Pablo Ignacio Calzadilla

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.

Transcriptional regulation of 9-cis-epoxycarotenoid dioxygenase (NCED) gene by putrescine accumulation positively modulates ABA synthesis and drought tolerance in Lotus tenuis plants

The accumulation of putrescine (Put) and increased arginine decarboxylase (ADC, EC 4.1.1.19) activity levels in response to osmotic stress has been reported; however, the biological meaning of this increase remains unclear. To obtain new insights into these questions, we studied the drought response of a transgenic Lotus tenuis line that expresses the oat ADC gene, which is driven by the stress-inducible pRD29A promoter. This line contains high levels of Put with no changes in spermidine and spermine contents, even under water deficits. Our results indicate that the biochemical and morphological responses to dehydration correlate with the Put level and provide evidence that Put controls the ABA content in response to drought by modulating ABA biosynthesis at the transcriptional 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.

The alkaline tolerance in Lotus japonicus is associated with mechanisms of iron acquisition and modification of the architectural pattern of the root

The response of fifty-four Lotus japonicus ecotypes, and of six selected ecotypes was investigated under alkaline conditions. Sensitive, but not tolerant ecotypes, showed interveinal chlorosis under all alkalinity conditions and high mortality under extreme alkalinity. Interveinal chlorosis was associated with Fe deficiency, as a reduced Fe2+ shoot content was observed in all sensitive ecotypes. In addition, some showed a decline in photosynthesis rate and PSII performance compared to the control. In contrast, some tolerant ecotypes did not change these parameters between treatments. Alkaline tolerance could be explained by a mechanism of Fe acquisition and a root structural modification. This conclusion was based on the fact that all tolerant, but not the sensitive ecotypes, presented high ferric reductase oxidase activity under alkaline stress compared to the control, and a Herringbone root pattern modification. On this basis, the analysis of these mechanisms of alkaline tolerance could be used in screening programs for the selection of new tolerant genotypes in the Lotus genus.

Photosynthetic responses mediate the adaptation of two Lotus japonicus ecotypes to low temperature

Lotus species are important forage legumes due to their high nutritional value and adaptability to marginal conditions. However, the dry matter production and regrowth rate of cultivable Lotus spp. is drastically reduced during colder seasons. In this work, we evaluated the chilling response of Lotus japonicus ecotypes MG-1 and MG-20. No significant increases were observed in reactive oxygen species and nitric oxide production or in lipid peroxidation, although a chilling-induced redox imbalance was suggested through NADPH/NADP(+) ratio alterations. Antioxidant enzyme catalase, ascorbate peroxidase, and superoxide dismutase activities were also measured. Superoxide dismutase, in particular the chloroplastic isoform, showed different activity for different ecotypes and treatments. Stress-induced photoinhibition also differentially influenced both ecotypes, with MG-1 more affected than MG-20. Data showed that the D2 PSII subunit was more affected than D1 after 1 d of low temperature exposure, although its protein levels recovered over the course of the experiment. Interestingly, D2 recovery was accompanied by improvements in photosynthetic parameters (Asat and Fv/Fm) and the NADPH/NADP(+) ratio. Our results suggest that the D2 protein is involved in the acclimation response of L. japonicus to low temperature. This may provide a deeper insight into the chilling tolerance mechanisms of the Lotus genus.

Rice tolerance to suboptimal low temperatures relies on the maintenance of the photosynthetic capacity

The purpose of this research was to identify differences between two contrasting rice cultivars in their response to suboptimal low temperatures stress. A transcriptomic analysis of the seedlings was performed and results were complemented with biochemical and physiological analyses. The microarray analysis showed downregulation of many genes related with PSII and particularly with the oxygen evolving complex in the sensitive cultivar IR50. Complementary studies indicated that the PSII performance, the degree of oxygen evolving complex coupling with the PSII core and net photosynthetic rate diminished in this cultivar in response to the stress. However, the tolerant cultivar Koshihikari was able to maintain its energy equilibrium by sustaining the photosynthetic capacity. The increase of oleic acid in Koshihikari could be related with membrane remodelling of the chloroplasts and hence contribute to tolerance. Overall, these results work as a ground for future analyses that look forward to characterize possible mechanisms to tolerate this stress.

LOTUS spp: BIOTECHNOLOGICAL STRATEGIES TO IMPROVE THE BIOECONOMY OF LOWLANDS IN THE SALADO RIVER BASIN (ARGENTINA)

The Salado River Basin region is the most important livestock breeding area in Argentina, wherethe Lotus species has been traditionally cultivated as forages. Nearly 60% of their land surface is dominated by salt-affected soils with severe constraints for crop cultivation. In order to cope with that limitation, farmers have utilized species such as non-native L. tenuis (ex- Lotus glaber), which shows a very good adaptation. As a result, inter-seeding of L. tenuis has been proposed as a strategy of choice for improving forage production in marginal areas. The increase in soil quality by these means is achieved by an increment of the organic matter content, improvement of soil fertility as well as microbial biodiversity. Thus, the introduction of L. tenuis and/or other Lotus genotypes could have enormous benefits for similar constrained lands around the world. We are developing an integrated analysis of the changes that occur in soils under legume production. We will not only analyze the microbial diversity associated, but also soil physical and chemical characteristics and the impact of different legume-microbes association on mitigation of GHG emissions. In addition, we are identifying the main genetic determinants associated with interesting agronomic traits such as plant tolerance against biotic and abiotic stresses and the content of condensed tannins. Our future and present research will build a solid base for the improvement of agronomicallyimportant species and the development of better strategies for the management of constrained lands such as the lowlands in the Argentinean Pampas.