The role of different PEPC isoforms in the growth and physiology of the model CAM plant, Kalanchoë fedtschenko

Abstract

Crassulacean acid metabolism (CAM) is one of plants' three types of photosynthesis. CAM evolved from C3 via changes in protein sequence and temporal gene expression. Compared to C3 and C4 photosynthesis types, CAM shows resilient photosynthetic ability under limited water availability. A key step in CAM photosynthesis involves phosphoenolpyruvate carboxylase (PEPC), which catalyzes nocturnal carboxylation of phosphoenolpyruvate (PEP) to form oxaloacetate which is reduced to malate. PEPC is controlled transcriptionally and posttranslationally. The two most abundant isoforms of PEPC in Kalanchoë fedtschenkoi are KfePEPC1 (kaladp0095s0055.1) and KfePEPC2 (Kaladp0048s0578.1), with the former being abundantly expressed in both light and dark periods while the latter is expressed abundantly in the dark period. In this study, we investigated the roles of different PEPC isoforms in the growth and physiology of the model CAM plant Kalanchoë fedtschenkoi. Our partners at Oak Ridge Laboratories USA created mutants of KfePEPC1 and KfePEPC2 genes using the novel gene-editing technique CRISPR. The aim of this thesis was to conduct physiological and biochemical analyses for independent lines of each of these mutated genes and thereby establish the physiological roles of PEPC1 and PEPC2. The effect of knocking down the KfePEPC1 gene includes a significant loss in PEPC enzyme activity and protein abundance along with a switch from nocturnal CO2 fixation and stomatal opening to daytime CO2 uptake and stomatal opening, as shown in the mutant kfepepc1 line1. In addition, the kfepepc1 line1 mutant had a significant drop in the amount of malic acid stored at dawn and increased starch accumulation at night compared to the wildtype. The same mutants also showed increased transpiration rate, reduced water use efficiency, reduced stomatal density with larger pore lengths and increased pigment content in drought conditions. In contrast, the kfepepc1 Line 2 mutant retained all the attributes of CAM observed in wildtype. Still, the mutation significantly perturbed PEPC protein abundance, altered stomatal regulation, reduced stomatal density, and increased saturated water content under drought conditions in the kfepepc1 Line 2 mutant. These different effects observed in the kfepepc1 mutant lines could be attributed to how completely the KfePEPC1 gene was knocked down in the lines. Overall, results indicated that the KfePEPC1 gene is essential for CAM in K. fedtschenkoi. The effect of knocking down the KfePEPC2 gene was also examined. Both lines of the kfepepc2 mutants retained all the attributes of CAM observed in wildtype, although downregulation of diel transcript abundance of the rbcL gene during the middle of the daytime was observed. Furthermore, the ability of the kfepepc2 mutants to regulate stomatal opening, especially towards the end of the light period, was significantly compromised, and growth was significantly suppressed. In addition, the KfePEPC2 mutation caused a reduction in stomatal density and increased pore length. These observations indicated that the KfePEPC2 gene isoform is implicated in other important non-photosynthetic functions, which are key to physiological performance in K. fedtschenkoi. At the same time, the effect of knocking down both KfePEPC1 and KfePEPC2 together was even more severe. In addition to losing the ability to do CAM, the mutation led to the loss of PEPC enzyme activity, protein abundance, inability to accumulate malic acid overnight, and highly suppressed growth in all the kfepepc1/2 mutant lines. Observations from this work indicate that the different isoforms of the PEPC gene investigated play pivotal but contrasting roles in optimizing CAM and photosynthetic activity of K. fedtschenkoi. To successfully bioengineer CAM into C3 plants, both PEPC genes must be functional in the bioengineered system.