Exploring C4-CAM plasticity within the Portulaca oleracea complex

Abstract

Portulaca oleracea is a C₄ herb capable of performing CAM under drought stress. It is distributed worldwide and is either considered a polymorphic species or a complex of subspecies, due to its numerous morphological variations. We evaluated CAM plasticity within P. oleracea genotypes since the complexity surrounding this species may be reflected in intraspecific variations in photosynthetic behavior. Eleven subspecies of P. oleracea from distant geographical locations and one cultivar were morphologically and physiologically characterized. C₄ and CAM photosynthesis were monitored in plants exposed to well-watered, droughted and rewatered treatments, and data obtained were compared among individual genotypes. All subspecies expressed CAM in a fully-reversible manner. Transcript abundance of C₄-CAM signature genes was shown to be a useful indicator of the C₄-CAM-C₄ switches in all genotypes. C₄-related genes were down-regulated and subsequently fully expressed upon drought and rewatering, respectively. CAM-marker genes followed the opposite pattern. A gradient of morphological traits and drought-induced nighttime malate accumulation was observed across genotypes. Therefore, different combinations of CAM expression levels, plant sizes and shapes are available within the P. oleracea complex, which can be a valuable tool in the context of C₄/CAM photosynthesis research.

Figure 1

Characterization of the Portulaca oleracea complex based on climatic and morphometric variables via principal component analysis (PCA) and hierarchical clustering analysis (HCA). (A,B) Variable contribution to each PC. The first two PCs explain 75% and 77% of the variance in A and B, respectively. The color scale indicates the relative variable contribution to each PC. (C,D) Groups of subspecies formed—the first two PCs harbor the most significant correlations to the variables. (E,F) HCA groups subspecies into clusters and values of approximately unbiased (AU) and bootstrap (BP) are presented in red and green, respectively (see Materials and methods section for details). Data for these analyses are presented in Tables S1 and S2. In (A,C,E), the following climatic variables were analyzed: latitude, longitutude, annual mean temperature (MeanTempY), mean diurnal range (MeanDiurnalR), isothermality (IsoTherm), temperature seasonality standard deviation (TempSeasonSD), max. temperature of warmest month (MTWM), min. temperature of coldest month (MTCM), temperature annual range (TempRangeY), mean temperature of wettest quarter (MTWeQ), mean temperature of driest quarter (MTDQ), mean temperature of warmest quarter (MTWaQ), mean temperature of coldest quarter (MTCQ), annual precipitation (PrecipY), precipitation of wettest month (PWM), precipitation of driest month (PDM), precipitation seasonality (PrecipS), precipitation of wettest quarter (PWeQ), precipitation of driest quarter (PDQ), precipitation of warmest quarter (PWaQ), precipitation of coldest quarter (PCQ).

Figure 2

Drought treatment impacts on soil volumetric water content (SVWC) and overall plant morphology. (A) Changes in SVWC during drought and rewatering treatments in P. oleracea, with red and blue arrows indicating partial (10 ml per pot) and full watering events, respectively (see Methods for details). Data are means ± SE for monitored genotypes. (B) Representative images of 2-month-old plants kept under well-watered (left) and droughted (right) conditions.

Figure 3

Similar drought-triggered changes in diel gas exchange are shared by Portulaca oleracea subspecies. (A) Net CO₂ exchange of shoots of representative subspecies after 16 days of drought. (B-D) Net CO₂ exchange by shoots of granulatostellulata in well-watered conditions (B), after the initial 10-day-period of water withholding (C), and after 20 days of drought and into rewatering (D). In (A–D), two-week-old individuals were used due to the size restriction of the cuvette. Also, data were normalized against the leaf area. Shaded areas indicate the dark period, red arrows indicate partial watering event (5 ml), and the blue arrow indicates full rewatering. Inserts schematically illustrate soil water content following water deprivation (see Methods for details), and the time points corresponding to the gas exchange measurements are highlighted in red.

Figure 4

Impacts of water availability P. oleracea on relative water content (RWC) and nocturnal malate accumulation. (A) Leaf RWC of well-watered, droughted and rewatered plants. (B) Nocturnal malate accumulation (Δ malate) in well-watered, droughted and rewatered plants. Data are means ± SE of at least three biological replicates, and different letters indicate statistically significant differences (p < 0.05) among the treatments for each subspecies. In (B), standard error = √((standard error_well-watered)² + (standard error_droughted)².

Figure 5

Impacts of water availability on transcript abundance of C₄- and CAM-marker genes. (A-C) Relative abundance of CAM-specific transcripts: PPC1E1c (A), ALMT-12E.1 (B), DIC-1.1 (C). (D–F) Relative abundance of CAM-specific transcripts: PPC1E1a’ (D), NADME-2E.1 (E), ASPAT-1E1 (F). Mean relative expression was normalized against well-watered trituberculata samples, and mRNA levels were determined in samples harvested at dawn (A–C) or dusk (D–F). Data are means ± SE of at least three biological replicates, and different letters indicate statistically significant differences (p < 0.05) among the treatments for each subspecies.