Elena Prats

Biotechnology approaches to overcome biotic and abiotic stress constraints in legumes

SummaryBiotic and abiotic stresses cause significant yield losses in legumes and can significantly affect their productivity. Biotechnology tools such as marker-assisted breeding, tissue culture, in vitro mutagenesis and genetic transformation can contribute to solve or reduce some of these constraints. However, only limited success has been achieved so far. The emergence of “omic” technologies and the establishment of model legume plants such as Medicago truncatula and Lotus japonicus are promising strategies for understanding the molecular genetic basis of stress resistance, which is an important bottleneck for molecular breeding. Understanding the mechanisms that regulate the expression of stress-related genes is a fundamental issue in plant biology and will be necessary for the genetic improvement of legumes. In this review, we describe the current status of biotechnology approaches in relation to biotic and abiotic stresses in legumes and how these useful tools could be used to improve resistance to important constraints affecting legume crops.

Methods of nitric oxide detection in plants: A commentary

Over the last decade nitric oxide (NO) has been shown to influence a range of processes in plants. However, when, where and even if NO production occurs is controversial in several physiological scenarios in plants. This arises from a series of causes: (a) doubts have arisen over the specificity of widely used 4,5-diaminofluorescein diacetate (DAF-2DA)/4-amino-5-methylamino-2,7-difluorofluorescein (DAF-FM) dyes for NO, (b) no plant nitric oxide synthase (NOS) has been cloned, so that the validity of using mammalian NOS inhibitors to demonstrate that NO is being measured is debatable, (c) the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (cPTIO) needs to be used with caution, and (d) some discrepancies between assays for in planta measurements and another based on sampling NO from the gas phase have been reported. This review will outline some commonly used methods to determine NO, attempt to reconcile differing results obtained by different laboratories and suggest appropriate approaches to unequivocally demonstrate the production of NO.

Nitric oxide contributes both to papilla-based resistance and the hypersensitive response in barley attacked by Blumeria graminis f. sp.hordei

SUMMARY Nonspecific penetration resistance due to papilla formation and race-specific hypersensitive response (HR) can both contribute to Blumeria graminis resistance in barley. Some effective papillae form even in the susceptible cv. Pallas and the isoline P01 carries the additional Mla1 allele conditioning HR. The NO-specific stain DAF-2DA (4,5-diaminofluorescein-2-diacetate) revealed a transient NO generation burst commencing 10 h after inoculation (h.a.i.) in close association with sites of papilla formation in both barley lines. In P01 a burst of NO production throughout some attacked cells was initiated around 10-12 h.a.i. and this preceded whole-cell autofluorescence indicative of HR. The specificity of DAF-2DA staining was demonstrated by the suppression of staining following application of the NO scavenger C-PTIO (1H-imidazol-1-yloxy-2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-3-oxide). In addition, C-PTIO application increased penetration frequencies in both barley lines, indicating a role for NO in papilla-based resistance. Furthermore, C-PTIO application slightly delayed HR in P01 whereas, conversely, application of an NO donor, sodium nitroprusside, slightly accelerated HR in P01 and increased cell death frequency in Pallas. Thus, NO generation is one of the earliest responses of barley epidermal cell defence against B. graminis attack and may be important in both the initiation and the development of effective papillae and cell death due to HR.

Abiotic Stress Responses in Legumes: Strategies Used to Cope with Environmental Challenges

Legumes are well recognized for their nutritional and health benefits as well as for their impact in the sustainability of agricultural systems. The threatening scenario imposed by climate change highlights the need for concerted research approaches in order to develop crops that are able to cope with environmental stresses, while increasing yield and quality. During the last decade, some physiological components and molecular players underlying abiotic stress responses of a broad range of legume species have been elucidated. Plant physiology approaches provided general outlines of plant responses, identifying stress tolerance-related traits or elite cultivars. A thorough identification of candidate genes and quantitative trait loci (QTLs) associated with these traits followed. Model legumes like Medicago truncatula, Lotus japonicus, and more recently, Glycine max provided valuable translational approaches for dissecting legume responses to abiotic stresses. The challenge now focuses on the translation of the information gained in model systems in controlled environments to crops grown under field conditions. In this review, we provide a general overview of the recent achievements on the study of abiotic stress responses in a broad range of model, grain and forage legumes species, highlighting the different approaches used. Major accomplishments, as well as limitations or drawbacks are discussed across the different sections. Some perspectives regarding new approaches for screening, breeding or engineering legumes with desirable abiotic stress resistance traits are anticipated. These advances will support the development of legumes better adapted to environmental constraints, tackling current demands on modern agriculture and food production presently exacerbated by global climate changes.

Breeding approaches for crenate broomrape (Orobanche crenata Forsk.) management in pea (Pisum sativum L.).

BACKGROUND Pea cultivation is strongly hampered in Mediterranean and Middle East farming systems by the occurrence of Orobanche crenata Forsk. Strategies of control have been developed, but only marginal successes have been achieved. Most control methods are either unfeasible, uneconomical, hard to achieve or result in incomplete protection. The integration of several control measures is the most desirable strategy. RESULTS [corrected] Recent developments in control are presented and re-evaluated in light of recent developments in crop breeding and molecular genetics. These developments are placed within a framework that is compatible with current agronomic practices. CONCLUSION The current focus in applied breeding is leveraging biotechnological tools to develop more and better markers to speed up the delivery of improved cultivars to the farmer. To date, however, progress in marker development and delivery of useful markers has been slow. The application of knowledge gained from basic genomic research and genetic engineering will contribute to more rapid pea improvement for resistance against O. crenata and/or the herbicide.

Integrating nitric oxide into salicylic acid and jasmonic acid/ ethylene plant defense pathways.

Plant defense against pests and pathogens is known to be conferred by either salicylic acid (SA) or jasmonic acid (JA)/ethylene (ET) pathways, depending on infection or herbivore-grazing strategy. It is well attested that SA and JA/ET pathways are mutually antagonistic allowing defense responses to be tailored to particular biotic stresses. Nitric oxide (NO) has emerged as a major signal influencing resistance mediated by both signaling pathways but no attempt has been made to integrate NO into established SA/JA/ET interactions. NO has been shown to act as an inducer or suppressor of signaling along each pathway. NO will initiate SA biosynthesis and nitrosylate key cysteines on TGA-class transcription factors to aid in the initiation of SA-dependent gene expression. Against this, S-nitrosylation of NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1) will promote the NPR1 oligomerization within the cytoplasm to reduce TGA activation. In JA biosynthesis, NO will initiate the expression of JA biosynthetic enzymes, presumably to over-come any antagonistic effects of SA on JA-mediated transcription. NO will also initiate the expression of ET biosynthetic genes but a suppressive role is also observed in the S-nitrosylation and inhibition of S-adenosylmethionine transferases which provides methyl groups for ET production. Based on these data a model for NO action is proposed but we have also highlighted the need to understand when and how inductive and suppressive steps are used.

Pathogen-derived nitric oxide influences formation of the appressorium infection structure in the phytopathogenic fungus Blumeria graminis

Nitric oxide (NO) is an important signal in plant resistance to pathogens. Here we report that NO is also generated by Blumeria graminis f.sp. hordei as a pathogenesis determinant on barley. Infection by B. graminis f.sp. hordei is dependent on appressorium formation in order to penetrate the host. Using fluorescent dye diaminofluorescein-2 diacetate (DAF-2DA) and confocal laser scanning microscopy, transient NO generation was detected within the B. graminis f.sp. hordei appressorium during its maturation. To confirm that NO was indeed being measured, DAF-2DA fluorescence was suppressed using a NO scavenger and a mammalian NO synthase inhibitor. Both chemicals affected the number of appressorial lobes produced by the fungus. These data indicate that NO plays a key role in formation of B. graminis f.sp. hordei appressoria.

Medicago truncatula as a Model for Nonhost Resistance in Legume-Parasitic Plant Interactions

Crenate broomrape (Orobanche crenata) is a root parasitic weed that represents a major constraint for grain legume production in Mediterranean and West Asian countries. Medicago truncatula has emerged as an important model plant species for structural and functional genomics. The close phylogenic relationship of M. truncatula with crop legumes increases its value as a resource for understanding resistance against Orobanche spp. Different cytological methods were used to study the mechanisms of resistance against crenate broomrape of two accessions of M. truncatula, showing early and late acting resistance. In the early resistance accession (SA27774) we found that the parasite died before a tubercle had formed. In the late resistance accession (SA4327) the parasite became attached without apparent problems to the host roots but most of the established tubercles turned dark and died before emergence. The results suggest that there are defensive mechanisms acting in both accessions but with a time gap that is crucial for a higher success avoiding parasite infection.

A metabolomic study in oats (Avena sativa) highlights a drought tolerance mechanism based upon salicylate signalling pathways and the modulation of carbon, antioxidant and photo-oxidative metabolism

Although a wealth of information is available on the induction of one or several drought-related responses in different species, little is known of how their timing, modulation and crucially integration influence drought tolerance. Based upon metabolomic changes in oat (Avena sativa L.), we have defined key processes involved in drought tolerance. During a time course of increasing water deficit, metabolites from leaf samples were profiled using direct infusion-electrospray mass spectroscopy (DI-ESI-MS) and high-performance liquid chromatography (HPLC) ESI-MS/MS and analysed using principal component analysis (PCA) and discriminant function analysis (DFA). The involvement of metabolite pathways was confirmed through targeted assays of key metabolites and physiological experiments. We demonstrate an early accumulation of salicylic acid (SA) influencing stomatal opening, photorespiration and antioxidant defences before any change in the relative water content. These changes are likely to maintain plant water status, with any photoinhibitory effect being counteracted by an efficient antioxidant capacity, thereby representing an integrated mechanism of drought tolerance in oats. We also discuss these changes in relation to those engaged at later points, consequence of the different water status in susceptible and resistant genotypes.

Genetic Diversity and Population Structure Among Oat Cultivars and Landraces

In this study, genetic diversity among 177 oat (Avena sativa L.) accessions including both white and red oat landraces and 36 commercial cultivars was studied for simple sequence repeat (SSR) loci. Thirty-one genomic and expressed sequence tags (EST)-derived primer pairs were selected according to high polymorphism from an initial 66 SSR batch. Markers revealed a high level of polymorphism, detecting a total of 454 alleles. The average gene diversity for the whole sample was 0.29. Genetic similarity, calculated using the Dice coefficient, was used for cluster analysis, and principal component analysis was also applied. In addition, population structure using a Bayesian clustering approach identified discrete subpopulation based on allele frequency and showed similar clustering of oat genotypes in four groups. Accessions could be classified into four main clusters that clearly separated the commercial cultivars, the red oat landraces and two clusters of white oat landraces. Cultivars showed less diversity than the landraces indicating a reduction of genetic diversity during breeding, whereas white oat landraces showed higher diversity than red ones. The average polymorphic information content of 0.80 for the SSR loci indicated the usefulness of many of the SSR for genotype identification. In particular, two markers, MAMA5 and AM04, with a total of 50 alleles and a high discrimination power (>0.90), were sufficient to discriminate among all commercial cultivars studied highlighting their potential use for variety identification.