Multiple mechanisms explain loss of anthocyanins from betalain-pigmented Caryophyllales, including repeated wholesale loss of a key anthocyanidin synthesis enzyme.

In this study, we investigate the genetic mechanisms responsible for the loss of anthocyanins in betalain-pigmented Caryophyllales, considering our hypothesis of multiple transitions to betalain pigmentation. Utilizing transcriptomic and genomic datasets across 357 species and 31 families, we scrutinize 18 flavonoid pathway genes and six regulatory genes spanning four transitions to betalain pigmentation. We examined evidence for hypotheses of wholesale gene loss, modified gene function, altered gene expression, and degeneration of the MBW (MYB-bHLH-WD40) trasnscription factor complex, within betalain-pigmented lineages. Our analyses reveal that most flavonoid synthesis genes remain conserved in betalain-pigmented lineages, with the notable exception of TT19 orthologs, essential for the final step in anthocyanidin synthesis, which appear to have been repeatedly and entirely lost. Additional late-stage flavonoid pathway genes upstream of TT19 also manifest strikingly reduced expression in betalain-pigmented species. Additionally, we find repeated loss and alteration in the MBW transcription complex essential for canonical anthocyanin synthesis. Consequently, the loss and exclusion of anthocyanins in betalain-pigmented species appear to be orchestrated through several mechanisms: loss of a key enzyme, downregulation of synthesis genes, and degeneration of regulatory complexes. These changes have occurred iteratively in Caryophyllales, often coinciding with evolutionary transitions to betalain pigmentation.

Epidermal bladder cells as a herbivore defense mechanism.

The aerial surfaces of quinoa (Chenopodium quinoa) and common ice plant (Mesembryanthemum crystallinum) are covered with a layer of epidermal bladder cells (EBCs), which are modified non-glandular trichomes previously considered to be key to the extreme salt and drought tolerance of these plants. Here, however, we find that EBCs of these plants play only minor roles, if any, in abiotic stress tolerance and in fact are detrimental under conditions of water deficit. We report that EBCs instead function as deterrents to a broad range of generalist arthropod herbivores, through their combined function of forming both a chemical and a physical barrier, and they also serve a protective function against a phytopathogen. Our study overturns current models that link EBCs to salt and drought tolerance and assigns new functions to these structures that might provide novel possibilities for protecting crops from arthropod pests.

Tissue succulence in plants: Carrying water for climate change.

Tissue succulence in plants involves the storage of water in one or more organs or tissues to assist in maintaining water potentials on daily or seasonal time scales. This drought-avoidance or drought-resistance strategy allows plants to occupy diverse environments including arid regions, regions with rocky soils, epiphytic habitats, and saline soils. Climate-resilient strategies are of increasing interest in the context of the global climate crisis, which is leading to hotter and drier conditions in many regions throughout the globe. Here, we describe a short history of succulent plants, the basic concepts of tissue succulence, the anatomical diversity of succulent morphologies and associated adaptive traits, the evolutionary, phylogenetic, and biogeographical diversity of succulent plants, extinction risks to succulents due to poaching from their natural environments, and the myriad uses and applications of economically important succulent species and the products derived from them. Lastly, we discuss current prospects for engineering tissue succulence to improve salinity and drought tolerance in crops.

Developmental dynamics of crassulacean acid metabolism (CAM) in Opuntia ficus-indica.

BACKGROUND AND AIMS: The relative contributions 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, δ13C 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 became highly significant by 75 days of development. Seedling cotyledons showed mean δ13C values of -21.4 and -17.1 ‰ at 30 and 100 days of age, respectively. Seedling cladodes showed mean δ13C values of -19.4 and -14.5 ‰ at 30 and 100 days of age, respectively. These values are 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 5 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 δ13C values of -14.5 to -15.6 ‰, typical of CAM plants. CO2 assimilation studies revealed that net CO2 uptake was negative in daughter cladodes <12 cm in length, but then exhibited net positive CO2 assimilation in both the day and the night, with net nocturnal CO2 assimilation predominating once the daughter cladode grew larger. 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.

The starch-deficient plastidic PHOSPHOGLUCOMUTASE mutant of the constitutive crassulacean acid metabolism (CAM) species Kalanchoë fedtschenkoi impacts diel regulation and timing of stomatal CO2 responsiveness.

BACKGROUND AND AIMS: Crassulacean 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. METHODS: Biochemical 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 RESULTS: Starch 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. CONCLUSIONS: Nocturnal 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.

The final piece of the Triangle of U: Evolution of the tetraploid Brassica carinata genome.

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.

Realizing the Potential of Camelina sativa as a Bioenergy Crop for a Changing Global Climate.

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.

Characterization of a microbial consortium with potential for biological degradation of cactus pear biomass for biofuel production.

Cactus pear (Opuntia ficus-indica) is a crassulacean acid metabolism (CAM) species that serves as a food, feed, and bioenergy crop. O. ficus-indica is an attractive alternative biofuel feedstock due to its low water demand and high biomass productivity. Current ethanol yields from O. ficus-indica are not commercially viable due to low concentrations of released fermentable carbohydrates. Axenic strains of bacteria and fungi were isolated and characterized from a soil microbial community consortium that effectively degrades cladodes into soluble components. The consortium consisted of species representing 14 genera of eubacteria and four genera of fungi. The digestion efficiency of each axenic isolate was evaluated by measuring the release of soluble material after aerobic digestion of cladodes and direct measurement of cellulase and pectinase activities in the culture supernatants. Pectobacterium cacticida was the most effective eubacterial species identified for degrading cladodes among all isolates evaluated. Thus, P. cacticida holds great promise for increasing the release of fermentable sugars and improving overall ethanol yields.

Membrane Profiling by Free Flow Electrophoresis and SWATH-MS to Characterize Subcellular Compartment Proteomes in Mesembryanthemum crystallinum.

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.

Metabolic profiling of epidermal and mesophyll tissues under water-deficit stress in Opuntia ficus-indica reveals stress-adaptive metabolic responses.

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.

Disentangling Sources of Gene Tree Discordance in Phylogenomic Data Sets: Testing Ancient Hybridizations in Amaranthaceae s.l.

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.].

Plant tissue succulence engineering improves water-use efficiency, water-deficit stress attenuation and salinity tolerance in Arabidopsis.

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.

Light-responsive expression atlas reveals the effects of light quality and intensity in Kalanchoë fedtschenkoi, a plant with crassulacean acid metabolism.

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.

Biosystems Design to Accelerate C3-to-CAM Progression.

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 C3 or C4 photosynthesis. CAM plants are derived from C3 photosynthesis ancestors. However, it is extremely unlikely that the C3 or C4 crop plants would evolve rapidly into CAM photosynthesis without human intervention. Currently, there is growing interest in improving WUE through transferring CAM into C3 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 C3 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 C3-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 C3-to-CAM transition in plants using synthetic biology toolboxes.

Evolution of l-DOPA 4,5-dioxygenase activity allows for recurrent specialisation to betalain pigmentation in Caryophyllales.

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.

The bracteatus pineapple genome and domestication of clonally propagated crops.

Domestication of clonally propagated crops such as pineapple from South America was hypothesized to be a 'one-step operation'. We sequenced the genome of Ananas comosus var. bracteatus CB5 and assembled 513 Mb into 25 chromosomes with 29,412 genes. Comparison of the genomes of CB5, F153 and MD2 elucidated the genomic basis of fiber production, color formation, sugar accumulation and fruit maturation. We also resequenced 89 Ananas genomes. Cultivars 'Smooth Cayenne' and 'Queen' exhibited ancient and recent admixture, while 'Singapore Spanish' supported a one-step operation of domestication. We identified 25 selective sweeps, including a strong sweep containing a pair of tandemly duplicated bromelain inhibitors. Four candidate genes for self-incompatibility were linked in F153, but were not functional in self-compatible CB5. Our findings support the coexistence of sexual recombination and a one-step operation in the domestication of clonally propagated crops. This work guides the exploration of sexual and asexual domestication trajectories in other clonally propagated crops.