Background: The ubiquitous, non-proteinaceous amino acid GABA (γ-aminobutyrate) accumulates in plants subjected to abiotic stresses such as chilling, O₂ deficiency and elevated CO₂. Recent evidence indicates that controlled atmosphere storage causes the accumulation of GABA in apple (Malus x domestica Borkh.) fruit, and now there is increasing interest in the biochemical mechanisms responsible for this phenomenon. Here, we investigated whether this phenomenon could be mediated via Ca²⁺/calmodulin (CaM) activation of glutamate decarboxylase (GAD) activity. Results: GAD activity in cell-free extracts of apple fruit was stimulated by Ca²⁺/CaM at physiological pH, but not at the acidic pH optimum. Based on bioinformatics analysis of the apple genome, three apple GAD genes were identified and their expression determined in various apple organs, including fruit. Like recombinant Arabidopsis GAD1, the activity and spectral properties of recombinant MdGAD1 and MdGAD2 were regulated by Ca²⁺/CaM at physiological pH and both enzymes possessed a highly conserved CaM-binding domain that was autoinhibitory. In contrast, the activity and spectral properties of recombinant MdGAD3 were not affected by Ca²⁺/CaM and they were much less sensitive to pH than MdGAD1, MdGAD2 and Arabidopsis GAD1; furthermore, the C-terminal region neither bound CaM nor functioned as an autoinhibitory domain. Conclusions: Plant GADs typically differ from microbial and animal GAD enzymes in possessing a C-terminal 30–50 amino acid residue CaM-binding domain. To date, rice GAD2 is the only exception to this generalization; notably, the C-terminal region of this enzyme still functions as an autoinhibitory domain. In the present study, apple fruit were found to contain two CaM-dependent GADs, as well as a novel CaM-independent GAD that does not possess a C-terminal autoinhibitory domain.

Edible dry beans (Phaseolus vulgaris L.) that darken during postharvest storage are graded lower and are less marketable than their non-darkened counterparts. Seed coat darkening in susceptible genotypes is dependent upon the availability of proanthocyanidins, and their subsequent oxidation to reactive quinones. Mature cranberry beans lacking this postharvest darkening trait tend to be proanthocyanidin-deficient, although the underlying molecular and biochemical determinants for this metabolic phenomenon are unknown. Seed coat proanthocyanidin levels increased with plant maturation in a darkening-susceptible cranberry bean recombinant inbred line (RIL), whereas these metabolites were absent in seeds of the non-darkening RIL plants. RNA sequencing (RNA-seq) analysis was used to monitor changes in the seed coat transcriptome as a function of bean development, where transcript levels were measured as fragments per kilobase of exon per million fragments mapped. A total of 1336 genes were differentially expressed between darkening and non-darkening cranberry bean RILs. Structural and regulatory genes of the proanthocyanidin biosynthesis pathway were upregulated in seed coats of the darkening RIL. A principal component analysis determined that changes in transcript levels for two genes of unknown function and three proanthocyanidin biosynthesis genes, FLAVANONE 3-HYDROXYLASE 1, DIHYDROFLAVONOL 4-REDUCTASE 1 and ANTHOCYANIDIN REDUCTASE 1 (PvANR1) were highly correlated with proanthocyanidin accumulation in seed coats of the darkening-susceptible cranberry bean RIL. HPLC-DAD analysis revealed that in vitro activity of a recombinant PvANR1 was NADPH-dependent and assays containing cyanidin yielded epicatechin and catechin; high cyanidin substrate levels inhibited the formation of both of these products. Proanthocyanidin oxidation is a pre-requisite for postharvest-related seed coat darkening in dicotyledonous seeds. In model plant species, the accumulation of proanthocyanidins is dependent upon upregulation of biosynthetic genes. In this study, proanthocyanidin production in cranberry bean seed coats was strongly associated with an increase in PvANR1 transcripts during seed maturation. In the presence of NADPH, PvANR1 converted the physiologically relevant substrate cyanidin to epicatechin and catechin.

Microgreens are specialty vegetables that contain human health-promoting phytochemicals. Typically, microgreens are cultivated in controlled environments under red and blue light-emitting diodes (LEDs). However, the impact of varying the proportions of these light qualities on the composition of diverse phytochemicals in indoor-grown microgreens is unclear. To address this problem, the levels of chlorophylls, carotenoids, ascorbates, phenolics, anthocyanins, and nitrate were examined in arugula ( Eruca sativa L.), ‘Red Russian’ kale [ Brassica napus L. subsp. napus var. pabularia (DC.) Alef.], ‘Mizuna’ mustard ( Brassica juncea L.), and red cabbage ( Brassica oleracea L. var. capitata f. rubra ) microgreens following cultivation under LEDs supplying varying proportions of blue light (5% to 30%) and red light (70% to 95%). Varying the proportion of blue light did not affect the extractable levels of total chlorophyll, total carotenoids, or nitrate in all four microgreen species. Generally, the levels of reduced and total ascorbate were greatest in arugula, kale, and mustard microgreens at 20% blue light, and a minor decrease was apparent at 30% blue light. These metabolite profiles were not impacted by the blue light percentage in red cabbage. Kale and mustard accumulated more total phenolics at 30% blue light than all other blue light regimens; however, this phytochemical attribute was unaffected in arugula and red cabbage. The total anthocyanin concentration increased proportionally with the percentage of supplied blue light up to 30% in all microgreens, with the exception of mustard. Our research showed that 20% blue light supplied from LED arrays is ideal for achieving optimal levels of both reduced and total ascorbate in all microgreens except red cabbage, and that 30% blue light promotes the greatest accumulation of total anthocyanin in indoor-grown Brassicaceae microgreens, with the exception of mustard.