Heat shock factor binding protein BrHSBP1 regulates seed and pod development in Brassica rapa.

Plant heat shock factor binding proteins (HSBPs) are well known for their implication in the negative regulation of heat stress response (HSR) pathways. Herein, we report on the hitherto unknown functions of HSBP1 in Brassica rapa (BrHSBP1). BrHBSP1 was found to be predominant in flower buds and young leaves, while its segmental duplicate, BrHSBP1-like, was abundant in green siliques. Exposure to abiotic stress conditions, such as heat, drought, cold, and H2O2, and to phytohormones was found to differentially regulate BrHSBP1. The activity of BrHSBP1-GFP fusion proteins revealed their cellular localization in nuclei and cytosols. Transgenic overexpression of BrHSBP1 (BrHSBP1OX) improved pod and seed sizes, while CRISPR-Cas BrHSBP1 knock-out mutants (Brhsbp1_KO) were associated with aborted seed and pod development. The transcriptomic signatures of BrHSBP1OX and Brhsbp1_KO lines revealed that 360 and 2381 genes, respectively, were differentially expressed (Log2FC≥2, padj<0.05) expressed relative to control lines. In particular, developmental processes, including plant reproductive structure development (RSD)-related genes, were relatively downregulated in Brhsbp1_KO. Furthermore, yeast two-hybrid assays confirmed that BrHSBP1 can physically bind to RSD and other genes. Taking the findings together, it is clear that BrHSBP1 is involved in seed development via the modulation of RSD genes. Our findings represent the addition of a new regulatory player in seed and pod development in B. rapa.

Optimization of extraction and quantification of Flavonoids from Averrhoa bilimbi fruits using RP-HPLC and its correlation between total flavonoids content against antimicrobial activity.

The present study was conducted to evaluate Averrhoa bilimbi fruits flavonoids extraction and quantification analysis through RP-HPLC and its comparison study on total flavonoids concentration versus antimicrobial activity analysis based on minimum inhibitory concentrations. Optimization of extraction was carried out using three different methods; among all methods the ultrasonic conventional assistant extraction (UCAE) showed an excellent recovery of flavonoids with solid phase elution. UCAE was performed with ethanol using different solvents ratio, the total flavonoid content was quantified through spectrophotometrically (850 ± 25 mg/kg) and flavonoids (myricetin and luteolin) were quantified by RP-HPLC. Optimized yield of myricetin and luteolin were presented in A. bilimbi fruits 336 ± 15 and 231 ± 18 mg/kg, respectively. Antimicrobial activity examined against E. coli, S. aureus and B. subtilis species using spectroscopically. The extracted sample with known quantity of total flavonoids content (10-200 µg/mL) used against E. coli, S. aureus and B. subtilis, MIC results shows 55.6 ± 6, 31 ± 3 and 28 ± 2 µg/mL respectively. Higher flavonoid content plays major role on antioxidant activities, which were evaluated and compared with commercial antioxidant butylated hydroxytoluene (BHT) employing superoxide anion scavenging activity and total reducing power IC50 value results shows 100 and 175 µg/mL, respectively. The maximum yield of flavonoid content results shows method suitability of flavonoids extraction and quantification.

Abiotic stress-induced secondary metabolite production in Brassica: opportunities and challenges.

Over the decades, extensive research efforts have been undertaken to understand how secondary plant metabolites are affected by genetic, environmental, and agronomic factors. Understanding the genetic basis of stress-response metabolite biosynthesis is crucial for sustainable agriculture production amidst frequent occurrence of climatic anomalies. Although it is known that environmental factors influence phytochemical profiles and their content, studies of plant compounds in relation to stress mitigation are only emerging and largely hindered by phytochemical diversities and technical shortcomings in measurement techniques. Despite these challenges, considerable success has been achieved in profiling of secondary metabolites such as glucosinolates, flavonoids, carotenoids, phenolic acids and alkaloids. In this study, we aimed to understand the roles of glucosinolates, flavonoids, carotenoids, phenolic acids and alkaloids in relation to their abiotic stress response, with a focus on the developing of stress-resilient crops. The focal genus is the Brassica since it (i) possesses variety of specialized phytochemicals that are important for its plant defense against major abiotic stresses, and (ii) hosts many economically important crops that are sensitive to adverse growth conditions. We summarize that augmented levels of specialized metabolites in Brassica primarily function as stress mitigators against oxidative stress, which is a secondary stressor in many abiotic stresses. Furthermore, it is clear that functional characterization of stress-response metabolites or their genetic pathways describing biosynthesis is essential for developing stress-resilient Brassica crops.

Phylogenomics-Based Reconstruction and Molecular Evolutionary Histories of Brassica Photoreceptor Gene Families.

Photosensory proteins known as photoreceptors (PHRs) are crucial for delineating light environments in synchronization with other environmental cues and regulating their physiological variables in plants. However, this has not been well studied in the Brassica genus, which includes several important agricultural and horticultural crops. Herein, we identified five major PHR gene families-phytochrome (PHY), cryptochrome (CRY), phototropin (PHOT), F-box containing flavin binding proteins (ZTL/FKF1/LKP2), and UV RESISTANCE LOCUS 8 (UVR8)-genomic scales and classified them into subfamilies based on their phylogenetic clustering with Arabidopsis homologues. The molecular evolution characteristics of Brassica PHR members indicated indirect expansion and lost one to six gene copies at subfamily levels. The segmental duplication was possibly the driving force of the evolution and amplification of Brassica PHRs. Gene replication retention and gene loss events of CRY, PHY, and PHOT members found in diploid progenitors were highly conserved in their tetraploid hybrids. However, hybridization events were attributed to quantitative changes in UVR8 and ZTL/FKF1/LKP2 members. All PHR members underwent purifying selection. In addition, the transcript expression profiles of PHR genes in different tissue and in response to exogenous ABA, and abiotic stress conditions suggested their multiple biological significance. This study is helpful in understanding the molecular evolution characteristics of Brassica PHRs and lays the foundation for their functional characterization.

Influence of Brevibacillus borestelensis strains on phytoremediation potential and biomolecules contents of Jatropha curcas on diluted chromium sludge soil.

This study was carried out in order to find an environmentally friendly solution to recover the abandoned Cr-enriched sludge soil, which causes a variety of environmental issues. Hence, in this research the influence of pre-identified Brevibacillus borstelensis UTM105 and Brevibacillus borstelensis AK2 coated Jatropha curcas seed in phytoremediation process with various treatment groups (group A to F) under greenhouse condition. Furthermore, their influence on growth, biomolecules (total proteins and total chlorophyll) content, and antioxidant activity of J. curcas during the phytoremediation process were analyzed. Surprisingly, the outstanding phytoremediation was recorded in group F treatment. In these groups, Group E. accompanied it, and the Cr was reduced by up to 31.17% and 25.65%, respectively, in treated soil after 90 days of treatment. Among these two bacterial strains, the B. borstelensis AK2 had greatest effect on J. curcas growth, the yield of biomass, total protein, total chlorophyll, and antioxidant activity and it followed by B. borstelensis UTM105. These phytoremediation potential of J. curcas was effective at soil diluted with fertile and xenobiotics free soil with dilution ratio of 50:50 and followed by 75:25 ratio. Because under undiluted Cr sludge soil condition seed germination has not occurred even though the seed has been coated with potential bacterial strains and soil blend with sterilized goat manure. Hence, under diluted conditions J. curcas seed coated with B. borstelensis AK2 showed an outstanding phytoremediation process. Hence, this approach can be applied to a field study to assess the metal removal potential of this sustainable approach.

Genome-wide identification, characterization of expansin gene family of banana and their expression pattern under various stresses.

Expansin, a cell wall-modifying gene family, has been well characterized and its role in biotic and abiotic stress resistance has been proven in many monocots, but not yet studied in banana, a unique model crop. Banana is one of the staple food crops in developing countries and its production is highly influenced by various biotic and abiotic factors. Characterizing the expansin genes of the ancestor genome (M. acuminata and M. balbisiana) of present day cultivated banana will enlighten their role in growth and development, and stress responses. In the present study, 58 (MaEXPs) and 55 (MbaEXPs) putative expansin genes were identified in A and B genome, respectively, and were grouped in four subfamilies based on phylogenetic analysis. Gene structure and its duplications revealed that EXPA genes are highly conserved and are under negative selection whereas the presence of more number of introns in other subfamilies revealed that they are diversifying. Expression profiling of expansin genes showed a distinct expression pattern for biotic and abiotic stress conditions. This study revealed that among the expansin subfamilies, EXPAs contributed significantly towards stress-resistant mechanism. The differential expression of MaEXPA18 and MaEXPA26 under drought stress conditions in the contrasting cultivar suggested their role in drought-tolerant mechanism. Most of the MaEXPA genes are differentially expressed in the root lesion nematode contrasting cultivars which speculated that this expansin subfamily might be the susceptible factor. The downregulation of MaEXPLA6 in resistant cultivar during Sigatoka leaf spot infection suggested that by suppressing this gene, resistance may be enhanced in susceptible cultivar. Further, in-depth studies of these genes will lead to gain insight into their role in various stress conditions in banana. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-021-03106-x.

Plant RNA Binding Proteins as Critical Modulators in Drought, High Salinity, Heat, and Cold Stress Responses: An Updated Overview.

Plant abiotic stress responses are tightly regulated by different players at multiple levels. At transcriptional or post-transcriptional levels, several RNA binding proteins (RBPs) regulate stress response genes through RNA metabolism. They are increasingly recognized as critical modulators of a myriad of biological processes, including stress responses. Plant RBPs are heterogeneous with one or more conservative RNA motifs that constitute canonical/novel RNA binding domains (RBDs), which can bind to target RNAs to determine their regulation as per the plant requirements at given environmental conditions. Given its biological significance and possible consideration as a potential tool in genetic manipulation programs to improve key agronomic traits amidst frequent episodes of climate anomalies, studies concerning the identification and functional characterization of RBP candidate genes are steadily mounting. This paper presents a comprehensive overview of canonical and novel RBPs and their functions in major abiotic stresses including drought, heat, salt, and cold stress conditions. To some extent, we also briefly describe the basic motif structure of RBPs that would be useful in forthcoming studies. Additionally, we also collected RBP genes that were modulated by stress, but that lacked functional characterization, providing an impetus to conduct further research.

BrPP5.2 Overexpression Confers Heat Shock Tolerance in Transgenic Brassica rapa through Inherent Chaperone Activity, Induced Glucosinolate Biosynthesis, and Differential Regulation of Abiotic Stress Response Genes.

Plant phosphoprotein phosphatases are ubiquitous and multifarious enzymes that respond to developmental requirements and stress signals through reversible dephosphorylation of target proteins. In this study, we investigated the hitherto unknown functions of Brassica rapa protein phosphatase 5.2 (BrPP5.2) by transgenic overexpression of B. rapa lines. The overexpression of BrPP5.2 in transgenic lines conferred heat shock tolerance in 65-89% of the young transgenic seedlings exposed to 46 °C for 25 min. The examination of purified recombinant BrPP5.2 at different molar ratios efficiently prevented the thermal aggregation of malate dehydrogenase at 42 °C, thus suggesting that BrPP5.2 has inherent chaperone activities. The transcriptomic dynamics of transgenic lines, as determined using RNA-seq, revealed that 997 and 1206 (FDR < 0.05, logFC ≥ 2) genes were up- and down-regulated, as compared to non-transgenic controls. Statistical enrichment analyses revealed abiotic stress response genes, including heat stress response (HSR), showed reduced expression in transgenic lines under optimal growth conditions. However, most of the HSR DEGs were upregulated under high temperature stress (37 °C/1 h) conditions. In addition, the glucosinolate biosynthesis gene expression and total glucosinolate content increased in the transgenic lines. These findings provide a new avenue related to BrPP5.2 downstream genes and their crucial metabolic and heat stress responses in plants.

Changes in Beneficial C-glycosylflavones and Policosanol Content in Wheat and Barley Sprouts Subjected to Differential LED Light Conditions.

The spectral quality and intensity of light, photoperiodism, and other environmental factors have profound impacts on the metabolic composition of light-dependent higher plants. Hence, we investigate the effects of fluorescent light (96 µmol m-2s-1) and white (100 µmol m-2s-1), blue (100 µmol m-2s-1), and red (93 µmol m-2s-1) light-emitting diode (LED) light irradiation on the C-glycosylflavone and policosanol contents in young seedlings of wheat and barley. Ultra-high-performance liquid chromatography (UHPLC) analyses of C-glycosylflavone contents in barley reveal that the saponarin content is significantly enhanced under blue LED light irradiation. Under similar conditions, isoorientin and isoschaftoside contents are improved in wheat seedlings. The contents of these C-glycosylflavones differed along with the light quality and growth period. The highest accumulation was observed in sprouts after three days under blue LED light irradiation. GC/MS analyses of policosanol contents showed that 1-hexacosanol (C26:o-OH) in barley and 1-octacosanol (C28:o-OH) in wheat seedlings were reduced under LED light irradiation, compared to seedlings under fluorescent light conditions. Nonetheless, the policosanol contents gradually improved with the extension of growth times and treatments, irrespective of the light quality. Additionally, a positive correlation was observed between the expression pattern of biosynthesis-related genes and the respective metabolite content in barley. This study demonstrates that blue LED light irradiation is useful in maximizing the C-glycosylflavone content in barley and wheat sprouts.

BrEXLB1, a Brassica rapa Expansin-Like B1 Gene is Associated with Root Development, Drought Stress Response, and Seed Germination.

Expansins are structural proteins prevalent in cell walls, participate in cell growth and stress responses by interacting with internal and external signals perceived by the genetic networks of plants. Herein, we investigated the Brassica rapa expansin-like B1 (BrEXLB1) interaction with phytohormones (IAA, ABA, Ethephon, CK, GA3, SA, and JA), genes (Bra001852, Bra001958, and Bra003006), biotic (Turnip mosaic Virus (TuMV), Pectobacterium carotovorum, clubroot disease), and abiotic stress (salt, oxidative, osmotic, and drought) conditions by either cDNA microarray or qRT-PCR assays. In addition, we also unraveled the potential role of BrEXLB1 in root growth, drought stress response, and seed germination in transgenic Arabidopsis and B. rapa lines. The qRT-PCR results displayed that BrEXLB1 expression was differentially influenced by hormones, and biotic and abiotic stress conditions; upregulated by IAA, ABA, SA, ethylene, drought, salt, osmotic, and oxidative conditions; and downregulated by clubroot disease, P. carotovorum, and TuMV infections. Among the tissues, prominent expression was observed in roots indicating the possible role in root growth. The root phenotyping followed by confocal imaging of root tips in Arabidopsis lines showed that BrEXLB1 overexpression increases the size of the root elongation zone and induce primary root growth. Conversely, it reduced the seed germination rate. Further analyses with transgenic B. rapa lines overexpressing BrEXLB1 sense (OX) and antisense transcripts (OX-AS) confirmed that BrEXLB1 overexpression is positively associated with drought tolerance and photosynthesis during vegetative growth phases of B. rapa plants. Moreover, the altered expression of BrEXLB1 in transgenic lines differentially influenced the expression of predicted BrEXLB1 interacting genes like Bra001852 and Bra003006. Collectively, this study revealed that BrEXLB1 is associated with root development, drought tolerance, photosynthesis, and seed germination.

Brassica Rapa SR45a Regulates Drought Tolerance via the Alternative Splicing of Target Genes.

The emerging evidence has shown that plant serine/arginine-rich (SR) proteins play a crucial role in abiotic stress responses by regulating the alternative splicing (AS) of key genes. Recently, we have shown that drought stress enhances the expression of SR45a (also known as SR-like 3) in Brassica rapa. Herein, we unraveled the hitherto unknown functions of BrSR45a in drought stress response by comparing the phenotypes, chlorophyll a fluorescence and splicing patterns of the drought-responsive genes of Arabidopsis BrSR45a overexpressors (OEs), homozygous mutants (SALK_052345), and controls (Col-0). Overexpression and loss of function did not result in aberrant phenotypes; however, the overexpression of BrSR45a was positively correlated with drought tolerance and the stress recovery rate in an expression-dependent manner. Moreover, OEs showed a higher drought tolerance index during seed germination (38.16%) than the control lines. Additionally, the overexpression of BrSR45a induced the expression of the drought stress-inducible genes RD29A, NCED3, and DREB2A under normal conditions. To further illustrate the molecular linkages between BrSR45a and drought tolerance, we investigated the AS patterns of key drought-tolerance and BrSR45a interacting genes in OEs, mutants, and controls under both normal and drought conditions. The splicing patterns of DCP5, RD29A, GOLS1, AKR, U2AF, and SDR were different between overexpressors and mutants under normal conditions. Furthermore, drought stress altered the splicing patterns of NCED2, SQE, UPF1, U4/U6-U5 tri-snRNP-associated protein, and UPF1 between OEs and mutants, indicating that both overexpression and loss of function differently influenced the splicing patterns of target genes. This study revealed that BrSR45a regulates the drought stress response via the alternative splicing of target genes in a concentration-dependent manner.

Sound waves affect the total flavonoid contents in Medicago sativa, Brassica oleracea and Raphanus sativus sprouts.

BACKGROUND: Sound waves are emerging as a potential biophysical alternative to traditional methods for enhancing plant growth and phytochemical contents. However, little information is available on the improvement of the concentration of functional metabolites like flavonoids in sprouts using sound waves. In this study, different frequencies of sound waves with short and long exposure times were applied to three important varieties to improve flavonoid content. The aim of this study was to investigate the effect of sound waves on flavonoid content on the basis of biochemical and molecular characteristics. RESULTS: We examined the effects of various sound wave treatments (250 Hz to 1.5 kHz) on flavonoid production in alfalfa (Medicago sativa), broccoli (Brassica oleracea) and red young radish (Raphanus sativus). The results showed that sound wave treatments differentially altered the total flavonoid contents depending upon the growth stages, species and frequency of and exposure time to sound waves. Sound wave treatments of alfalfa (250 Hz), broccoli sprouts (800 Hz) and red young radish sprouts (1 kHz) increased the total flavonoid content by 200%, 35% and 85%, respectively, in comparison with untreated control. Molecular analysis showed that sound waves induce the expression of genes of the flavonoid biosynthesis pathway, which positively corresponds to the flavonoid content. Moreover, the sound wave treatment significantly improves the antioxidant efficiency of sprouts. CONCLUSIONS: The significant improvement of flavonoid content in sprouts with sound waves makes their use a potential and promising technology for the production of agriculture-based functional foods. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Genome-wide analysis of spatiotemporal gene expression patterns during floral organ development in Brassica rapa.

Flowering is a key agronomic trait that directly influences crop yield and quality and serves as a model system for elucidating the molecular basis that controls successful reproduction, adaptation, and diversification of flowering plants. Adequate knowledge of continuous series of expression data from the floral transition to maturation is lacking in Brassica rapa. To unravel the genome expression associated with the development of early small floral buds (< 2 mm; FB2), early large floral buds (2-4 mm; FB4), stamens (STs) and carpels (CPs), transcriptome profiling was carried out with a Br300K oligo microarray. The results showed that at least 6848 known nonredundant genes (30% of the genes of the Br300K) were differentially expressed during the floral transition from vegetative tissues to maturation. Functional annotation of the differentially expressed genes (DEGs) (fold change ≥ 5) by comparison with a close relative, Arabidopsis thaliana, revealed 6552 unigenes (4579 upregulated; 1973 downregulated), including 131 Brassica-specific and 116 functionally known floral Arabidopsis homologs. Additionally, 1723, 236 and 232 DEGs were preferentially expressed in the tissues of STs, FB2, and CPs. These DEGs also included 43 transcription factors, mainly AP2/ERF-ERF, NAC, MADS-MIKC, C2H2, bHLH, and WRKY members. The differential gene expression during flower development induced dramatic changes in activities related to metabolic processes (23.7%), cellular (22.7%) processes, responses to the stimuli (7.5%) and reproduction (1%). A relatively large number of DEGs were observed in STs and were overrepresented by photosynthesis-related activities. Subsequent analysis via semiquantitative RT-PCR, histological analysis performed with in situ hybridization of BrLTP1 and transgenic reporter lines (BrLTP promoter::GUS) of B. rapa ssp. pekinensis supported the spatiotemporal expression patterns. Together, these results suggest that a temporally and spatially regulated process of the selective expression of distinct fractions of the same genome leads to the development of floral organs. Interestingly, most of the differentially expressed floral transcripts were located on chromosomes 3 and 9. This study generated a genome expression atlas of the early floral transition to maturation that represented the flowering regulatory elements of Brassica rapa.

Differential proteome analysis during early somatic embryogenesis in Musa spp. AAA cv. Grand Naine.

KEY MESSAGE: Endogenous hormone secretion proteins along with stress and defense proteins play predominant role in banana embryogenesis. This study reveals the underlying molecular mechanism during transition from vegetative to embryogenic state. Banana (Musa spp.) is well known globally as a food fruit crop for millions. The requirement of quality planting material of banana is enormous. Although mass multiplication through tissue culture is in vogue, high-throughput techniques like somatic embryogenesis (SE) as a mass multiplication tool needs to be improved. Apart from clonal propagation, SE has extensive applications in genetic improvement and mutation. SE in banana is completely genome-dependent and most of the commercial cultivars exhibit recalcitrance. Thus, understanding the molecular basis of embryogenesis in Musa will help to develop strategies for mass production of quality planting material. In this study, differentially expressed proteins between embryogenic calli (EC) and non-embryogenic calli (NEC) with respect to the explant, immature male flower buds (IMFB), of cv. Grand Naine (AAA) were determined using two-dimensional gel electrophoresis (2DE). The 2DE results were validated through qRT-PCR. In total, 65 proteins were identified: 42 were highly expressed and 23 were less expressed in EC compared to NEC and IMFB. qRT-PCR analysis of five candidate proteins, upregulated in EC, were well correlated with expression at transcript level. Further analysis of proteins showed that embryogenesis in banana is associated with the control of oxidative stress. The regulation of ROS scavenging system and protection of protein structure occurred in the presence of heat shock proteins. Alongside, high accumulation of stress-related cationic peroxidase and plant growth hormone-related proteins like indole-3-pyruvate monooxygenase and adenylate isopentenyltransferase in EC revealed the association with the induction of SE.

Transcriptomic Changes of Drought-Tolerant and Sensitive Banana Cultivars Exposed to Drought Stress.

In banana, drought responsive gene expression profiles of drought-tolerant and sensitive genotypes remain largely unexplored. In this research, the transcriptome of drought-tolerant banana cultivar (Saba, ABB genome) and sensitive cultivar (Grand Naine, AAA genome) was monitored using mRNA-Seq under control and drought stress condition. A total of 162.36 million reads from tolerant and 126.58 million reads from sensitive libraries were produced and mapped onto the Musa acuminata genome sequence and assembled into 23,096 and 23,079 unigenes. Differential gene expression between two conditions (control and drought) showed that at least 2268 and 2963 statistically significant, functionally known, non-redundant differentially expressed genes (DEGs) from tolerant and sensitive libraries. Drought has up-regulated 991 and 1378 DEGs and down-regulated 1104 and 1585 DEGs respectively in tolerant and sensitive libraries. Among DEGs, 15.9% are coding for transcription factors (TFs) comprising 46 families and 9.5% of DEGs are constituted by protein kinases from 82 families. Most enriched DEGs are mainly involved in protein modifications, lipid metabolism, alkaloid biosynthesis, carbohydrate degradation, glycan metabolism, and biosynthesis of amino acid, cofactor, nucleotide-sugar, hormone, terpenoids and other secondary metabolites. Several, specific genotype-dependent gene expression pattern was observed for drought stress in both cultivars. A subset of 9 DEGs was confirmed using quantitative reverse transcription-PCR. These results will provide necessary information for developing drought-resilient banana plants.