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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 ofTT19orthologs, essential for the final step in anthocyanidin synthesis, which appear to have been repeatedly and entirely lost. Additional late‐stage flavonoid pathway genes upstream ofTT19also 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.

 

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.

 

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. 

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 fromRBOHDorZAT12to 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 asRbohD,ZAT12,SOS2andPR5suggesting these ROS bioreporters provide a robust indicator of increased ROS related to stress responses. Based upon the spatiotemporal response patterns of signal increase,ZAT12promoter‐dependent ROS (Zat12p‐ROS) bioreporter appears to be suitable for cellular mapping of ROS changes in response to abiotic and biotic stresses.

 

The evolution ofl‐DOPA4,5‐dioxygenase activity, encoded by the geneDODA, was a key step in the origin of betalain biosynthesis in Caryophyllales. We previously proposed thatl‐DOPA4,5‐dioxygenase activity evolved via a single Caryophyllales‐specific neofunctionalisation event within theDODAgene lineage. However, this neofunctionalisation event has not been confirmed and theDODAgene lineage exhibits numerous gene duplication events, whose evolutionary significance is unclear.

To address this, we functionally characterised 23 distinctDODAproteins forl‐DOPA4,5‐dioxygenase activity, from four betalain‐pigmented and five anthocyanin‐pigmented species, representing key evolutionary transitions across Caryophyllales. By mapping these functional data to an updatedDODAphylogeny, we then explored the evolution ofl‐DOPA4,5‐dioxygenase activity.

We find that lowl‐DOPA4,5‐dioxygenase activity is distributed across theDODAgene lineage. In this context, repeated gene duplication events within theDODAgene lineage give rise to polyphyletic occurrences of elevatedl‐DOPA4,5‐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 elevatedl‐DOPA4,5‐dioxygenase activity is consistent with recurrent specialisation to betalain synthesis in Caryophyllales.

 

The altered carbon assimilation pathway of crassulacean acid metabolism (CAM) photosynthesis results in an up to 80% higher water‐use efficiency than C₃photosynthesis in plants making it a potentially useful pathway for engineering crop plants with improved drought tolerance. Here we surveyed detailed temporal (diel time course) and spatial (across a leaf gradient) gene and microRNA(miRNA) expression patterns in the obligateCAMplant pineapple [Ananas comosus(L.) Merr.]. The high‐resolution transcriptome atlas allowed us to distinguish betweenCAM‐related and non‐CAMgene copies. A differential gene co‐expression network across green and white leaf diel datasets identified genes with circadian oscillation,CAM‐related functions, and source‐sink relations. Gene co‐expression clusters containingCAMpathway genes are enriched with clock‐associatedcis‐elements, suggesting circadian regulation ofCAM. About 20% of pineapple microRNAs have diel expression patterns, with several that target keyCAM‐related genes. Expression and physiology data provide a model forCAM‐specific carbohydrate flux and long‐distance hexose transport. Together these resources provide a list of candidate genes for targeted engineering ofCAMinto C₃photosynthesis crop species.