Anthocyanin Production Research Articles

Anthocyanin Production from Plant Cell and Organ Cultures In Vitro.

Anthocyanins are water-soluble pigments found in plants. They exist in various colors, including red, purple, and blue, and are utilized as natural colorants in the food and cosmetics industries. The pharmaceutical industry uses anthocyanins as therapeutic compounds because they have several medicinal qualities, including anti-obesity, anti-cancer, antidiabetic, neuroprotective, and cardioprotective effects. Anthocyanins are conventionally procured from colored fruits and vegetables and are utilized in the food, pharmaceutical, and cosmetic industries. However, the composition and concentration of anthocyanins from natural sources vary quantitively and qualitatively; therefore, plant cell and organ cultures have been explored for many decades to understand the production of these valuable compounds. A great deal of research has been carried out on plant cell cultures using varied methods, such as the selection of suitable cell lines, medium optimization, optimization culture conditions, precursor feeding, and elicitation for the production of anthocyanin pigments. In addition, metabolic engineering technologies have been applied for the hyperaccumulation of these compounds in varied plants, including tobacco and arabidopsis. In this review, we describe various strategies applied in plant cell and organ cultures for the production of anthocyanins.

Unveiling the evolutionary history of lingonberry (Vaccinium vitis-idaea L.) through genome sequencing and assembly of European and North American subspecies.

Lingonberry (Vaccinium vitis-idaea L.) produces tiny red berries that are tart and nutty in flavor. It grows widely in the circumpolar region, including Scandinavia, northern parts of Eurasia, Alaska, and Canada. Although cultivation is currently limited, the plant has a long history of cultural use among indigenous communities. Given its potential as a food source, genomic resources for lingonberry are significantly lacking. To advance genomic knowledge, the genomes for 2 subspecies of lingonberry (V. vitis-idaea ssp. minus and ssp. vitis-idaea var. 'Red Candy') were sequenced and de novo assembled into contig-level assemblies. The assemblies were scaffolded using the bilberry genome (Vaccinium myrtillus) to generate a chromosome-anchored reference genome consisting of 12 chromosomes each with a total length of 548.07 Mb [contig N50 = 1.17 Mb, BUSCO (C%) = 96.5%] for ssp. vitis-idaea and 518.70 Mb [contig N50 = 1.40 Mb, BUSCO (C%) = 96.9%] for ssp. minus. RNA-seq-based gene annotation identified 27,243 and 25,718 genes on the respective assembly, and transposable element detection methods found that 45.82 and 44.58% of the genome were repeats. Phylogenetic analysis confirmed that lingonberry was most closely related to bilberry and was more closely related to blueberries than cranberries. Estimates of past effective population size suggested a continuous decline over the past 1-3 MYA, possibly due to the impacts of repeated glacial cycles during the Pleistocene leading to frequent population fragmentation. The genomic resource created in this study can be used to identify industry-relevant genes (e.g. anthocyanin production), infer phylogeny, and call sequence-level variants (e.g. SNPs) in future research.

Exogenous abscisic acid and sugar induce a cascade of ripening events associated with anthocyanin accumulation in cultured Pinot Noir grape berries.

Fruit quality is dependent on various factors including flavour, texture and colour. These factors are determined by the ripening process, either climacteric or non-climacteric. In grape berry, which is non-climacteric, the process is signalled by a complex set of hormone changes. Abscisic acid (ABA) is one of the key hormones involved in ripening, while sugar availability also plays a significant role in certain ripening aspects such as anthocyanin production. To understand the relative influence of hormone and sugar signalling in situ can prove problematic due to the physiological and environmental (abiotic and biotic) factors at play in vineyards. Here we report on the use of in vitro detached berry culture to investigate the comparative significance of ABA and sugar in the regulation of Pinot noir berry anthocyanin production under controlled conditions. Using a factorial experimental design, pre-véraison berries were cultured on media with various concentrations of sucrose and ABA. After 15 days of in vitro culture, the berries were analysed for changes in metabolites, hormones and gene expression. Results illustrated a stimulatory effect of sucrose and ABA on enhancing berry colour and a corresponding increase in anthocyanins. Increased ABA concentration was able to boost anthocyanin production in berries when sucrose supply was low. The sucrose and ABA effects on berry anthocyanins were primarily manifested through the up-regulation of transcription factors and other genes in the phenylpropanoid pathway, while in other parts of the pathway a down-regulation of key proanthocyanindin transcription factors and genes corresponded to sharp reduction in berry proanthocyanidins, irrespective of sucrose supply. Similarly, increased ABA was correlated with a significant reduction in berry malic acid and associated regulatory genes. These findings suggest a predominance of berry ABA over berry sugar in coordinating the physiological and genetic regulation of anthocyanins and proanthocyanins in Pinot noir grape berries.

Morpho-physiological, biochemical, and molecular responses of lettuce (Lactuca sativa L.) seedlings to chlortetracycline stress

Antibiotic contamination in soil is an emerging environmental problem due to its potential threat to the soil ecosystem. Among antibiotics, chlortetracycline (CTC) is widely used for livestock and accumulates in agricultural soil, leading to detrimental effects on various physiological and biochemical metabolic processes in crops. However, the adverse impacts of CTC on plants and their defensive mechanisms remain poorly understood. We aimed to investigate the physiological, biochemical, and molecular responses of lettuce to CTC toxicity and to understand better the regulatory mechanism influencing the growth and development of plants. In this study, lettuce seedlings were exposed to different CTC doses (0, 0.01, 0.1, 0.5, and 1 mg L–1) and grown for 4 weeks in a hydroponic system. The results showed that an initial CTC dose (0.01 mg L–1) induced a hormesis phenomenon in the seedling's growth accompanied by upregulation of anthocyanin production, and their biosynthesis and regulation-related genes (e.g., F3H, F3′H, DFR, UFGT, ANS, and MYB) were found upregulated. While increasing CTC doses displayed stimulated phenolic acid and flavonoid contents, it had negative impacts on plant growth and leaf fresh/dry biomass. A progressive increase in the contents of H2O2 and MDA in the plants was found as the CTC dose increased, while the relative water content (RWC) level decreased. The activities of antioxidant enzymes, like superoxide dismutase, catalase, ascorbate peroxidase, and peroxidase, displayed differential responses to CTC. In addition, CTC doses, especially at 0.5 and 1 mg L–1, tended to promote the production of abscisic acid and jasmonic acid, but salicylic acid remained unaltered. Overall, this study provides a new perspective insight into the toxic effects of CTC and the resilience mechanisms interlinked in lettuce plants' survival against antibiotic contamination.

A transient expression tool box for anthocyanin biosynthesis in Nicotiana benthamiana.

Transient expression in Nicotiana benthamiana offers a robust platform for the rapid production of complex secondary metabolites. It has proven highly effective in helping identify genes associated with pathways responsible for synthesizing various valuable natural compounds. While this approach has seen considerable success, it has yet to be applied to uncovering genes involved in anthocyanin biosynthetic pathways. This is because only a single anthocyanin, delphinidin 3-O-rutinoside, can be produced in N. benthamiana by activation of anthocyanin biosynthesis using transcription factors. The production of other anthocyanins would necessitate the suppression of certain endogenous flavonoid biosynthesis genes while transiently expressing others. In this work, we present a series of tools for the reconstitution of anthocyanin biosynthetic pathways in N. benthamiana leaves. These tools include constructs for the expression or silencing of anthocyanin biosynthetic genes and a mutant N. benthamiana line generated using CRISPR. By infiltration of defined sets of constructs, the basic anthocyanins pelargonidin 3-O-glucoside, cyanidin 3-O-glucoside and delphinidin 3-O-glucoside could be obtained in high amounts in a few days. Additionally, co-infiltration of supplementary pathway genes enabled the synthesis of more complex anthocyanins. These tools should be useful to identify genes involved in the biosynthesis of complex anthocyanins. They also make it possible to produce novel anthocyanins not found in nature. As an example, we reconstituted the pathway for biosynthesis of Arabidopsis anthocyanin A5, a cyanidin derivative and achieved the biosynthesis of the pelargonidin and delphinidin variants of A5, pelargonidin A5 and delphinidin A5.

Production of species-specific anthocyanins through an inducible system in plant hairy roots

Anthocyanins are widely distributed pigments in flowering plants with red, purple or blue colours. Their properties in promoting heath make anthocyanins perfect natural colourants for food additives. However, anthocyanins with strong colour and stability at neutral pH, suitable as food colourants are relatively rare in nature. Acylation increases anthocyanin stability and confers bluer colour. In this study, we isolated two anthocyanin regulators SbMyb75 and SbDel from S. baicalensis, and showed that constitutive expression of the two TFs led to accumulation of anthocyanins at high levels in black carrot hairy roots. However, these hairy roots had severe growth problems. We then developed a β-estradiol inducible system using XVE and a Lex-35S promoter, to initiate expression of the anthocyanin regulators and induced this system in hairy roots of black carrot, tobacco and morning glory. Anthocyanins with various decorations were produced in these hairy roots without any accompanying side-effects on growth. We further produced highly acylated anthocyanins with blue colour in a 5 L liquid culture in a bioreactor of hairy roots from morning glory. We provide here a strategy to produce highly decorated anthocyanins without the need for additional engineering of any of the genes encoding decorating enzymes. This strategy could be transferred to other species, with considerable potential for natural colourant production for the food industries.

Functional Fruit Trees from the Atlantic and Amazon Forests: Selection of Potential Chestnut Trees Rich in Antioxidants, Nutrients, and Fatty Acids.

The Amazon rainforest and the biodiversity hotspot of the Atlantic Forest are home to fruit trees that produce functional foods, which are still underutilized. The present study aimed to select potential functional nut donor trees from two Brazilian chestnuts, by evaluating the nutritional and antioxidant composition of the nuts and the fatty acid profile of the oil. The nutritional characteristics, antioxidants, oil fatty acid profile, and X-ray densitometry of the nuts were evaluated, as well as the characterization of leaf and soil nutrients for each parent tree. The nut oil was evaluated through Brix (%), mass (g), yield (%), and the fatty acid profile. For L. pisonis, the most nutritious nuts were produced by L. pisonis tree 4 (N > P > K > Mg > Ca > Zn > Fe) and L. pisonis tree 6 (P > Ca > Mg > Mn > Zn > Cu > Fe), and for the species L. lanceolata, L. lanceolata tree 6 (N > P > Ca > Mg > Zn > Fe > Cu) and L. lanceolata tree 2 (P > K > Mg > Zn > Cu). In L. pisonis, the highest production of anthocyanins, DPPH, total phenolics, and flavonoids was obtained from the nuts of L. pisonis tree 4 as well as for L. lanceolata, from L. lanceolata tree 1, except for flavonoids. The Brix of the oil from the nuts of both species showed no difference between the trees and the fatty acid profile with a similar amount between saturated (48-65%) and unsaturated (34-57%) fatty acids. Both species have nuts rich in nutrients and antioxidant compounds and can be considered unconventional functional foods. The data collected in the present study confirm that the nuts of these species can replace other foods as a source of selenium.

Extraction, isolation, and standardization of anthocyanin from Vaccinium myrtillus to evaluate their potential in glaucoma

In order to preserve the structural integrity and bioactivity of anthocyanins while improving their extraction, identification, and isolation from Vaccinium myrtillus berries is the goal of this study. The following three goals were pursued: · Creating effective extraction procedures, · Using cutting-edge chromatographic techniques for identification, and · Isolating certain anthocyanin chemicals for possible bioactivity research. Three extraction techniques (A, B, and C) were contrasted with a fresh berry control for Objective 1. The maximum anthocyanin production (14.2 mg/g) and antioxidant activity (90%) were produced by Method C, which can be attributable to the high concentration of Petunidin-3-glucoside (55%). Method A produced anthocyanins with a concentration of 12.5 mg/g, predominantly made up of cyanidin-3-glucoside (45%), compared to Method B's 9.8 mg/g, primarily made up of delphinidin-3-glucoside (30%). The structural integrity of each approach was kept to a satisfactory level, as shown by the absorbance tests. Objective 2 was to identify anthocyanins using sophisticated chromatographic methods. Cyanidin-3-glucoside (5.21 min, 12500), Delphinidin-3-glucoside (7.46 min, 9800), and Petunidin-3-glucoside (9.83 min, 14200) were measured for retention times and peak areas. Anthocyanin molecules were identified using purification methods to work towards Objective 3. Three fractions were collected, and the anthocyanin chemicals in each fraction were identified: Fraction 1, 2.5 mg, 95% purity of Cyanidin-3-glucoside; Fraction 2, 1.8 mg, 90% purity of Delphinidin-3-glucoside; and Fraction 3, 3.2 mg, 92% purity of Petunidin-3-glucoside. Analysing UV-Vis spectra showed the purity, and chromatographic retention durations matched benchmark values. Further confirmation of chemical identities was obtained by mass spectrometry and NMR investigations, with detected peaks matching recognised references. This thorough investigation contributes to our understanding of how to extract and characterise anthocyanins from V. myrtillus berries. The findings demonstrate that Method C is a better extraction technique, producing high anthocyanin content while maintaining bioactivity.

SlMYB7, an AtMYB4-Like R2R3-MYB Transcription Factor, Inhibits Anthocyanin Accumulation in Solanum lycopersicum Fruits.

Tomato is a horticultural crop with an incomplete flavonoid metabolic pathway that does not typically accumulate anthocyanins in the fruit. In recent years, intensive studies of the loci Anthocyanin fruit (Aft) and atroviolacium (atv) have clarified the functions of positive regulators (R2R3-MYBs) and a negative regulator (CPC-MYB) in anthocyanin biosynthesis in the fruits. However, little is known about the R2R3-MYB repressors. Here, we used transient overexpression analysis to show that SlMYB7, a subgroup 4 AtMYB4-like R2R3-MYB, inhibited anthocyanin accumulation and reduced expression of anthocyanin synthase genes in the 'black pearl' tomato fruits, which usually accumulate high concentrations of anthocyanins. These findings revealed that SlMYB7 served as a repressor of anthocyanin production. Furthermore, SlMYB7 actively repressed SlANS expression by binding its promoter and passively inhibited anthocyanin synthesis by interacting with the basic helix-loop-helix (bHLH) proteins SlJAF13 and SlAN1, which are involved in the formation of MBW complexes. Thus, SlMYB7 and the MBW complex may coregulate the anthocyanin content of 'black pearl' tomato fruits via a negative feedback loop. These findings provide a theoretical basis for the future enhancement of tomato anthocyanin contents through genetic manipulation of the biosynthetic regulatory network.

Molecular insights into the VIRESCENS amino acid sequence and its implication in anthocyanin production in red- and yellow-fruited cultivars of date palm

This study examined the amino acid sequence of the VIRESCENS gene (VIR), which regulates the production of anthocyanin in 12 cultivars of the date palm (Phoenix dactylifera L.), grown in Al-Madinah Al-Munawarah of the Kingdom of Saudi Arabia. The gene products were amplified via polymerase chain reactions, amplifying both exons and introns. The products were sequenced for the reconstruction of a phylogenetic tree, which used the associated amino acid sequences. The ripening stages of Khalal, Rutab, and Tamar varied among the cultivars. Regarding VIR genotype, the red date had the wild-type gene (VIR+), while the yellow date carried a dominant mutation (VIRIM), i.e., long terminal repeat retrotransposons (LTR-RTs). The DNA sequence of VIRIM revealed that the insertion length of the LTR-RTs ranged between 386 and 476 bp. The R2 and R3 motifs in both VIR+ and VIRIM were conserved. The C-terminus motifs S6A, S6B, and S6C were found in the VIR+ protein sequence. However, the amino acids at positions 123, 161, 166, and 168 differed between VIR+ and VIRIM, and were not included in the C-terminus motifs. Within the VIR+ allele, the lysine at position 187 in the C-terminus was located immediately after S6B, with a protein binding score of 0.3, which was unique to the dark, red-fruited cultivars Ajwah, Anbarah, and Safawi. In the lighter, red-fruited cultivars, the presence of glutamic acid at the same position suggested that the anthocyanin regulation of date palm might be outside the R2 and R3 domains in the N-terminus.

Antioxidant Properties and Geroprotective Potential of Wheat Bran Extracts with Increased Content of Anthocyanins.

In recent years, there has been a focus on breeding wheat with high anthocyanin levels in order to improve food quality and human health. The objective of this study was to examine the antioxidant and geroprotective properties of wheat bran extracts using both in vitro and in vivo research methods. Two wheat lines were used: one with uncolored pericarp (anthocyanin-free) and another with colored pericarp (anthocyanin-containing). These lines differed in a specific region of chromosome 2A containing the Pp3/TaMyc1 gene, which regulates anthocyanin production. High-performance liquid chromatography-mass spectrometry revealed the presence of cyanidin glucoside and cyanidin arabinoside in the anthocyanin-containing wheat bran extract (+AWBE), while no anthocyanins were found in the anthocyanin-free wheat bran extract (-AWBE). The +AWBE showed higher radical scavenging activity (DPPH and ABTS assays) and membrane protective activity (AAPH oxidative hemolysis model) compared to the -AWBE. Both extracts extended the lifespan of female Drosophila, indicating geroprotective properties. This study demonstrates that wheat bran extracts with high anthocyanin levels have antioxidant and geroprotective effects. However, other secondary metabolites in wheat bran can also contribute to its antioxidant and geroprotective potential.

DlMYB113 mutation affects anthocyanin accumulation in red pericarp longan (Dimocarpus longan Lour.)

Fruit color influences fruit quality and commodity value. Most longan (Dimocarpus longan Lour.) varieties have a yellowish-brown or grayish-yellow pericarp, and the discovery of red pericarp (RP) longan expanded the color varieties of longan fruit. Our previous research showed that the red pericarp of RP fruit was mainly caused by anthocyanin accumulation; however, its underlying regulatory mechanism remains unknown. Herein, DlMYB113, an R2R3-MYB transcription factor, was discovered by examining differentially expressed genes in two longan cultivars. Dimocarpus longan MYB (v-myb avian myeloblastosis viral) 113 (DlMYB113) expression was significantly higher in the pericarp and leaves of RP longan than in ‘Shixia’ longan. Sequence alignment analysis revealed two amino acid substitutions in the R3 domain between DlMYB113rp in RP longan and DlMYB113sx in ‘Shixia’ longan. Transient expression of DlMYB113rp significantly increased anthocyanin accumulation in tobacco leaves, whereas DlMYB113sx had negligible effect. Meanwhile, DlMYB113 overexpression promotes anthocyanin accumulation in Arabidopsis and longan calli. Site-directed mutation detection revealed divergence in DlMYB113 function when the R3 repeat 197-position base T was replaced with G, and the 317- and 318-position AT bases were replaced with GA. Our findings indicate that DlMYB113 can regulate anthocyanin production in RP longan, and three mutations in its nucleic acid sequence lead to anthocyanin accumulation, thereby developing molecular markers associated with the anthocyanin accumulation trait in RP longan. This study will facilitate early screening of longan hybrids with desirable fruit color and be significant for breeding new characteristic varieties.

Molecular mechanism of different flower color formation of Cymbidium ensifolium.

Cymbidium ensifolium is one of the national orchids in China, which has high ornamental value with changeable flower colors. To understand the formation mechanism of different flower colors of C. ensifolium, this research conducted transcriptome and metabolome analyses on four different colored sepals of C. ensifolium. Metabolome analysis detected 204 flavonoid metabolites, including 17 polyphenols, 27 anthocyanins, 75 flavones, 34 flavonols, 25 flavonoids, 18 flavanones, and 8 isoflavones. Among them, purple-red and red sepals contain a lot of anthocyanins, including cyanidin, pelargonin, and paeoniflorin, while yellow-green and white sepals have less anthocyanins detected, and their metabolites are mainly flavonols, flavanones and flavonoids. Transcriptome sequencing analysis showed that the expression levels of the anthocyanin biosynthetic enzyme genes in red and purple-red sepals were significantly higher than those in white and yellow-green sepals of C. ensifolium. The experimental results showed that CeF3'H2, CeDFR, CeANS, CeF3H and CeUFGT1 may be the key genes involved in anthocyanin production in C. ensifolium sepals, and CeMYB104 has been proved to play an important role in the flower color formation of C. ensifolium. The results of transformation showed that the CeMYB104 is involved in the synthesis of anthocyanins and can form a purple-red color in the white perianth of Phalaenopsis. These findings provide a theoretical reference to understand the formation mechanism of flower color in C. ensifolium.

Combined Metabolome and Transcriptome Analysis of Creamy Yellow and Purple Colored Panax notoginseng Roots.

Panax notoginseng (Burk.) F.H. Chen is a species of the Araliaceae family that inhabits southwestern China, Burma, and Nepal. It is cultivated on a commercial scale in Yunnan province, China, owing to its significance in traditional Chinese medicine. Panax notoginseng roots are usually yellow-white (HS); however, purple roots (ZS) have also been reported. The majority of P. notoginseng research has concentrated on the identification and production of natural chemicals in HS; however, there is little to no information about the composition of ZS. Using UPLC-MS/MS, we investigated the global metabolome profile of both ZS- and HS-type roots and discovered 834 metabolites from 11 chemical groups. There were 123 differentially accumulated metabolites (DAM) in the HS and ZS roots, which were classified as lipids and lipid-like molecules, polyketides, organoheterocyclic chemicals, and organooxygen compounds. We investigated the associated compounds in the DAMs because of the importance of anthocyanins in color and saponins and ginsenosides in health benefits. In general, we discovered that pigment compounds such as petunidin 3-glucoside, delphinidin 3-glucoside, and peonidin-3-O-beta-galactoside were more abundant in ZS. The saponin (eight compounds) and ginsenoside (26 compounds) content of the two varieties of roots differed as well. Transcriptome sequencing revealed that flavonoid and anthocyanin production genes were more abundant in ZS than in HS. Similarly, we found differences in gene expression in genes involved in terpenoid production and related pathways. Overall, these findings suggest that the purple roots of P. notoginseng contain varying amounts of ginsenosides and anthocyanins compared to roots with a creamy yellow color.

HaMYBA-HabHLH1 regulatory complex and HaMYBF fine-tune red flower coloration in the corolla of sunflower (Helianthus annuus L.)

Sunflowers are well-known ornamental plants, while sunflowers with red corolla are rare and the mechanisms underlying red coloration remain unclear. Here, a comprehensive analysis of metabolomics and transcriptomics on flavonoid pathway was performed to investigate the molecular mechanisms underlying the differential color formation between red sunflower Pc103 and two yellow sunflowers (Yr17 and Y35). Targeted metabolomic analysis revealed higher anthocyanin levels but lower flavonol content in Pc103 compared to the yellow cultivars. RNA-sequencing and phylogenetic analysis identified multiple genes involved in the flavonoid pathway, including series of structural genes and three MYB and bHLH genes. Specifically, HaMYBA and HabHLH1 were up-regulated in Pc103, whereas HaMYBF exhibited reduced expression. HaMYBA was found to interact with HabHLH1 in vivo and in vitro, while HaMYBF does not. Transient expression analysis further revealed that HabHLH1 and HaMYBA cooperatively regulate increased expression of dihydroflavonol 4-reductase (DFR), leading to anthocyanin accumulation. On the other hand, ectopic expression of HaMYBF independently modulates flavonol synthase (FLS) expression, but hindered anthocyanin production. Collectively, our findings suggest that the up-regulation of HaMYBA and HabHLH1, as well as the down-regulation of HaMYBF, contribute to the red coloration in Pc103. It offers a theoretical basis for improving sunflower color through genetic engineering.

Extraction by maceration, ultrasound, and pressurized liquid methods for the recovery of anthocyanins present in the peel of guabiju (Myrcianthes pungens): Maximizing the extraction of anthocyanins from the peel of guabiju

Guabiju (Myrcianthes pungens) is a fruit that stands out for its chemical composition, especially anthocyanins, which bring health benefits and can be used as natural dyes. However, there are no studies comparing different methods of extracting anthocyanins from guabiju peel. The objective of this work was to maximize the extraction of anthocyanins from guabiju peels by comparing the extraction methods of maceration, assisted by ultrasound, and pressurized liquid to characterize the obtained extracts. The extracts were characterized in terms of anthocyanin content, extract yield and productivity, soluble solids, color, acidity, total phenolic compounds, flavonoids, antioxidant activity, and the cost analysis involved in each methodology. The maximized conditions were absolute ethanol pH 1.0, fractional extraction (240 min) with solvent renewal in half the extraction time, 60 °C, 100 rpm of agitation in maceration, and 70 kHz by using ultrasound. The ultrasound assisted extraction method obtained the highest amount of anthocyanins (248.96 mg CE - cyanidin-3-glucoside equivalent/100 g) and an extract yield of 11.96%. For phenolic compounds and flavonoids (10,544.04 mg GAE - gallic acid equivalent/100 g and 1621.78 mg QE - quercetin equivalent/100 g), respectively, with good antioxidant capacity. Its properties and color allow for possible applications in different products. Ultrasound-assisted extraction showed a slightly lower cost of extraction than by maceration, while by PLE had a final cost approximately 2.4 times higher for 1 kg of anthocyanins.

Testing Cultivar-specific Tipburn Sensitivity of Lettuce for Indoor Vertical Farms

Low evaporative conditions in indoor (vertical) farms reduce mass-flow–driven transport of calcium (Ca), resulting in tipburn of lettuce. Lettuce tipburn symptoms develop along the margins of young leaves and the growing shoot tip, where necrotic tissue forms as a result of Ca deficiency. For indoor farms, lettuce tipburn poses a major economic risk because the crop becomes unmarketable as a result of its appearance. Difference in tipburn sensitivity among cultivars has been thought to be related to differences in growth rate, morphology, or anthocyanin production, whereas most commercial lettuce cultivars have been known to express tipburn symptoms in the indoor farm setting. We created a tipburn-inducing growing condition in walk-in growth chambers that limits plant potential transpiration rate while achieving relatively high growth rates, and examined 10 commercial cultivars selected for tipburn sensitivity. Selected cultivars differ in morphology (butterhead, romaine, and leafy type) and color (red or green; resulting from anthocyanin production). All cultivars expressed visually detectable tipburn symptoms 22 ± 2.6 days after transplanting, and varied tipburn rates of 7% to 41% of all leaves at the time of harvest (28 days after transplanting). Despite cultivar-specific variation, neither lettuce morphology nor anthocyanin content were significantly correlated with the incidence or severity of tipburn. However, cultivars recommended for “indoor” production by seed suppliers had less tipburn severity than those recommended for outdoor or both indoor and outdoor production systems. Although tipburn risk may vary under other environmental conditions, low evaporative conditions in this experiment caused tipburn symptoms in all tested cultivars at varying degrees of severity. Cultivar-specific average yield and tipburn severity were not correlated with the Ca concentrations in the inner leaves, suggesting that the amount of tissue Ca required to prevent tipburn is cultivar specific and not related to yield. Our selected tipburn-inducing condition was found to be effective in comparing tipburn sensitivity of lettuce cultivars for indoor farm settings, and similar fast-growing but low-evaporative conditions should be used to assess cultivars for indoor farm production.

Analysis of Antioxidant Capacity Variation among Thai Holy Basil Cultivars (Ocimum tenuiflorum L.) Using Density-Based Clustering Algorithm

Holy basil (Ocimum tenuiflorum L.) is a widely renowned herb for its abundance of bioactive compounds and medicinal applications. Nevertheless, there exists a dearth of knowledge regarding the variability among holy basil cultivars capable of yielding substantial bioactive compounds. This study aims to address this gap by shedding light on the diversity of antioxidant capacities within different accessions of Thai holy basil by employing a density-based clustering algorithm to categorize the holy basil cultivars that demonstrate notable antioxidant potential. The study involves the analysis of the anthocyanin, flavonoid, phenolic, and terpenoid content, as well as DPPH antioxidant activity, in 26 Thai holy basil accessions collected from diverse locations in Thailand. Among the 26 tested Thai holy basil cultivars, terpenoids were found to be the dominant class of compounds, with average values of 707 mg/gDW, while the levels of flavonoids and phenolic compounds remained below 65 mg rutin/gDW and 46 mg GAE/gDW, respectively. The DPPH assay in holy basil cultivars demonstrated that the antioxidant activity ranged between 50% and 93%. After standardizing the data, the clustering results revealed four distinct groups of cultivars: the first group, with low antioxidant levels; the second group, with high terpenoid content; the third group, with high flavonoid, DPPH antioxidant activity, and phenolic content; and the fourth group, with elevated levels of anthocyanin, DPPH antioxidant activity, and phenolic content. A strong positive correlation was observed among DPPH antioxidant activity, flavonoids, and phenolics. Specific cultivars: The Red, OC108, and OC106 holy basil cultivars in cluster 4 exhibited high anthocyanin and phenolic production. In cluster 3, the accessions OC113, OC057, OC063, and OC059 showed high DPPH antioxidant activity, flavonoids, and phenolics, while, in cluster 2, only accessions from Udon Thani, Thailand—namely OC194 and OC195—displayed high terpenoid content. Ultimately, this study significantly contributes to the inherent diversity in the antioxidant capacities among various Thai holy basil cultivars. It lays the foundation for targeted breeding strategies and informed choices regarding consumption. The comprehensive insights from this analysis hold the potential to accurately identify holy basil cultivars with promising applications in medicine, functional foods, and the nutraceutical industry.

Genome-based identification of the CYP75 gene family in Orchidaceae and its expression patterns in Cymbidium goeringii.

With a great diversity of species, Orchidaceae stands out as an essential component of plant biodiversity, making it a primary resource for studying angiosperms evolution and genomics. This study focuses on 13 published orchid genomes to identify and analyze the CYP75 gene family belonging to the cytochrome P450 superfamily, which is closely related to flavonoid biosynthetic enzymes and pigment regulation. We found 72 CYP75s in the 13 orchid genomes and further classified them into two classes: CYP75A and CYP75B subfamily, the former synthesizes blue anthocyanins, while the latter is involved in the production of red anthocyanins. Furthermore, the amount of CYP75Bs (53/72) greatly exceeds the amount of CYP75As (19/72) in orchids. Our findings suggest that CYP75B genes have a more important evolutionary role, as red plants are more common in nature than blue plants. We also discovered unique conserved motifs in each subfamily that serve as specific recognition features (motif 19 belong to CYP75A; motif 17 belong to CYP75B). Two diverse-colored varieties of C. goeringii were selected for qRT-PCR experiments. The expression of CgCYP75B1 was significantly higher in the purple-red variant compared to the yellow-green variant, while CgCYP75A1 showed no significant difference. Based on transcriptomic expression analysis, CYP75Bs are more highly expressed than CYP75As in floral organs, especially in colorful petals and lips. These results provide valuable information for future studies on CYP75s in orchids and other angiosperms.

CsTT8 regulates anthocyanin accumulation in blood orange through alternative splicing transcription.

A homologous gene of basic-helix-loop-helix AtTT8 in Arabidopsis thaliana was identified in juice sac cells of pulp tissues from blood orange (Citrus sinensis cv 'Tarocco'), which was designated as CsTT8 in this study. Additionally, the mRNA levels of TT8 with the full-length open reading frame were significantly higher in 'Tarocco' than in mutant fruit lacking pigment in pulp or peel tissues. However, an alternative splicing transcript, Δ15-TT8, with the fourth exon skipped, was also identified from transcripts different in length from that in 'Tarocco'. The mRNA levels of Δ15-TT8 were higher in mutant fruit lacking pigment in pulp or peel tissues than in the wild type. Therefore, the TT8/Δ15-TT8 mRNA level ratio was found to be crucial for sufficient pigment in either pulp or peel tissues. TT8 from blood orange fruit demonstrated the capacity for nucleus localization and binding to other proteins. In contrast, Δ15-TT8, lacking the fourth exon, lost its ability to interact with RUBY1 and to localize at the nucleus. Using a dual luciferase reporter assay and transient overexpression in tobacco, we proved that two regulatory complexes formed by a functional TT8 with different MYB(v-myb avian myeloblastosis viral oncogene homolog)-type partners significantly promoted expression of an anthocyanin biosynthetic gene and a proton pumping gene, leading to anthocyanin and citrate production. Our findings suggest that TT8, rather than dysfunctional Δ15-TT8, is possibly involved in modulating anthocyanin biosynthesis and its transport into vacuoles by proton gradients. However, increased mRNA levels of the dysfunctional alternative splicing transcript may act as a negative feedback to downregulate TT8 expression and limit anthocyanin accumulation in blood oranges.