Timing of gene expression and recruitment in independent origins of CAM in the Agavoideae (Asparagaceae).

CAM photosynthesis has evolved repeatedly across the plant tree of life, yet our understanding of the genetic convergence across independent origins remains hampered by the lack of comparative studies. CAM is furthermore thought to be closely linked to the circadian clock in order to achieve temporal separation of carboxylation and sugar production. Here, we explore gene expression profiles in eight species from the Agavoideae (Asparagaceae) encompassing three independent origins of CAM. Using comparative physiology and transcriptomics, we examined the variable modes of CAM in this subfamily and the changes in gene expression across time of day and between well-watered and drought-stressed treatments. We further assessed gene expression and molecular evolution of genes encoding phosphoenolpyruvate carboxylase (PPC), an enzyme required for primary carbon fixation in CAM. Most time-of-day expression profiles are largely conserved across all eight species and suggest that large perturbations to the central clock are not required for CAM evolution. In contrast, transcriptional response to drought is highly lineage specific. Yucca and Beschorneria have CAM-like expression of PPC2, a copy of PPC that has never been shown to be recruited for CAM in angiosperms, and evidence of positive selection in PPC genes implicates mutations that may have facilitated the recruitment for CAM function early in the evolutionary history of the Agavoideae. Together the physiological and transcriptomic comparison of closely related C3 and CAM species reveals similar gene expression profiles, with the notable exception of differential recruitment of carboxylase enzymes for CAM function.

Stomatal Responses to Light, CO2, and Mesophyll Tissue in Vicia faba and Kalanchoë fedtschenkoi

The responses of stomatal aperture to light intensity and CO2 concentration were studied in both Vicia faba (C3) and Kalanchoë fedtschenkoi (Crassulacean acid metabolism; CAM), in material sampled from both light and dark periods. Direct comparison was made between intact leaf segments, epidermises grafted onto exposed mesophyll, and isolated epidermal peels, including transplantations between species and between diel periods. We reported the stomatal opening in response to darkness in isolated CAM peels from the light period, but not from the dark. Furthermore, we showed that C3 mesophyll has stimulated CAM stomata in transplanted peels to behave as C3 in response to light and CO2. By using peels and mesophyll from plants sampled in the dark and the light period, we provided clear evidence that CAM stomata behaved differently from C3. This might be linked to stored metabolites/ions and signalling pathway components within the guard cells, and/or a mesophyll-derived signal. Overall, our results provided evidence for both the involvement of guard cell metabolism and mesophyll signals in stomatal responses in both C3 and CAM species.

Transcriptome and Degradome Profiling Reveals a Role of miR530 in the Circadian Regulation of Gene Expression in Kalanchoë marnieriana

Crassulacean acid metabolism (CAM) is an important photosynthetic pathway for plant adaptation to dry environments. CAM plants feature a coordinated interaction between mesophyll and epidermis functions that involves refined regulations of gene expression. Plant microRNAs (miRNAs) are crucial post-transcription regulators of gene expression, however, their roles underlying the CAM pathway remain poorly investigated. Here, we present a study characterizing the expression of miRNAs in an obligate CAM species Kalanchoë marnieriana. Through sequencing of transcriptome and degradome in mesophyll and epidermal tissues under the drought treatments, we identified differentially expressed miRNAs that were potentially involved in the regulation of CAM. In total, we obtained 84 miRNA genes, and eight of them were determined to be Kalanchoë-specific miRNAs. It is widely accepted that CAM pathway is regulated by circadian clock. We showed that miR530 was substantially downregulated in epidermal peels under drought conditions; miR530 targeted two tandem zinc knuckle/PLU3 domain encoding genes (TZPs) that were potentially involved in light signaling and circadian clock pathways. Our work suggests that the miR530-TZPs module might play a role of regulating CAM-related gene expression in Kalanchoë.

Correlations between morphological fruit types, fruit and seed colors, and functional groups

The associations between morphological fruit types, fruit and seed colors, and functional plant traits: life forms, epiphytism, physiology, nutritional relationships, fruit phenology, and successional stage, were determined for 1,139 plant species from contrasting plant communities. Texture and dehiscence were closely related. Dehiscence is largely associated with dry tissues; indehiscence, however, is an attribute of both dry and fleshy fruits. The number of morphological fruit types was 28 or 55 for Gray's and Spjut's classifications, respectively. Fruits were predominantly dark in color (brown, purple-black, black or green), whilst seeds had both dark and light colors (brown, beige, or black). The most representative associations were mainly found between the more abundant fruit types and the colors most common. Asymmetries in the level of specialization, whereby less common fruit and seed colors tended to be associated with the most common fruit types, were also found. Fleshy fruits showed more variation as regards their coloration, and only drupes and berries showed a tendency towards a specific color: purple-black. The relationships among fruit type and color, seed color, and functional plant traits revealed the following trends: trees produced both fleshy and dry fruits; shrubs produced fleshy fruits; and herbaceous species, dry fruits. Woody species tended to have dark or bright colors, depending on their seed dispersal mechanisms and phylogenetic relations. Epiphytes were associated with dry-dehiscent fruits and brown seeds, and parasitic-hemiparasitic species had predominantly fleshy-indehiscent fruits. Pioneer species were more likely to have dry fruits, whereas fleshy fruits tended to be more frequent in late successional stage species. The C4 species, mostly herbs, had mainly one-seeded dry fruits, but multi-seeded fruits in succulent-CAM species showed morphologically diverse fruit types. Unripe and ripe fruits showed seasonal changes, especially during the rainy-dry transition period for the most abundant morphological fruit types, dry fruits during the dry period and fleshy fruited species was positively associated with the rainy season. All these trends are discussed with regard to their environmental significance and the relationships between fruit morphology, colors and functional groups. .

Pyrogenic carbon improves the physiological performance of a C3 species planted on a green roof

Plants utilizing C3 physiology have a more difficult time establishing in rooftop environments than plants with more heat and drought adapted constitutions, such as species that employ crassulacean acid metabolism (CAM). CAM species are much less susceptible to limitations of shallow, infertile soil-less media under abiotic and biotic stress. It is thought that soil amendments might improve rooftop media in a way that allows for C3 species to prosper in rooftop environments. While compost is typically added to media to achieve this goal, we hypothesized that the addition of an anthropogenic pyrogenic carbon (PyC) supplement, instead, would enable better organic and mineral sorption and water retention, resulting in improved physiological performance of C3 species. To test this, we grew a C3 legume species, wild indigo (Baptisia tinctoria L R.Br. ex), in control compost-amended media and media amended by PyC on a rooftop in Massachusetts, USA. We found PyC-amended media had greater mean organic and mineral nutrient sorption. We also found 16% greater soil water holding capacity (GWL/ψ g) than control media. In addition, wild indigo photosynthetic intrinsic water use efficiency (iWUE) was significantly increased by 19% when grown in PyC-amended as compared to control media. We conclude that amending green roof media with PyC provides greater benefits than compost amendments for colonization of a C3 legume, wild indigo. Our results gathered over seven years suggest that PyC from converted waste stream cardboard could be used to improve the rooftop performance of other leguminous species, including agricultural crops.

Leaf anatomy is not correlated to CAM function in a C3+CAM hybrid species, Yucca gloriosa

Background and Aims Crassulacean acid metabolism (CAM) is often considered to be a complex trait, requiring orchestration of leaf anatomy and physiology for optimal performance. However, the observation of trait correlations is based largely on comparisons between C3 and strong CAM species, resulting in a lack of understanding as to how such traits evolve and the level of intraspecific variability for CAM and associated traits. Methods To understand intraspecific variation for traits underlying CAM and how these traits might assemble over evolutionary time, we conducted detailed time course physiological screens and measured aspects of leaf anatomy in 24 genotypes of a C3+CAM hybrid species, Yucca gloriosa (Asparagaceae). Comparisons were made to Y. gloriosa’s progenitor species, Y. filamentosa (C3) and Y. aloifolia (CAM). Key Results Based on gas exchange and measurement of leaf acids, Y. gloriosa appears to use both C3 and CAM, and varies across genotypes in the degree to which CAM can be upregulated under drought stress. While correlations between leaf anatomy and physiology exist when testing across all three Yucca species, such correlations break down at the species level in Y. gloriosa. Conclusions The variation in CAM upregulation in Y. gloriosa is a result of its relatively recent hybrid origin. The lack of trait correlations between anatomy and physiology within Y. gloriosa indicate that the evolution of CAM, at least initially, can proceed through a wide combination of anatomical traits, and more favourable combinations are eventually selected for in strong CAM plants.

Are thick leaves, large mesophyll cells and small intercellular air spaces requisites for CAM?

Background and Aims It is commonly accepted that the leaf of a crassulacean acid metabolism (CAM) plant is thick, with large mesophyll cells and vacuoles that can accommodate the malic acid produced during the night. The link between mesophyll characteristics and CAM mode, whether obligate or C3/CAM, was evaluated. Methods Published values of the carbon isotopic ratio (δ 13C) as an indicator of CAM, leaf thickness, leaf micrographs and other evidence of CAM operation were used to correlate cell density, cell area, the proportion of intercellular space in the mesophyll (IAS) and the length of cell wall facing the intercellular air spaces (Lmes/A) with CAM mode. Key Results Based on 81 species and relatively unrelated families (15) belonging to nine orders, neither leaf thickness nor mesophyll traits helped explain the degree of CAM expression. A strong correlation was found between leaf thickness and δ 13C in some species of Crassulaceae and between leaf thickness and nocturnal acid accumulation in a few obligate CAM species of Bromeliaceae but, when all 81 species were pooled together, no significant changes with δ 13C were observed in cell density, cell area, IAS or Lmes/A. Conclusions An influence of phylogeny on leaf anatomy was evidenced in a few cases but this precluded generalization for widely separate taxa containing CAM species. The possible relationships between leaf anatomy and CAM mode should be interpreted cautiously.

The role of cis-elements in the evolution of crassulacean acid metabolism photosynthesis

Crassulacean acid metabolism (CAM) photosynthesis is an innovation of carbon concentrating mechanism that is characterized by nocturnal CO2 fixation. Recent progresses in genomics, transcriptomics, proteomics, and metabolomics of CAM species yielded new knowledge and abundant genomic resources. In this review, we will discuss the pattern of cis-elements in stomata movement-related genes and CAM CO2 fixation genes, and analyze the expression dynamic of CAM related genes in green leaf tissues. We propose that CAM photosynthesis evolved through the re-organization of existing enzymes and associated membrane transporters in central metabolism and stomatal movement-related genes, at least in part by selection of existing circadian clock cis-regulatory elements in their promoter regions. Better understanding of CAM evolution will help us to design crops that can thrive in arid or semi-arid regions, which are likely to expand due to global climate change.

The role of shaded cocoa plantations in the maintenance of epiphytic orchids and their interactions with phorophytes

Aims Habitat loss and fragmentation are the main threats to biodiversity in tropical forests. Agroecosystems such as shaded cocoa plantations (SCP) provide refuge for tropical forest biota. However, it is poorly known whether the interspecific ecological interactions are also maintained in these transformed habitats. We evaluated the diversity, reproductive status and photosynthetic metabolism (CAM or C3) of the epiphytic orchid community, and their interactions with host trees (phorophytes) in SCP compared to tropical rainforest (TRF). Methods In southeastern Mexico, three sites each in TRF and SCP were studied, with four 400 m2 plots established at each site to record all orchids and their phorophytes. We determined the reproductive (adult) or non-reproductive (juvenile) status of each orchid individual in relation to the presence or absence, respectively, of flowers/fruits (or remnants), and assigned the photosynthetic pathway of each orchid species based in literature. We used true diversity and ecological networks approaches to analyze orchid diversity and orchid–phorophyte interactions, respectively. Important Findings In total, 607 individuals belonging to 47 orchid species were recorded. Orchid diversity was higher in TRF (19 effective species) than in SCP (11 effective species) and only seven species were shared between the two habitats. CAM orchid species were more frequent in SCP (53%) than in TRF (14%). At the community level the proportion of non-reproductive and reproductive orchid species and the nested structure and specialization level of the TRF orchid–phorophyte network were maintained in SCP. However, only a subset of TRF epiphytic orchids remains in SCP, highlighting the importance of protecting TRF. Despite this difference, shaded agroecosystems such as SCP can maintain some of the diversity and functions of natural forests, since the SCP epiphytic orchid community, mainly composed of CAM species, and its phorophytes constitute a nested interaction network, which would confer robustness to disturbances.