Summary Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that features nocturnal CO2 uptake, facilitates increased water‐use efficiency (WUE), and enables CAM plants to inhabit water‐limited environments such as semi‐arid deserts or seasonally dry forests. Human population growth and global climate change now present challenges for agricultural production systems to increase food, feed, forage, fiber, and fuel production. One approach to meet these challenges is to increase reliance on CAM crops, such as Agave and Opuntia, for biomass production on semi‐arid, abandoned, marginal, or degraded agricultural lands. Major research efforts are now underway to assess the productivity of CAM crop species and to harness the WUE of CAM by engineering this pathway into existing food, feed, and bioenergy crops. An improved understanding of CAM has potential for high returns on research investment. To exploit the potential of CAM crops and CAM bioengineering, it will be necessary to elucidate the evolution, genomic features, and regulatory mechanisms of CAM. Field trials and predictive models will be required to assess the productivity of CAM crops, while new synthetic biology approaches need to be developed for CAM engineering. Infrastructure will be needed for CAM model systems, field trials, mutant collections, and data management.

Silencing phosphoenolpyruvate carboxylase in a Crassulacean acid metabolism species prevented nocturnal CO 2 fixation and malate accumulation and perturbed the circadian clock and guard cell signalling.

Short Title: Silencing PPC1 in an obligate CAM plant One Sentence Summary: Silencing phosphoenolpyruvate carboxylase in an obligate CAM species prevented dark period CO2 fixation and malate accumulation, caused arrhythmia of some components within the central circadian clock and enhanced rhythms of others, and adjusted the temporal regulation and relative abundance of guard cell signalling genes. ABSTRACT Unlike C3 plants, Crassulacean acid metabolism (CAM) plants fix CO2 in the dark using phosphoenolpyruvate carboxylase (PPC; EC 4.1.1.31). PPC combines PEP with CO2(as HCO3 -), forming oxaloacetate that is rapidly converted to malate, leading to vacuolar malic acid accumulation that peaks phased to dawn. In the light period, malate decarboxylation concentrates CO2 around RuBisCO for secondary fixation.CAM mutants lacking PPC have not been described. Here, RNAi was employed totranscripts, PPC activity, dark period CO2 fixation, and nocturnal malate accumulation.Light period stomatal closure was also perturbed, and the plants displayed reduced but detectable dark period stomatal conductance, and arrhythmia of the CAM CO2 fixation circadian rhythm under constant light and temperature (LL) free-running conditions. By contrast, the rhythm of delayed fluorescence was enhanced in plants lacking PPC1. Furthermore, a subset of gene transcripts within the central circadian oscillator were up-regulated and oscillated robustly. The regulation guard cell genes involved controlling stomatal movements was also altered in rPPC1-B. This provided direct evidence that altered regulatory patterns of key guard cell signaling genes are linked with the characteristic inverse pattern of stomatal opening and closing during CAM. RESULTS Initial Screening and Characterisation of PPC1 RNAi Lines of K. laxifloraAs K. laxiflora is relatively slow to grow, flower and set seed, taking around 9-months seed-to-seed (Hartwell et al., 2016), data are presented for independent primary transformants that were propagated clonally via leaf margin plantlets and/ or stem cuttings. Primary transformants were screened initially using high-throughput leaf disc tests for starch and acidity at dawn and dusk (Cushman et al., 2008; Dever et al., 2015). Independent transgenic lines that acidified less during the dark period were screened for the steady-state abundance of PPC1 transcripts using RT-qPCR. Line rPPC1-B displayed a complete loss of PPC1 transcripts, whereas rPPC1-A had an intermediate level of PPC1 transcripts ( Figure 1A). The other plant-type PPC genes (PPC2, PPC3 and PPC4) were up-regulated relative to the wild type, with their peak