Identification and expression analysis of WRKY gene family in eukaryotic algae / 生物工程学报

WRKY is a superfamily of plant-specific transcription factors, playing a critical regulatory role in multiple biological processes such as plant growth and development, metabolism, and responses to biotic and abiotic stresses. Although WRKY genes have been characterized in a variety of higher plants, little is known about them in eukaryotic algae, which are close to higher plants in evolution. To fully characterize algal WRKY family members, we carried out multiple sequence alignment, phylogenetic analysis, and conserved domain prediction to identify the WRKY genes in the genomes of 30 algal species. A total of 24 WRKY members were identified in Chlorophyta, whereas no WRKY member was detected in Rhodophyta, Glaucophyta, or Bacillariophyta. The 24 WRKY members were classified into Ⅰ, Ⅱa, Ⅱb and R groups, with a conserved heptapeptide domain WRKYGQ(E/A/H/N)K and a zinc finger motif C-X4-5-C-X22-23-H-X-H. Haematococcus pluvialis, a high producer of natural astaxanthin, contained two WRKY members (HaeWRKY-1 and HaeWRKY-2). Furthermore, the coding sequences of HaeWRKY-1 and HaeWRKY-2 genes were cloned and then inserted into prokaryotic expression vector. The recombinant vectors were induced to express in Escherichia coli BL21(DE3) cells and the fusion proteins were purified by Ni-NTA affinity chromatography. HaeWRKY-1 had significantly higher expression level than HaeWRKY-2 in H. pluvialis cultured under normal conditions. High light stress significantly up-regulated the expression of HaeWRKY-1 while down-regulated that of HaeWRKY-2. The promoters of HaeWRKY genes contained multiple cis-elements responsive to light, ethylene, ABA, and stresses. Particularly, the promoter of HaeWRKY-2 contained no W-box specific for WRKY binding. However, the W-box was detected in the promoters of HaeWRKY-1 and the key enzyme genes HaeBKT (β-carotene ketolase) and HaePSY (phytoene synthase) responsible for astaxanthin biosynthesis. Considering these findings and the research progress in the related fields, we hypothesized that the low expression of HaeWRKY-2 under high light stress may lead to the up-regulation of HaeWRKY-1 expression. HaeWRKY-1 may then up-regulate the expression of the key genes (HaeBKT, HaePSY, etc.) for astaxanthin biosynthesis, consequently promoting astaxanthin enrichment in algal cells. The findings provide new insights into further analysis of the regulatory mechanism of astaxanthin biosynthesis and high light stress response of H. pluvialis.

Alteration in the acceptor side of photosystem II of chloroplast by high light.

The effect of high light on the acceptor side of photosystem II of chloroplasts and core particles of spinach was studied. Both Vmax and apparent Km for DCIP were altered in photoinhibited photosystem II core particles. The double reciprocal plot analysis as a function of actinic light showed increased slope in chloroplasts photoinhibited in the presence of DCMU. Exposure of chloroplasts to high light in the presence of DCMU did not protect the chloroplast against high light induced decrease in Fm, level. Further the high light stress induced decrease in Fm level was not restored by the addition of DCMU. These results suggest that the high light stress induced damage to chloroplast involves alteration in the binding site for QB on the DI protein on the acceptor side of photosystem II.