A split GAL4 RUBY assay for visual in planta detection of protein-protein interactions.

Protein-protein interactions (PPIs) are a fundamental process in cellular biogenesis. Here we have developed a split GAL4 RUBY assay that enables macroscopically visual PPI detection in plant leaves in real time. Candidate interacting protein partners are fused to specific domains of the yeast GAL4 and herpes simplex virus VP16 transcription factors and transiently expressed in Nicotiana benthamina leaves by Agrobacterium infiltration. PPI, that may be either direct or indirect, results in transcriptional activation of a RUBY reporter gene leading to the production of the highly visual metabolite, betalain, in leaf tissue of living plants. Samples require no processing for in planta visual qualitative assessment, but with very simple processing steps the assay is quantitative. Its accuracy is demonstrated using a series of known interacting protein partners and mutant derivatives including transcription factors, signalling molecules and plant resistance proteins with cognate pathogen effectors. Using this assay, association between the wheat Sr27 stem rust disease resistance protein and corresponding AvrSr27 avirulence effector family produced by the rust pathogen is detected. Interaction is also observed between this resistance protein and the effector encoded by the corresponding avrSr27-3 virulence allele. However, this association appears weaker in the split GAL4 RUBY assay, which coupled with lower avrSr27-3 expression during stem rust infection, likely enables virulent races of the rust pathogen to avoid Sr27-mediated detection.

Straightforward and affordable agroinfiltration with RUBY accelerates RNA silencing research.

Transient expression and induction of RNA silencing by agroinfiltration is a fundamental method in plant RNA biology. Here, we introduce a new reporter assay using RUBY, which encodes three key enzymes of the betalain biosynthesis pathway, as a polycistronic mRNA. The red pigmentation conferred by betalains allows visual confirmation of gene expression or silencing levels without tissue disruption, and the silencing levels can be quantitatively measured by absorbance in as little as a few minutes. Infiltration of RUBY in combination with p19, a well-known RNA silencing suppressor, induced a fivefold higher accumulation of betalains at 7 days post infiltration compared to infiltration of RUBY alone. We demonstrated that co-infiltration of RUBY with two RNA silencing inducers, targeting either CYP76AD1 or glycosyltransferase within the RUBY construct, effectively reduces RUBY mRNA and betalain levels, indicating successful RNA silencing. Therefore, compared to conventional reporter assays for RNA silencing, the RUBY-based assay provides a simple and rapid method for quantitative analysis without the need for specialized equipment, making it useful for a wide range of RNA silencing studies.

Optimising promoters and subcellular localisation for constitutive transgene expression in Marchantia polymorpha.

Marchantia polymorpha has become an important model system for comparative studies and synthetic biology. The systematic characterisation of genetic elements would make heterologous gene expression more predictable in this testbed for gene circuit assembly and bioproduction. Yet, the toolbox of genetic parts for Marchantia includes only a few constitutive promoters that need benchmarking to assess their utility. We compared the expression patterns of previously characterized and new constitutive promoters. We found that driving expression with the double enhancer version of the cauliflower mosaic virus 35S promoter (pro35S×2) provided the highest yield of proteins although it also inhibits the growth of transformants. In contrast, promoters derived from the Marchantia ETHYLENE RESPONSE FACTOR 1 (MpERF1) and the CLASS II HOMEODOMAIN-LEUCINE ZIPPER (MpC2HDZ) genes drove expression to higher levels across all tissues without growth penalty and can provide intermediate levels of gene expression. In addition, we showed that the cytosol is the best subcellular compartment to target heterologous proteins for higher levels of expression without a significant growth burden. To demonstrate the potential of these promoters in Marchantia, we expressed the polycistronic RUBY betalain synthesis cassette to demonstrate coordinated expression of metabolic enzymes. A heat-shock inducible promoter was used to further mitigate growth burdens associated with high amounts of betalain accumulation. We have expanded the existing toolkit for gene expression in Marchantia and provide new resources for the Marchantia research community.

Flower color modification in Torenia fournieri by genetic engineering of betacyanin pigments.

BACKGROUND: Betalains are reddish and yellow pigments that accumulate in a few plant species of the order Caryophyllales. These pigments have antioxidant and medicinal properties and can be used as functional foods. They also enhance resistance to stress or disease in crops. Several plant species belonging to other orders have been genetically engineered to express betalain pigments. Betalains can also be used for flower color modification in ornamental plants, as they confer vivid colors, like red and yellow. To date, betalain engineering to modify the color of Torenia fournieri-or wishbone flower-a popular ornamental plant, has not been attempted. RESULTS: We report the production of purple-reddish-flowered torenia plants from the purple torenia cultivar "Crown Violet."  Three betalain-biosynthetic genes encoding CYP76AD1, dihydroxyphenylalanine (DOPA) 4,5-dioxygenase (DOD), and cyclo-DOPA 5-O-glucosyltransferase (5GT) were constitutively ectopically expressed under the cauliflower mosaic virus (CaMV) 35S promoter, and their expression was confirmed by quantitative real-time PCR (qRT-PCR) analysis. The color traits, measured by spectrophotometric colorimeter and spectral absorbance of fresh petal extracts, revealed a successful flower color modification from purple to reddish. Red pigmentation was also observed in whole plants. LC-DAD-MS and HPLC analyses confirmed that the additional accumulated pigments were betacyanins-mainly betanin (betanidin 5-O-glucoside) and, to a lesser extent, isobetanin (isobetanidin 5-O-glucoside). The five endogenous anthocyanins in torenia flower petals were also detected. CONCLUSIONS: This study demonstrates the possibility of foreign betacyanin accumulation in addition to native pigments in torenia, a popular garden bedding plant. To our knowledge, this is the first report presenting engineered expression of betalain pigments in the family Linderniaceae. Genetic engineering of betalains would be valuable in increasing the flower color variation in future breeding programs for torenia.

Genomic basis of seed colour in quinoa inferred from variant patterns using extreme gradient boosting.

Quinoa is an agriculturally important crop species originally domesticated in the Andes of central South America. One of its most important phenotypic traits is seed colour. Seed colour variation is determined by contrasting abundance of betalains, a class of strong antioxidant and free radicals scavenging colour pigments only found in plants of the order Caryophyllales. However, the genetic basis for these pigments in seeds remains to be identified. Here we demonstrate the application of machine learning (extreme gradient boosting) to identify genetic variants predictive of seed colour. We show that extreme gradient boosting outperforms the classical genome-wide association approach. We provide re-sequencing and phenotypic data for 156 South American quinoa accessions and identify candidate genes potentially controlling betalain content in quinoa seeds. Genes identified include novel cytochrome P450 genes and known members of the betalain synthesis pathway, as well as genes annotated as being involved in seed development. Our work showcases the power of modern machine learning methods to extract biologically meaningful information from large sequencing data sets.

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.

Quantitative RUBY reporter assay for gene regulation analysis.

Various reporter genes have been developed to study gene expression pattern and gene regulation. The RUBY reporter gene was recently developed and widely used, because of its visible and noninvasive advantages. However, quantitative analysis of RUBY gene expression levels was lacking. In this study, we introduce a novel betalain quantification method in combination with the tobacco transient expression system. The betalain produced in tobacco leaves was extracted and purified, and its concentration was quantitatively measured. We successfully applied this approach in studying the transcriptional regulation of ARC5 gene by transcription factors CPD25 and CPD45. Furthermore, with this method, we showed that the gene expression of RCA and Rbcs1A gene were regulated by light, transcription factors HY5 and PIFs through G-box and I-box elements. The development of this betalain quantification approach with the tobacco transient expression system offers a cost-effective and intuitive strategy for studying the regulatory mechanism of gene expression.

Robust soybean leaf agroinfiltration.

KEY MESSAGE: A robust agroinfiltration-mediated transient gene expression method for soybean leaves was developed. Plant genotype, developmental stage and leaf age, surfactant, and Agrobacterium culture conditions are important for successful agroinfiltration. Agroinfiltration of Nicotiana benthamiana has emerged as a workhorse transient assay for plant biotechnology and synthetic biology to test the performance of gene constructs in dicot leaves. While effective, it is nonetheless often desirable to assay transgene constructs directly in crop species. To that end, we innovated a substantially robust agroinfiltration method for Glycine max (soybean), the most widely grown dicot crop plant in the world. Several factors were found to be relevant to successful soybean leaf agroinfiltration, including genotype, surfactant, developmental stage, and Agrobacterium strain and culture medium. Our optimized protocol involved a multi-step Agrobacterium culturing process with appropriate expression vectors, Silwet L-77 as the surfactant, selection of fully expanded leaves in the VC or V1 stage of growth, and 5 min of vacuum at - 85 kPa followed by a dark incubation period before plants were returned to normal growth conditions. Using this method, young soybean leaves of two lines-V17-0799DT, and TN16-5004-were high expressors for GUS, two co-expressed fluorescent protein genes, and the RUBY reporter product, betalain. This work not only represents a new research tool for soybean biotechnology, but also indicates critical parameters for guiding agroinfiltration optimization for other crop species. We speculate that leaf developmental stage might be the most critical factor for successful agroinfiltration.

Betalain biosynthesis in red pulp pitaya is regulated via HuMYB132: a R-R type MYB transcription factor.

BACKGROUND: Multiple MYB transcription factors (TFs) are involved in the regulation of plant coloring. Betalain is a kind of natural plant pigment and its biosynthesis is regulated by a number of enzymes. Despite this, little is known about the molecular properties and roles of MYB TFs in pitaya betalain biosynthesis. RESULTS: In the present study, we identified a 1R-MYB gene, HuMYB132, which is preferentially expressed in red-pulp pitaya at the mature stage. It was clustered with Arabidopsis R-R-type genes and had two DNA-binding domains and a histidine-rich region. The expression assays in N. benthamiana and yeast indicated that HuMYB132 is a nucleus-localized protein with transcriptional activation activity. Dual luciferase reporter assay and electrophoretic mobility shift assays (EMSA) demonstrated that HuMYB132 could promote the transcriptional activities of HuADH1, HuCYP76AD1-1, and HuDODA1 by binding to their promoters. Silencing HuMYB132 reduced betalain accumulation and the expression levels of betalain biosynthetic genes in pitaya pulps. CONCLUSIONS: According to our findings, HuMYB132, a R-R type member of 1R-MYB TF subfamily, positively regulates pitaya betalain biosynthesis by regulating the expression of HuADH1, HuCYP76AD1-1, and HuDODA1. The present study provides a new theoretical reference for the management of pitaya betalain biosynthesis and also provides an essential basis for future regulation of betalain biosynthesis in Hylocereus.

BpCYP76AD15 is involved in betaxanthin biosynthesis in bougainvillea callus.

MAIN CONCLUSION: BpCYP76AD15 is involved in betaxanthin biosynthesis in callus, but not in bracts, in bougainvillea. Bougainvillea (Bougainvillea peruviana) is a climbing tropical ornamental tree belonging to Nyctaginaceae. Pigments that are conferring colorful bracts in bougainvillea are betalains, and that conferring yellow color are betaxanthins. In general, for red-to-purple betacyanin biosynthesis, α clade CYP76AD that has tyrosine hydroxylase and DOPA oxygenase activity is required, while for betaxanthin biosynthesis, ß clade CYP76AD that has only tyrosine hydroxylase is required. To date, betaxanthin biosynthesis pathway genes have not been identified yet in bougainvillea. Since bougainvillea is phylogenetically close to four-O-clock (Mirabilis jalapa), and it was reported that ß clade CYP76AD, MjCYP76AD15, is involved in floral betaxanthin biosynthesis in four-O-clock. Thus, we hypothesized that orthologous gene of MjCYP76AD15 in bougainvillea might be involved in bract betaxanthin biosynthesis. To test the hypothesis, we attempted to identify ß clade CYP76AD gene from yellow bracts by RNA-seq; however, we could not. Instead, we found that callus accumulated betaxanthin and that ß clade CYP76AD gene, BpCYP76AD15, were expressed in callus. We validated BpCYP76AD15 function by transgenic approach (agro-infiltration and over-expression in transgenic tobacco), and it was suggested that BpCYP76AD15 is involved in betaxanthin biosynthesis in callus, but not in bracts in bougainvillea. Interestingly, our data also indicate the existence of two pathways for betaxanthin biosynthesis (ß clade CYP76AD-dependent and -independent), and the latter pathway is important for betaxanthin biosynthesis in bougainvillea bracts.

The RUBY reporter enables efficient haploid identification in maize and tomato.

In vivo haploid induction has been extended from maize to monocotyledonous plants like rice, wheat, millet and dicotyledonous plants such as tomato, rapeseed, tobacco and cabbage. Accurate identification of haploids is a crucial step of doubled haploid technology, where a useful identification marker is very pivotal. R1-nj is an extensively used visual marker for haploid identification in maize. RFP and eGFP have been shown to be feasible in identifying haploid. However, these methods are either limited to specific species, or require specific equipment. It still lacks an efficient visual marker that is practical across different crop species. In this study, we introduced the RUBY reporter, a betalain biosynthesis system, into maize and tomato haploid inducers as a new marker for haploid identification. Results showed that expression of RUBY could result in deep betalain pigmentation in maize embryos as early as 10 days after pollination, and enabled 100% accuracy of immature haploid embryo identification. Further investigation in tomato revealed that the new marker led to deep red pigmentation in radicles and haploids can be identified easily and accurately. The results demonstrated that the RUBY reporter is a background-independent and efficient marker for haploid identification and would be promising in doubled haploid breeding across different crop species.

Production of yellow-flowered gentian plants by genetic engineering of betaxanthin pigments.

Genetic engineering of flower color provides biotechnological products such as blue carnations or roses by accumulating delphinidin-based anthocyanins not naturally existing in these plant species. Betalains are another class of pigments that in plants are only synthesized in the order Caryophyllales. Although they have been engineered in several plant species, especially red-violet betacyanins, the yellow betaxanthins have yet to be engineered in ornamental plants. We attempted to produce yellow-flowered gentians by genetic engineering of betaxanthin pigments. First, white-flowered gentian lines were produced by knocking out the dihydroflavonol 4-reductase (DFR) gene using CRISPR/Cas9-mediated genome editing. Beta vulgaris BvCYP76AD6 and Mirabilis jalapa MjDOD, driven by gentian petal-specific promoters, flavonoid 3',5'-hydroxylase (F3'5'H) and anthocyanin 5,3'-aromatic acyltransferase (AT), respectively, were transformed into the above DFR-knockout white-flowered line; the resultant gentian plants had vivid yellow flowers. Expression analysis and pigment analysis revealed petal-specific expression and accumulation of seven known betaxanthins in their petals to c. 0.06-0.08 µmol g FW-1 . Genetic engineering of vivid yellow-flowered plants can be achieved by combining genome editing and a suitable expression of betaxanthin-biosynthetic genes in ornamental plants.

Metabolic engineering of betacyanin in vegetables for anti-inflammatory therapy.

Betalains, which consist of the subgroups betaxanthins and betacyanins, are hydrophilic pigments that have classically been used for food colorants. Owing to their strong antioxidant property, their usefulness for application for therapeutic use is also expected. In addition, as betalains are mainly naturally available from plants of the order Caryophyllales, including beet (Beta vulgaris), metabolic engineering for betalain production in crops such as vegetables, fruits and cereals may provide new food resources useful for healthcare. Here we conducted metabolic engineering of betacyanins in tomato fruits and potato tubers. The transgenic tomato fruits and potato tubers with coexpression of betacyanin biosynthesis genes, CYP76AD1 from B. vulgaris, DOD (DOPA 4,5-dioxygenase) and 5GT (cyclo-DOPA 5-O-glucosyltransferase) from Mirabilis jalapa, under control of suitable specific promoters, possessed dark red tissues with enriched accumulation of betacyanins (betanin and isobetanin). The anti-inflammatory activity of transgenic tomato fruit extract was superior to that of wild-type fruit extract on macrophage RAW264.7 cells stimulated with lipopolysaccharide (LPS), as a result of decreased LPS-stimulated transcript levels of proinflammatory genes. These findings were in accord with the observation that administration of the transgenic tomato fruits ameliorated dextran sulfate sodium (DSS)-induced colitis as well as body weight loss and disease activity index in mice, via suppression of DSS-stimulated transcript levels of pro-inflammatory genes, including Tnf (encoding TNF-alpha), Il6, and Ptgs2 (encoding cyclooxygenae 2). Intriguingly, given the fact that the transgenic potato tuber extract failed to enrich the anti-inflammatory activity of macrophage cells, it is likely that metabolic engineering of betacyanins will be a powerful way of increasing the anti-inflammatory property of ordinary foods such as tomato.

Traversing through the cell signaling pathways of neuroprotection by betanin: therapeutic relevance to Alzheimer's Disease and Parkinson's Disease.

Modulation of cell signaling pathways is the key area of research towards the treatment of neurodegenerative disorders. Altered Nrf2-Keap1-ARE (Nuclear factor erythroid-2-related factor 2-Kelch-like ECH-associated protein 1-Antioxidant responsive element) and SIRT1 (Sirtuin 1) cell signaling pathways are considered to play major role in the etiology and pathogenesis of Alzheimer's disease (AD) and Parkinson's disease (PD). Strikingly, betanin, a betanidin 5-O-ß-D-glucoside compound is reported to show commendable anti-oxidative, anti-inflammatory and anti-apoptotic effects in several disease studies including AD and PD. The present review discusses the pre-clinical studies demonstrating the neuroprotective effects of betanin by virtue of its potential to ameliorate oxidative stress, neuroinflammation, abnormal protein aggregation and cell death. It highlights the direct linkage between the neuroprotective abilities of betanin and upregulation of the Nrf2-Keap1-ARE and SIRT1 signaling pathways. The review further hypothesizes the involvement of the betanin-Nrf2-ARE route in the inhibition of beta-amyloid aggregation through beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), one of the pivotal hallmarks of AD. The present review hereby for the first time elaborately discusses the reported neuroprotective abilities of betanin and decodes the Nrf2 and SIRT1 modulating potential of betanin as a primary mechanism of action behind, hence highlighting it as a novel drug candidate for the treatment of neurodegenerative diseases in the near future.

A Comparative Study of Ethanol and Citric Acid Solutions for Extracting Betalains and Total Phenolic Content from Freeze-Dried Beetroot Powder.

This research compares the extraction of betalains (betacyanin and betaxanthin) and total phenolic content using citric acid and aqueous-ethanol solutions. The aim is to find an environmentally sustainable alternative solvent for extracting these compounds from dried beetroot powder. Using citric acid solution as a solvent offers several benefits over ethanol. Citric acid is a weak organic acid found naturally in citrus fruits, making it a safe and environmentally friendly choice for certain extraction processes. Moreover, the use of citric acid as solvent offers biodegradability, non-toxicity, non-flammability, and is cost effective. A full factorial design and response surface methodology (RSM) were employed to assess the effects of extraction parameters (extraction time (5-30 min), extraction temperature (20, 30, 40 °C), pH of citric acid solution (3, 4, 5) and ethanol concentration (10, 20, 30% v/v)). The yield was determined spectrophotometrically and expressed as mg/g of dry powder. The results showed that citric acid solution yielded 85-90% of the ethanolic extract under identical conditions. The maximum yields of betacyanin, betaxanthin, and total phenolic content in citric acid solution were 3.98 ± 0.21 mg/g dry powder, 3.64 ± 0.26 mg/g dry powder, and 8.28 ± 0.34 mg/g dry powder, respectively, while aqueous-ethanol yielded 4.38 ± 0.17 mg/g dry powder, 3.95 ± 0.22 mg/g dry powder, and 8.45 ± 0.45 mg/g dry powder. Optimisation resulted in maximum extraction yields of 90% for betalains and 85% for total phenolic content. The study demonstrates the potential of citric acid as a viable alternative to polar organic solvents for extracting phytochemicals from plant material, providing comparable results to aqueous-ethanol. Artificial Neural Network (ANN) models outperformed RSM in predicting extraction yields. Overall, this research highlights the importance of exploring bio-solvents to enhance the environmental sustainability of phytochemical extraction.

A straight-forward seed production technology system for foxtail millet (Setaria italica).

For autogamous crops, a precondition for using heterosis is to produce sufficient pure male-sterile female parents that can be used to produce hybrid seeds. To date, cytoplasmic male sterility (CMS) and environment-sensitive genic male sterility (EGMS) have been used commercially to exploit heterosis for autogamous species. However, neither CMS nor EGMS has been established for foxtail millet (Setaria italica). Here, we report on the establishment and application of a seed production technology (SPT) system for this crop. First, we established a DsRed-based SPT system, but found that it was unsuitable because it required the use of a fluorescent device for seed sorting. Instead, we constructed an SPT system with de novo betalain biosynthesis as the selection marker. This allowed us to distinguish transgenic seeds with the naked eye, thereby facilitating the identification of SPT maintainer line seeds. In this system, a seed sorter was not required to obtain sufficient seeds. The key point of the strategy is that the seed pool of the SPT maintainer line is propagated by artificial identification and harvesting of male-fertile individuals in the field, and the male-sterile line seed pool for hybrid production is produced and propagated by free pollination of male-sterile plants with the SPT maintainer line. In a field experiment, we obtained 423.96 kg male-sterile line seeds per acre, which is sufficient to plant 700.18 acres of farmland for hybrid seed production or male-sterile line reproduction. Our study therefore describes a powerful tool for hybrid seed production in foxtail millet, and demonstrates how the SPT system can be used for a small-grained crop with high reproduction efficiency.

Transcriptome analyses shed light on floral organ morphogenesis and bract color formation in Bougainvillea.

BACKGROUND: Bougainvillea is a popular ornamental plant with brilliant color and long flowering periods. It is widely distributed in the tropics and subtropics. The primary ornamental part of the plant is its colorful and unusual bracts, rich in the stable pigment betalain. The developmental mechanism of the bracts is not clear, and the pathway of betalain biosynthesis is well characterized in Bougainvillea. RESULTS: At the whole-genome level, we found 23,469 protein-coding genes by assembling the RNA-Seq and Iso-Seq data of floral and leaf tissues. Genome evolution analysis revealed that Bougainvillea is related to spinach; the two diverged approximately 52.7 million years ago (MYA). Transcriptome analysis of floral organs revealed that flower development of Bougainvillea was regulated by the ABCE flower development genes; A-class, B-class, and E-class genes exhibited high expression levels in bracts. Eight key genes of the betalain biosynthetic pathway were identified by homologous alignment, all of which were upregulated concurrently with bract development and betalain accumulation during the bract initiation stage of development. We found 47 genes specifically expressed in stamens, including seven highly expressed genes belonging to the pentose and glucuronate interconversion pathways. BgSEP2b, BgSWEET11, and BgRD22 are hub genes and interacted with many transcription factors and genes in the carpel co-expression network. CONCLUSIONS: We assembled protein-coding genes of Bougainvilea, identified the floral development genes, and constructed the gene co-expression network of petal, stamens, and carpel. Our results provide fundamental information about the mechanism of flower development and pigment accumulation in Bougainvillea, and will facilitate breeding of cultivars with high ornamental value.

A comprehensive review of beetroot (Beta vulgaris L.) bioactive components in the food and pharmaceutical industries.

Beetroot is rich in various bioactive phytochemicals, which are beneficial for human health and exert protective effects against several disease conditions like cancer, atherosclerosis, etc. Beetroot has various therapeutic applications, including antioxidant, antibacterial, antiviral, and analgesic functions. Besides the pharmacological effects, food industries are trying to preserve beetroots or their phytochemicals using various food preservation methods, including drying and freezing, to preserve their antioxidant capacity. Beetroot is a functional food due to valuable active components such as minerals, amino acids, phenolic acid, flavonoid, betaxanthin, and betacyanin. Due to its stability, nontoxic and non-carcinogenic and nonpoisonous capabilities, beetroot has been used as an additive or preservative in food processing. Beetroot and its bioactive compounds are well reported to possess antioxidant, anti-inflammatory, antiapoptotic, antimicrobial, antiviral, etc. In this review, we provided updated details on (i) food processing, preservation and colorant methods using beetroot and its phytochemicals, (ii) synthesis and development of several nanoparticles using beetroot and its bioactive compounds against various diseases, (iii) the role of beetroot and its phytochemicals under disease conditions with molecular mechanisms. We have also discussed the role of other phytochemicals in beetroot and their health benefits. Recent technologies in food processing are also updated. We also addressed on molecular docking-assisted biological activity and screening for bioactive chemicals. Additionally, the role of betalain from different sources and its therapeutic effects have been listed. To the best of our knowledge, little or no work has been carried out on the impact of beetroot and its nanoformulation strategies for phytocompounds on antimicrobial, antiviral effects, etc. Moreover, epigenetic alterations caused by phytocompounds of beetroot under several diseases were not reported much. Thus, extensive research must be carried out to understand the molecular effects of beetroot in the near future.

Evolution and function of red pigmentation in land plants.

BACKGROUND: Land plants commonly produce red pigmentation as a response to environmental stressors, both abiotic and biotic. The type of pigment produced varies among different land plant lineages. In the majority of species they are flavonoids, a large branch of the phenylpropanoid pathway. Flavonoids that can confer red colours include 3-hydroxyanthocyanins, 3-deoxyanthocyanins, sphagnorubins and auronidins, which are the predominant red pigments in flowering plants, ferns, mosses and liverworts, respectively. However, some flowering plants have lost the capacity for anthocyanin biosynthesis and produce nitrogen-containing betalain pigments instead. Some terrestrial algal species also produce red pigmentation as an abiotic stress response, and these include both carotenoid and phenolic pigments. SCOPE: In this review, we examine: which environmental triggers induce red pigmentation in non-reproductive tissues; theories on the functions of stress-induced pigmentation; the evolution of the biosynthetic pathways; and structure-function aspects of different pigment types. We also compare data on stress-induced pigmentation in land plants with those for terrestrial algae, and discuss possible explanations for the lack of red pigmentation in the hornwort lineage of land plants. CONCLUSIONS: The evidence suggests that pigment biosynthetic pathways have evolved numerous times in land plants to provide compounds that have red colour to screen damaging photosynthetically active radiation but that also have secondary functions that provide specific benefits to the particular land plant lineage.

Increased rates of gene-editing events using a simplified RNAi configuration designed to reduce gene silencing.

KEY MESSAGE: An optimal RNAi configuration that could restrict gene expression most efficiently was determined. This approach was also used to target PTGS and yielded higher rates of gene-editing events. Although it was characterized long ago, transgene silencing still strongly impairs transgene overexpression, and thus is a major barrier to plant crop gene-editing. The development of strategies that could prevent transgene silencing is therefore essential to the success of gene editing assays. Transgene silencing occurs via the RNA silencing process, which regulates the expression of essential genes and protects the plant from viral infections. The RNA silencing machinery thereby controls central biological processes such as growth, development, genome integrity, and stress resistance. RNA silencing is typically induced by aberrant RNA, that may lack 5' or 3' processing, or may consist in double-stranded or hairpin RNA, and involves DICER and ARGONAUTE family proteins. In this study, RNAi inducing constructs were designed in eleven different configurations and were evaluated for their capacity to induce silencing in Nicotiana spp. using transient and stable transformation assays. Using reporter genes, it was found that the overexpression of a hairpin consisting of a forward tandem inverted repeat that started with an ATG and that was not followed downstream by a transcription terminator, could downregulate gene expression most potently. Furthermore, using this method, the downregulation of the NtSGS3 gene caused a significant increase in transgene expression both in transient and stable transformation assays. This SGS3 silencing approach was also employed in gene-editing assays and caused higher rates of gene-editing events. Taken together, these findings suggested the optimal genetic configuration to cause RNA silencing and showed that this strategy may be used to restrict PTGS during gene-editing experiments.