Plant virus evolution under strong drought conditions results in a transition from parasitism to mutualism.

Environmental conditions are an important factor driving pathogens' evolution. Here, we explore the effects of drought stress in plant virus evolution. We evolved turnip mosaic potyvirus in well-watered and drought conditions in Arabidopsis thaliana accessions that differ in their response to virus infection. Virus adaptation occurred in all accessions independently of watering status. Drought-evolved viruses conferred a significantly higher drought tolerance to infected plants. By contrast, nonsignificant increases in tolerance were observed in plants infected with viruses evolved under standard watering. The magnitude of this effect was dependent on the plant accessions. Differences in tolerance were correlated to alterations in the expression of host genes, some involved in regulation of the circadian clock, as well as in deep changes in the balance of phytohormones regulating defense and growth signaling pathways. Our results show that viruses can promote host survival in situations of abiotic stress, with the magnitude of such benefit being a selectable trait.

Characterization of the Copper Transporters from Lotus spp. and Their Involvement under Flooding Conditions.

Forage legumes are an important livestock nutritional resource, which includes essential metals, such as copper. Particularly, the high prevalence of hypocuprosis causes important economic losses to Argentinian cattle agrosystems. Copper deficiency in cattle is partially due to its low content in forage produced by natural grassland, and is exacerbated by flooding conditions. Previous results indicated that incorporation of Lotus spp. into natural grassland increases forage nutritional quality, including higher copper levels. However, the biological processes and molecular mechanisms involved in copper uptake by Lotus spp. remain poorly understood. Here, we identify four genes that encode putative members of the Lotus copper transporter family, denoted COPT in higher plants. A heterologous functional complementation assay of the Saccharomyces cerevisiae ctr1∆ctr3∆ strain, which lacks the corresponding yeast copper transporters, with the putative Lotus COPT proteins shows a partial rescue of the yeast phenotypes in restrictive media. Under partial submergence conditions, the copper content of L. japonicus plants decreases and the expression of two Lotus COPT genes is induced. These results strongly suggest that the Lotus COPT proteins identified in this work function in copper uptake. In addition, the fact that environmental conditions affect the expression of certain COPT genes supports their involvement in adaptive mechanisms and envisages putative biotechnological strategies to improve cattle copper nutrition.

Characterization of the responses to saline stress in the symbiotic green microalga Trebouxia sp. TR9.

MAIN CONCLUSION: For the first time we provide a study on the physiological, ultrastructural and molecular effects of salt stress on a terrestrial symbiotic green microalga, Trebouxia sp. TR9. Although tolerance to saline conditions has been thoroughly studied in plants and, to an extent, free-living microalgae, scientific data regarding salt stress on symbiotic lichen microalgae is scarce to non-existent. Since lichen phycobionts are capable of enduring harsh, restrictive and rapidly changing environments, it is interesting to study the metabolic machinery operating under these extreme conditions. We aim to determine the effects of prolonged exposure to high salt concentrations on the symbiotic phycobiont Trebouxia sp. TR9, isolated from the lichen Ramalina farinacea. Our results suggest that, when this alga is confronted with extreme saline conditions, the cellular structures are affected to an extent, with limited chlorophyll content loss and photosynthetic activity remaining after 72 h of exposure to 5 M NaCl. Furthermore, this organism displays a rather different molecular response compared to land plants and free-living halophile microalgae, with no noticeable increase in ABA levels and ABA-related gene expression until the external NaCl concentration is raised to 3 M NaCl. Despite this, the ABA transduction pathway seems functional, since the ABA-related genes tested are responsive to exogenous ABA. These observations could suggest that this symbiotic green alga may have developed alternative molecular pathways to cope with highly saline environments.

Lotus tenuis x L. corniculatus interspecific hybridization as a means to breed bloat-safe pastures and gain insight into the genetic control of proanthocyanidin biosynthesis in legumes.

BACKGROUND: Proanthocyanidins (PAs) are secondary metabolites that strongly affect plant quality traits. The concentration and the structure of these metabolites influence the palatability and nutritional value of forage legumes. Hence, modulating PAs in the leaves of forage legumes is of paramount relevance for forage breeders worldwide. The lack of genetic variation in the leaf PA trait within the most important forage species and the difficulties in engineering this pathway via the ectopic expression of regulatory genes, prompted us to pursue alternative strategies to enhance this trait in forage legumes of agronomic interest. The Lotus genus includes forage species which accumulate PAs in edible organs and can thus be used as potential donor parents in breeding programs. RESULTS: We recovered a wild, diploid and PA-rich population of L. corniculatus and crossed with L. tenuis. The former grows in an alkaline-salty area in Spain while the latter is a diploid species, grown extensively in South American pastures, which does not accumulate PAs in the herbage. The resulting interspecific hybrids displayed several traits of outstanding agronomic relevance such as rhizome production, PA levels in edible tissues sufficient to prevent ruminal bloating (around 5 mg of PAs/g DW), biomass production similar to the cultivated parent and potential for adaptability to marginal lands. We show that PA levels correlate with expression levels of the R2R3MYB transcription factor TT2 and, in turn, with those of the key structural genes of the epicatechin and catechin biosynthetic pathways leading to PA biosynthesis. CONCLUSIONS: The L. tenuis x L. corniculatus hybrids, reported herein, represent the first example of the introgression of the PA trait in forage legumes to levels known to provide nutritional and health benefits to ruminants. Apart from PAs, the hybrids have additional traits which may prove useful to breed forage legumes with increased persistence and adaptability to marginal conditions. Finally, our study suggests the hybrids and their progeny are an invaluable tool to gain a leap forward in our understanding of the genetic control of PA biosynthesis and tolerance to stresses in legumes.

Plant triterpenoid saponins function as susceptibility factors to promote the pathogenicity of Botrytis cinerea.

The gray mold fungus Botrytis cinerea is a necrotrophic pathogen that causes diseases in hundreds of plant species, including high-value crops. Its polyxenous nature and pathogenic success are due to its ability to perceive host signals in its favor. In this study, we found that laticifer cells of Euphorbia lathyris are a source of susceptibility factors required by B. cinerea to cause disease. Consequently, poor-in-latex (pil) mutants, which lack laticifer cells, show full resistance to this pathogen, whereas lot-of-latex mutants, which produce more laticifer cells, are hypersusceptible. These S factors are triterpenoid saponins, which are widely distributed natural products of vast structural diversity. The downregulation of laticifer-specific oxydosqualene cyclase genes, which encode the first committed step enzymes for triterpene and, therefore, saponin biosynthesis, conferred disease resistance to B. cinerea. Likewise, the Medicago truncatula lha-1 mutant, compromised in triterpenoid saponin biosynthesis, showed enhanced resistance. Interestingly, the application of different purified triterpenoid saponins pharmacologically complemented the disease-resistant phenotype of pil and hla-1 mutants and enhanced disease susceptibility in different plant species. We found that triterpenoid saponins function as plant cues that signal transcriptional reprogramming in B. cinerea, leading to a change in its growth habit and infection strategy, culminating in the abundant formation of infection cushions, the multicellular appressoria apparatus dedicated to plant penetration and biomass destruction in B. cinerea. Taken together, these results provide an explanation for how plant triterpenoid saponins function as disease susceptibility factors to promote B. cinerea pathogenicity.

Opposing roles of plant laticifer cells in the resistance to insect herbivores and fungal pathogens.

More than 12,000 plant species (ca. 10% of flowering plants) exude latex when their tissues are injured. Latex is produced and stored in specialized cells named "laticifers". Laticifers form a tubing system composed of rows of elongated cells that branch and create an internal network encompassing the entire plant. Laticifers constitute a recent evolutionary achievement in ecophysiological adaptation to specific natural environments; however, their fitness benefit to the plant still remains to be proven. The identification of Euphorbia lathyris mutants (pil mutants) deficient in laticifer cells or latex metabolism, and therefore compromised in latex production, allowed us to test the importance of laticifers in pest resistance. We provided genetic evidence indicating that laticifers represent a cellular adaptation for an essential defense strategy to fend off arthropod herbivores with different feeding habits, such as Spodoptera exigua and Tetranychus urticae. In marked contrast, we also discovered that a lack of laticifer cells causes complete resistance to the fungal pathogen Botrytis cinerea. Thereafter, a latex-derived factor required for conidia germination on the leaf surface was identified. This factor promoted disease susceptibility enhancement even in the non-latex-bearing plant Arabidopsis. We speculate on the role of laticifers in the co-evolutionary arms race between plants and their enemies.

The Rhizobia-Lotus Symbioses: Deeply Specific and Widely Diverse.

The symbiosis between Lotus and rhizobia has been long considered very specific and only two bacterial species were recognized as the microsymbionts of Lotus: Mesorhizobium loti was considered the typical rhizobia for the L. corniculatus complex, whereas Bradyrhizobium sp. (Lotus) was the symbiont for L. uliginosus and related species. As discussed in this review, this situation has dramatically changed during the last 15 years, with the characterization of nodule bacteria from worldwide geographical locations and from previously unexplored Lotus spp. Current data support that the Lotus rhizobia are dispersed amongst nearly 20 species in five genera (Mesorhizobium, Bradyrhizobium, Rhizobium, Ensifer, and Aminobacter). As a consequence, M. loti could be regarded an infrequent symbiont of Lotus, and several plant-bacteria compatibility groups can be envisaged. Despite the great progress achieved with the model L. japonicus in understanding the establishment and functionality of the symbiosis, the genetic and biochemical bases governing the stringent host-bacteria compatibility pairships within the genus Lotus await to be uncovered. Several Lotus spp. are grown for forage, and inoculation with rhizobia is a common practice in various countries. However, the great diversity of the Lotus rhizobia is likely squandered, as only few bacterial strains are used as inoculants for Lotus pastures in very different geographical locations, with a great variety of edaphic and climatic conditions. The agroecological potential of the genus Lotus can not be fully harnessed without acknowledging the great diversity of rhizobia-Lotus interactions, along with a better understanding of the specific plant and bacterial requirements for optimal symbiotic nitrogen fixation under increasingly constrained environmental conditions.

Transcriptome Response Mediated by Cold Stress in Lotus japonicus.

Members of the Lotus genus are important as agricultural forage sources under marginal environmental conditions given their high nutritional value and tolerance of various abiotic stresses. However, their dry matter production is drastically reduced in cooler seasons, while their response to such conditions is not well studied. This paper analyzes cold acclimation of the genus by studying Lotus japonicus over a stress period of 24 h. High-throughput RNA sequencing was used to identify and classify 1077 differentially expressed genes, of which 713 were up-regulated and 364 were down-regulated. Up-regulated genes were principally related to lipid, cell wall, phenylpropanoid, sugar, and proline regulation, while down-regulated genes affected the photosynthetic process and chloroplast development. Together, a total of 41 cold-inducible transcription factors were identified, including members of the AP2/ERF, NAC, MYB, and WRKY families; two of them were described as putative novel transcription factors. Finally, DREB1/CBFs were described with respect to their cold stress expression profiles. This is the first transcriptome profiling of the model legume L. japonicus under cold stress. Data obtained may be useful in identifying candidate genes for breeding modified species of forage legumes that more readily acclimate to low temperatures.

Differential growth of Spartina densiflora populations under saline flooding is related to adventitious root formation and innate root ion regulation.

Global change anticipates scenarios of sea level rise that would provoke long lasting floods, especially in lowland areas of salt marshes. Our aim was to evaluate the morpho-physiological adjustment ability to deal with continuous saline flooding of Spartina densiflora Brogn. plants from lowlands and uplands along a subtle topographical gradient (0.2m differential altitude). Plants from both origins were subjected to continuous saline flooding (300mM NaCl) for 35 days. Responses associated to adventitious rooting, aerenchyma formation, concentration of Na+, K+ and Cl- in roots and shoots tissues, tillering and growth were assessed. Root responses differentiated populations given that lowland plants showed higher ability for adventitious root formation and innate superior root ion regulation than upland plants. High constitutive K+ concentration plus high Na+ exclusion in root tissues led to significant low values of Na+:K+ ratios in lowland plants. Better root functioning was, in turn, related with more consistent shoot performance as lowland plants maintained plant tiller number and shoot relative growth rate unaltered while upland plants decreased both parameters by 35 and 18%, respectively, when in saline flooding. The superior performance of lowland plants indicates that locally adapted populations can be promoted in salt marsh habitats with subtle differences at topographic level.

Response to long-term NaHCO3-derived alkalinity in model Lotus japonicus Ecotypes Gifu B-129 and Miyakojima MG-20: transcriptomic profiling and physiological characterization.

The current knowledge regarding transcriptomic changes induced by alkalinity on plants is scarce and limited to studies where plants were subjected to the alkaline salt for periods not longer than 48 h, so there is no information available regarding the regulation of genes involved in the generation of a new homeostatic cellular condition after long-term alkaline stress. Lotus japonicus is a model legume broadly used to study many important physiological processes including biotic interactions and biotic and abiotic stresses. In the present study, we characterized phenotipically the response to alkaline stress of the most widely used L. japonicus ecotypes, Gifu B-129 and MG-20, and analyzed global transcriptome of plants subjected to 10 mM NaHCO3 during 21 days, by using the Affymetrix Lotus japonicus GeneChip®. Plant growth assessment, gas exchange parameters, chlorophyll a fluorescence transient (OJIP) analysis and metal accumulation supported the notion that MG-20 plants displayed a higher tolerance level to alkaline stress than Gifu B-129. Overall, 407 and 459 probe sets were regulated in MG-20 and Gifu B-129, respectively. The number of probe sets differentially expressed in roots was higher than that of shoots, regardless the ecotype. Gifu B-129 and MG-20 also differed in their regulation of genes that could play important roles in the generation of a new Fe/Zn homeostatic cellular condition, synthesis of plant compounds involved in stress response, protein-degradation, damage repair and root senescence, as well as in glycolysis, gluconeogenesis and TCA. In addition, there were differences between both ecotypes in the expression patterns of putative transcription factors that could determine distinct arrangements of flavonoid and isoflavonoid compounds. Our results provided a set of selected, differentially expressed genes deserving further investigation and suggested that the L. japonicus ecotypes could constitute a useful model to search for common and distinct tolerance mechanisms to long-term alkaline stress response in plants.

Defense responses in two ecotypes of Lotus japonicus against non-pathogenic Pseudomonas syringae.

Lotus japonicus is a model legume broadly used to study many important processes as nitrogen fixing nodule formation and adaptation to salt stress. However, no studies on the defense responses occurring in this species against invading microorganisms have been carried out at the present. Understanding how this model plant protects itself against pathogens will certainly help to develop more tolerant cultivars in economically important Lotus species as well as in other legumes. In order to uncover the most important defense mechanisms activated upon bacterial attack, we explored in this work the main responses occurring in the phenotypically contrasting ecotypes MG-20 and Gifu B-129 of L. japonicus after inoculation with Pseudomonas syringae DC3000 pv. tomato. Our analysis demonstrated that this bacterial strain is unable to cause disease in these accessions, even though the defense mechanisms triggered in these ecotypes might differ. Thus, disease tolerance in MG-20 was characterized by bacterial multiplication, chlorosis and desiccation at the infiltrated tissues. In turn, Gifu B-129 plants did not show any symptom at all and were completely successful in restricting bacterial growth. We performed a microarray based analysis of these responses and determined the regulation of several genes that could play important roles in plant defense. Interestingly, we were also able to identify a set of defense genes with a relative high expression in Gifu B-129 plants under non-stress conditions, what could explain its higher tolerance. The participation of these genes in plant defense is discussed. Our results position the L. japonicus-P. syringae interaction as a interesting model to study defense mechanisms in legume species.

Ecological and agronomic importance of the plant genus Lotus. Its application in grassland sustainability and the amelioration of constrained and contaminated soils.

The genus Lotus comprises around 100 annual and perennial species with worldwide distribution. The relevance of Lotus japonicus as a model plant has been recently demonstrated in numerous studies. In addition, some of the Lotus species show a great potential for adaptation to a number of abiotic stresses. Therefore, they are relevant components of grassland ecosystems in environmentally constrained areas of several South American countries and Australia, where they are used for livestock production. Also, the fact that the roots of these species form rhizobial and mycorrhizal associations makes the annual L. japonicus a suitable model plant for legumes, particularly in studies directed to recognize the mechanisms intervening in the tolerance to abiotic factors in the field, where these interactions occur. These properties justify the increased utilization of some Lotus species as a strategy for dunes revegetation and reclamation of heavy metal-contaminated or burned soils in Europe.