Abscisic Acid And Nitrate Transporter Mtlatd/nip Signaling In Root And Nodule Development In Medicago Truncatula

Abscisic acid (ABA) is a plant hormone that regulates various developmental processes and environmental stress responses. ABA modulates growth of both primary roots and lateral roots, helping to shape root architecture. The lateral root organ defective (latd) mutants, disrupted in the MtLATD/NIP gene, encoding a nitrate transporter, have severe root growth defects that can be rescued by applying ABA. However, the way in which ABA stimulates latd root growth is unclear, and the downstream components of MtLATD/NIP and ABA signaling are completely unknown. To answer these questions, this dissertation focuses on two major potential downstream regulators: Reactive Oxygen Species (ROS) and transcription factors (TFs). ROS are important signaling molecules required in ABA-induced stomatal closure under drought or osmotic stresses, but their role in ABA regulation of root development is unclear. I found that latd mutant roots have increased ROS levels, and the expression level of several MtRboh genes, which encode major ROS-producing enzymes, the NADPH oxidases, is also increased. ABA decreases the amount of ROS in latd roots and also reduces expression of MtRbohC, in particular. In addition, I observed that latd mutant roots have cell elongation defects, which can also be rescued by exogenous ABA. I demonstrated that pharmaceutically decreasing ROS levels using an NADPH oxidase inhibitor, or reducing the expression of MtRbohC using RNA interference can increase cell elongation and stimulate lateral root elongation in latd roots. These findings have revealed a mechanism by which ABA restores root growth in latd mutant roots via regulating ROS levels, and identified MtRbohC as an important downstream target of ABA signaling mediated by MtLATD/NIP. TFs act as regulatory nodes controlling the transcription of gene clusters and playing a crucial role in plant growth and development. Using a high-throughput TF profiling approach, I have identified 20 TFs that exhibit altered expression levels in latd mutant roots as compared to wild type, 60% of which can be restored to normal levels by ABA. My analysis also revealed that ABA regulates the expression of a different set of TFs in latd roots, suggesting that MtLATD/NIP is crucial for ABA regulation of TF expression. Moreover, ABA changes the TFs regulated by MtLATD/NIP almost completely, indicating a tight control of ABA on TFs regulated by MtLATD/NIP. Surprisingly, I found that the expression of NODULATION SIGNALING PATHWAY 2 (MtNSP2), a GRAS family TF required for nodulation, is regulated by MtLATD/NIP, ABA and nitrate in non-symbiotic roots. In symbiotic roots, MtLATD/NIP is required for the transcriptional signaling pathway downstream of MtNSP2 in the epidermis as well as induction of MtNSP2 expression by cytokinin and subsequent activation of its downstream targets in the cortex. These findings indicate that MtLATD/NIP functions in nodulation signal transduction upstream of MtNSP2, and mediates crosstalk with cytokinin. Together, these two approaches have begun to characterize a signaling pathway downstream of ABA and MtLATD/NIP that involves ROS, MtNSP2, and a core group of TFs in the regulation of root development and nodulation in M. truncatula

Plant Responses and Tolerance to Salt Stress: Physiological and Molecular Interventions

Overall, the 19 contributions in this Special Issue “Plant Responses and Tolerance to Salt Stress: Physiological and Molecular Interventions” discuss the various aspects of salt stress responses in plants. It also discusses various mechanisms and approaches to conferring salt tolerance on plants. These types of research studies provide further directions in the development of crop plants for the saline environment in the era of climate change

NF-YB family transcription factors in Arabidopsis: Structure, phylogeny, and expression analysis in biotic and abiotic stresses

Nuclear factor-Y (NF-Y) transcription factors (TFs) are conserved heterotrimeric complexes present and widespread across eukaryotes. Three main subunits make up the structural and functional aspect of the NF-Y TFs: NF-YA, NF-YB and NF-YC, which bind to the conserved CCAAT- box of the promoter region of specific genes, while also interacting with each other, thereby forming myriad combinations. The NF-YBs are expressed differentially in various tissues and plant development stages, likely impacting many of the cellular processes constitutively and under stress conditions. In this study, ten members of NF-YB family from Arabidopsis thaliana were identified and expression profiles were mined from microarray data under different biotic and abiotic conditions, revealing key insights into the involvement of this class of proteins in the cellular and biological processes in Arabidopsis. Analysis of cis-acting regulatory elements (CAREs) indicated the presence of abiotic and biotic stress-related transcription factor binding sites (TFBs), shedding light on the multifaceted roles of these TFs. Microarray data analysis inferred distinct patterns of expression in various tissues under differing treatments such as drought, cold and heat stress as well as bacterial, fungal, and viral stress, indicating their likelihood of having an expansive range of regulatory functions under native and stressed conditions; while quantitative real-time PCR (qRT-PCR) based expression analysis revealed that these TFs get real-time-modulated in a stress dependent manner. This study, overall, provides an understanding of the AtNF-YB family of TFs in their regulation and participation in various morphogenetic and defense- related pathways and can provide insights for development of transgenic plants for trait dependent studies

Identifying signal transduction components acting downstream of reactive oxygen species (ROS) in Arabidopsis thaliana

Traditionally, reactive oxygen species (ROS) have been regarded as toxic by-products of aerobic metabolism. However, in recent years it has become apparent that plants actively produce ROS as signalling molecules. ROS are able to mediate adaptive responses to various environmental stresses as well as processes such as stomatal closure and development. Downstream signalling events that are modulated by ROS include calcium mobilisation, protein phosphorylation and gene expression. This study investigated signalling proteins acting downstream of ROS, in order to understand how ROS are perceived and transduced to elicit such a wide range of responses. To establish a molecular profile provoked by ROS, a microarray experiment of Arabidopsis plants exposed to exogenous H(_2)O(_2) was analysed. Of the 895 differentially expressed transcripts, a substantial proportion had predicted functions in cell rescue and defence, including heat shock, disease resistance and antioxidant genes. Genes encoding candidate H(_2)O(_2) signalling components were identified from this microarray experiment and their H(_2)O(_2) - induced expression was verified by northern RNA-blot analysis. Two transcription factors of the ethylene response factor (ERF) family (AtERFS [At5g47230]) and AtERF6 [At4g17490])and an ankyrin protein kinase (APK [At4g18950]) were selected for further study. Northern blot analysis and comparison with publicly available transcriptome data sets demonstrated that the expression of these three genes was induced by various stress treatments, such as UV-B irradiation, cold and elicitor challenge. To unravel the potential in vivo function of these proteins, loss- and gain-of-function lines were generated and analysed. No abnormal plant phenotypes were observed during development or in response to the stress and hormone treatments tested. A high level of functional redundancy may exist between AtERFS and AtERF6. Microarray analyses were performed on the over-expression lines. Genes that were differentially regulated in APK over-expressor lines gave no indication of its function. However, the microarray analyses revealed that AtERFS and AtERF6 have roles in the plant pathogen defence response, since their over-expression induced defence gene expression. Analysis of cis elements in the promoters of the ERF-differentially regulated genes revealed that both transcription factors displayed GCC box binding activity. However, the GCC box was not over-represented in the promoters of H202-differentially regulated genes, which suggests that this element has a ROS independent regulation

Protomyces Comparative Genomics and Modulation of Arabidopsis Immunity

The plant phyllosphere environment offers a habitat for multiple kinds of microbes, including bacteria, fungi, yeast, etc. Microbes can be beneficial, pathogenic, or mostly neutral to plants. Increasingly the interaction patterns and related plant immunity signaling pathways against bacteria and filamentous fungi have been extensively studied. However, the interaction between plants and yeast or yeast-like fungi is largely unclear. Phyllosphere yeast-like fungi from wild Arabidopsis were isolated and characterized in this study. Around a hundred yeast isolates, including ascomycete Protomyces species, were identified and cultured. Protomyces species have been described as pathogens of plants in the Umbelliferae and Compositae families, however, with questionable phylogeny and little genomic information. We isolated and investigated the interaction of a strain Protomyces sp. SC29 (SC29) with Arabidopsis. SC29 can persist in the Arabidopsis phylloplane, and activate Arabidopsis immune responses with MAPKs (mitogen-activated protein kinases) activation and upregulation of salicylic acid signaling and camalexin biosynthesis marker genes. Additionally, indolic compounds produced by Protomyces species are able to activate plant auxin responses. The genomes of SC29 and all currently available Protomyces species were sequenced, assembled, and annotated. Comparative genomic analysis revealed genomic characters of SC29 related to adaptation to the phyllosphere environment. Genomic insights into the pathogenesis of Protomyces species were also discovered. The phylogenetic relationships of both the genus Protomyces and the subphylum Taphrinomycotina were re-constructed with genome-wide single-copy protein sequences. Small secreted proteins from the genomes of Protomyces spp. were analyzed as candidate effectors. Physiological, phylogenetic, and genomic evidence supported SC29 to be a novel species distinct from currently accepted Protomyces species. Thus, the study of SC29 and its interaction with Arabidopsis represents a new model system for the exploration of the genetics of plant interactions with phyllosphere resident yeasts.Kasvin fyllosfääri tarjoaa elinympäristön monenlaisille mikrobeille, kuten bakteereille, sienille ja hiivoille. Mikrobit voivat olla hyödyllisiä, patogeenisiä tai lähinnä neutraaleja kasveille. Kasvien vuorovaikutuskuvioita mikrobien kanssa ja siihen liittyviä kasvien immuniteettisignaloinnin polkuja bakteereja sekä rihmasieniä vastaan on tutkittu enenevässä määrin, mutta vuorovaikutus kasvien ja hiivojen tai hiivan kaltaisten sienien välillä on kuitenkin suurelta osin epäselvää. Tässä tutkimuksessa eristettiin ja karakterisoitiin hiivan kaltaisia sieniä villinä kasvaneiden lituruohojen fyllosfääreistä. Noin sata hiivaisolaattia, sisältäen kotelosieniin kuuluvia Protomyces-lajeja, tunnistettiin ja viljeltiin. Protomyces-lajeja on kuvailtu patogeenisiksi sarjakukkais- (Umbelliferae) ja mykerökukkaiskasveille (Compositae), mutta tähän liittyvä fylogenetiikka on ollut kyseenalaista ja genomitietoa on ollut vähän. Eristimme kannan Protomyces sp. SC29 (SC29) ja tutkimme sen vuorovaikutusta lituruohon kanssa. SC29 kykenee selviytymään lituruohon lehdistön pinnalla ja se aktivoi lituruohon immuunivasteita mitogeeniaktivoituvia proteiinikinaaseja aktivoimalla sekä sääntelemällä ylös salisyylihapposignalointia ja kamaleksiinin biosynteesin markkerigeenejä. Lisäksi, Protomyces-lajien tuottamat indoliyhdisteet kykenevät aktivoimaan kasvin auksiinivasteita. SC29:n ja kaikkien tällä hetkellä saatavilla olevien Protomyces-lajien genomit sekvensoitiin, koottiin ja annotoitiin. Vertaileva genomianalytiikka paljasti SC29:stä genomisia piirteitä, jotka liittyvät sopeutumiseen fyllosfääriympäristöön. Myös genomin yhteyksiä Protomyces-lajien patogeenisyyteen löydettiin. Sekä Protomyces-suvun että Taphrinomycotina-alajakson fylogeneettiset suhteet estimoitiin käyttäen proteiinisekvenssejä, joita löytyi vain yhtenä kopiona koko genomista. Protomyces-lajien genomeista tunnistettuja pieniä erittyviä proteiineja analysoitiin mahdollisina efektoreina. Fysiologinen, fylogeneettinen ja genominen aineisto antoi tukea sille, että SC29 olisi uusi laji ja erillinen tällä hetkellä hyväksytyistä Protomyces-lajeista. Tämä SC29:ään ja sen interaktioon lituruohon kanssa kohdistettu tutkimus tarjoaa uuden mallijärjestelmän, jolla voidaan tutkia kasvin ja fyllosfääriä asuttavien hiivojen välillä tapahtuvien vuorovaikutusten genetiikkaa

Salt-tolerant genes from halophytes are potential key players of salt tolerance in glycophytes

Crop productivity strongly depends on several biotic and abiotic factors. Salinity is one of the most important abiotic factors, besides drought, extreme temperatures, light and metal stress. The enhanced burden of secondary salinization induced through anthropogenic activities increases pressure on glycophytic crop plants. The recent isolation and characterization of salt tolerance genes encoding signaling components from halophytes, which naturally grow in high salinity, has provided tools for the development of transgenic crop plants with improved salt tolerance and economically beneficial traits. In addition understanding of the differences between glycophytes and halophytes with respect to levels of salinity tolerance is also one of the prerequisite to achieve this goal. Based on the recent developments in mechanisms of salt tolerance in halophytes, we will explore the potential of introducing salt tolerance by choosing the available genes from both dicotyledonous and monocotyledonous halophytes, including the salt overly sensitive system (SOS)-related cation/proton antiporters of plasma (NHX/SOS1) and vacuolar membranes (NHX), energy-related pumps, such as plasma membrane and vacuolar H+ adenosine triphosphatase (PM& V-H+ATPase), vacuolar H+ pyrophosphatases (V-H+PPase) and potassium transporter genes. Various halophyte genes responsible for other processes, such as crosstalk signaling, osmotic solutes production and reactive oxygen species (ROS) suppression, which also enhance salt tolerance will be described. In addition, the transgenic overexpression of halophytic genes in crops (rice, peanut, finger millet, soybean, tomato, alfalfa, jatropha, etc.) will be discussed as a successful mechanism for the induction of salt tolerance. Moreover, the advances in genetic engineering technology for the production of genetically modified crops to achieve the improved salinity tolerance under field conditions will also be discussed

Redox homeostasis in photosynthetic organisms: Novel and established thiol-based molecular mechanisms

Redox homeostasis consists of an intricate network in which reactive molecular species (RMS), redox modifications and redox proteins act in concert to allow both physiological responses and adaptation to stress conditions. This review highlights established and novel thiol-based regulatory pathways underlying the functional facets and significance of redox biology in photosynthetic organisms. This cannot be all-encompassing, but is intended to provide a comprehensive overview on the structural/molecular mechanisms governing the most relevant thiol switching modifications with emphasis on the large genetic and functional diversity of redox controllers (i.e. redoxins). We also summarize the different proteomic-based approaches aimed at investigating the dynamics of redox modifications and the recent evidence that extends the possibility to monitor the cellular redox state in vivo. Lastly, the physiological relevance of redox transitions is discussed based on reverse genetic studies confirming the importance of redox homeostasis in plant growth, development, and stress responses

Advances in Plant Tolerance to Abiotic Stresses

During the last 50 years, it has been shown that abiotic stresses influence plant growth and crop production greatly, and crop yields have evidently stagnated or decreased in economically important crops, where only high inputs assure high yields. The recent manifesting effects of climate change are considered to have aggravated the negative effects of abiotic stresses on plant productivity. On the other hand, the complexity of plant mechanisms controlling important traits and the limited availability of germplasm for tolerance to certain stresses have restricted genetic advances in major crops for increased yields or for improved other traits. However, some level of success has been achieved in understanding crop tolerance to abiotic stresses; for instance, identification of abscisic acid (ABA) receptors (e.g., ABA-responsive element (ABRE) binding protein/ABRE binding factor (AREB/ABF) transcription factors), and other regulons (e.g., WRKYs, MYB/MYCs, NACs, HSFs, bZIPs and nuclear factor-Y (NF-Y)), has shown potential promise to improve plant tolerance to abiotic stresses. Apart from these major regulons, studies on the post-transcriptional regulation of stress-responsive genes have provided additional opportunities for addressing the molecular basis of cellular stress responses in plants. This chapter focuses on the progress in the study of plant tolerance to abiotic stresses, and describes the major tolerance pathways and implicated signaling factors that have been identified, so far. To link basic and applied research, genes and proteins that play functional roles in mitigating abiotic stress damage are summarized and discussed