Functional analysis of a novel cryptochrome gene (GbCRY1) from Ginkgo biloba

Abstract

Cryptochrome (CRY) is a blue light receptor that is widely distributed in animals, plants, and microorganisms. CRY as a coding gene of cryptochrome that regulates the organism gene expression and plays an important role in organism growth and development. In this study, we identified four photolyase/cryptochrome (PHR/CRY) members from the genome of Ginkgo biloba. Phylogenetic tree analysis showed that the Ginkgo PHR/CRY family members were closely related to Arabidopsis thaliana and Solanum lycopersicum. We isolated a cryptochrome gene, GbCRY1, from G. biloba and analyzed its structure and function. GbCRY1 shared high similarity with AtCRY1 from A. thaliana. GbCRY1 expression level was higher in stems and leaves and lower in roots, male strobili, female strobili. GbCRY1 expression level fluctuated periodically within 24 h, gradually increased in the dark, and decreased under blue light. The newly germinated ginkgo seedlings were cultured under dark, white light, and blue light conditions. The blue light normally induced photomorphogenesis of ginkgo seedlings, which included hypocotyl elongation inhibition, leaf expansion inhibition, and chlorophyll formation. Treating dark-adapted ginkgo leaves with blue light could induce stomatal opening. At the same time, blue light reduced the expression level of GbCRY1 in the process of inducing photomorphogenesis and stoma opening. Our results provide evidence that GbCRY1 expression is affected by space, circadian cycle and light, and also proves that GbCRY1 is related to ginkgo circadian clock, photomorphogenesis and stoma opening process.

Keywords: CRY1; Ginkgo biloba; blue light; expression.

Figure 1.

Phylogenetic tree analysis and function prediction of CRY1. (a) Phylogenetic analysis of ginkgo PHR/CRY family members. The color indicates the different subgroups. (b) Prediction of CRY1 protein-protein interaction network. Green represents the predicted results of protein-protein interaction in plants, and blue represents the predicted results of protein-protein interaction in animals. (c) GO enrichment analysis of CRY1 interacting protein in plants and (d) animals. The color indicates the q-value, with a lower q-value indicating more significant enrichment and the point size indicating the DEG number

Figure 2.

Abundance of CRY1 expression in different tissues of G. biloba. The error bars represent the standard error of three biological replicates. Bar and line charts represent the qRT-PCR and FPKM values of the genes, respectively. The expression in female strobili of G. biloba was set as 1 and the different letters indicates values are significantly different at P < .05. The R² value represents the correlation between the qRT-PCR and FPKM values

Figure 3.

The effect of blue light mediated GbCRY1 on the stoma opening and Circadian clock of ginkgo seedlings. (a) Stomatal conductance changes. (b) The relative expression of GbCRY1 during blue light durations. (c) The relative expression of GbCRY1 during the periodic changes of blue light (12h blue light/12 dark). The different letters indicates values are significantly different at P < .05

Figure 4.

The effect of blue light mediated GbCRY1 on the photomorphogenesis of ginkgo seedlings. (a) Growth of ginkgo seedlings under different light conditions. (b) The length of shoot, internodes and hypocotyls of ginkgo seedlings under different light conditions. (c) Abundance of CRY1 expression under different light conditions. (d) Correlation plot of the shoot length and the relative expression of GbCRY1. (e) Correlation plot of the internode length and the relative expression of GbCRY1. (f) Correlation plot of the hypocotyl length and the relative expression of GbCRY1. (g) Observation on the anatomical structure of ginkgo seedling cells under different light conditions. The different letters indicates values are significantly different at P < .05