Identification of regulatory factors promoting embryogenic callus formation in barley through transcriptome analysis

Abstract

Background: Barley is known to be recalcitrant to tissue culture, which hinders genetic transformation and its biotechnological application. To date, the ideal explant for transformation remains limited to immature embryos; the mechanism underlying embryonic callus formation is elusive.

Results: This study aimed to uncover the different transcription regulation pathways between calli formed from immature (IME) and mature (ME) embryos through transcriptome sequencing. We showed that incubation of embryos in an auxin-rich medium caused dramatic changes in gene expression profiles within 48 h. Overall, 9330 and 11,318 differentially expressed genes (DEGs) were found in the IME and ME systems, respectively. 3880 DEGs were found to be specific to IME_0h/IME_48h, and protein phosphorylation, regulation of transcription, and oxidative-reduction processes were the most common gene ontology categories of this group. Twenty-three IAA, fourteen ARF, eight SAUR, three YUC, and four PIN genes were found to be differentially expressed during callus formation. The effect of callus-inducing medium (CIM) on IAA genes was broader in the IME system than in the ME system, indicating that auxin response participates in regulating cell reprogramming during callus formation. BBM, LEC1, and PLT2 exhibited a significant increase in expression levels in the IME system but were not activated in the ME system. WUS showed a more substantial growth trend in the IME system than in the ME system, suggesting that these embryonic, shoot, and root meristem genes play crucial roles in determining the acquisition of competency. Moreover, epigenetic regulators, including SUVH3A, SUVH2A, and HDA19B/703, exhibited differential expression patterns between the two induction systems, indicating that epigenetic reprogramming might contribute to gene expression activation/suppression in this process. Furthermore, we examined the effect of ectopic expression of HvBBM and HvWUS on Agrobacterium-mediated barley transformation. The transformation efficiency in the group expressing the PLTPpro:HvBBM + Axig1pro:HvWUS construct was increased by three times that in the control (empty vector) because of enhanced plant regeneration capacity.

Conclusions: We identified some regulatory factors that might contribute to the differential responses of the two explants to callus induction and provide a promising strategy to improve transformation efficiency in barley.

Keywords: Auxin response; Barley (Hordeum vulgare); Callus induction; Plant regeneration.

Fig. 1

Characteristics of calli formed from immature (IME) and mature (ME) embryos. a: Schematic diagram of sample collection for RNA-seq analysis. After surface-sterilisation of mature seeds and embryonic axis removal from immature seeds (14 days post-anthesis), embryos were isolated and cultured on callus-inducing medium (CIM). The samples for RNA-seq were collected at three time points from immature embryo-derived callus (IME_0h, IME_24h and IME_48h), and two time points from mature embryo-derived callus (ME_0h and ME_24h). b: Scutellum-induced callus formation in Golden Promise barley after 7 days of culture. Bar =500 μm. c: Scutellum-induced callus formation in Golden Promise barley after 4 weeks of culture. Bar =500 mm. d: The number of DEGs up- or downregulated during embryo-derived callus formation and between two callus induction systems. e: Venn diagram showing overlap and specific DEGs between two samples

Fig. 2

Gene Ontology (GO) and KEGG analysis of differentially expressed genes in different groups. a: The selected 15 most enriched GO biological processes categories among DEGs in IME_0h/IME_48h, ME_0h/IME_24h, IME_0h/ME_0h, and IME_48h/ME_24h. b: The top 15 most enriched KEGG pathways [67]. P value≤0.05, sorted by DEGs number. c: The top 10 most enriched GO in the IME-specific DEGs (refer to DEGs only found in the contrast IME_0h/IME_48h, but not included in ME_0h/ME_24h). d: The top 19 most enriched KEGG pathway categories in the IME-specific DEGs

Fig. 3

Expression of a set of callus-inducing medium (CIM)-induced transcription factors (TFs) during - callus formation from immature and mature embryos. a: The number of differentially expressed TFs detected only in the IME system (IME specific), only in the ME system (ME specific), and in both systems (IME∩ME). b: Upregulated (upper) and downregulated (lower) TF families specific to the IME system. Numbers represent the gene members associated with a given TF family. c: The top 10 differentially expressed TFs in the IME system (IME specific). Genes marked in blue are upregulated TFs, and TFs marked in black are downregulated

Fig. 4

Expression of genes involved in the auxin pathway during callus-inducing medium (CIM)-mediated callus formation. The expression levels were visualised by using OmicStudio tools at https://www.omicstudio.cn/tool based on RNA-seq datasets (Additional file 4). Numbers beneath the heat map indicate the relative expression intensities, and the higher expression intensities are indicated by more reddish colours. Genes are grouped by auxin response, biosynthesis, and transport genes. Note that only genes with FPKM > 1 are shown

Fig. 5

Heat map showing expression changes of key developmental genes for embryos and meristems during callus induction. The expression levels were visualised by using OmicStudio tools at https://www.omicstudio.cn/tool based on RNA-seq datasets (Additional file 4). a: Clustering display of expression intensities of the embryonic, shoot, and root meristem genes based on RNA-seq datasets. b: The transcript levels of LEC1 and PLT5 in five samples were revealed by qRT-PCR and RNA-seq data. The data shown are means ± S.D. of three biological replicates

Fig. 6

Identification of BBM and WUS candidate genes in barley and their expression response to callus-inducing medium (CIM). a: Sequence alignment and domain analysis of BBM and WUS in Arabidopsis, rice, maize, and barley. b: The transcript levels of BBM and WUS in the five samples were revealed by qRT-PCR and RNA-seq data. The data shown are means ± S.D. of three biological replicates

Fig. 7

The effect of BBM and WUS ectopic expression on callus-inducing medium (CIM)-induced callus formation and transformation efficiency. a Schematic representation of the construct used for Agrobacterium-mediated barley transformation. The proZmPLTP:HvBBM + proZmAxig1:HvWUS construct contained two cassettes: the first one included the maize phospholipid transferase promoter (proZmPLTP) driving HvBBM with a Nos terminator, and the second one included the maize Axig1 promoter (proZmAxig1) driving HvWUS with a Nos terminator. b The callus-forming and plant regeneration phenotype after Agrobacterium inoculation. The group using an empty vector was set as control. c Fresh weight analysis of callus in the control and proZmPLTP:HvBBM + proZmAxig1:HvWUS transformation group. Error bars indicate the SE of the mean (n = 30). The experiments were performed in three independent replicates. d The effect of BBM and WUS ectopic expression on the genes in the LEC1-ABI3-FUS3-LEC2 network. The data shown are means ± S.D. of three biological replicates.**, P < 0.05; ***, P <0.01 (Student’s t-test).

Fig. 8

Transcriptional changes of genes regulating DNA methylation and histone modification. The expression levels were visualised by using OmicStudio tools at https://www.omicstudio.cn/tool based on RNA-seq datasets (Additional file 4). Numbers beneath the heat map indicate the relative expression intensities, and the higher expression intensities are indicated by more reddish colours. Note that only genes with FPKM > 1 are shown

Fig. 9

A schematic diagram describing gene expression regulation during callus formation from immature and mature barley embryos. Dicamba (synthetic auxin) induces cell fate transition through the auxin signalling pathway, and more genes are included in the IME system (left) than in the IM system (right). Embryonic genes BBM, LEC1, and FUS3, shoot meristem gene WUS, and root meristem gene PLT2 displayed differential expression patterns between the two systems, resulted in the production of different types of callus. Embryonic callus (left) and non-embryonic callus (right) exhibit differential regeneration potential on shoot-inducing medium (SIM). Orange represents significantly upregulated genes, while blue represents genes that were activated slightly or remained unchanged. Epigenetic modification might be involved in regulating the expression status of regulatory genes in different explants and their responses to callus induction