PYK10 myrosinase reveals a functional coordination between endoplasmic reticulum bodies and glucosinolates in Arabidopsis thaliana.

The endoplasmic reticulum body (ER body) is an organelle derived from the ER that occurs in only three families of the order Brassicales and is suggested to be involved in plant defense. ER bodies in Arabidopsis thaliana contain large amounts of ß-glucosidases, but the physiological functions of ER bodies and these enzymes remain largely unclear. Here we show that PYK10, the most abundant ß-glucosidase in A. thaliana root ER bodies, hydrolyzes indole glucosinolates (IGs) in addition to the previously reported in vitro substrate scopolin. We found a striking co-expression between ER body-related genes (including PYK10), glucosinolate biosynthetic genes and the genes for so-called specifier proteins affecting the terminal products of myrosinase-mediated glucosinolate metabolism, indicating that these systems have been integrated into a common transcriptional network. Consistent with this, comparative metabolite profiling utilizing a number of A. thaliana relatives within Brassicaceae identified a clear phylogenetic co-occurrence between ER bodies and IGs, but not between ER bodies and scopolin. Collectively, our findings suggest a functional link between ER bodies and glucosinolate metabolism in planta. In addition, in silico three-dimensional modeling, combined with phylogenomic analysis, suggests that PYK10 represents a clade of 16 myrosinases that arose independently from the other well-documented class of six thioglucoside glucohydrolases. These findings provide deeper insights into how glucosinolates are metabolized in cruciferous plants and reveal variation of the myrosinase-glucosinolate system within individual plants.

A stable and efficient nuclear transformation system for the diatom Chaetoceros gracilis.

Chaetoceros gracilis belongs to the centric diatoms, and has recently been used in basic research on photosynthesis. In addition, it has been commercially used in fisheries and is also attracting interest as a feedstock for biofuels production and biorefinery. In this study, we developed an efficient genetic transformation system for C. gracilis. The diatom cells were transformed via multi-pulse electroporation using plasmids containing various promoters to drive expression of the nourseothricin acetyltransferase gene (nat) as a selectable marker. The transformation efficiency reached ~400 positive transgenic clones per 10(8) recipient cells, which is the first example of successful transformation with electroporation in a centric diatom species. We further produced two expression vectors: the vector pCgLhcr5p contains the light-dependent promoter of a fucoxanthin chlorophyll a/c binding protein gene and the vector pCgNRp contains the inducible promoter of a nitrate reductase gene to drive the expression of introduced genes. In both vectors, an acetyl-CoA acetyltransferase promoter drives nat gene expression for antibiotic selection. Stable integration and expression of reporter genes, such as the firefly luciferase and green fluorescent protein Azami-Green genes, were observed in transformed C. gracilis cells. This efficient and stable transformation system for C. gracilis will enable both functional analysis of diatom-specific genes and strain improvement for further biotechnological applications.

Highly efficient transformation of the diatom Phaeodactylum tricornutum by multi-pulse electroporation.

A highly efficient nuclear transformation method was established for the pennate diatom Phaeodactylum tricornutum using an electroporation system that drives multi-sequence pulses to introduce foreign DNAs into the cells. By optimizing pulse conditions, the diatom cells can be transformed without removing rigid silica-based cell walls, and high transformation efficiency (about 4,500 per 10(8) cells) is achieved.

Quantitative analysis of ER body morphology in an Arabidopsis mutant.

Although fluorescence microscopy screening has proven useful in the identification of genes involved in plant organelle biogenesis and integrity, the quantitative and statistical study of the geometric phenotype is highly limited. This situation could generate unconscious bias in the understanding and presentation of a mutant phenotype. Therefore, we have developed an automated quantification system for green fluorescent protein (GFP) images, which enabled us to easily obtain quantitative data on ER bodies (an endoplasmic reticulum-derived organelle). We isolated an ER body morphology mutant of Arabidopsis thaliana, leb-1 (long ER body). The leb-1 mutant had significantly fewer and larger ER bodies than the wild-type. An amino acid substitution of Cys29 with tyrosine (C29Y) on PYK10, a major component protein of ER bodies, was found in leb-1. Non-reducing SDS-PAGE revealed that the electrophoretic mobility of PYK10 in the leb-1 mutant was clearly different from that in the wild type. This difference suggests that the C29Y amino acid substitution caused a tertiary structural change of the PYK10 protein. While the bglu21-1 and pyk10-1 single mutations slightly affected the number and morphology of the ER bodies, a bglu21-1 pyk10-1 double mutant had fewer and larger ER bodies than the wild type. The quantitative ER body phenotypes of leb-1 were similar to those of bglu21-1 pyk10-1 and bglu21-1 leb-1, suggesting that the leb-1 mutation allele acts dominantly to the BGLU21 wild-type allele. The leb-1 type PYK10 protein, which has an abnormal structure, may competitively inhibit interactions between the wild-type BGLU21/PYK10 protein and an unknown partner.