The WRINKLED1 (WRI1) protein is an important regulator of oil accumulation in maturing Arabidopsis seeds. WRI1 is a member of a plant-specific family of transcription factors (AP2/EREBP) that share either one or two copies of a DNA-binding domain called the AP2 domain. Here, it is shown that WRI1 acts as a transcriptional enhancer of genes involved in carbon metabolism in transgenic seeds overexpressing this transcription factor. PKp-beta1 and BCCP2, two genes encoding enzymes of the glycolysis and fatty acid biosynthetic pathway, respectively, have been chosen to investigate the regulatory action exerted by WRI1 over these pathways. Using the reporter gene uidA, it was possible to demonstrate in planta that WRI1 regulates the activity of both PKp-beta1 and BCCP2 promoters. Electrophoretic mobility-shift assays and yeast one-hybrid experiments showed that WRI1 was able to interact with the BCCP2 promoter. To further elucidate the regulatory mechanism controlling the transcription of these genes, functional dissections of PKp-beta1 and BCCP2 promoters were performed. Two enhancers, of 54 and 79 bp, respectively, have thus been isolated that are essential to direct the activity of these promoters in oil-accumulating tissues of the embryo. A consensus site is present in these enhancers as well as in other putative target promoters of WRI1. Loss of this consensus sequence in the BCPP2 promoter decreases both the strength of the interaction between WRI1 and this promoter in yeast and the activity of the promoter in planta.

SummaryAcetyl-CoA carboxylase (ACCase) catalyses the carboxylation of acetyl-CoA, forming malonyl-CoA, which is used in the plastid for fatty acid synthesis and in the cytosol in various biosynthetic pathways including fatty acid elongation. In Arabidopsis thaliana, ACC1 and ACC2, two genes located in a tandem repeat within a 25-kbp genomic region near the centromere of chromosome 1, encode two multifunctional ACCase isoforms. Both genes, ACC1 and ACC2, appear to be ubiquitously expressed, but little is known about their respective function and importance. Here, we report the isolation and characterisation of two allelic mutants disrupted in the ACC1 gene. Both acc1-1 and acc1-2 mutations are recessive and embryo lethal. Embryo morphogenesis is impaired and both alleles lead to cucumber-like structures lacking in cotyledons, while the shortened hypocotyl and root exhibit a normal radial pattern organisation of the body axis. In this abnormal embryo, the maturation process still occurs. Storage proteins accumulate normally, while triacylglycerides (TAG) are synthesised at a lower concentration than in the wild-type seed. However, these TAG are totally devoid of very long chain fatty acids (VLCFA) and consequently enriched in C18:1, like all lipid fractions analysed in the mutant seed. These data demonstrate, in planta, the role of ACCase 1 in VLCFA elongation. Furthermore, this multifunctional enzyme also plays an unexpected and central function in embryo morphogenesis, especially in apical meristem development.

Summary Pyruvate kinase (PK) catalyses the irreversible synthesis of pyruvate and ATP, which are both used in multiple biochemical pathways. These compounds are essential for sustained fatty acid production in the plastids of maturing Arabidopsis embryos. Using a real‐time quantitative reverse transcriptase (RT)‐PCR approach, the three genes encoding putative plastidial PKs (PKps) in Arabidopsis, namely PKp1 (At3g22960), PKp2 (At5g52920) and PKp3 (At1g32440), were shown to be ubiquitously expressed. However, only PKp1 and PKp2 exhibited significant expression in maturing seeds. The activity of PKp1 and PKp2 promoters was consistent with this pattern, and the study of the PKp1:GFP and PKp2:GFP fusion proteins confirmed the plastidial localization of these enzymes. To further investigate the function of these two PKp isoforms in seeds comprehensive functional analyses were carried out, including the cytological, biochemical and molecular characterization of two pkp1 and two pkp2 alleles, together with a pkp1pkp2 double mutant. The results obtained outlined the importance of these PKps for fatty acid synthesis and embryo development. Mutant seeds were depleted of oil, their fatty acid content was drastically modified, embryo elongation was retarded and, finally, seed germination was also affected. Together, these results provide interesting insights concerning the carbon fluxes leading to oil synthesis in maturing Arabidopsis seeds. The regulation of this metabolic network by the WRINKLED1 transcription factor is discussed, and emphasizes the role of plastidial metabolism and the importance of its tight regulation.

SummaryTo investigate regulatory processes and protective mechanisms leading to desiccation tolerance (DT) in seeds, 16086-element microarrays were used to monitor changes in the transcriptome of desiccation-sensitive 3-mmlong radicles of Medicago truncatula seeds at different time points during incubation in a polyethylene glycol (PEG) solution at )1.7 MPa, resulting in a gradual re-establishment of DT. Gene profiling was also performed on embryos before and after the acquisition of DT during maturation. More than 1300 genes were differentially expressed during the PEG incubation. A large number of genes involved in C metabolism are expressed during the re-establishment of DT. Quantification of C reserves confirms that lipids, starch and oligosaccharides were mobilised, coinciding with the production of sucrose during the early osmotic adjustment. Several clusters of gene profiles were identified with different time-scales. Genes expressed early during the PEG incubation belonged to classes involved in early stress and adaptation responses. Interestingly, several regulatory genes typically expressed during abiotic/drought stresses were also upregulated during maturation, arguing for the partial overlap of ABA-dependent and -independent regulatory pathways involved in both drought and DT. At later time points, in parallel to the re-establishment of DT, upregulated genes are comparable with those involved in late seed maturation. Concomitantly, a massive repression of genes belonging to numerous classes occurred, including cell cycle, biogenesis, primary and energy metabolism. The re-establishment of DT in the germinated radicles appears to concur with a partial return to the quiescent state prior to germination.