Immacolata Coraggio

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.

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.

Nitrate assimilation during the anaerobic germination of rice: expression of ferredoxin-dependent glutamate synthase

Ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) is the last enzyme involved in the pathway of nitrate assimilation in higher plants. This paper describes the synthesis and expression of the enzyme in anaerobic coleoptiles of rice (Oryza sativa L.) and its regulation by exogenous nitrate. The activity of Fd-GOGAT was strongly inhibited by cycloheximide between 4 and 9 d of anaerobic germination. The addition of nitrate slightly increased, in the first 5 h, the specific activity of Fd-GOGAT as well as the amount of a 160-kDa protein specifically immunoprecipitated with anti-Fd-GOGAT serum. Northern blot analysis, performed with a specific riboprobe, showed the presence of mRNA of the expected size and the inductive effect of nitrate. The role of Fd-GOGAT is discussed in relation to the anaerobic assimilation of nitrate by rice coleoptiles.

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.

Early Phenylpropanoid Biosynthetic Steps in Cannabis sativa: Link between Genes and Metabolites

Phenylalanine ammonia-lyase (PAL), Cinnamic acid 4-hydroxylase (C4H) and 4-Coumarate: CoA ligase (4CL) catalyze the first three steps of the general phenylpropanoid pathway whereas chalcone synthase (CHS) catalyzes the first specific step towards flavonoids production. This class of specialized metabolites has a wide range of biological functions in plant development and defence and a broad spectrum of therapeutic activities for human health. In this study, we report the isolation of hemp PAL and 4CL cDNA and genomic clones. Through in silico analysis of their deduced amino acid sequences, more than an 80% identity with homologues genes of other plants was shown and phylogenetic relationships were highlighted. Quantitative expression analysis of the four above mentioned genes, PAL and 4CL enzymatic activities, lignin content and NMR metabolite fingerprinting in different Cannabis sativa tissues were evaluated. Furthermore, the use of different substrates to assay PAL and 4CL enzymatic activities indicated that different isoforms were active in different tissues. The diversity in secondary metabolites content observed in leaves (mainly flavonoids) and roots (mainly lignin) was discussed in relation to gene expression and enzymatic activities data.

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.

Post-Transcriptional Regulation by Anoxia of a myb Gene in Rice (Oryza sativa)

We isolated a myb coding cDNA expressed in anaerobic rice coleoptiles. Sequence analysis revealed some peculiar features, suggesting the presence of post-transcriptional regulation events: an upstream ORF (uORF) in the long leader sequence, an unspliced intron and the presence of a short putative leucine zipper in the ORF encoded by the unspliced RNA. Analyses of mRNA in rice roots and coleoptiles by RT-PCR demonstrated that both RNA forms, spliced and unspliced, are present in vivo and that anaerobiosis strongly induced the presence of spliced transcript in roots. Northern blot analysis indicated that in rice roots anoxia affects expression of a set of genes belonging to the myb family. Moreover, we demonstrated the negative influence of the myb7 uORF on the expression of the downstream ORF, and that the inhibition was depended, at least in part, on the uORF-AUG.