Lessons from the history of Agave: ecological and cultural context for valuation of CAM.

BACKGROUND AND SCOPE: Crassulacean acid metabolism (CAM) is an intriguing physiological adaptation in plants that are widespread throughout many ecosystems. Despite the relatively recent mechanistic understanding of CAM in plant physiology, evidence from historical records suggests that ancient cultures in the Americas also recognized the value of CAM plants. Agave species, in particular, have a rich cultural legacy that provides a foundation for commercially valued products. Here, we review that legacy and potential relationships between ancient values and the needs of modern-day climate adaptation strategies. CONCLUSIONS: There are many products that can be produced from Agave species, including food, sugar, fibre and medicines. Traditional knowledge about agricultural management and preparation of plant products can be combined with new ecophysiological knowledge and agronomic techniques to develop these resources in the borderland region of the southwestern USA and Mexico. Historical records of pre-Columbian practices in the Sonoran desert and remnants of centuries-old agriculture in Baja California and Sonora demonstrate the climate resilience of Agave agriculture. Commercial growth of both tequila and bacanora indicates the potential for large-scale production today, but also underscores the importance of adopting regenerative agricultural practices to accomplish environmentally sustainable production. Recent international recognition of the Appellation of Origin for several Agave species produced for spirits in Mexico might provide opportunities for agricultural diversification. In contrast, fibre is currently produced from several Agave species on many continents. Projections of growth with future climate change suggest that Agave spp. will be viable alternatives for commodity crops that suffer declines during drought and increased temperatures. Historical cultivation of Agave affirms that these CAM plants can supply sugar, soft and hard fibres, medicines and food supplements.

Weak crassulacean acid metabolism and other xerophytic adaptive traits in the genus Beaucarnea Lem. (Asparagaceae).

The genus Beaucarnea (Asparagaceae) has economic value as an ornamental plant but also has ecological importance. Nonetheless, habitat, physiological traits and growth parameters of this genus remain largely unknown. We characterized the environmental ranges of Beaucarnea inermis, B. gracilis and B. pliabilis; and screened for the presence of physiological adaptations to drought (biomass allocation, presence of crassulacean acid metabolism [CAM] and its effect on plant water use). We performed experiments in 3- and 5-year-old nursery-grown plants of the three species, measured dry mass accumulation in leaves/stems/roots and screened for CAM using gas exchange, titratable acidity and δ13 C. We performed a second experiment on the water and light use responses of B. pliabilis under drought treatment. We found that B. gracilis was limited to xerophytic scrubs (precipitation >400 mm yr-1 ), while B. pliabilis and B. inermis (precipitation >500 and 700 mm year-1 , respectively) inhabited dry forests. Beaucarnea gracilis had the lowest dry mass and allocation to leaves, while B. inermis showed the opposite pattern. Only B. pliabilis exhibited small but significant acid fluctuations, characterized as weak CAM, along with high proline content. Acid concentration contributed in 2.7% of the daily carbon during the wet season but represented most of the carbon in the dry season, under closed stomata and had an important contribution to osmolality. Thus, CAM is described for the first time in the genus Beaucarnea, but was only present in one of three species, warranting exploration of this metabolism in the remaining species of this genus.

A matter of time: regulatory events behind the synchronization of C4 and crassulacean acid metabolism in Portulaca oleracea.

Portulaca species can switch between C4 and crassulacean acid metabolism (CAM) depending on environmental conditions. However, the regulatory mechanisms behind this rare photosynthetic adaptation remain elusive. Using Portulaca oleracea as a model system, here we investigated the involvement of the circadian clock, plant hormones, and transcription factors in coordinating C4 and CAM gene expression. Free-running experiments in constant conditions suggested that C4 and CAM gene expression are intrinsically connected to the circadian clock. Detailed time-course, drought, and rewatering experiments revealed distinct time frames for CAM induction and reversion (days versus hours, respectively), which were accompanied by changes in abscisic acid (ABA) and cytokinin metabolism and signaling. Exogenous ABA and cytokinins were shown to promote and repress CAM expression in P. oleracea, respectively. Moreover, the drought-induced decline in C4 transcript levels was completely recovered upon cytokinin treatment. The ABA-regulated transcription factor genes HB7, NFYA7, NFYC9, TT8, and ARR12 were identified as likely candidate regulators of CAM induction following this approach, whereas NFYC4 and ARR9 were connected to C4 expression patterns. Therefore, we provide insights into the signaling events controlling C4-CAM transitions in response to water availability and over the day/night cycle, highlighting candidate genes for future functional studies in the context of facultative C4-CAM photosynthesis.

CAM photosynthesis: the acid test.

There is currently considerable interest in the prospects for bioengineering crassulacean acid metabolism (CAM) photosynthesis - or key elements associated with it, such as increased water-use efficiency - into C3 plants. Resolving how CAM photosynthesis evolved from the ancestral C3 pathway could provide valuable insights into the targets for such bioengineering efforts. It has been proposed that the ability to accumulate organic acids at night may be common among C3 plants, and that the transition to CAM might simply require enhancement of pre-existing fluxes, without the need for changes in circadian or diurnal regulation. We show, in a survey encompassing 40 families of vascular plants, that nocturnal acidification is a feature entirely restricted to CAM species. Although many C3 species can synthesize malate during the light period, we argue that the switch to night-time malic acid accumulation requires a fundamental metabolic reprogramming that couples glycolytic breakdown of storage carbohydrate to the process of net dark CO2 fixation. This central element of the CAM pathway, even when expressed at a low level, represents a biochemical capability not seen in C3 plants, and so is better regarded as a discrete evolutionary innovation than as part of a metabolic continuum between C3 and CAM.

CAM photosynthesis in desert blooming Cistanthe of the Atacama, Chile.

When plants of the Atacama desert undergo episodic blooms, among the most prominent are succulent-leaved Cistanthe (Montiaceae). We demonstrate that two Cistanthe species, the perennial Cistanthe sp. aff. crassifolia and the annual/biannual Cistanthe sp. aff. longiscapa, can exhibit net CO2 uptake and leaf acidification patterns typical of crassulacean acid metabolism (CAM). In C. sp. aff. crassifolia leaves, CAM expression was facultative. CAM-type nocturnal net CO2 uptake and acid accumulation occurred in drought-stressed but not in well-watered plants. By contrast, CAM expression in C. sp. aff. longiscapa was largely constitutive. Nocturnal acid accumulation was present in leaves of well-watered and in droughted plants. Following water-deficit stress, net nocturnal CO2 uptake was induced and the level of acid accumulated increased. Neither nocturnal CO2 uptake nor acid accumulation was reduced when the plants were re-watered. δ13C values of a further nine field-collected Cistanthe species are consistent with a contribution of CAM to their carbon pools. In the Portulacinae, a suborder with eight CAM-containing families, Cistanthe becomes the sixth genus with CAM within the family Montiaceae, and it is likely that the ancestor of all Portulacineae also possessed CAM photosynthesis. In the stochastic rainfall landscape of the Atacama, carbon uptake in the dark is a water-use efficient mechanism that increases the carbon pool available for seed production or dormancy. The next rain event may be years away.

Crassulacean acid metabolism (CAM) supersedes the turgor loss point (TLP) as an important adaptation across a precipitation gradient, in the genus Clusia.

As future climates continue to change, precipitation deficits are expected to become more severe across tropical ecosystems. As a result, it is important that we identify plant physiological traits that act as adaptations to drought, and determine whether these traits act synergistically or independently of each other. In this study, we assessed the role of three leaf-level putative adaptations to drought: crassulacean acid metabolism (CAM), the turgor loss point (TLPΨ) and water storage hydrenchyma tissue. Using the genus Clusia as a model, we were able to explore the extent to which these leaf physiological traits co-vary, and also how they contribute to species' distributions across a precipitation gradient in Central and South America. We found that CAM is independent of the TLPΨ and hydrenchyma depth in Clusia. In addition, we provide evidence that constitutive CAM is an adaptation to year-long water deficits, whereas facultative CAM appears to be more important for surviving acute dry seasons. Finally, we find that the other leaf traits tested did not correlate with environmental precipitation, suggesting that the reduced transpirational rates associated with CAM obviate the need to adapt the TLPΨ and hydrenchyma depth in this genus.

Constitutive and facultative crassulacean acid metabolism (CAM) in Cuban oregano, Coleus amboinicus (Lamiaceae).

Plants exhibiting the water-conserving crassulacean acid metabolism (CAM) photosynthetic pathway provide some of the most intriguing examples of photosynthetic diversity and plasticity. Here, a largely unnoticed facet of CAM-plant photosynthesis is highlighted: the co-occurrence of ontogenetically controlled constitutive and environmentally controlled facultative CAM in a species. Both forms of CAM are displayed in leaves of Coleus amboinicus Lour. (Lamiaceae), a semi-succulent perennial plant with oregano-like flavour that is native to southern and eastern Africa and naturalised elsewhere in the tropics. Under well-watered conditions, leaves assimilate CO2 predominantly by the C3 pathway. They also display low levels of CO2 uptake at night accompanied by small nocturnal increases in leaf tissue acidity. This indicates the presence of weakly expressed constitutive CAM. CAM expression is strongly enhanced in response to drought stress. The drought-enhanced component of CAM is reversible upon rewatering and thus considered to be facultative. In contrast to C. amboinicus, the thin-leaved closely related Coleus scutellarioides (L.) Benth. exhibits net CO2 fixation solely in the light via the C3 pathway, both under well-watered and drought conditions. However, low levels of nocturnal acidification detected in leaves and stems indicate that the CAM cycle is present. The highly speciose mint family, which contains few known CAM-exhibiting species and is composed predominantly of C3 species, appears to be an excellent group of plants for studying the evolutionary origins of CAM and for determining the position of facultative CAM along the C3-full CAM trajectory.

Developing Portulaca oleracea as a model system for functional genomics analysis of C4/CAM photosynthesis.

Previously regarded as an intriguing photosynthetic curiosity, the occurrence of C4 and Crassulacean acid metabolism (CAM) photosynthesis within a single organism has recently emerged as a source of information for future biotechnological use. Among C4/CAM facultative species, Portulaca oleracea L. has been used as a model for biochemical and gene expression analysis of C4/CAM under field and laboratory conditions. In the present work, we focussed on developing molecular tools to facilitate functional genomics studies in this species, from the optimisation of RNA isolation protocols to a method for stable genetic transformation. Eleven variations of RNA extraction procedures were tested and compared for RNA quantity and quality. Also, 7 sample sets comprising total RNA from hormonal and abiotic stress treatments, distinct plant organs, leaf developmental stages, and subspecies were used to select, among 12 reference genes, the most stable reference genes for RT-qPCR analysis of each experimental condition. Furthermore, different explant sources, Agrobacterium tumefaciens strains, and regeneration and antibiotic selection media were tested in various combinations to optimise a protocol for stable genetic transformation of P. oleracea. Altogether, we provide essential tools for functional gene analysis in the context of C4/CAM photosynthesis, including an efficient RNA isolation method, preferred reference genes for RT-qPCR normalisation for a range of experimental conditions, and a protocol to produce P. oleracea stable transformants using A. tumefaciens.

Exploring C4-CAM plasticity within the Portulaca oleracea complex.

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.

C4 and crassulacean acid metabolism within a single leaf: deciphering key components behind a rare photosynthetic adaptation.

Although biochemically related, C4 and crassulacean acid metabolism (CAM) systems are expected to be incompatible. However, Portulaca species, including P. oleracea, operate C4 and CAM within a single leaf, and the mechanisms behind this unique photosynthetic arrangement remain largely unknown. Here, we employed RNA-seq to identify candidate genes involved exclusively or shared by C4 or CAM, and provided an in-depth characterization of their transcript abundance patterns during the drought-induced photosynthetic transitions in P. oleracea. Data revealed fewer candidate CAM-specific genes than those recruited to function in C4 . The putative CAM-specific genes were predominantly involved in night-time primary carboxylation reactions and malate movement across the tonoplast. Analysis of gene transcript-abundance regulation and photosynthetic physiology indicated that C4 and CAM coexist within a single P. oleracea leaf under mild drought conditions. Developmental and environmental cues were shown to regulate CAM expression in stems, whereas the shift from C4 to C4 -CAM hybrid photosynthesis in leaves was strictly under environmental control. Moreover, efficient starch turnover was identified as part of the metabolic adjustments required for CAM operation in both organs. These findings provide insights into C4 /CAM connectivity and compatibility, contributing to a deeper understanding of alternative ways to engineer CAM into C4 crop species.