Gene co-expression reveals the modularity and integration of C₄and CAM inPortulaca

Abstract

C4 and Crassulacean acid metabolism (CAM) have been considered as largely independent photosynthetic adaptations in spite of sharing key biochemical modules. Portulaca is a geographically widespread clade of over 100 annual and perennial angiosperm species that primarily use C4 photosynthesis, but facultatively exhibit CAM when drought stressed, a photosynthetic system known as C4+CAM. It has been hypothesized that C4+CAM is rare because of pleiotropic constraints, but these have not been deeply explored. We generated a chromosome-level genome assembly of P. amilis and sampled mRNA from P. amilis and P. oleracea during CAM induction. Gene co-expression network analyses identified C4 and CAM gene modules shared and unique to both Portulaca species. A conserved CAM module linked phosphoenolpyruvate carboxylase (PEPC) to starch turnover during the day-night transition and was enriched in circadian clock regulatory motifs in the P. amilis genome. Preservation of this co-expression module regardless of water status suggests that Portulaca constitutively operate a weak CAM cycle that is transcriptionally and post-transcriptionally upregulated during drought. C4 and CAM mostly used mutually exclusive paralogs for primary carbon fixation and, although it is likely that nocturnal CAM malate stores are shuttled into diurnal C4 decarboxylation pathways, we find some evidence that metabolite cycling may occur at low levels. C4 likely evolved in Portulaca through co-option of redundant paralogs and integration of the diurnal portion of CAM. Thus, the ancestral CAM system did not strongly constrain C4 evolution because photosynthetic gene networks are not co-regulated for both daytime and nighttime functions.

Spatial resolution of an integrated C4+CAM photosynthetic metabolism

“…After quality filtering and trimming, a mean of 68.97% of the reads were kept. The P. oleracea Trinity assembled transcriptome retrieved from Gilman et al (2021) consisted of 444307 contigs and 230895…”

“…We annotated 629 trinity unigenes with a known or hypothesized function in CCMs, including starch metabolism, circadian rhythm, and associated transcription factors as in (Christin et al, 2014;Ferrari et al, 2020;Gilman et al, 2021), (online Table S3). For most core CCM enzymes, unigenes were annotated to eudicot gene lineages as in (Christin et al, 2013(Christin et al, , 2014Moreno-Villena et al, 2017;Ferrari et al, 2020;Gilman et al, 2021). When available, eudicot gene lineage nomenclature is indicated after the gene names followed by a dash.…”

“…Future work should confirm the benefits of facultative CAM in Portulaca experimentally via gene editing. Portulaca is highly amenable to development as a model system for photosynthesis research (Callegari Ferrari et al, 2021): it has a short life cycle, is self-compatible, and a high-quality genome of P. amilis is already available (Gilman et al, 2021).…”

“…We mapped reads from both LCM and Visium experiments to a de novo Portulaca oleracea transcriptome assembly from (Gilman et al, 2021), Before that, we reduced the dataset to contigs containing coding sequences. For that, we first used TransDecoder v5.5.0 [http://transdecoder.github.io] to predict coding sequences within the transcripts, providing the software with precomputed BLASTX (Camacho et al, 2009) alignments to the UniProt protein sequences database (Consortium, 2019) to improve prediction.…”

“…Recent genomic information for Portulaca has provided an opportunity to resolve the spatial configuration of C4 and CAM reactions. Multiple studies have now identified C4 and CAM-specific homologs of PEPC and some accompanying CCM-affiliated enzymes (Christin et al, 2014;Ferrari et al, 2020;Gilman et al, 2021), providing key markers to identify the cell populations where CAM and C4 CO2 fixation occur. The lack of significant upregulation of distinct homologs of CAM decarboxylation genes in multiple Portulaca species is notable (Gilman et al, 2021), and suggests that some elements of C4 and CAM .…”