현 변화 범위, 히스톤 H3의 27번째 라이신 아세틸화 (H3 lysine 27 acetylation) 및 낮은 DNA 메틸화 수준과 연관되어 있음을 확인하였다. 유전자로부터 먼 거리에 위치하는 열린 염색질 지역은 배추와 순무 간에 DNA 염기서열이 높은 수준으로 보존되어 있었으나, 염색질 접근성과 전이인자 모티프는 상이한 양상을 보였다. 이러한 연구 결과는 아종 간의 염색질 접근성의 차이가 형태학적 다양성에 기여할 수 있음을 의미한다. 본 연구는 외부 환경 요인과 내부 발달 신호에 반응하여 일어나는 DNA 탈메틸화 효소에 의한 유전자 발현 조절 기작에 대한 이해를 높이고, 진화 과정 동안 염색질 구조 차이에서 기인하는 아종 간 형태적 차이에 대한 새로운 관점을 제시할 수 있을 것으로 기대된다.GENERAL INTRODUCTION 1 CHAPTER 1. Plant-Specific DNA Demethylation in Response to Abscisic Acid 16 ABSTRACT 17 INTRODUCTION 19 MATERIALS AND METHODS 25 RESULTS 30 DISCUSSION 53 REFERENCES 57 CHAPTER 2. Regulation of Reproductive Development by DNA Demethylases in Arabidopsis 64 ABSTRACT 65 INTRODUCTION 67 MATERIALS AND METHODS 72 RESULTS 77 DISCUSSION 112 REFERENCES 116 CHAPTER 3. Comparative Analysis of Genome and Epigenome Landscapes in Brassica rapa subspecies 121 ABSTRACT 122 INTRODUCTION 124 MATERIALS AND METHODS 130 RESULTS 138 DISCUSSION 177 REFERENCES 182 ABSTRACT IN KOREAN 193박

Polyamine signaling pathway during environmental stress: Metabolomic approaches to elucidate spermine down-stream targets

[spa] El estrés medioambiental está afectando de forma paulatina la productividad de los cultivos. En la búsqueda de soluciones, uno de los principales objetivos de la investigación en fisiología de plantas, es dilucidar los mecanismos de tolerancia que se presentan ante diversos estreses, con la finalidad de generar plantas con fenotipos resistentes. En referencia a este asunto, las poliaminas y sus rutas señalizadoras son de importancia crucial. Las más abundantes en plantas (putrescina, espermidina y espermina) tienden a acumularse en respuesta al estrés por lo cual se les asocia a un rol protector, sin embargo, las tendencias de acumulación dependen del tipo de estrés que la planta es capaz de percibir. De hecho, su ruta biosintética se activa a diferentes niveles dependiendo del estímulo, lo cual les confiere un carácter selectivo. La Espermina (una de las poliaminas superiores) no es esencial para el crecimiento de la planta, no obstante, actualmente se sabe que esta molécula ejerce diversos roles protectores en una gran variedad de condiciones y además activa cascadas señalizadoras implicadas en la respuesta defensiva de la planta. En consecuencia, para dilucidar los mecanismos de tolerancia, se ha hecho necesario profundizar en las dianas de señalización por parte de la espermina. Empleando como modelo experimental Arabidopsis thaliana, el presente estudio ha demostrado la implicación de este policatión en el aumento de la capacidad anti-oxidativa a través de conexiones con metabolitos centrales en el metabolismo de azucares, lípidos y aminoácidos como es el caso del piruvato y el mio-Inositol, así como también, la implicación de esta poliamina en la morfología y ramificación de las raíces, reforzando la noción de implicación esencial por parte de esta poliamina, en la fisiología del estrés en plantas.[eng] Environmental stress is increasingly wearing down crop productivity. Nowadays, one of the main aims of plant research is to elucidate tolerance mechanisms to diverse stresses, in order to provide solutions by generating stress-tolerant plants. In regard with this matter, polyamine signaling pathway is of crucial importance. The major polyamines in plants (putrescine, spermidine and spermine) tend to accumulate in response to stress and are associated with a protective role. The trend of their accumulation is related to the stress the plant is sensing; in fact, polyamine biosynthetic pathway is activated at different levels depending on the stimulus, which gives a selective role to these molecules. Spermine, one of the higher polyamines, is not essential for plant growth; however, it is presently known that this molecule plays diverse protective roles under several stress factors and triggers signaling cascades implicated in plant defense. Therefore, research on spermine down-stream targets has become necessary towards the elucidation of plant tolerance responses. By the use of model plant Arabidopsis thaliana this study demonstrated the implication of this polycation on enhancement of anti-oxidative capacity by signaling connections to central hub metabolites for sugar, lipid and amino acid metabolism such as pyruvate or myo-Inositol, as well as its involvement on root morphology

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