Developing Portulaca oleracea as a model system for functional genomics analysis of C-4/CAM photosynthesis

Author(s)

Ferrari, Renata Callegari ^[1] ; Bittencourt, Priscila Pires ^[1] ; Nagumo, Paula Yumi ^[1] ; Oliveira, Willian Silva ^[1] ; Rodrigues, Maria Aurineide ^[1] ; Hartwell, James ^[2] ; Freschi, Luciano ^[1]

Affiliation

^[1] Univ Sao Paulo, Inst Biociencias, Dept Bot, BR-05508090 Sao Paulo - Brazil

^[2] Univ Liverpool, Inst Syst Mol & Integrat Biol, Dept Biochem & Syst Biol, Liverpool L69 7ZB, Merseyside - England

• Document type: Journal article
• Source: FUNCTIONAL PLANT BIOLOGY; v. 48, n. 7 DEC 2020.
• Web of Science Citations: 1

Abstract

Previously regarded as an intriguing photosynthetic curiosity, the occurrence of C-4 and Crassulacean acid metabolism (CAM) photosynthesis within a single organism has recently emerged as a source of information for future biotechnological use. Among C-4/CAM facultative species, Portulaca oleracea L. has been used as a model for biochemical and gene expression analysis of C-4/CAM under field and laboratory conditions. In the present work, we focussed on developing molecular tools to facilitate functional genomics studies in this species, from the optimisation of RNA isolation protocols to a method for stable genetic transformation. Eleven variations of RNA extraction procedures were tested and compared for RNA quantity and quality. Also, 7 sample sets comprising total RNA from hormonal and abiotic stress treatments, distinct plant organs, leaf developmental stages, and subspecies were used to select, among 12 reference genes, the most stable reference genes for RT-qPCR analysis of each experimental condition. Furthermore, different explant sources, Agrobacterium tumefaciens strains, and regeneration and antibiotic selection media were tested in various combinations to optimise a protocol for stable genetic transformation of P. oleracea. Altogether, we provide essential tools for functional gene analysis in the context of C-4/CAM photosynthesis, including an efficient RNA isolation method, preferred reference genes for RT-qPCR normalisation for a range of experimental conditions, and a protocol to produce P. oleracea stable transformants using A. tumefaciens. (AU)

• FAPESP's process: 16/04755-4 - Exploring C4 and Crassulacean Acid Metabolism (CAM) compatibility within a single organism: changes in global transcriptional profile, hormonal regulation, and tissue localization of key C4 and CAM components in Portulaca oleracea
• Grantee: Renata Callegari Ferrari
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)