An Optimized Transformation System and Functional Test of CYC-Like TCP Gene CpCYC in Chirita pumila (Gesneriaceae)

Abstract

The development of an ideal model plant located at a key phylogenetic node is critically important to advance functional and regulatory studies of key regulatory genes in the evolutionary developmental (evo-devo) biology field. In this study, we selected Chirita pumila in the family Gesneriaceae, a basal group in Lamiales, as a model plant to optimize its genetic transformation system established previously by us through investigating a series of factors and further conduct functional test of the CYC-like floral symmetry gene CpCYC. By transforming a RNAi:CpCYC vector, we successfully achieved the desired phenotypes of upright actinomorphic flowers, which suggest that CpCYC actually determines the establishment of floral zygomorphy and the horizontal orientation of flowers in C. pumila. We also confirmed the activities of CpCYC promoter in dorsal petals, dorsal/lateral staminodes, as well as the pedicel by transferring a CpCYC promoter:GUS vector into C. pumila. Furthermore, we testified the availability of a transient gene expression system using C. pumila mesophyll protoplasts. The improved transformation system together with the inherent biological features would make C. pumila an attractive new model in functional and regulatory studies for a broad range of evo-devo issues.

An Efficient and Universal Protoplast Isolation Protocol Suitable for Transient Gene Expression Analysis and Single-Cell RNA Sequencing

“…This activation was dramatically decreased in the mABRE-mini:GFP reporter plasmids ( Figure 5 a,b). In addition, the protoplasts prepared from this protocol are also suitable for subcellular localization and protein–protein interaction assays, such as bimolecular fluorescent complimentary (BiFC), as described previously [ 55 ].…”

“…The seeds were then germinated on 1/2 Murashige and Skoog (MS) basal medium supplied with 1% sucrose, 0.4% gelzan, and 0.02 mg/L α-Naphthalene Acetic Acid (NAA). The 4-week-old seedlings were transplanted on 1/2 MS medium in a glass tissue culture bottle at 26 °C under 10 h light/14 h dark conditions and light intensity of ~100 μmoL/m 2 /s in the growth chamber for 4–6 weeks [ 55 ].…”

“…The Ct value of each gene was determined by normalizing the fluorescence threshold. The relative expression level of the target gene was determined using the ratio = 2 −ΔΔCt method, and GUS was used as an internal control for protoplasts resulting from transient expression analysis and CpACTIN was used as a reference gene for expression analysis [ 55 , 57 ]. All primers used for gene expression analysis are listed in Supplemental Table S1 .…”

Advances in Research on the Regulation of Floral Development by CYC-like Genes

“…The second variation involved floral organ morphology (e.g., a lobe incision towards the base of the corolla, and stamen, pistil, and calyx petalization). Liu et al functionally characterized the CYC -like flower symmetry-related gene CpCYC in C. pumila [ 124 ]. By transforming plants with a RNAi:CpCYC vector, vertically radially symmetrical flowers were obtained, implying that CpCYC determines the establishment of zygomorphy and the horizontal plane of flowers.…”

Genomic survey of TCP transcription factors in plants: Phylogenomics, evolution and their biology

“…Expression analysis of G. hirsutum TCP62 have proved these findings, and showed a high enrichment in the auxiliary buds and phyllophores. Nonetheless, overexpression of GhTCP62 in A. thaliana has shown a negative regulation of total number of shoots and reduced growth vigor ( Liu J. et al, 2021 ). The Branched and Indeterminate spikelet 1 (BDI1), which encodes a TCP transcription factor, and highly conserved in both wild and cultivated barley has been shown to play a crucial role in determining barley inflorescence architecture and spikelet development; through regulating the gene transcription of cell wall modification and known Trehalose-6-phosphate homeostasis ( Shang et al, 2020 ).…”