Identification of Respiratory Burst Oxidase Homolog (Rboh) Family Genes From Pyropia yezoensis and Their Correlation With Archeospore Release

Abstract

Reactive oxygen species (ROS) play important regulatory roles in plant growth and development, as well as in cell differentiation and stress responses. Respiratory burst oxidase homolog (RBOH) is the key enzyme in ROS production. So far, the Rboh family genes in Pyropia yezoensis have not been comprehensively characterized, and whether their function was involved in the formation of archeospores is still unknown. In this study, a total of 11 PyRboh genes were identified from the P. yezoensis genome by homology mining. Through phylogenetic analysis, it is suggested that the PyRboh genes were evolutionarily conserved among the lineages of red algae, but a few genes exhibited a species-specific manner. The treatment of P. yezoensis blades with NADPH oxidase inhibitor diphenylene iodonium (DPI) could significantly inhibit the formation of archeospores, suggesting that RBOH may be involved in the formation of archeospores. According to PyRboh gene expression analysis using the P. yezoensis strains with obvious differences in releasing archeospores, it is showed that the expression trends of most genes were consistent, with no significant difference between strains, whereas the expression pattern of the two P. yezoensis-specific genes (PyRbohJ and PyRbohK) was positively correlated with the amount of archeospores. Furthermore, as treatment of blades with allantoin resulted in a significant increase in the release of archeospores, the expression levels of PyRbohJ and PyRbohK were also consistently upregulated, further confirming the relationship between the two genes and archeospore formation. These findings provide insights into the molecular mechanism of P. yezoensis archeospore formation.

Keywords: DPI treatment; Pyropia yezoensis; RBOH gene; archeospore; evolutionary analysis.

Figure 1

The chromosomal locations of the PyRboh genes. The long red bars represent the chromosomes of P. yezoensis. The chromosome numbers are labeled on the left of the bars, and the black fonts represent the PyRboh genes.

Figure 2

Phylogenetic tree (A) and conserved motifs (B) of Rboh proteins from representative species of red algae, green algae, and land plants. Species' name abbreviations are used as prefixes: Ph, Pyropia haitanensis; Py, Pyropia yezoensis; At, Arabidopsis thaliana; Os, Oryza sativa; Cre, Chlamydomonas reinhardtii; Ccr, Chondrus crispus; Cme, Cyanidioschyzon merolae; Gch, Gracilariopsis chorda; Pum, Porphyra umbilicalis; Ppu, Porphyridium purpureum.

Figure 3

Phylogenetic analysis and sequence alignment of FAD and NAD conserved regions of Rboh proteins in P. yezoensis, Pyropia haitanensis, and Porphyra umbilicalis. The colored shapes next to the tree branches represent different species. FAD, FAD binding domain; NAD, NAD binding domain.

Figure 4

Numbers of the archeospores released from the blades of P. yezoensis Py-332 strain after being cultured in normal medium (CK) and DPI (0.05 μM) medium (DPI) for 3 days and then cultured in normal medium for another 7 days. **Extremely significant difference (P < 0.01).

Figure 5

Expression trends of PyRboh genes for blades of P. yezoensis at the age of 20 and 25 days in Py26W and Py26W' strains. *Significant difference (P < 0.05); **Extremely significant difference (P < 0.01).

Figure 6

Expression trends of PyRboh genes for blades of P. yezoensis at the age of 20 and 25 days in Py-LS and Py-332 strains. *Significant difference (P < 0.05); **Extremely significant difference (P < 0.01).

Figure 7

Numbers of the released archeospores from the 22-day-aged blades after being cultured in normal medium (CK) and allantoin (5 mM) medium (Allantoin) for 7 days (A) and the relative expression of PyRbohJ (B) and PyRbohK (C) of the blades in Py-332 strain of P. yezoensis. **Extremely significant difference (P < 0.01).