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Ethiopian mustard (Brassica carinata) is an ancient crop with remarkable stress resilience and a desirable seed fatty acid profile for biofuel uses. Brassica carinata is one of six Brassica species that share three major genomes from three diploid species (AA, BB, and CC) that spontaneously hybridized in a pairwise manner to form three allotetraploid species (AABB, AACC, and BBCC). Of the genomes of these species, that of B. carinata is the least understood. Here, we report a chromosome scale 1.31-Gbp genome assembly with 156.9-fold sequencing coverage for B. carinata, completing the reference genomes comprising the classic Triangle of U, a classical theory of the evolutionary relationships among these six species. Our assembly provides insights into the hybridization event that led to the current B. carinata genome and the genomic features that gave rise to the superior agronomic traits of B. carinata. Notably, we identified an expansion of transcription factor networks and agronomically important gene families. Completion of the Triangle of U comparative genomics platform has allowed us to examine the dynamics of polyploid evolution and the role of subgenome dominance in the domestication and continuing agronomic improvement of B. carinata and other Brassica species.

Background and Aims The relative contribution of C3 photosynthesis and crassulacean acid metabolism (CAM) during the earliest stages of development were investigated to assess how much each might contribute to cactus pear (Opuntia ficus-indica) productivity. Methods The developmental progression of C3 photosynthesis and CAM was assessed in seedlings and daughter cladodes of mature plants by titratable acidity, δ 13C carbon isotopic values, and diel gas exchange measurements. Key Results Nocturnal acidification was observed in seedling cladodes and cotyledons at the earliest stages of development and which became highly significant by 75 days of development. Seedlings cotyledons showed mean δ 13C values of -21.4‰ to -17.1‰ from 30 to 100 days of age, respectively. Seedlings cladodes showed mean δ 13C values of -19.4‰ to -14.5‰ from 30 to 100 days of age, respectively. These values were typical of CAM plants. Net CO2 assimilation was negative, then occurred in both the day and the night, with nighttime fixation becoming predominant once the primary cladode reached five cm in size. Emergent daughter cladodes growing on mature plants showed nocturnal titratable acidity at the earliest stages of development, which became significant when daughter cladodes were >2.5-5 cm in height. Emergent daughter cladodes showed mean δ 13C values of -14.5‰ to -15.6‰ typical of CAM plants. CO2 assimilation studies revealed that net CO2 uptake was negative in daughter cladodes Conclusions Developing O. ficus-indica primary and daughter cladodes begin as respiring sink tissues that transition directly to performing CAM once net positive CO2 fixation is observed. Overall, these results demonstrate that CAM is the primary form of photosynthetic carbon assimilation for O. ficus-indica even at the earliest stages of seedling or daughter cladode development.

Background and AimsCrassulacean acid metabolism (CAM) is a specialized type of photosynthesis characterized by a diel pattern of stomatal opening at night and closure during the day, which increases water-use efficiency. Starch degradation is a key regulator of CAM, providing phosphoenolpyruvate as a substrate in the mesophyll for nocturnal assimilation of CO2. Growing recognition of a key role for starch degradation in C3 photosynthesis guard cells for mediating daytime stomatal opening presents the possibility that starch degradation might also impact CAM by regulating the provision of energy and osmolytes to increase guard cell turgor and drive stomatal opening at night. In this study, we tested the hypothesis that the timing of diel starch turnover in CAM guard cells has been reprogrammed during evolution to enable nocturnal stomatal opening and daytime closure.MethodsBiochemical and genetic characterization of wild-type and starch-deficient RNAi lines of Kalanchoë fedtschenkoi with reduced activity of plastidic phosphoglucomutase (PGM) constituted a preliminary approach for the understanding of starch metabolism and its implications for stomatal regulation in CAM plants.Key ResultsStarch deficiency reduced nocturnal net CO2 uptake but had negligible impact on nocturnal stomatal opening. In contrast, daytime stomatal closure was reduced in magnitude and duration in the starch-deficient rPGM RNAi lines, and their stomata were unable to remain closed in response to elevated concentrations of atmospheric CO2 administered during the day. Curtailed daytime stomatal closure was linked to higher soluble sugar contents in the epidermis and mesophyll.ConclusionsNocturnal stomatal opening is not reliant upon starch degradation, but starch biosynthesis is an important sink for carbohydrates, ensuring daytime stomatal closure in this CAM species.

Cactus pear (Opuntia ficus-indica) is a high productivity species within the Cactaceae grown in many semiarid parts of the world for food, fodder, forage, and biofuels. O. ficus-indica utilises obligate crassulacean acid metabolism (CAM), an adaptation that greatly improves water-use efficiency (WUE) and reduces crop water usage. To better understand CAM-related metabolites and water-deficit stress responses of O. ficus-indica, comparative metabolic profiling was performed on mesophyll and epidermal tissues collected from well-watered and water-deficit stressed cladodes at 50% relative water content (RWC). Tissues were collected over a 24-h period to identify metabolite levels throughout the diel cycle and analysed using a combination of acidic/basic ultra-high-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) and gas chromatography/mass spectrometry (GC/MS) platforms. A total of 382 metabolites, including 210 (55%) named and 172 (45%) unnamed compounds, were characterised across both tissues. Most tricarboxylic acid (TCA) cycle and glycolysis intermediates were depleted in plants undergoing water-deficit stress indicative of CAM idling or post-idling, while the raffinose family oligosaccharides (RFO) accumulated in both mesophyll and epidermal tissues as osmoprotectants. Levels of reduced glutathione and other metabolites of the ascorbate cycle as well as oxylipins, stress hormones such as traumatic acid, and nucleotide degradation products were increased under water-deficit stress conditions. Notably, tryptophan accumulation, an atypical response, was significantly (24-fold) higher during all time points in water-deficit stressed mesophyll tissue compared with well-watered controls. Many of the metabolite increases were indicative of a highly oxidising environment under water-deficit stress. A total of 34 unnamed metabolites also accumulated in response to water-deficit stress indicating that such compounds might play important roles in water-deficit stress tolerance.

Climate‐resilient and highly water‐use efficient crops are needed to meet the future food, feed, fiber, and fuel demands of a growing human population. Cactus pear (Opuntia spp.) are highly productive yet have crop water demands that are approximately 20% that of traditional crops due to their ability to perform crassulacean acid metabolism (CAM). In the first long‐term field trial of Opuntia for bioenergy in the United States, the aboveground biomass (cladode and fruit) productivity and response to different water inputs of O. cochenillifera, O. ficus‐indica, and O. streptacantha were evaluated at a planting density of 1418 plants ha−1 under three irrigation levels over a 5‐year period to investigate their response to different water inputs. Mean cladode fresh weight, cladode dry weight, cladode count, fruit fresh weight, and fruit dry weight increased linearly by 1.26‐, 1.88‐, 1.53‐, 1.89‐, and 2.13‐fold, respectively, with increasing irrigation from 300 to 716 mm year−1. Significant differences in irrigation response appeared in years 4 and 5 as the cactus trees grew larger. However, no significant differences were observed among the three Opuntia spp. for all parameters measured. Biomass productivity increased over 5 years by 140.8‐ and 132.5‐fold for cladode fresh and dry weight, respectively. Mean annual total aboveground biomass at 300, 407, and 716 mm year−1 water input resulted in 8.25, 11.16, and 15.52 Mg dry mass ha−1 year−1, respectively, at the sparse planting density used here to separate irrigation blocks, which could have been easily quadrupled. O. ficus‐indica performed better than O. cochenillifera and O. streptacantha in terms of cladode biomass and fruit count and quality. Biomass productivity was consistent with previous studies for Opuntia spp. demonstrating that cactus pear displays very high biomass and food/forage potential using substantially lower rates of supplemental irrigation than conventional crops grown in semi‐arid and arid regions.

Ethiopian mustard (Brassica carinata) is an ancient crop with significant potential for expanded cultivation as a biodiesel feedstock. The remarkable stress resilience of B. carinata and desirable seed fatty acid profile addresses the ongoing food vs. fuel debate as the crop is productive on marginal lands otherwise not suitable for even closely related species. B. carinata is one of six key Brassica spp. that share three major genomes: three diploid species (AA, BB, CC) that spontaneously hybridized in a pairwise manner, forming three allotetraploid species (AABB, AACC, and BBCC). Each of these genomes has been researched extensively, except for that of B. carinata. In the present study, we report a high-quality, 1.31 Gbp genome with 156.9-fold sequencing coverage for B. carinata var. Gomenzer, completing and confirming the classic Triangle of U, a theory of the evolutionary relationships among these six species that arose almost a century ago. Our assembly provides insights into the genomic features that give rise to B. carinata’s superior agronomic traits for developing more climate-resilient Brassica crops with excellent oil production. Notably, we identified an expansion of transcription factor networks and agronomically-important gene families. Completing the Triangle of U comparative genomics platform allowed us to examine the dynamics of polyploid evolution and the role of subgenome dominance in domestication and agronomical improvement.

• The evolution of l ‐DOPA 4,5‐dioxygenase activity, encoded by the gene DODA , was a key step in the origin of betalain biosynthesis in Caryophyllales. We previously proposed that l ‐DOPA 4,5‐dioxygenase activity evolved via a single Caryophyllales‐specific neofunctionalisation event within the DODA gene lineage. However, this neofunctionalisation event has not been confirmed and the DODA gene lineage exhibits numerous gene duplication events, whose evolutionary significance is unclear. • To address this, we functionally characterised 23 distinct DODA proteins for l ‐DOPA 4,5‐dioxygenase activity, from four betalain‐pigmented and five anthocyanin‐pigmented species, representing key evolutionary transitions across Caryophyllales. By mapping these functional data to an updated DODA phylogeny, we then explored the evolution of l ‐DOPA 4,5‐dioxygenase activity. • We find that low l ‐DOPA 4,5‐dioxygenase activity is distributed across the DODA gene lineage. In this context, repeated gene duplication events within the DODA gene lineage give rise to polyphyletic occurrences of elevated l ‐DOPA 4,5‐dioxygenase activity, accompanied by convergent shifts in key functional residues and distinct genomic patterns of micro‐synteny. • In the context of an updated organismal phylogeny and newly inferred pigment reconstructions, we argue that repeated convergent acquisition of elevated l ‐DOPA 4,5‐dioxygenase activity is consistent with recurrent specialisation to betalain synthesis in Caryophyllales., New Phytologist, 227 (3), ISSN:0028-646X, ISSN:1469-8137

Increased demand for food requires us to investigate livestock forage and fodder crops that can be grown over a wide range of locations where their cultivation will not compete with that of the food supply. A large portion of the southwestern United States consists of underutilized semi‐arid land. Crops typically used for livestock fodder or forage have high‐water demands that make them uneconomical or unsustainable for semi‐arid and arid regions. The growth rate and low‐input requirements of prickly pear cactus (Opuntia ficus‐indica) make it an excellent candidate for forage or fodder supplementation or replacement in these regions. Previous reports about forage quality data on Opuntia have been scattered across multiple locations, growing conditions and cultivars. Here, we report on the forage quality and mineral content of Opuntia ficus‐indica grown under both field and greenhouse conditions. Crude protein was 71 and 264 g/kg of dry mass for field and greenhouse conditions, respectively. Field‐grown plants showed higher acid and neutral detergent fibre content than greenhouse‐grown plants reflecting higher cellulose, hemicellulose and lignin accumulation. Nutritional values were also compared to requirements of cattle to determine what deficiencies might need to be addressed through supplementation. These data suggest that Opuntia can be used in combination with other feed sources to reduce the demand of resource‐intensive forage crops for raising livestock in dryland areas.

This paper presents a simultaneous isolation of pure, intact chloroplasts and mitochondria from mature leaves of Ice plant (Mesembryanthemum crystallinum) and mitochondrial protein preparation for two-dimensional electrophoresis (2DE) analysis under well watered and water -deficit stressed treatments. The washed chloroplasts and mitochondria were purified with Percoll gradients prepared using a Master flex R pump. The chloroplast and mitochondrial proteins were extracted in lysis buffer containing a protease inhibitor mix supplemented with 1 μM Leupeptin and 1 μM E64, followed by precipitation with ice-cold acetone. The protein contents were determined by an EZQ protein quantitation kit. The results show that chloroplast and mitochondria isolated from Ice plant leaves via this protocol have pure and intact. The shape of chloroplast and mitochondria observed by microscopy were clear and sharp. This procedure was employed for assessing the significant differences in mitochondrial protein expression patterns from the well watered and water-deficit stressed treatment leaves collected at dawn (6 a.m.) and dusk (6 p.m.). The results showed 71 and 20 differentially abundant spots between control and CAM for 6 a.m. and 6 p.m., respectively. In addition, 32 protein spots were differentially abundant for 6 a.m. control compared with 6 p.m. control, and 45 protein spots were differentially abundant for 6 a.m. CAM compared with 6 p.m. CAM. Spots that displayed differential abundance for control compared with CAM likely included proteins involved in mitochondrial processes necessary for CAM function. Through further analysis, these proteins will be identified and characterized in the near future using mass-spectrometry-based techniques.

The study of subcellular membrane structure and function facilitates investigations into how biological processes are divided within the cell. However, work in this area has been hampered by the limited techniques available to fractionate the different membranes. Free Flow Electrophoresis (FFE) allows for the fractionation of membranes based on their different surface charges, a property made up primarily of their varied lipid and protein compositions. In this study, high-resolution plant membrane fractionation by FFE, combined with mass spectrometry-based proteomics, allowed the simultaneous profiling of multiple cellular membranes from the leaf tissue of the plant Mesembryanthemum crystallinum. Comparisons of the fractionated membranes’ protein profile to that of known markers for specific cellular compartments sheds light on the functions of proteins, as well as provides new evidence for multiple subcellular localization of several proteins, including those involved in lipid metabolism.

Global climatic change is predicted to result in certain areas of the earth’s surface becoming hotter and drier. These spatially durable changes in climate will likely have negative impacts on the productivity of many plant species that are poorly adapted to increasing air:leaf vapor pressure deficits. However, more that 6% of vascular plants have evolved crassulacean acid metabolism (CAM) as a photosynthetic adaptation to hotter and drier climates. This specialized mode of photosynthesis exploits a temporal CO2 pump with nocturnal CO2 uptake and concentration to reduce photorespiration, improve water-use efficiency (WUE), and optimize the adaptability of plants to hotter and drier climates. CAM species have a suite of physiological and anatomical adaptations that make them resilient to extreme heat and high insolation including tissue succulence, water-storage and water-capture strategies to attenuate drought, and thick cuticles, epicuticular wax, low stomatal density, high stomatal responsiveness, UV-light protection, and shallow rectifier-like roots to limit water loss under conditions of water deficit. Various modeling approaches have been developed to estimate the growth potential of CAM species under current and future climatic conditions.

Cactus pear (Opuntia ficus-indica) is a high productivity species within the Cactaceae grown in many semiarid parts of the world for food, fodder, forage, and biofuels. O. ficus-indica utilises obligate crassulacean acid metabolism (CAM), an adaptation that greatly improves water-use efficiency (WUE) and reduces crop water usage. To better understand CAM-related metabolites and water-deficit stress responses of O. ficus-indica, comparative metabolic profiling was performed on mesophyll and epidermal tissues collected from well-watered and water-deficit stressed cladodes at 50% relative water content (RWC). Tissues were collected over a 24-h period to identify metabolite levels throughout the diel cycle and analysed using a combination of acidic/basic ultra-high-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) and gas chromatography/mass spectrometry (GC/MS) platforms. A total of 382 metabolites, including 210 (55%) named and 172 (45%) unnamed compounds, were characterised across both tissues. Most tricarboxylic acid (TCA) cycle and glycolysis intermediates were depleted in plants undergoing water-deficit stress indicative of CAM idling or post-idling, while the raffinose family oligosaccharides (RFO) accumulated in both mesophyll and epidermal tissues as osmoprotectants. Levels of reduced glutathione and other metabolites of the ascorbate cycle as well as oxylipins, stress hormones such as traumatic acid, and nucleotide degradation products were increased under water-deficit stress conditions. Notably, tryptophan accumulation, an atypical response, was significantly (24-fold) higher during all time points in water-deficit stressed mesophyll tissue compared with well-watered controls. Many of the metabolite increases were indicative of a highly oxidising environment under water-deficit stress. A total of 34 unnamed metabolites also accumulated in response to water-deficit stress indicating that such compounds might play important roles in water-deficit stress tolerance.

This paper originates from a presentation at the IIIrd International Congress on Crassulacean Acid Metabolism, Cape Tribulation, Queensland, Australia, August 2001. Expression of crassulacean acid metabolism (CAM) is characterized by the extreme plasticity observed within and between species. Switches between C3 photosynthesis and CAM, and subsequent 24-h patterns of day/night CO2 uptake, are tightly controlled by a variety of environmental and metabolic factors that optimize the response of CAM plants to the most challenging environments over seasonal and daily time scales. Regulation of the genes and enzymes involved in CAM and connected metabolic pathways occurs at a number of levels (transcriptional through to post-translational). Such multiple levels of control are considered to be the key to the photosynthetic plasticity of CAM. Here, we review some of the primary environmental and hormonal factors controlling CAM plasticity in different CAM-inducible species, with emphasis on the regulatory signalling circuits responsible for this control. We also examine the inherent circadian regulation of the pathway, mainly in the context of the diel regulation of phosphoenolpyruvate carboxylase and the dedicated kinase that modulates its activity. We then consider the role of secondary signals, with emphasis on changes in cytosolic [Ca2+]i and the downstream signalling pathways, based on studies conducted on Mesembryanthemum crystallinum L. Besides representing an important metabolic adaptation, CAM provides an intriguing paradigm for studying the complex signalling mechanisms that control and coordinate the expression of genes under a variety of short- and long-term environmental perturbations.

Camelina sativa (L.) Crantz. is an annual oilseed crop within the Brassicaceae family. C. sativa has been grown since as early as 4000 BCE. In recent years, C. sativa received increased attention as a climate-resilient oilseed, seed meal, and biofuel (biodiesel and renewable or green diesel) crop. This renewed interest is reflected in the rapid rise in the number of peer-reviewed publications (>2300) containing “camelina” from 1997 to 2021. An overview of the origins of this ancient crop and its genetic diversity and its yield potential under hot and dry growing conditions is provided. The major biotic barriers that limit C. sativa production are summarized, including weed control, insect pests, and fungal, bacterial, and viral pathogens. Ecosystem services provided by C. sativa are also discussed. The profiles of seed oil and fatty acid composition and the many uses of seed meal and oil are discussed, including food, fodder, fuel, industrial, and medical benefits. Lastly, we outline strategies for improving this important and versatile crop to enhance its production globally in the face of a rapidly changing climate using molecular breeding, rhizosphere microbiota, genetic engineering, and genome editing approaches.

While the accumulation of reactive oxygen species (ROS) through spontaneous generation or as the by-products of aerobic metabolism can be toxic to plants, recent findings demonstrate that ROS act as signaling molecules that play a critical role in adapting to various stress conditions. Tight regulation of ROS homeostasis is required to adapt to stress and survive, yet in vivo spatiotemporal information of ROS dynamics are still largely undefined. In order to understand the dynamics of ROS changes and their biological function in adapting to stresses, two quantitative ROS transcription-based bioreporters were developed. These reporters use ROS-responsive promoters from RBOHD or ZAT12 to drive green fluorescent protein (GFP) expression. The resulting GFP expression is compared to a constitutively expressed mCherry that is contained on the same cassette with the ROS-responsive promoter: This allows for the generation of ratiometric images comparing ROS changes (GFP) to the constitutively expressed mCherry. Both reporters were used to assess ROS levels to oxidative stress, salt stress, and the pathogen defense elicitor flg22. These bioreporters showed increases in the ratio values of GFP to mCherry signals between 10 and 30 min poststress application. Such stress-associated ROS signals correlated with the induction of abiotic/biotic stress responsive markers such as RbohD, ZAT12, SOS2 and PR5 suggesting these ROS bioreporters provide a robust indicator of increased ROS related to stress responses. Based upon the spatiotemporal response patterns of signal increase, ZAT12 promoter-dependent ROS (Zat12p-ROS) bioreporter appears to be suitable for cellular mapping of ROS changes in response to abiotic and biotic stresses.

Polyacetylenic lipids accumulate in various Apiaceae species after pathogen attack, suggesting that these compounds are naturally occurring pesticides and potentially valuable resources for crop improvement. These compounds also promote human health and slow tumor growth. Even though polyacetylenic lipids were discovered decades ago, the biosynthetic pathway underlying their production is largely unknown. To begin filling this gap and ultimately enable polyacetylene engineering, we studied polyacetylenes and their biosynthesis in the major Apiaceae crop carrot (Daucus carota subsp. sativus). Using gas chromatography and mass spectrometry, we identified three known polyacetylenes and assigned provisional structures to two novel polyacetylenes. We also quantified these compounds in carrot leaf, petiole, root xylem, root phloem, and root periderm extracts. Falcarindiol and falcarinol predominated and accumulated primarily in the root periderm. Since the multiple double and triple carbon-carbon bonds that distinguish polyacetylenes from ubiquitous fatty acids are often introduced by Δ12 oleic acid desaturase (FAD2)-type enzymes, we mined the carrot genome for FAD2 genes. We identified a FAD2 family with an unprecedented 24 members and analyzed public, tissue-specific carrot RNA-Seq data to identify coexpressed members with root periderm-enhanced expression. Six candidate genes were heterologously expressed individually and in combination in yeast and Arabidopsis (Arabidopsis thaliana), resulting in the identification of one canonical FAD2 that converts oleic to linoleic acid, three divergent FAD2-like acetylenases that convert linoleic into crepenynic acid, and two bifunctional FAD2s with Δ12 and Δ14 desaturase activity that convert crepenynic into the further desaturated dehydrocrepenynic acid, a polyacetylene pathway intermediate. These genes can now be used as a basis for discovering other steps of falcarin-type polyacetylene biosynthesis, to modulate polyacetylene levels in plants, and to test the in planta function of these molecules.

Summary Strategies for improving plant size are critical targets for plant biotechnology to increase vegetative biomass or reproductive yield. To improve biomass production, a codon‐optimized helix–loop–helix transcription factor (VvCEB1 opt) from wine grape was overexpressed in Arabidopsis thaliana resulting in significantly increased leaf number, leaf and rosette area, fresh weight and dry weight. Cell size, but typically not cell number, was increased in all tissues resulting in increased vegetative biomass and reproductive organ size, number and seed yield. Ionomic analysis of leaves revealed the VvCEB1 opt‐overexpressing plants had significantly elevated, K, S and Mo contents relative to control lines. Increased K content likely drives increased osmotic potential within cells leading to greater cellular growth and expansion. To understand the mechanistic basis of VvCEB1 opt action, one transgenic line was genotyped using RNA‐Seq mRNA expression profiling and revealed a novel transcriptional reprogramming network with significant changes in mRNA abundance for genes with functions in delayed flowering, pathogen–defence responses, iron homeostasis, vesicle‐mediated cell wall formation and auxin‐mediated signalling and responses. Direct testing of VvCEB1 opt‐overexpressing plants showed that they had significantly elevated auxin content and a significantly increased number of lateral leaf primordia within meristems relative to controls, confirming that cell expansion and organ number proliferation were likely an auxin‐mediated process. VvCEB1 opt overexpression in Nicotiana sylvestris also showed larger cells, organ size and biomass demonstrating the potential applicability of this innovative strategy for improving plant biomass and reproductive yield in crops.

Opuntia ficus-indica (prickly pear) and Grindelia squarrosa (gumweed) are two exceptionally drought tolerant plant species capable of growing in arid and semiarid environments. Additionally, they have unique cell wall structures. Prickly pear contains pectin and high levels of ash (16.1%) that is predominantly Ca and K. Gumweed has high levels of extractives that contain grindelic acid and monoterpenoids. The objective of this paper was to evaluate how these unique cell wall components alter the pyrolysis performance of prickly pear and gumweed. Using a tandem micropyrolyzer with GC-MS/FID/TCD, a detailed account of the product slate is given for products generated between 450 and 650 °C. Pyrolysis of prickly pear showed that the high levels of ash increase the amount of organics volatilized and shifted product pools, making it possible to generate up to 7.3% carbonyls vs 3.8% for Pinus taeda (loblolly pine) and 10.5% hydrocarbons vs 1.8% for pine depending on reaction conditions. Pyrolysis of gumweed sho...

Global demand for food and bioenergy production has increased rapidly, while the area of arable land has been declining for decades due to damage caused by erosion, pollution, sea level rise, urban development, soil salinization, and water scarcity driven by global climate change. In order to overcome this conflict, there is an urgent need to adapt conventional agriculture to water-limited and hotter conditions with plant crop systems that display higher water-use efficiency (WUE). Crassulacean acid metabolism (CAM) species have substantially higher WUE than species performing C 3 or C 4 photosynthesis. CAM plants are derived from C 3 photosynthesis ancestors. However, it is extremely unlikely that the C 3 or C 4 crop plants would evolve rapidly into CAM photosynthesis without human intervention. Currently, there is growing interest in improving WUE through transferring CAM into C 3 crops. However, engineering a major metabolic plant pathway, like CAM, is challenging and requires a comprehensive deep understanding of the enzymatic reactions and regulatory networks in both C 3 and CAM photosynthesis, as well as overcoming physiometabolic limitations such as diurnal stomatal regulation. Recent advances in CAM evolutionary genomics research, genome editing, and synthetic biology have increased the likelihood of successful acceleration of C 3 -to-CAM progression. Here, we first summarize the systems biology-level understanding of the molecular processes in the CAM pathway. Then, we review the principles of CAM engineering in an evolutionary context. Lastly, we discuss the technical approaches to accelerate the C 3 -to-CAM transition in plants using synthetic biology toolboxes.

Tissue succulence (ratio of tissue water/leaf area or dry mass) or the ability to store water within living tissues is among the most successful adaptations to drought in the plant kingdom. This taxonomically widespread adaptation helps plants avoid the damaging effects of drought, and is often associated with the occupancy of epiphytic, epilithic, semi-arid and arid environments. Tissue succulence was engineered in Arabidopsis thaliana by overexpression of a codon-optimized helix-loop-helix transcription factor (VvCEB1opt ) from wine grape involved in the cell expansion phase of berry development. VvCEB1opt -overexpressing lines displayed significant increases in cell size, succulence and decreased intercellular air space. VvCEB1opt -overexpressing lines showed increased instantaneous and integrated water-use efficiency (WUE) due to reduced stomatal conductance caused by reduced stomatal aperture and density resulting in increased attenuation of water-deficit stress. VvCEB1opt -overexpressing lines also showed increased salinity tolerance due to reduced salinity uptake and dilution of internal Na+ and Cl- as well as other ions. Alterations in transporter activities were further suggested by media and apoplastic acidification, hygromycin B tolerance and changes in relative transcript abundance patterns of various transporters with known functions in salinity tolerance. Engineered tissue succulence might provide an effective strategy for improving WUE, drought avoidance or attenuation, salinity tolerance, and for crassulacean acid metabolism biodesign.

Gene tree discordance in large genomic data sets can be caused by evolutionary processes such as incomplete lineage sorting and hybridization, as well as model violation, and errors in data processing, orthology inference, and gene tree estimation. Species tree methods that identify and accommodate all sources of conflict are not available, but a combination of multiple approaches can help tease apart alternative sources of conflict. Here, using a phylotranscriptomic analysis in combination with reference genomes, we test a hypothesis of ancient hybridization events within the plant family Amaranthaceae s.l. that was previously supported by morphological, ecological, and Sanger-based molecular data. The data set included seven genomes and 88 transcriptomes, 17 generated for this study. We examined gene-tree discordance using coalescent-based species trees and network inference, gene tree discordance analyses, site pattern tests of introgression, topology tests, synteny analyses, and simulations. We found that a combination of processes might have generated the high levels of gene tree discordance in the backbone of Amaranthaceae s.l. Furthermore, we found evidence that three consecutive short internal branches produce anomalous trees contributing to the discordance. Overall, our results suggest that Amaranthaceae s.l. might be a product of an ancient and rapid lineage diversification, and remains, and probably will remain, unresolved. This work highlights the potential problems of identifiability associated with the sources of gene tree discordance including, in particular, phylogenetic network methods. Our results also demonstrate the importance of thoroughly testing for multiple sources of conflict in phylogenomic analyses, especially in the context of ancient, rapid radiations. We provide several recommendations for exploring conflicting signals in such situations. [Amaranthaceae; gene tree discordance; hybridization; incomplete lineage sorting; phylogenomics; species network; species tree; transcriptomics.]

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that exploits a temporal CO2 pump with nocturnal CO2 uptake and concentration to reduce photorespiration, improve water-use efficiency (WUE), and optimize the adaptability of plants to climates with seasonal or intermittent water limitations. CAM plants display a plastic continuum in the extent to which species engage in net nocturnal CO2 uptake that ranges from 0 to 100%. CAM plants also display diverse enzyme and organic acid and carbohydrate storage systems, which likely reflect the multiple, independent evolutionary origins of CAM. CAM is often accompanied by a diverse set of anatomical traits, such as tissue succulence and water-storage and water-capture strategies to attenuate drought. Other co-adaptive traits, such as thick cuticles, epicuticular wax, low stomatal density, high stomatal responsiveness, and shallow rectifier-like roots limit water loss under conditions of water deficit. Recommendations for future research efforts to better explore and understand the diversity of traits associated with CAM and CAM Biodesign efforts are presented.

Background Crassulacean acid metabolism (CAM), a specialized mode of photosynthesis, enables plant adaptation to water-limited environments and improves photosynthetic efficiency via an inorganic carbon-concentrating mechanism. Kalanchoë fedtschenkoi is an obligate CAM model featuring a relatively small genome and easy stable transformation. However, the molecular responses to light quality and intensity in CAM plants remain understudied. Results Here we present a genome-wide expression atlas of K. fedtschenkoi plants grown under 12 h/12 h photoperiod with different light quality (blue, red, far-red, white light) and intensity (0, 150, 440, and 1,000 μmol m–2 s–1) based on RNA sequencing performed for mature leaf samples collected at dawn (2 h before the light period) and dusk (2 h before the dark period). An eFP web browser was created for easy access of the gene expression data. Based on the expression atlas, we constructed a light-responsive co-expression network to reveal the potential regulatory relationships in K. fedtschenkoi. Measurements of leaf titratable acidity, soluble sugar, and starch turnover provided metabolic indicators of the magnitude of CAM under the different light treatments and were used to provide biological context for the expression dataset. Furthermore, CAM-related subnetworks were highlighted to showcase genes relevant to CAM pathway, circadian clock, and stomatal movement. In comparison with white light, monochrome blue/red/far-red light treatments repressed the expression of several CAM-related genes at dusk, along with a major reduction in acid accumulation. Increasing light intensity from an intermediate level (440 μmol m−2 s−1) of white light to a high light treatment (1,000 μmol m–2 s–1) increased expression of several genes involved in dark CO2 fixation and malate transport at dawn, along with an increase in organic acid accumulation. Conclusions This study provides a useful genomics resource for investigating the molecular mechanism underlying the light regulation of physiology and metabolism in CAM plants. Our results support the hypothesis that both light intensity and light quality can modulate the CAM pathway through regulation of CAM-related genes in K. fedtschenkoi.

An efficient plant regeneration procedure has been established from hypocotyl explants of the common ice plant, Mesembryanthemum crystallinum L, a halophytic leaf succulent that exhibits a stress-induced switch from C3 photosynthesis to crassulacean acid metabolism (CAM). Somatic embryos were initiated and developed up to globular and heart stages in Murashige and Skoog (MS) media supplemented with 3% sucrose, 0.6% bacto-agar, 80 mM NaCl, 5 μM 2,4-D and 1 μM kinetin. High frequency regeneration occurred when somatic embryos were germinated on media that lacked 2,4-D. High cytokinin treatment suppressed normal growth of embryos and favored abnormal embryo proliferation. Without growth regulators, regenerated plants rooted on MS medium with 100% efficiency. Mature, regenerated plants were fertile and morphologically identical to seed-derived plants.

Mesembryanthemum crystallinum L., a halophytic species, displays modified trichomes, epidermal bladder cells (EBC), on the surfaces of its aerial organs. EBCs serve to sequester excessive salt from underlying metabolically active tissues. To elucidate the molecular determinants governing EBC development in the common ice plant, we constructed a cDNA-based suppression subtractive hybridization library and identified genes differentially expressed between the wild-type and the EBC-less mutant. After hybridization, 38 clones were obtained. Among them, 24 clones had homology with plant genes of known functions, whose roles might not be directly related to EBC-morphology, while 14 clones were homologous to genes of unknown functions. After confirmation by northern blot analysis, 12 out of 14 clones of unknown functions were chosen for semi-quantitative RT-PCR analysis, and the results revealed that three clones designated as MW3, MW21, and MW31 preferentially expressed in the EBC-less mutant, whereas the other two designated as WM10 and WM28 preferentially expressed in the wild type. Among these genes, the expression of a putative jasmonate-induced gene, designated as WM28 was completely suppressed in the EBC-mutant. In addition, the deletion of C-box cis-acting element was found in the promoter region of WM28 in the EBC-less mutant. Overexpression of WM28 in Arabidopsis resulted in increased trichome number due to the upregulation of key trichome-related genes GLABRA1 (GL1), and GLABRA3 (GL3). These results demonstrate that WM28 can be an important factor responsible for EBC formation, and also suggest the similarity of developmental mechanism between trichome in Arabidopsis and EBC in common ice plant.

Life in high salinity environments poses challenges to cells in a variety of ways: maintenance of ion homeostasis and nutrient acquisition, often while concomitantly enduring saturating irradiances. Dunaliella salina has an exceptional ability to thrive even in saturated brine solutions. This ability has made it a model organism for studying responses to abiotic stress factors. Here we describe the occurrence of unique gene families, expansion of gene families, or gene losses that might be linked to osmoadaptive strategies. We discovered multiple unique genes coding for several of the homologous superfamily of the Ser-Thr-rich glycosyl-phosphatidyl-inositol-anchored membrane family and of the glycolipid 2-alpha-mannosyltransferase family, suggesting that such components on the cell surface are essential to life in high salt. Gene expansion was found in families that participate in sensing of abiotic stress and signal transduction in plants. One example is the patched family of the Sonic Hedgehog receptor proteins, supporting a previous hypothesis that plasma membrane sterols are important for sensing changes in salinities in D. salina. We also investigated genome-based capabilities regarding glycerol metabolism and present an extensive map for core carbon metabolism. We postulate that a second broader glycerol cycle exists that also connects to photorespiration, thus extending the previously described glycerol cycle. Further genome-based analysis of isoprenoid and carotenoid metabolism revealed duplications of genes for 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and phytoene synthase (PSY), with the second gene copy of each enzyme being clustered together. Moreover, we identified two genes predicted to code for a prokaryotic-type phytoene desaturase (CRTI), indicating that D. salina may have eukaryotic and prokaryotic elements comprising its carotenoid biosynthesis pathways. In brief, our genomic data provide the basis for further gene discoveries regarding sensing abiotic stress, the metabolism of this halophilic alga, and its potential in biotechnological applications.

Sustainable production of lignocellulosic biofuels requires a sufficient supply of biomass feedstocks. Agave and Opuntia represent highly water-use efficient bioenergy crops that are suitable for expanding feedstock production into semi-arid marginal lands. These feedstocks have garnered interest as dedicated biofuel feedstocks because of their high water- and fertilizer-use efficiency and not competing with major food crops or conventional biofuel feedstocks. To better understand the potential of these feedstocks, the biomass composition of Agave tequilana and Opuntia ficus-indica was analyzed. Previous extraction procedures and analytical methods have led to variable estimates of the chemical compositions of the biomass of these species. Therefore, National Renewable Energy Laboratory (NREL) standard methods were used in the present study. A. tequilana showed higher mass fractions of water-soluble constituents, structural carbohydrates, cellulose, hemicellulose, and lignin than O. ficus-indica. In contrast, O. ficus-indica had higher protein, water, and ash mass fractions than A. tequilana. Both species had lower lignin mass fractions, thus yielding lower heating values, but had higher water and ash mass fractions than most woody biomass feedstocks. The high water mass fractions of these species (85–94%) could prove advantageous for biomass deconstruction and aqueous phase catalytic conversion processes as less exogenous water inputs would be needed. Lastly, solid-state NMR analysis revealed that both A. tequilana and O. ficus-indica had high amorphous and para-crystalline cellulose mass fractions (>80%), indicating that these biomass feedstocks would be far less recalcitrant to deconstruction than traditional lignocellulosic biomass feedstocks.

Microalgae can serve as useful feedstocks for biofuel production as they can be grown with fresh, brackish, or salt water and their lipid and starch contents can be manipulated to create customized feedstocks for different classes of biofuels. Continuous buoyant density gradient centrifugation (CBDGC) was used to perform reiterative, transgressive selection to isolate wildtype and ethyl methanesulfonate-mutagenized Dunaliella salina cells with enhanced lipid and starch production. Sixty rounds of transgressive selection resulted in the isolation of cell populations with significantly lower or higher buoyant densities. Lipid content in the low-density populations was enhanced by 1.2- to 2.9-fold in wildtype cells and 1.3- to 2.3-fold in mutagenized cells as measured by Nile Red dye staining, but the lipid content differences were not significant when quantified by liquid chromatography–tandem mass spectroscopy possibly due to the composition of the lipid pools measured by these contrasting techniques. In contrast, starch content in the high-density populations was increased by 2-fold in wild type cells and 1.4- to 1.6-fold in mutagenized cells, respectively. The observed alterations in lipid and starch contents appeared to be stable after more than 70 weeks (392 cell generations). CBDGC-based selection provides a useful and accessible technological alternative to genetic engineering approaches for the customization of microbial biofuel feedstocks.

Global climate change is predicted to increase heat, drought, and soil-drying conditions, and thereby increase crop sensitivity to water vapour pressure deficit, resulting in productivity losses. Increasing competition between agricultural freshwater use and municipal or industrial uses suggest that crops with greater heat and drought durability and greater water-use efficiency will be crucial for sustainable biomass production systems in the future. Agave (Agavaceae) and Opuntia (Cactaceae) represent highly water-use efficient bioenergy crops that could diversify bioenergy feedstock supply yet preserve or expand feedstock production into semi-arid, abandoned, or degraded agricultural lands, and reclaim drylands. Agave and Opuntia are crassulacean acid metabolism species that can achieve high water-use efficiencies and grow in water-limited areas with insufficient precipitation to support traditional C3 or C4 bioenergy crops. Both Agave and Opuntia have the potential to produce above-ground biomass rivalling that of C3 and C4 crops under optimal growing conditions. The low lignin and high amorphous cellulose contents of Agave and Opuntia lignocellulosic biomass will be less recalcitrant to deconstruction than traditional feedstocks, as confirmed by pretreatments that improve saccharification of Agave. Refined environmental productivity indices and geographical information systems modelling have provided estimates of Agave and Opuntia biomass productivity and terrestrial sequestration of atmospheric CO2; however, the accuracy of such modelling efforts can be improved through the expansion of field trials in diverse geographical settings. Lastly, life cycle analysis indicates that Agave would have productivity, life cycle energy, and greenhouse gas balances comparable or superior to those of traditional bioenergy feedstocks, but would be far more water-use efficient.

Due to public concerns about the decreasing supply of blue water and increasing heat and drought stress on plant growth caused by urbanization, increasing human population and climate change, interest in crassulacean acid metabolism (CAM), a specialized type of photosynthesis enhancing water-use efficiency (WUE) and drought tolerance, has increased markedly. Significant progress has been achieved in both basic and applied research in CAM plants since the beginning of this century. Here we provide a brief overview of the current status of CAM research, and discuss future needs and opportunities in a wide range of areas including systems biology, synthetic biology, and utilization of CAM crops for human benefit, with a focus on the following aspects: 1) application of genome-editing technology and high-throughput phenotyping to functional genomics research in model CAM species and genetic improvement of CAM crops, 2) challenges for multi-scale metabolic modeling of CAM systems, 3) opportunities and new strategies for CAM pathway engineering to enhance WUE and drought tolerance in C3 (and C4) photosynthesis crops, 4) potential of CAM species as resources for food, feed, natural products, pharmaceuticals and biofuels, and 5) development of CAM crops for ecological and aesthetic benefits.

The halotolerant alga Dunaliella salina is a model for stress tolerance and is used commercially for production of beta-carotene (=pro-vitamin A). The presented draft genome of the genuine strain CCAP19/18 will allow investigations into metabolic processes involved in regulation of stress responses, including carotenogenesis and adaptations to life in high-salinity environments.

RLSB occurs in photosynthetic tissues and is post-transcriptionally regulated by light. Modification to bundle sheath specificity correlated with minor sequence divergence and the ontogeny of Kranz development during C4 evolution, Nuclear-encoded RLSB protein binds chloroplastic rbcL mRNA encoding the Rubisco large subunit. RLSB is highly conserved across all groups of land plants and is associated with positive post-transcriptional regulation of rbcL expression. In C3 leaves, RLSB and Rubisco occur in all chlorenchyma cell chloroplasts, while in C4 leaves these accumulate only within bundle sheath (BS) chloroplasts. RLSB’s role in rbcL expression makes modification of its localization a likely prerequisite for the evolutionary restriction of Rubisco to BS cells. Taking advantage of evolutionarily conserved RLSB orthologs in several C3, C3–C4, C4-like, and C4 photosynthetic types within the genus Flaveria, we show that low level RLSB sequence divergence and modification to BS specificity coincided with ontogeny of Rubisco specificity and Kranz anatomy during C3 to C4 evolution. In both C3 and C4 species, Rubisco production reflected RLSB production in all cell types, tissues, and conditions examined. Co-localization occurred only in photosynthetic tissues, and both proteins were co-ordinately induced by light at post-transcriptional levels. RLSB is currently the only mRNA-binding protein to be associated with rbcL gene regulation in any plant, with variations in sequence and acquisition of cell type specificity reflecting the progression of C4 evolution within the genus Flaveria.

Nocturnal degradation of transitory starch is a limiting factor for the optimal function of crassulacean acid metabolism and must be coordinated with phosphoenolypyruvate carboxylase (PEPC)-mediated CO2 uptake to optimise carbon gain over the diel cycle. The aim of this study was to test the hypothesis that nocturnal carboxylation is coordinated with starch degradation in CAM via a mechanism whereby the products of these pathways regulate diel transcript abundance and enzyme activities for both processes. To test this hypothesis, a starch and CAM-deficient mutant of Mesembryanthemum crystallinum was compared with wild type plants under well-watered and saline (CAM-inducing) conditions. Exposure to salinity increased the transcript abundance of genes required for nocturnal carboxylation, starch and sucrose degradation in both wild type and mutant, but the transcript abundance of several of these genes was not sustained over the dark period in the low-carbohydrate, CAM-deficient mutant. The diel pattern of transcript abundance for PEPC mirrored that of PEPC protein, as did the transcripts, protein, and activity of chloroplastic starch phosphorylase in both wild type and mutant, suggesting robust diel coordination of these metabolic processes. Activities of several amylase isoforms were low or lacking in the mutant, whilst the activity of a cytosolic isoform of starch phosphorylase was significantly elevated, indicating contrasting modes of metabolic regulation for the hydrolytic and phosphorylytic routes of starch degradation. Externally supplied sucrose resulted in an increase in nocturnal transcript abundance of genes required for nocturnal carboxylation and starch degradation. These results demonstrate that carbohydrates impact on transcriptional and post-transcriptional regulation of nocturnal carboxylation and starch degradation in CAM.

Global climate change threatens the sustainability of agriculture and agroforestry worldwide through increased heat, drought, surface evaporation and associated soil drying. Exposure of crops and forests to warmer and drier environments will increase leaf:air water vapour-pressure deficits (VPD), and will result in increased drought susceptibility and reduced productivity, not only in arid regions but also in tropical regions with seasonal dry periods. Fast-growing, short-rotation forestry (SRF) bioenergy crops such as poplar (Populus spp.) and willow (Salix spp.) are particularly susceptible to hydraulic failure following drought stress due to their isohydric nature and relatively high stomatal conductance. One approach to sustaining plant productivity is to improve water-use efficiency (WUE) by engineering crassulacean acid metabolism (CAM) into C3 crops. CAM improves WUE by shifting stomatal opening and primary CO2 uptake and fixation to the night-time when leaf:air VPD is low. CAM members of the tree genus Clusia exemplify the compatibility of CAM performance within tree species and highlight CAM as a mechanism to conserve water and maintain carbon uptake during drought conditions. The introduction of bioengineered CAM into SRF bioenergy trees is a potentially viable path to sustaining agroforestry production systems in the face of a globally changing climate.

Summary Multiple isoforms of phosphoenolpyruvate carboxylase genes were sequenced from related orchid species with distinct photosynthesic types. Phylogenetic analysis indicated that CAM-associated isoforms originated from gene duplications and adaptive sequence divergence., Phosphoenolpyruvate carboxylase (PEPC) catalyses the initial fixation of atmospheric CO2 into oxaloacetate and subsequently malate. Nocturnal accumulation of malic acid within the vacuole of photosynthetic cells is a typical feature of plants that perform crassulacean acid metabolism (CAM). PEPC is a ubiquitous plant enzyme encoded by a small gene family, and each member encodes an isoform with specialized function. CAM-specific PEPC isoforms probably evolved from ancestral non-photosynthetic isoforms by gene duplication events and subsequent acquisition of transcriptional control elements that mediate increased leaf-specific or photosynthetic-tissue-specific mRNA expression. To understand the patterns of functional diversification related to the expression of CAM, ppc gene families and photosynthetic patterns were characterized in 11 closely related orchid species from the subtribe Oncidiinae with a range of photosynthetic pathways from C3 photosynthesis (Oncidium cheirophorum, Oncidium maduroi, Rossioglossum krameri, and Oncidium sotoanum) to weak CAM (Oncidium panamense, Oncidium sphacelatum, Gomesa flexuosa and Rossioglossum insleayi) and strong CAM (Rossioglossum ampliatum, Trichocentrum nanum, and Trichocentrum carthagenense). Phylogenetic analysis revealed the existence of two main ppc lineages in flowering plants, two main ppc lineages within the eudicots, and three ppc lineages within the Orchidaceae. Our results indicate that ppc gene family expansion within the Orchidaceae is likely to be the result of gene duplication events followed by adaptive sequence divergence. CAM-associated PEPC isoforms in the Orchidaceae probably evolved from several independent origins.

Climatic extremes threaten agricultural sustainability worldwide. One approach to increase plant water-use efficiency (WUE) is to introduce crassulacean acid metabolism (CAM) into C3 crops. Such a task requires comprehensive systems-level understanding of the enzymatic and regulatory pathways underpinning this temporal CO2 pump. Here we review the progress that has been made in achieving this goal. Given that CAM arose through multiple independent evolutionary origins, comparative transcriptomics and genomics of taxonomically diverse CAM species are being used to define the genetic 'parts list' required to operate the core CAM functional modules of nocturnal carboxylation, diurnal decarboxylation, and inverse stomatal regulation. Engineered CAM offers the potential to sustain plant productivity for food, feed, fiber, and biofuel production in hotter and drier climates.

To meet future food and energy security needs, which are amplified by increasing population growth and reduced natural resource availability, metabolic engineering efforts have moved from manipulating single genes/proteins to introducing multiple genes and novel pathways to improve photosynthetic efficiency in a more comprehensive manner. Biochemical carbon-concentrating mechanisms such as crassulacean acid metabolism (CAM), which improves photosynthetic, water-use, and possibly nutrient-use efficiency, represent a strategic target for synthetic biology to engineer more productive C3 crops for a warmer and drier world. One key challenge for introducing multigene traits like CAM onto a background of C3 photosynthesis is to gain a better understanding of the dynamic spatial and temporal regulatory events that underpin photosynthetic metabolism. With the aid of systems and computational biology, vast amounts of experimental data encompassing transcriptomics, proteomics, and metabolomics can be related in a network to create dynamic models. Such models can undergo simulations to discover key regulatory elements in metabolism and suggest strategic substitution or augmentation by synthetic components to improve photosynthetic performance and water-use efficiency in C 3 crops. Another key challenge in the application of synthetic biology to photosynthesis research is to develop efficient systems for multigene assembly and stacking. Here, we review recent progress in computational modelling as applied to plant photosynthesis, with attention to the requirements for CAM, and recent advances in synthetic biology tool development. Lastly, we discuss possible options for multigene pathway construction in plants with an emphasis on CAM-into-C3 engineering.