Franca Locatelli

Osmyb4 expression improves adaptive responses to drought and cold stress in transgenic apples

Constitutive expression of the rice cold-inducible Osmyb4 gene in transgenic Arabidopsis (Arabidopsisthaliana) plants improves adaptive responses to cold and drought stress, most likely due to the constitutive activation of several stress-inducible pathways and to the accumulation of several compatible solutes (e.g., glucose, fructose, sucrose, proline, glycine betaine and some aromatic compounds). Although the Osmyb4 gene seems able to activate stress responsive pathways in different species, we previously reported that its specific effect on stress tolerance depends on the transformed species. In the present work, we report the effects of the Osmyb4 expression for improving the stress response in apple (Maluspumila Mill.) plants. Namely, we found that the ectopic expression of the Myb4 transcription factor improved physiological and biochemical adaptation to cold and drought stress and modified metabolite accumulation. Based on these results it may be of interest to use Osmyb4 as a tool for improving the productivity of woody perennials under environmental stress conditions.

Metabolic response to cold and freezing of Osteospermum ecklonis overexpressing Osmyb4

The constitutive expression of the rice Osmyb4 gene in Arabidopsis plants gives rise to enhanced abiotic and biotic stress tolerance, probably by activating several stress-inducible pathways. However, the effect of Osmyb4 on stress tolerance likely depends on the genetic background of the transformed species. In this study, we explored the potential of Osmyb4 to enhance the cold and freezing tolerance of Osteospermum ecklonis, an ornamental and perennial plant native to South Africa, because of an increasing interest in growing this species in Europe where winter temperatures are low. Transgenic O. ecklonis plants were obtained through transformation with the Osmyb4 rice gene under the control of the CaMV35S promoter. We examined the phenotypic adaptation of transgenic plants to cold and freezing stress. We also analysed the ability of wild-type and transgenic Osteospermum to accumulate several solutes, such as proline, amino acids and sugars. Using nuclear magnetic resonance, we outlined the metabolic profile of this species under normal growth conditions and under stress for the first time. Indeed, we found that overexpression of Osmyb4 improved the cold and freezing tolerance and produced changes in metabolite accumulation, especially of sugars and proline. Based on our data, it could be of agronomic and economic interest to use this gene to produce Osteospermum plants capable of growing in open field, even during the winter season in climatic zone Z9.

Efficiency of transient transformation in tobacco protoplasts is independent of plasmid amount

We describe an optimized protocol for the transient transformation of tobacco protoplasts mediated by polyethylene–glycol (PEG). As expected, the quantitative β–glucuronidase (Gus) activity driven by pCaMVGus was dependent on the amount of plasmid used. Nevertheless, we demonstrate by an immunodetection method that transformation efficiency did not depend on the amount of plasmid used but on the limitation imposed by cell competence. In fact, we obtained the same percentage of transformed cells (about 60%) using a wide range of plasmid concentrations (0.1–10 μg per test). Finally, we show that, when we used two plasmid types in a mixture at a concentration ranging from 0.1 to 10 μg for each, all transformed cells expressed proteins encoded by both plasmids. Transient expression and co-transformation experiments are routinely used methods and, probably, the major results from this work were assumed by many researchers in this field, but our data experimentally support this assumption.

The Product of the Rice myb7 Unspliced mRNA Dimerizes with the Maize Leucine Zipper Opaque2 and Stimulates Its Activity in a Transient Expression Assay

myb7 mRNA is present in rice in spliced and unspliced forms, splicing being enhanced by anoxia. The protein (Mybleu) encoded by the unspliced mRNA is composed of an incomplete Myb domain followed by a leucine zipper; however, it lacks canonical sequences for DNA binding, transcriptional activation, and nuclear localization. We show here that in transiently transformed tobacco protoplasts, Mybleu is able to enhance the transcriptional activity of the maize leucine zipper Opaque2 on its targetb32 promoter. The Mybleu transactivation effect is strictly dependent on the presence of Opaque2 and is driven by Mybleu-Opaque2 heterodimers. Mybleu is located in the nucleus, both in rice and in transformed tobacco protoplasts. In rice, the protein is expressed in regions corresponding to undifferentiated cells of roots and coleoptiles. Therefore, myb7 mRNA encodes, depending on its splicing, two transcription factors belonging to separate classes. One of them, Mybleu, has novel structural characteristics, suggesting the existence of new mechanisms acting in the activation of transcription.

Plant Metabolomics: A Characterisation of Plant Responses to Abiotic Stresses

As with all organisms, plants thrive within a range of environmental conditions that are optimal for their growth and development. They must, however, respond and adapt to conditions that deviate from the optimal, such as low/high temperature, dehydration, high salinity, oxidative stress, heavy metals and nutrient deficiency; these deviations are often responsible for losses in productivity and for spatial (geographical) and temporal (growing season) limitations in the cultivation of crops. Although plants and animals share some responsive mechanisms to unfavourable environmental conditions, plants, as sessile organisms, have developed highly sophisticated and efficient strategies of response. Because of the great interest for both basic and applied research, many scientific endeavours have long addressed the understanding of the mechanisms underlying the stress response and the identification of the specific genes/metabolites that are responsible for tolerance phenotypes. In recent years, the “omics” approaches have allowed high-throughput analyses of the changes that are induced by environmental stresses, confirming data previously obtained with targeted analysis and extending the scope of investigation. It is noteworthy that the metabolomic changes that have been observed in plants subjected to stress conditions depend on different causes; therefore, they have different significance and are expected to differently correlate with tolerance/sensitivity phenotypes. Namely, changes in the metabolome composition due to adverse environmental conditions may depend on i) the stability and catalytic activity of enzymes involved in the production/degradation of specific metabolites, ii) the production of abnormal compounds (or abnormal concentrations of normal compounds) as a result of cell damage, iii) the adjustment of concentration of some metabolites to restore homeostasis and normal metabolic fluxes and iv) the synthesis and/or accumulation of compounds involved in mediating tolerance mechanisms. The main goal of studying metabolic changes during stress responses is to identify metabolites belonging to the (iii) and (iv) groups that are responsible for stress tolerance. Upon exposure to osmotic stress as a result of low temperature, drought and high salinity, plants accumulate a range of osmolytes with the primary function of turgor maintenance.

The rice Mybleu transcription factor increases tolerance to oxygen deprivation in Arabidopsis plants

Mybleu is a natural incomplete transcription factor of rice (Oryza sativa), consisting of a partial Myb repeat followed by a short leucine zipper. We previously showed its localization to the apical region of rice roots and coleoptiles. Specifically, in coleoptiles, Mybleu is expressed under both aerobic and anaerobic conditions, whereas in roots, it is expressed only under aerobic conditions. Mybleu is able to dimerize with canonical leucine zippers and to activate transcription selectively. To investigate Mybleu function in vivo, we transformed Arabidopsis thaliana and evaluated several morphological, physiological and biochemical parameters. In agreement with a hypothesized role of Mybleu in cell elongation in the differentiation zone, we found that the constitutive expression of this transcription factor in Arabidopsis induced elongation in the primary roots and in the internodal region of the floral stem; we also observed a modification of the root apex morphology in transformed lines. Based on the high expression of Mybleu in anaerobic rice coleoptiles, we studied the role of this transcription factor in transgenic plants grown under low-oxygen conditions. We found that overexpression of this transcription factor increased tolerance to oxygen deficit. In transgenic plants, this effect may depend both on the maintenance of a higher metabolism during stress and on the higher expression levels of certain genes involved in the anaerobic response.

Hd3a, RFT1 and Ehd1 integrate photoperiodic and drought stress signals to delay the floral transition in rice

Plants show a high degree of developmental plasticity in response to external cues, including day length and environmental stress. Water scarcity in particular can interfere with photoperiodic flowering, resulting in the acceleration of the switch to reproductive growth in several species, a process called drought escape. However, other strategies are possible and drought stress can also delay flowering, albeit the underlying mechanisms have never been addressed at the molecular level. We investigated these interactions in rice, a short day species in which drought stress delays flowering. A protocol that allows the synchronization of drought with the floral transition was set up to profile the transcriptome of leaves subjected to stress under distinct photoperiods. We identified clusters of genes that responded to drought differently depending on day length. Exposure to drought stress under floral-inductive photoperiods strongly reduced transcription of EARLY HEADING DATE 1 (Ehd1), HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1), primary integrators of day length signals, providing a molecular connection between stress and the photoperiodic pathway. However, phenotypic and transcriptional analyses suggested that OsGIGANTEA (OsGI) does not integrate drought and photoperiodic signals as in Arabidopsis, highlighting molecular differences between long and short day model species.

Analysis of transcript and metabolite levels in Italian rice (Oryza sativa L.) cultivars subjected to osmotic stress or benzothiadiazole treatment

One of the major objectives of rice (Oryza sativa L.) breeding programs is the development of new varieties with higher tolerance/resistance to both abiotic and biotic stresses. In this study, Italian rice cultivars were subjected to osmotic stress or benzothiadiazole (BTH) treatments. An analysis of the expression of selected genes known to be involved in the stress response and (1)H nuclear magnetic resonance ((1)H NMR) metabolic profiling were combined with multivariate statistical analyses to elucidate potential correlations between gene expression or metabolite content and observed tolerant/resistant phenotypes. We observed that the expression of three chosen genes (two WRKY genes and one peroxidase encoding gene) differed between susceptible and resistant cultivars in response to BTH treatments. Moreover, the analysis of metabolite content, in particular in the osmotic stress experiment, enabled discrimination between selected cultivars based on differences in the accumulation of some primary metabolites, primarily sugars. This research highlights the potential usefulness of this approach to characterise rice varieties based on transcriptional or metabolic changes due to adverse environmental conditions.

Ectopic expression of a rice transcription factor, Mybleu, enhances tolerance of transgenic plants of Carrizo citrange to low oxygen stress

Oxygen deficit, which occurs in flooded or poorly drained soils, can limit plant growth and development. Low-oxygen environmental conditions also limit the distribution of many woody plants, such as citrus trees, which are considered flood-sensitive crops, although tolerance to this stress varies among genotypes and rootstocks. In this study, the rice transcription factor Mybleu was inserted into the pGA470 plant cloning vector and transferred into the epicotyl explants of the Carrizo citrange rootstock (Citrus sinensis × Poncirus trifoliata) using Agrobacterium tumefaciens-mediated transformation. The transgenic lines were confirmed for the presence and expression of the transgene, and physiological, biochemical and molecular parameters were evaluated for adaptation to hypoxic and anoxic stress conditions. The ectopic expression of Mybleu increased tolerance to oxygen deprivation in the transgenic lines, contributing to increased viability under this stress condition. This improved tolerance correlates with, and may depend on, the induction of genes and the activation of enzymes from various fermentation and carbohydrate metabolic pathways, antioxidant systems and nonsymbiotic haemoglobin-nitric oxide homeostasis mechanisms. Together, our data suggest a key role for Mybleu in coordinating the multifaceted plant response to low oxygen stress and the conservation of Mybleu-regulated pathways among species.