Transcriptomic analysis of Medicago truncatula calli with MtWOX9-1 overexpression

Abstract

Somatic embryogenesis (SE) is the development of embryo-like structures from somatic plant tissues. This process rarely can be observed in nature, but for many plant species, in vitro protocols are developed, which allow to obtain somatic embryos formation directly from tissues of plant explant or from the embryogenic callus. SE is widely used for plant propagation and transformation; therefore, the search for SE stimulators and revealing of the mechanisms of their functioning are very important for biotechnology. Among the SE regulators, proteins of the WOX family play significant roles. WOX (WUSCHEL-RELATED HOMEOBOX) is a homeodomain-containing transcription factor family. Different WOX genes function in different plant organs and tissues, maintaining meristem activity and regulating cell proliferation and differentiation. Recently, we have shown that transcription factor MtWOX9-1, belonging to the WOX family, can stimulate SE in the Medicago truncatula callus culture. In this research, transcriptomic analysis of highly embryogenic calli with MtWOX9-1 overexpression was performed in comparison to wildtype calli. It was shown that MtWOX9-1 overexpression led to the activation of several groups of genes, including genes related to cell division, tissue diff erentiation, and seed development. Enriched GO pathways included several groups related to histone methyltransferase activity as well as DNA methylation and chromatin binding, suggesting major epigenetic changes that occur in call overexpressing MtWOX9-1. Using Medicago Truncatula Gene Expression Atlas, we also iden tified a group of genes coding for transcription factors that were both coexpressed with MtWOX9-1 in different plant organs and differentially expressed in our samples. These genes are putative targets of MtWOX9-1, and they may act in the same pathway with this regulator during SE.

The WOX Genes from the Intermediate Clade: Influence on the Somatic Embryogenesis in Medicago truncatula

“…The primers gave PCR product of corresponding size when used with genomic DNA of M. truncatula; therefore, they should be functional in qPCR with cDNA. However, according to the transcriptomic analysis, MtWOX11-2 had a rather high expression level in the embryogenic calli [ 36 ]. At the same time, MtWOX11-2 mRNA is known to have at least two different isoforms that differ in their 3′ parts.…”

MtCLE08, MtCLE16, and MtCLE18 Transcription Patterns and Their Possible Functions in the Embryogenic Calli of Medicago truncatula

“…MtWOX9-1 was found to be expressed during SE [ 16 ] and to stimulate this process [ 15 ]. Recently, transcriptomic analysis of calli with MtWOX9-1 overexpression was performed [ 17 ]. We analyzed these data and found that MtCLE18 gene expression level increases substantially in the calli with MtWOX9-1 overexpression in comparison with the wildtype calli ( Figure 1 A, Table S1 ).…”

“… Expression levels of MtCLE18 ( A ) and MtCLE08 ( B ) in control (R108) calli and calli with MtWOX9-1 overexpression according to the transcriptomic analysis [ 17 ]. Error bars represent the standard error for two biological replicates.…”

MtWOX2 and MtWOX9-1 Effects on the Embryogenic Callus Transcriptome in Medicago truncatula

“…We compared the MtWOX2oe DEGs with DEGs in calli with MtWOX9-1 overexpression (in comparison with R108 calli) [ 27 ]. Unlike MtWOX2, which stimulated callus development according to our data, MtWOX9-1 can stimulate development of somatic embryos.…”

“…These data suggest that MtWOX9-1 carries out both WOX8 and WOX9 functions in M. truncatula development. MtWOX2 transcription is stimulated by MtWOX9-1 overexpression, according to the transcriptome analysis [ 27 ]. Our data on the antagonistic effects of MtWOX2 and MtWOX9-1 in the embryogenic calli suggest the existence of a negative feedback loop between these two genes, reminiscent of WOX2 and WOX8 relations in the zygotic embryos [ 21 ].…”