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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

Grant number: 16/04755-4
Support type:Scholarships in Brazil - Doctorate (Direct)
Effective date (Start): July 01, 2016
Effective date (End): June 30, 2020
Field of knowledge:Biological Sciences - Botany
Principal Investigator:Luciano Freschi
Grantee:Renata Callegari Ferrari
Home Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

C4 and CAM photosynthesis are two metabolic pathways that have evolved from C3 photosynthesis multiple times in plants. Though biochemically related, they represent two ecologically distinct adaptations, with the C4 pathway affording high performance in high light and hot temperatures, and CAM providing extreme water use efficiency and drought tolerance. Certain plant lineages show many C4 and CAM origins that are also phylogenetically intermingled, suggesting that some early steps in the evolutionary trajectories of these syndromes may be shared. The most extreme example of this connectivity is the Portulaca lineage, the only plants known to operate both C4 and CAM cycles within a single leaf. There are various reasons to expect C4 and CAM systems to be incompatible, thus Portulaca tells us that our knowledge of how these syndromes work is incomplete. Recent work suggests that ancestral Portulaca was a facultative CAM plant, and evolved a fully functional C4 system while maintaining CAM capability. Our laboratory has selected P. oleracea as a model to explore the biochemical, physiological and genetic regulation of CAM expression. Our studies revealed that this species also has a clear C3-to-CAM switch taking place in the stems, which perform significant photosynthesis, but do not possess bundle sheath and cannot perform classical C4 photosynthesis. Recently, we have developed a stable genic transformation protocol for this species, thereby allowing future manipulations and functional analysis of genes of interest. Therefore, P. oleracea represents a unique model system for exploring the biochemical and genetic basis that have allowed the presence of C3, C4 and CAM photosynthesis in a single organism. The proposed research will explore key features of this complex photosynthetic scenario and begin to develop Portulaca as a model system for the study of both C4 and CAM pathways. This project aims to: 1) identify the subspecies of the P. oleracea complex with the highest plasticity in CAM expression and compare this trait with leaf and stem anatomical differences among these subspecies; 2) utilize RNA-seq analysis to identify key components of the C4 and CAM machineries as well as key regulatory elements controlling the transitions between C3, C4 and CAM photosynthesis; 3) explore the signalling events responsible for the up- and down-regulation of CAM expression in leaf and stem tissues of P. oleracea plants subjected to contrasting water availability conditions; 4) identify, for the first time, the spatial configuration of C4 and CAM cycles within the leaf, using in-situ hybridization of CAM and C4-specific mRNA probes. We have developed three primary hypotheses that will be tested here: 1) due to its cosmopolite distribution, P. oleracea may possess a high plasticity in CAM expression, particularly when comparing subspecies that have evolved under contrasting environmental conditions; 2) the evolution of a C4 cycle on top of a CAM-performing organism may have required recruiting new isogenes specifically enrolled in the C4 cycle, which might be differently regulated by environmental and/or endogenous signals than those enrolled in the CAM pathway; 3) C4 and CAM cycles are able to operate within the same leaf due to the innovation of a novel 2-cell CAM system, where 4-C acids are produced and stored in mesophyll cells overnight and then transported to the bundle sheath cells for decarboxylation. Alternatively, C4 and CAM cycles may even operate within the same mesophyll cells via drastic modifications in the classical functioning of these mechanisms. Finally, it is important to highlight that CAM and C4 pathways play important roles in plant evolution and function, and are also important to humanity as key food and biofuel crops. Therefore, Portulaca provides an evolutionary blueprint for a supercrop, one that performs C4 when resources are plenty, but can persist through droughts with facultative CAM. (AU)