Glutathione S-transferase CrGST24 in the differentiation of adventitious buds from Camellia reticulata callus

Abstract

Introduction: Camellia reticulata holds cultural and horticultural significance in traditional Chinese gardens, as a regional endemic species in Yunnan Province. However, during the in vitro regeneration process of C. reticulata, there is often a phenomenon of low efficiency of adventitious bud differentiation or no adventitious bud differentiation at all.

Methods: In previous study, we observed significant morphological differences between the callus tissues of C. reticulata 'Zipao' and wild species. The callus of 'Zipao' was white and loosely textured, the wild species was green and hard. To investigate the differences between these two types of callus, we conducted transcriptome analysis and identified a differentially expressed gene GST24, which is closely related to the synthesis of glutathione (GSH). Heterologous transformation of CrGST24 into tobacco (Nicotiana tabacum) was conducted.

Results and discussion: The CrGST24 gene promoted the synthesis of endogenous auxin and cytokinin in transgenic tobacco by regulating the expression of transcription factors related to auxin and cytokinin. Exogenous IBA, 6-BA, and red-blue light treatment increased the levels of auxins and cytokinins in C. reticulata callus, thereby promoting adventitious bud differentiation. Additionally, CrGST24 interacted with CrGSHB and CrDHAR2 genes, facilitating GSH and AsA synthesis and clearing ROS.

Keywords: ASA; Camellia reticulata; CrGST24; GSH; ROS; auxin; cytokinin.

Figure 1

Bioinformatics analysis of the GST from C reticulata. Note: (a) Phylogenetic tree analysis of GST family members; (b) Protein-protein interaction network.

Figure 2

Expression analysis of CrGST24. (a) CrGST24 expression under different concentrations of IBA, and 6-BA (2 h); (b) CrGST24 expression under different concentrations of IBA, and 6-BA (1 week); (c) CrGST24 expression under different light conditions (8 h); (d) CrGST24 expression under different light quality (8 h); (e) CrGST24 expression under different light quality (3 d). Note: The bar chart represents the mean of three biological replicates, with error bars showed standard deviations. Asterisks indicate statistical significance based on one-way analysis of variance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 3

Genetic transformation, positive seedling screening, and phenotypic observation of transgenic tobacco. (a) Tobacco leaf discs infected with Agrobacterium tumefaciens; (b) Induction of resistant buds; (c) Expansion of resistant buds; (d) Isolation of resistant buds; (e) Formation of independent lines; (f) Expression analysis of transgenic lines; (g) Transgenic tobacco after transplanting; (h) Seeds harvested from mature transgenic tobacco for subsequent experiments; (i, m) WT tobacco plants; (j, n) CrGST24-OE6; (k, o) CrGST24-OE7; (l, p) CrGST24-OE22. The bar chart represents the mean of three biological replicates, with error bars showing standard deviations. Asterisks indicate statistical significance based on one-way analysis of variance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 4

Observations on in vitro regeneration of CrGST24 transgenic tobacco and expression of auxin and cytokinin-related genes in CrGST24 transgenic tobacco. (a) in vitro regeneration (b-h) auxin-related genes; (i-n) cytokinin-related genes. The bar chart represents the mean of three biological replicates, with error bars showing standard deviations. Asterisks indicate statistical significance based on one-way analysis of variance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 5

Expression of auxin and cytokinin-related genes in C. reticulata callus. (a-g) Auxin-related genes; (h-l) Cytokinin-related genes. The bar chart represents the mean of three biological replicates, with error bars showing standard deviations. Asterisks indicate statistical significance based on one-way analysis of variance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 6

Expression of auxin, cytokinin, ROS, GSH, and AsA contents in CrGST24 transgenic tobacco. Note: (a) Auxin content; (b) Cytokinin content; (c) Auxin/cytokinin content; (d) WT; (e) CrGST24-OE6; (f) CrGST24-OE7; (g) CrGST24-OE22; (h) ROS content; (i) GSH content; (j) AsA content; (d-g) the blue part is the area of ROS accumulation. The bar chart represents the mean of three biological replicates, with error bars showing standard deviations. Asterisks indicate statistical significance based on one-way analysis of variance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 7

CrGST24 interact with CrGSHB and CrDHAR2. (a) Subcellular localization assay; (b, c) Y2H assay; (d, e) LUC assay.

Figure 8

Growth status of callus from C. reticulata. (a, b) Callus in medium contaning different concentrations of IBA, 6-BA, and different light quality treatments; (c, d) Callus under red light, blue light, and red-blue light; (e, f) Callus under GSH and AsA treatments. (a, c, e) ‘Zipao’; (b, d, f) wild species.

Figure 9

The mechanism of CrGST24 gene in C. reticulata promoting the formation of adventitious buds from callus differentiation.