A Rapid Method for the Determination of Fucoxanthin in Diatom.

Fucoxanthin is a natural pigment found in microalgae, especially diatoms and Chrysophyta. Recently, it has been shown to have anti-inflammatory, anti-tumor, and anti-obesityactivity in humans. Phaeodactylum tricornutum is a diatom with high economic potential due to its high content of fucoxanthin and eicosapentaenoic acid. In order to improve fucoxanthin production, physical and chemical mutagenesis could be applied to generate mutants. An accurate and rapid method to assess the fucoxanthin content is a prerequisite for a high-throughput screen of mutants. In this work, the content of fucoxanthin in P. tricornutum was determined using spectrophotometry instead of high performance liquid chromatography (HPLC). This spectrophotometric method is easier and faster than liquid chromatography and the standard error was less than 5% when compared to the HPLC results. Also, this method can be applied to other diatoms, with standard errors of 3-14.6%. It provides a high throughput screening method for microalgae strains producing fucoxanthin.

A pollen-specific RALF from tomato that regulates pollen tube elongation.

Rapid Alkalinization Factors (RALFs) are plant peptides that rapidly increase the pH of plant suspension cell culture medium and inhibit root growth. A pollen-specific tomato (Solanum lycopersicum) RALF (SlPRALF) has been identified. The SlPRALF gene encodes a preproprotein that appears to be processed and released from the pollen tube as an active peptide. A synthetic SlPRALF peptide based on the putative active peptide did not affect pollen hydration or viability but inhibited the elongation of normal pollen tubes in an in vitro growth system. Inhibitory effects of SlPRALF were detectable at concentrations as low as 10 nm, and complete inhibition was observed at 1 mum peptide. At least 10-fold higher levels of alkSlPRALF, which lacks disulfide bonds, were required to see similar effects. A greater effect of peptide was observed in low-pH-buffered medium. Inhibition of pollen tube elongation was reversible if peptide was removed within 15 min of exposure. Addition of 100 nm SlPRALF to actively growing pollen tubes inhibited further elongation until tubes were 40 to 60 mum in length, after which pollen tubes became resistant to the peptide. The onset of resistance correlated with the timing of the exit of the male germ unit from the pollen grain into the tube. Thus, exogenous SlPRALF acts as a negative regulator of pollen tube elongation within a specific developmental window.

The maize O2 and PBF proteins act additively to promote transcription from storage protein gene promoters in rice endosperm cells.

A transient expression assay system was employed to investigate the possible use of the maize Opaque 2 (O2) and prolamin box binding factor (PBF) proteins as transcriptional activators of rice and wheat storage protein gene promoters. When assayed in developing rice endosperm cells, either O2 or PBF alone could increase transcription from the promoter of the rice glutelin gene, Gt1. However, mutant forms of O2 and PBF that are defective in DNA binding could not. Co-transfection with both transcriptional activators resulted in an additive increase in transactivation of the Gt1 promoter. Co-bombardment of a Gt1::GUS construct with plasmids expressing the DNA binding domains of O2 and PBF in antisense orientation resulted in a decrease of GUS expression below background levels. Similar stimulatory and additive effects of O2 and PBF could be observed on the promoters from other storage protein genes including rice globulin (Glb), prolamins (RP6 and PG5a) and a wheat glutenin (Bx7). However, responsiveness of the promoters from non-storage protein genes like rice actin and CaMV 35S to O2 and PBF was insignificant. Our results indicate that the maize O2 and PBF proteins can act singly or additively as effective stimulators of heterologous storage protein promoters in developing rice endosperm cells. These data support the use of well-characterized transcription factors from maize as an effective means of increasing the expression level of recombinant proteins in developing rice seeds.

Whole-genome comparison of leucine-rich repeat extensins in Arabidopsis and rice. A conserved family of cell wall proteins form a vegetative and a reproductive clade.

We have searched the Arabidopsis and rice (Oryza sativa) genomes for homologs of LRX1, an Arabidopsis gene encoding a novel type of cell wall protein containing a leucine-rich repeat (LRR) and an extensin domain. Eleven and eight LRX (LRR/EXTENSIN) genes have been identified in these two plant species, respectively. The LRX gene family encodes proteins characterized by a short N-terminal domain, a domain with 10 LRRs, a cysteine-rich motif, and a variable C-terminal extensin-like domain. Phylogenetic analysis performed on the conserved domains indicates the existence of two major clades of LRX proteins that arose before the eudicot/monocot divergence and then diversified independently in each lineage. In Arabidopsis, gene expression studies by northern hybridization and promoter::uidA fusions showed that the two phylogenetic clades represent a specialization into "reproductive" and "vegetative" LRXs. The four Arabidopsis genes of the "reproductive" clade are specifically expressed in pollen, whereas the seven "vegetative" genes are predominantly expressed in various sporophytic tissues. This separation into two expression classes is also supported by previous studies on maize (Zea mays) and tomato (Lycopersicon esculentum) LRX homologs and by information on available rice ESTs. The strong conservation of the amino acids responsible for the putative recognition specificity of the LRR domain throughout the family suggests that the LRX proteins interact with similar ligands.