The production of cereal plants with enhanced tolerance to drought stress

Resumen

Polyamines are ubiquitous, nitrogen-rich compounds implicated in many stress responses in different organisms. Putrescine, spermidine and spermine are the most common polyamines and all are derived from arginine. In plants, putrescine is synthesized via two pathways, one in which arginine is converted into putrescine via the intermediate agmatine through the activity of arginine decarboxylase (ADC), and through a second pathway in which arginine is converted first to ornithine and subsequently to putrescine through the activity of ornithine decarboxylase (ODC). Putrescine is further converted into spermidine and then spermine by the enzymes spermidine synthase (SPDS) and spermine synthase (SPMS), respectively. These reactions require aminopropyl groups which are supplied from S-adenosylmethionine (SAM) by SAM decarboxylase (SAMDC). We generated transgenic rice plants constitutively expressing the Samdc gene from Datura stramonium (Ds) in order to dissect the roles of putrescine from the higher polyamines spermidine and spermine in the drought stress tolerance response. These plants exhibited drought-induced symptoms typical of wild type plants under stress. However a much more robust recovery on return to normal conditions, compared to wild type plants. In transgenic plants, there was no spike in putrescine but a smooth increase in spermine levels at the expense of spermidine. These results confirm and extend the threshold model for polyamine activity in drought stress, and attribute individual roles to the three polyamines individually. In other to understand how the corresponding rice Adc homologues might influence the drought related response we cloned and characterize the rice Adc genes. Rice (Oryza sativa; Os) has a small family of Adc genes and OsAdc1 expression has been shown to fluctuate under drought and chilling stress. We identified and characterized a second rice Adc gene (OsAdc2) which encodes a 629-amino-acid protein with a predicted molecular mass of 67 kDa. Sequence comparisons showed that OsAdc2 is more closely related to the oat (Avena sativa; As) Adc gene than to OsAdc1 or its dicot homologs. mRNA analysis demonstrated that the two rice Adc genes are regulated differently; whereas OsAdc1 is expressed in leaves, roots and stems, OsAdc2 gene expression is restricted to the stem. Protein expression was investigated with specific antibodies for ADC1 and ADC2, corroborating the mRNA data. In rice, several stress-induced genes are found to be strongly inducible by wounding, methyl jasmonate (MeJa) or pathogen derived signals. However, even though polyamines play an essential role in wound healing responses in animals, not much experimental evidence has been reported for the involvement of polyamines in the plant wound response. I investigated the effect of MeJa on the expression of the OsAdc1 gene and polyamine metabolism in wild type and transgenic rice plants expressing the DsAdc cDNA. Transgenic plants accumulated two- to three-fold higher levels of putrescine over wild type and exhibited a normal phenotype. Exogenous application of MeJa negatively regulated expression of the endogenous Adc gene as well as the introduced DsAdc transgene, at the mRNA level. The effect was more pronounced in the expression of the endogenous OsAdc1 homolog. The free polyamine content was reduced significantly in leaves in wild type and transgenic plants, following MeJa application. We discuss our findings in mechanistic terms consistent with the presence of a jasmonate responsive region in the Adc1 promoter (endogenous and heterologous). The identification of transposon or T-DNA tagged mutants is a useful tool to investigate the phenotypic consequences of the perturbed pathway. Such experiments will aid in clarifying the biological function of polyamines further. In a lethal embryo (lem2) mutant identified from a collection of different maize (Zea mays; Zm) lines, the Ac (Activator) transposon tagged a copy of the Adc gene. We report the identification and isolation of the maize ZmAdc1 gene and a second maize Adc gene (ZmAdc2), homologous to rice OsAdc2 and AsAdc. mRNA analysis showed differential profile expression of ZmAdc1 in leaves, coleoptiles, roots and seeds of maize plants. We introduced the oat Adc cDNA into maize under the control of the constitutive maize ubiquitin 1 promoter to carry out complementation analysis with the Ac mutant. A specific lineage will be selected to carry out cross-pollinations with the mutant line to investigate the possibility of restoring the initial phenotype.