A Novel Role of Medicago truncatula KNAT3/4/5-like Class 2 KNOX Transcription Factors in Drought Stress Tolerance.

Class 2 KNOX homeobox transcription factors (KNOX2) play a role in promoting cell differentiation in several plant developmental processes. In Arabidopsis, they antagonize the meristematic KNOX1 function during leaf development through the modulation of phytohormones. In Medicago truncatula, three KNOX2 genes belonging to the KNAT3/4/5-like subclass (Mt KNAT3/4/5-like or MtKNOX3-like) redundantly works upstream of a cytokinin-signaling module to control the symbiotic root nodule formation. Their possible role in the response to abiotic stress is as-of-yet unknown. We produced transgenic M. truncatula lines, in which the expression of four MtKNOX3-like genes was knocked down by RNA interference. When tested for response to water withdrawal in the soil, RNAi lines displayed a lower tolerance to drought conditions compared to the control lines, measured as increased leaf water loss, accelerated leaf wilting time, and faster chlorophyll loss. Reanalysis of a transcriptomic M. truncatula drought stress experiment via cluster analysis and gene co-expression networks pointed to a possible role of MtKNOX3-like transcription factors in repressing a proline dehydrogenase gene (MtPDH), specifically at 4 days after water withdrawal. Proline measurement and gene expression analysis of transgenic RNAi plants compared to the controls confirmed the role of KNOX3-like genes in inhibiting proline degradation through the regulation of the MtPDH gene.

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.

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.

The rice Osmyb4 gene enhances tolerance to frost and improves germination under unfavourable conditions in transgenic barley plants.

The Osmyb4 rice gene, coding for a transcription factor, proved to be efficient against different abiotic stresses as a trans(cis)gene in several plant species, although the effectiveness was dependent on the host genomic background. Eight barley transgenic lines carrying the rice Osmyb4 gene under the control of the Arabidopsis cold inducible promoter cor15a were produced to test the efficiency of this gene in barley. After a preliminary test, the best performing lines were subjected to freezing at -11°C and -12°C. Frost tolerance was assessed measured the F(v)/F(m) parameter widely used to indicate the maximum quantum yield of photosystem II photochemistry in the dark adapted state. Three transgenic lines showed significantly increased tolerance. These selected lines were further studied under a complex stress applying cold and hypoxia at germinating stage. In these conditions the three selected transgenic lines outperformed the wild type barley in terms of germination vigour. The transgenic plants also showed a significant modification of their metabolism under cold/hypoxia conditions as demonstrated through the assessment of the activity of key enzymes involved in anoxic stress response. None of the transgenic lines showed dwarfism, just a slight retarded growth. These results provide evidence that the cold dependent expression of Osmyb4 can efficiently improved frost tolerance and germination vigour at low temperature without deleterious effect on plant growth.

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.

Inhibitory effect of myb7 uORF on downstream gene expression in homologous (rice) and heterologous (tobacco) systems.

The rice myb7 mRNA contains in its long leader an upstream open reading frame (uORF) putatively coding for a 40 amino acid peptide. uORFs have been found in the leader of mRNAs encoding transcriptional factors or other proteins involved in cellular growth and development. They are thought to translationally regulate the expression of downstream ORFs. Here, we showed the ability of the myb7 uORF to inhibit the expression of downstream reporter genes both in homologous (rice) and heterologous (tobacco) systems. This effect seems partially related to its translation, as indicated by the comparison with the mutagenized uORF. In both systems most of the inhibitory effect was due to the presence of the intercistronic region, in disagreement with the Kozak model. Moreover, replacing the uORF or the intercistronic region with a different one, we demonstrated that the inhibitory effect strictly depends on their co-presence. Finally, in vitro assays showed that the myb7 uORF is translated and inhibits the downstream ORF translation.

Overexpression of the rice Osmyb4 gene increases chilling and freezing tolerance of Arabidopsis thaliana plants.

The expression of the gene Osmyb4, detected at low level in rice (Oryza sativa) coleoptiles grown for 3 days at 29 degrees C, is strongly induced by treatments at 4 degrees C. At sublethal temperatures of 10 and 15 degrees C, its expression in rice seedlings is already evident, but this effect cannot be vicariated by other stresses or ABA treatment. We demonstrate by transient expression that Myb4 transactivates the PAL2, ScD9 SAD and COR15a cold-inducible promoters. The Osmyb4 function in vivo is demonstrated overexpressing its cDNA in Arabidopsis thaliana plants (ecotype Wassilewskija) under the control of the constitutive CaMV 35S promoter. Myb4 overexpressing plants show a significant increased cold and freezing tolerance, measured as membrane or Photosystem II (PSII) stability and as whole plant tolerance. Finally, in Osmyb4 transgenic plants, the expression of genes participating in different cold-induced pathways is affected, suggesting that Myb4 represents a master switch in cold tolerance.