Role of the ELONGATED GYNOPHORE/ELONGATA2 Protein in Fruit and Root Development in Arabidopsis Thaliana

Abstract

In order to identify new players in fruit development, a forward genetic screen was performed on EMS mutagenized plants. A mutant named elongated gynophore (egy) was identified in the M2 population based on altered fruit morphology. Genetic analysis established that the egy phenotype is due to a monogenic and recessive mutation. The egy plants show additional developmental defects including shorter root, narrower cotyledons and malformed leaf lamina. Molecular mapping and whole genome sequencing analyses showed a G/C deletion at the position 4414980 on the AT5G13680 gene locus which is predicted to encode the ELONGATA2 (ELO2) protein. ELO2 is a constituent member of the elongator complex which helps in transcriptional elongation in association with the phosphorylated form of RNA polymerase II. This complex has been implicated in controlling development, abiotic stress and biotic stress. Genetic complementation test confirmed that egy is indeed allelic to elo2-3. Surprisingly, the EGY overexpression line 35S::EGY showed loss-of-function phenotype, suggesting transgene silencing. In angiosperms, fruit is derived from the fertilized ovary. The initiation of the female reproductive organ commences with a lump of cells which eventually develops into the gynoecium with a stigma, a style, two fused ovaries and a gynophore, arranged from the apical to basal axis in that order. Genetic networks faithfully shapes up the carpel primordium into predetermined gynoecium shape. Following fertilization, siliques elongate concomitantly with developing embryos. Here we show that the egy mutant has apical basal patterning defect with longer gynophore at the base. This gynophore phenotype resembles the phenotype found in the mutants with altered auxin and cytokinin levels/signaling. We show that egy is hypersensitive to cytokinin treatment; egy fruits treated with cytokinin display phenotype similar to the plants expressing IPT7 under fruit-specific promoter. These results suggest that broadened shoulders at the apical region of egy gynoecium possibly results from higher cytokinin level/response. Genetic interaction studies have shown that EGY act independent of AGAMOUS and PEAPOD to suppress the medio-lateral growth of the apical gynoecium region. Genetic and expression studies suggest that PINOID and TMO5/T5L1 work downstream to EGY, while ETTIN acts in parallel to EGY. We also observed larger seeds in the egy mutant and show that this is controlled maternally. Thus, the gametic lethality in egy can possibly be accounted for by the defective ovules. We show that egy primary roots are shorter compared to Col-0, though egy seeds have longer embryonic root to begin with, suggesting a defect in cell division. The root cells are arranged radially in a stereotypic pattern in root meristem from the outer epidermis to the inner stele specific the vascular bundles. The four QC cells are also surrounded by stem cells of various identities. This stereotypic pattern of cell arrangement is perturbed in the egy root. The stele, composed of pericycle and vascular bundles is reduced in the egy mutant, suggesting a positive role of EGY in vascular cell division. Confocal microscopic studies and real-time PCR data suggest that TMO5/T5L1 work downstream to EGY. Thus, the Arabidopsis „ELONGATOR‟ complex regulates the transcription of target genes that are necessary for plant growth and development. A proposed genetic network for the role of EGY in fruit and root development. Based on the genetic interaction studies and expression analysis, we have placed EGY in the existing molecular network that control fruit and root vascular development in Arabidopsis