Role of WRINKLED1 in the transcriptional regulation of glycolytic and fatty acid biosynthetic genes in Arabidopsis.

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.

Study of AtSUS2 localization in seeds reveals a strong association with plastids.

Sucrose synthase (SUS) is a key enzyme in sucrose metabolism. This enzyme catalyzes the reversible conversion of sucrose and UDP to UDP-glucose and fructose. In the Arabidopsis SUS gene family (six members), SUS2 is strongly and specifically expressed in Arabidopsis seeds during the maturation phase. Using specific antibodies, we have shown that SUS2 is localized in the embryo, endosperm and seed coat with differential patterns. During the maturation phase, the SUS2 protein seems to be mainly co-localized with plastids in the embryo. This novel finding is discussed in relation to the role of this enzyme in storage organs.

Function of plastidial pyruvate kinases in seeds of Arabidopsis thaliana.

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.

Transcriptome profiling uncovers metabolic and regulatory processes occurring during the transition from desiccation-sensitive to desiccation-tolerant stages in Medicago truncatula seeds.

To 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-mm-long 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.

The AtSUC5 sucrose transporter specifically expressed in the endosperm is involved in early seed development in Arabidopsis.

The sucrose transporter gene AtSUC5 was studied as part of a programme aimed at identifying and studying the genes involved in seed maturation in Arabidopsis. Expression profiling of AtSUC5 using the technique of real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) showed that the gene was specifically and highly induced during seed development between 4 and 9 days after flowering (DAF). Analysis of the activity of the AtSUC5 promoter in planta was consistent with this timing, and suggested that AtSUC5 expression is endosperm specific, spreading from the micropylar to the chalazal pole of the filial tissue. To demonstrate the function of AtSUC5, the corresponding cDNA was used to complement a sucrose uptake-deficient yeast mutant, thus confirming its sucrose transport capacity. To investigate the function in planta, three allelic mutants disrupted in the AtSUC5 gene were isolated and characterized. A strong but transient reduction in fatty acid concentration was observed in mutant seeds 8 DAF. This biochemical phenotype was associated with a slight delay in embryo development. Taken together, these data demonstrated the role of the AtSUC5 carrier in the nutrition of the filial tissues during early seed development. However, additional sugar uptake systems, which remain to be characterized, must be functional in developing seeds, especially during maturation of the embryo.

Multifunctional acetyl-CoA carboxylase 1 is essential for very long chain fatty acid elongation and embryo development in Arabidopsis.

Acetyl-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.

Characterization of a tissue-specific and developmentally regulated beta-1,3-glucanase gene in pea (Pisum sativum).

As part of a search for seed coat-specific expressed genes in Pisum sativum cv. Finale by PCR-based methods, we identified and isolated a cDNA encoding a beta- 1,3-glucanase, designated PsGNS2. The deduced peptide sequence of PsGNS2 is similar to a subfamily of beta-1,3-glucanases, which is characterized by the presence of a long amino acid extension at the C-terminal end compared to the other beta-1,3-glucanases. PsGNS2 is expressed in young flowers and in the seed coat and is weakly expressed in vegetative tissues (roots and stems) during seedling development. It is not inducible by environmental stress or in response to fungal infection. In developing pea flowers the transcript is detectable in all four whirls. In the seed coat the expression is temporally and spatially regulated. High abundance of the transcript became visible in the seed coat when the embryo reached the late heart stage and remained until the mid seed-filling stage. In situ hybridization data demonstrated that the expression of PsGNS2 is restricted to a strip of the inner parenchyma tissue of the seed coat, which is involved in temporary starch accumulation and embryo nutrition. This tissue showed also less callose deposits than the other ones. The 5' genomic region of PsGNS2 was isolated and promoter activity studies in transgenic Medicago truncatula showed a seed-specific expression. Highest activity of the promoter was found in the seed coat and in the endosperm part of the seed.