Expression of Chlamydomonas reinhardtii chloroplast diacylglycerol acyltransferase 3 is induced by light in concert with triacylglycerol accumulation.

Considerable progress has been made towards the understanding of triacylglycerol (TAG) accumulation in algae. One key aspect is finding conditions that trigger TAG production without reducing cell division. Previously, we identified a soluble diacylglycerol acyltransferase (DGAT), related to plant DGAT3, with heterologous DGAT activity. In this work, we demonstrate that Chlamydomonas reinhardtii DGAT3 localizes to the chloroplast and that its expression is induced by light, in correspondence with TAG accumulation. Dgat3 mRNAs and TAGs increase in both wild-type and starch-deficient cells grown with acetate upon transferring them from dark or low light to higher light levels, albeit affected by the particularities of each strain. The response of dgat3 mRNAs and TAGs to light depends on the pre-existing levels of TAGs, suggesting the existence of a negative regulatory loop in the synthesis pathway, although an effect of TAG turnover cannot be ruled out. Altogether, these results hint towards a possible role of DGAT3 in light-dependent TAG accumulation in C. reinhardtii.

Signs of a phyllospheric lifestyle in the genome of the stress-tolerant strain Azospirillum brasilense Az19.

Azospirillum brasilense Az19 is a plant-beneficial bacterium capable of protecting plants from the negative effects of drought. The objective of this study was to determine and analyze the genomic sequence of strain Az19 as a means of identifying putative stress-adaptation mechanisms. A high-quality draft genome of ca. 7 Mb with a predicted coding potential of 6710 genes was obtained. Phylogenomic analyses confirmed that Az19 belongs to the brasilense clade and is closely related to strains Az39 and REC3. Functional genomics revealed that the denitrification pathway of Az19 is incomplete, which was in agreement with a reduced growth on nitrate under low O2 concentrations. Putative genes of the general stress response and oxidative stress-tolerance, as well as synthesis of exopolysaccharides, carotenoids, polyamines and several osmolytes, were detected. An additional poly-beta-hydroxybutyrate (PHB) synthase coding gene was found in Az19 genome, but the accumulation of PHB did not increase under salinity. The detection of exclusive genes related to DNA repair led to discover that strain Az19 also has improved UV-tolerance, both in vitro and in planta. Finally, the analysis revealed the presence of multiple kaiC-like genes, which could be involved in stress-tolerance and, possibly, light responsiveness. Although A. brasilense has been a model for the study of beneficial plant-associated rhizobacteria, the evidence collected in this current study suggests, for the first time in this bacterial group, an unexpected possibility of adaptation to the phyllosphere.

Interspecific cooperation: enhanced growth, attachment and strain-specific distribution in biofilms through Azospirillum brasilense-Pseudomonas protegens co-cultivation.

Plant-growth-promoting bacteria belonging to Azospirillum and Pseudomonas genera are major inhabitants of the rhizosphere. Both are increasingly commercialized as crops inoculants. Interspecific interaction in the rhizosphere is critical for inoculants aptness. The objective of this work was to evaluate Azospirillum and Pseudomonas interaction in mixed biofilms by co-cultivation of the model strains Azospirillum brasilense Sp245 and Pseudomonas protegens CHA0. The results revealed enhanced growth of both strains when co-cultured in static conditions. Moreover, Sp245 biofilm formed in plastic surfaces was increased 2-fold in the presence of CHA0. Confocal microscopy revealed highly structured mixed biofilms showing Sp245 mainly on the bottom and CHA0 towards the biofilm surface. In addition, A. brasilense biofilm was thicker and denser when co-cultured with P. protegens. In a colony-colony interaction assay, Sp245 changed nearby CHA0 producing small colony phenotype, which accounts for a diffusible metabolite mediator; though CHA0 spent medium did not affect Sp245 colony phenotype. Altogether, these results point to a cooperative interaction between A. brasilense Sp245 and P. protegens CHA0 in which both strains increase their static growth and produce structured mixed biofilms with a strain-specific distribution.

On the role of a Lipid-Transfer Protein. Arabidopsis ltp3 mutant is compromised in germination and seedling growth.

Plant Lipid-Transfer Proteins (LTPs) exhibit the ability to reversibly bind/transport lipids in vitro. LTPs have been involved in diverse physiological processes but conclusive evidence on their role has only been presented for a few members, none of them related to seed physiology. Arabidopsis seeds rely on storage oil breakdown to supply carbon skeletons and energy for seedling growth. Here, Arabidopsis ltp3 mutant was analyzed for its ability to germinate and for seedling establishment. Ltp3 showed delayed germination and reduced germination frequency. Seedling growth appeared reduced in the mutant but this growth restriction was rescued by the addition of an exogenous carbon supply, suggesting a defective oil mobilization. Lipid breakdown analysis during seedling growth revealed a differential profile in the mutant compared to the wild type. The involvement of LTP3 in germination and seedling growth and its relationship with the lipid transfer ability of this protein is discussed.

Extracellular sunflower proteins: evidence on non-classical secretion of a jacalin-related lectin.

Extracellular proteins from sunflower seedlings were analyzed by electrophoresis followed by peptide mass fingerprinting. Tentative identification revealed novel proteins for this crop. A significant number of those proteins were not expected to be extracellular because they lacked the typical signal peptide responsible for secretion. In silico analysis showed that some members of this group presented the characteristic disordered structures of certain non-classical and leaderless mammalian secretory proteins. Among these proteins, a putative jacalin-related lectin (Helja) with a mannose binding domain was further isolated from extracellular fluids by mannose-affinity chromatography, thus validating its identification. Besides, immunolocalization assays confirmed its extracellular location. These results showed that a lectin, not predicted to be secreted in strict requirement of the N-terminal signal peptide, occurs in a sunflower extracellular compartment. The implications of this finding are discussed.

Unexpected localization of a lipid transfer protein in germinating sunflower seeds.

Plant lipid transfer proteins (LTPs) are low-molecular-mass proteins whose biological function still remains elusive. They are synthesized with a signal peptide that drives them to the secretory pathway. We have previously described the occurrence of an apoplastic LTP named Ha-AP10, present in sunflower seeds. Using a biochemical approach we now demonstrate that a fraction of Ha-AP10 is perispherically bound to membranes of germinating seeds. Purification of plasma membranes revealed the presence of Ha-AP10 in this fraction. Fluorimmunolocalization studies on germinating sunflower seeds demonstrated that in addition to the apoplastic and plasma membrane localization, Ha-AP10 is also present intracellularlly associated to unidentified structures. This varied distribution of Ha-AP10 in sunflower seeds may give novel clues to understand the role of LTPs in seed physiology.