SQUAMOSA PROMOTER BINDING PROTEIN Research Articles

MicroRNA (miRNA) profiling of maize genotypes with differential response to Aspergillus flavus implies zma-miR156–squamosa promoter binding protein (SBP) and zma-miR398/zma-miR394–F -box combinations involved in resistance mechanisms

Maize (Zea mays), a major food crop worldwide, is susceptible to infection by the saprophytic fungus Aspergillus flavus that can produce the carcinogenic metabolite aflatoxin (AF) especially under climate change induced abiotic stressors that favor mold growth. Several studies have used “-omics” approaches to identify genetic elements with potential roles in AF resistance, but there is a lack of research identifying the involvement of small RNAs such as microRNAs (miRNAs) in maize-A. flavus interaction. In this study, we compared the miRNA profiles of three maize lines (resistant TZAR102, moderately resistant MI82, and susceptible Va35) at 8 h, 3 d, and 7 d after A. flavus infection to investigate possible regulatory antifungal role of miRNAs. A total of 316 miRNAs (275 known and 41 putative novel) belonging to 115 miRNA families were identified in response to the fungal infection across all three maize lines. Eighty-two unique miRNAs were significantly differentially expressed with 39 miRNAs exhibiting temporal differential regulation irrespective of the maize genotype, which targeted 544 genes (mRNAs) involved in diverse molecular functions. The two most notable biological processes involved in plant immunity, namely cellular responses to oxidative stress (GO:00345990) and reactive oxygen species (GO:0034614) were significantly enriched in the resistant line TZAR102. Coexpression network analysis identified 34 hubs of miRNA-mRNA pairs where nine hubs had a node in the module connected to their target gene with potentially important roles in resistance/susceptible response of maize to A. flavus. The miRNA hubs in resistance modules (TZAR102 and MI82) were mostly connected to transcription factors and protein kinases. Specifically, the module of miRNA zma-miR156b-nb – squamosa promoter binding protein (SBP), zma-miR398a-3p – SKIP5, and zma-miR394a-5p – F-box protein 6 combinations in the resistance-associated modules were considered important candidates for future functional studies.

Identification and expression profiling of flowering-related microRNAs and their targets reveals complex regulatory networks associated with bolting time in spinach

Bolting is one of the most important limiting traits in vegetable crops, like spinach, when spurt to flowering before harvesting. The evidence indicates bolting is controlled by various environmental factors including temperature, photoperiod, stresses, and genetic factors such as lncRNAs and miRNAs, but there is no information about miRNA-mediated gene expressions and their roles in flowering-time regulatory networks in spinach. Therefore, to gain more insights into the molecular mechanisms of flowering and bolting, we undertook a comprehensive analysis of miRNAs and their expression profiles in two distinct spinach accessions, namely Kashan (early bolting) and Viroflay (late bolting) by employing cutting-edge high-throughput sequencing technology. Our investigation identified 720 and 750 miRNAs in Kashan and Viroflay, respectively. Remarkably, amid the conserved miRNAs, a convergence of 15 families, exemplified by miR156, miR160, miR164, miR166, miR169, and miR399, renowned for their pivotal roles in flowering/bolting temporal control, was discerned across both accessions. Concerning differentially expressed miRNAs (DEMs), 64 target genes were identified, of which 40 genes were classified into seven flowering-related pathways including age, autonomous, circadian clock, hormone, integrator, photoperiodism, and vernalization. Moreover, novel crosstalks between miRNAs and various flowering-associated genes operating in flowering-related pathways, such as miR156 and squamosa promoter-binding proteins, and miR159 and GAMYB genes in the GA flowering pathway, were identified. Additionally, miR172 influenced MADS-box and AP2-like, while miR319 family, miR164, miR160, and miR169 played distinct roles in modulating key factors associated with flowering processes, including Flowering locus T, FLOWERING PROMOTING FACTOR 1, NAC domain, auxin response factor genes, zinc finger genes, and inorganic phosphate transporter genes, showcasing their intricate regulatory involvement in flowering and bolting pathways. The findings of this study will extend our knowledge of miRNA-mediated flowering modulation and can be used in breeding programs aimed at manipulating the spinach floral regulation system.

Identification and expression analysis of SBP-Box-like (SPL) gene family disclose their contribution to abiotic stress and flower budding in pigeon pea (Cajanus cajan).

The SPL gene family (for Squamosa Promoter-binding like Proteins) represents specific transcription factors that have significant roles in abiotic stress tolerance, development and the growth processes of different plants, including initiation of the leaf, branching and development of shoot and fruits. The SPL gene family has been studied in different plant species; however, its role is not yet fully explored in pigeon pea (Cajanus cajan ). In the present study, 11 members of the CcSPL gene family were identified in C. cajan . The identified SPLs were classified into nine groups based on a phylogenetic analysis involving SPL protein sequences from C. cajan , Arabidopsis thaliana , Cicer arietinum , Glycine max , Phaseolus vulgaris , Vigna unguiculata and Arachis hypogaea . Further, the identification of gene structure, motif analysis, domain analysis and presence of cis -regulatory elements in the SPL family members were studied. Based on RNA-sequencing data, gene expression analysis was performed, revealing that CcSPL2.1, 3 and 13A were significantly upregulated for salt-tolerance and CcSPL14 and 15 were upregulated in a salt-susceptible cultivar. Real-time qPCR validation indicated that CcSPL3, 4, 6 and 13A were upregulated under salt stress conditions. Therefore, molecular docking was performed against the proteins of two highly expressed genes (CcSPL3 and CcSPL14 ) with three ligands: abscisic acid, gibberellic acid and indole-3-acetic acid. Afterward, their binding affinity was obtained and three-dimensional structures were predicted. In the future, our study may open avenues for harnessing CcSPL genes in pigeon pea for enhanced abiotic stress resistance and developmental traits.

Glutathione-dependent redox homeostasis is critical for chlorothalonil detoxification in tomato leaves

Glutathione plays a critical role in plant growth, development and response to stress. It is a major cellular antioxidant and is involved in the detoxification of xenobiotics in many organisms, including plants. However, the role of glutathione-dependent redox homeostasis and associated molecular mechanisms regulating the antioxidant system and pesticide metabolism remains unclear. In this study, endogenous glutathione levels were manipulated by pharmacological treatments with glutathione synthesis inhibitors and oxidized glutathione. The application of oxidized glutathione enriched the cellular oxidation state, reduced the activity and transcript levels of antioxidant enzymes, upregulated the expression level of nitric oxide and Ca2+ related genes and the content, and increased the residue of chlorothalonil in tomato leaves. Further experiments confirmed that glutathione-induced redox homeostasis is critical for the reduction of pesticide residues. RNA sequencing analysis revealed that miRNA156 and miRNA169 that target transcription factor SQUAMOSA-Promoter Binding Proteins (SBP) and NUCLEAR FACTOR Y (NFY) potentially participate in glutathione-mediated pesticide degradation in tomato plants. Our study provides important clues for further dissection of pesticide degradation mechanisms via miRNAs in plants.

Comprehensive characterization of SBP genes revealed their role under multifactorial stress combinations in dragon fruit (Selenicereus undatus L.)

The SQUAMOSA PROMOTER BINDING PROTEIN (SBP) gene family is essential for plant growth and development. However, little is known about the SBP gene family in pitaya (Selenicereus undatus L.); a fruit crop cultivated in tropical climates, severely affected by abiotic stress factors. These stresses produce excessive reactive oxygen species (ROS), affecting pitaya growth and development. In this study, we identified 17 SBP genes in the pitaya genome (HuSBP) and classified them into seven distinct clusters based on their phylogeny. The HuSBP genes exhibited a conserved intron-exon structure and motif domain organization. These SBP genes were differentially expressed in 15 developmental stages of flower (F), flower buds (FB), pericarp (PeriC), and pulp. In addition, specific HuSBP genes exhibited higher expression under single-factor, double-factor, and multifactorial stresses, revealing their diverse role in the growth and development of pitaya. Exogenous application of melatonin with known antioxidant and stress-protective properties, was also applied. The expression pattern of RNA-Seq data was validated by RT-qPCR analysis. Based on genome-wide results and transcriptome-based data under multifactorial stresses, we predicted five putative candidate genes (HuSBP1/3/7/11/14) that might play diverse roles in pitaya growth and development. These genes contain vital enzymes that play a role in the production of phenylpropanoid and glutathione metabolism. These results provide valuable insights into the role of HuSBP genes in pitaya stress tolerance and could be used to develop innovative strategies to create resilient and adaptable pitaya genotypes using conventional breeding and modern biotechnological tools.

Genome-wide identification and analysis of the evolution and expression pattern of the SBP gene family in two Chimonanthus species.

Chimonanthus praecox and Chimonanthus salicifolius are closely related species that diverged approximately six million years ago. While both C. praecox and C. salicifolius could withstand brief periods of low temperatures of -15°C. Their flowering times are different, C. praecox blooms in early spring, whereas C. salicifolius blooms in autumn. The SBP-box (SQUAMOSA promoter-binding protein) is a plant-specific gene family that plays a crucial vital role in regulating plant flowering. Although extensively studied in various plants, the SBP gene family remains uncharacterized in Calycanthaceae. We conducted genome-wide identification of SBP genes in both C. praecox and C. salicifolius and comprehensively characterized the chromosomal localization, gene structure, conserved motifs, and domains of the identified SBP genes. In total, 15 and 18 SBP genes were identified in C. praecox and C. salicifolius, respectively. According to phylogenetic analysis, the SBP genes from Arabidopsis, C. praecox, and C. salicifolius were clustered into eight groups. Analysis of the gene structure and conserved protein motifs showed that SBP proteins of the same subfamily have similar motif structures. The expression patterns of SBP genes were analyzed using transcriptome data. The results revealed that more than half of the genes exhibited lower expression levels in leaves than in flowers, suggesting their potential involvement in the flower development process and may be linked to the winter and autumn flowering of C. praecox and C. salicifolius. Thirty-three SBPs were identified in C. praecox and C. salicifolius. The evolutionary characteristics and expression patterns were examined in this study. These results provide valuable information to elucidate the evolutionary relationships of the SBP family and help determine the functional characteristics of the SBP genes in subsequent studies.

Evolutionary assessment of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE genes in citrus relatives with a specific focus on flowering

Phase transition and floral induction in citrus requires several years of juvenility after germination. Such a long period of juvenility has been a major hindrance to its genetic improvement program. Studies have shown that miR156 along with its downstream genes SQUAMOSA PROMOTER BINDING PROTEINS (SBP) and SBP-LIKE (SPL) mediate the phase transition and floral induction process in plants. Our current study has systematically analyzed SPLs in 15 different citrus-related species, systematically annotated them based on their close homology to their respective Arabidopsis orthologs, and confirmed the functional attributes of the selected members in floral precocity. The majority of the species harbored 15 SPLs. Their cis-element assessment suggested the involvement of the SPLs in diverse developmental and physiological processes in response to different biotic and abiotic cues. Among all, SPL5, SPL9, and SPL11 stood out as consistently differentially expressed SPLs in the adult and young tissues of different citrus-related species. Independent overexpression of their F. hindsii orthologs (FhSPL5, FhSPL9, and FhSPL11) brought an enhanced expression of endogenous FLOWERING LOCUS T leading to the significantly precocious flowering in transgenic Arabidopsis lines. Future study of the genes in the citrus plant itself is expected to conclude the assessments made in the current study.Graphical

Molecular and functional dissection of LIGULELESS1 (LG1) in plants.

Plant architecture is a culmination of the features necessary for capturing light energy and adapting to the environment. An ideal architecture can promote an increase in planting density, light penetration to the lower canopy, airflow as well as heat distribution to achieve an increase in crop yield. A number of plant architecture-related genes have been identified by map cloning, quantitative trait locus (QTL) and genome-wide association study (GWAS) analysis. LIGULELESS1 (LG1) belongs to the squamosa promoter-binding protein (SBP) family of transcription factors (TFs) that are key regulators for plant growth and development, especially leaf angle (LA) and flower development. The DRL1/2-LG1-RAVL pathway is involved in brassinosteroid (BR) signaling to regulate the LA in maize, which has facilitated the regulation of plant architecture. Therefore, exploring the gene regulatory functions of LG1, especially its relationship with LA genes, can help achieve the precise regulation of plant phenotypes adapted to varied environments, thereby increasing the yield. This review comprehensively summarizes the advances in LG1 research, including its effect on LA and flower development. Finally, we discuss the current challenges and future research goals associate with LG1.

Identification and expression analysis of SQUAMOSA promoter-binding protein (SBP) genes in mungbean

Identification and expression analysis of SQUAMOSA promoter-binding protein (SBP) genes in mungbean

Overexpression of soybean microRNA156b enhanced tolerance to phosphorus deficiency and seed yield in Arabidopsis

microRNAs (miRNAs) are endogenous small RNAs that are key regulatory factors participating in various biological activities such as the signaling of phosphorus deficiency in the plant. Previous studies have shown that miR156 expression was modulated by phosphorus starvation in Arabidopsis and soybean. However, it is not clear whether the over-expression of soybean miR156b (GmmiR156b) can improve a plant’s tolerance to phosphorus deficiency and affect yield component traits. In this study, we generated Arabidopsis transgenic lines overexpressing GmmiR156b and investigated the plant’s response to phosphorus deficiency. Compared with the wild type, the transgenic Arabidopsis seedlings had longer primary roots and higher phosphorus contents in roots under phosphorus-deficit conditions, but lower fresh weight root/shoot ratios under either phosphorus-deficient or sufficient conditions. Moreover, the GmmiR156b overexpression transgenic lines had higher phosphorus content in shoots of adult plants and grew better than the wide type under phosphorus-deficient conditions, and exhibited increased seed yields as well as strong pleiotropic developmental morphology such as dwarfness, prolonged growth period, bushy shoot/branching, and shorter silique length, suggesting that the transgenic lines were more tolerant to phosphorus deficiency. In addition, the expression level of four SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes (i.e., AtSPL4/5/6/15) were markedly suppressed in transgenic plants, indicating that they were the main targets negatively regulated by GmmiR156b (especially AtSPL15) and that the enhanced tolerance to phosphorus deficiency and seed yield is conferred mainly by the miR156-mediated downregulation of AtSPL15.

Identification of SBP Gene Family and Analysis of Expression Patterns Under Salt Stress in Quinoa

This study aimed to identify the Squamosa promoter Binding Protein (SBP) transcription factor from the whole genome of quinoa by analyzing its phylogenetic relationship, gene structure, chromosome location, gene replication, upstream cis-regulatory elements, tissue expression, and construction of the SBP protein interaction network, as well as the characteristics of its tissue expression pattern under salt stress. The researchers found that 23 genes of the CqSBP family were identified through bioinformatics analysis and showed diversity in their amino acid physical and chemical properties. These proteins were hydrophilic and all 23 CqSBPs were located in the nucleus. The SBP family genes were unevenly distributed across the 12 chromosomes of quinoa, mostly on the B chromosome group. The upstream cis-acting element analysis revealed the presence of 49 elements with plant hormones, stress, light response, and tissue-specific expression, and all CqSBPs contained one or more Tata box elements. Protein interaction network analysis showed that all CqSBP proteins appeared in the known interaction network of Arabidopsis. Different SBP genes were differently expressed in different organs and periods of quinoa, and SBP genes were expressed with certain tissue specificity. The expression of CqSBP showed many changes under salt stress.

UB2/UB3/TSH4-anchored transcriptional networks regulate early maize inflorescence development in response to simulated shade.

Increasing planting density has been adopted as an effective means to increase maize (Zea mays) yield. Competition for light from neighbors can trigger plant shade avoidance syndrome, which includes accelerated flowering. However, the regulatory networks of maize inflorescence development in response to high-density planting remain poorly understood. In this study, we showed that shade-mimicking treatments cause precocious development of the tassels and ears. Comparative transcriptome profiling analyses revealed the enrichment of phytohormone-related genes and transcriptional regulators among the genes co-regulated by developmental progression and simulated shade. Network analysis showed that three homologous Squamosa promoter binding protein (SBP)-like (SPL) transcription factors, Unbranched2 (UB2), Unbranched3 (UB3), and Tasselsheath4 (TSH4), individually exhibited connectivity to over 2,400 genes across the V3-to-V9 stages of tassel development. In addition, we showed that the ub2 ub3 double mutant and tsh4 single mutant were almost insensitive to simulated shade treatments. Moreover, we demonstrated that UB2/UB3/TSH4 could directly regulate the expression of Barren inflorescence2 (BIF2) and Zea mays teosinte branched1/cycloidea/proliferating cell factor30 (ZmTCP30). Furthermore, we functionally verified a role of ZmTCP30 in regulating tassel branching and ear development. Our results reveal a UB2/UB3/TSH4-anchored transcriptional regulatory network of maize inflorescence development and provide valuable targets for breeding shade-tolerant maize cultivars.

Genome-Wide Identification and Characterization of the SBP Gene Family in Passion Fruit (Passiflora edulis Sims).

The SQUAMOSA promoter binding proteins (SBPs) gene family plays important roles in plant growth and development. The SBP gene family has been identified and reported in many species, but it has not been well studied in passion fruit. In this study, a total of 14 SBP genes were identified in passion fruit and named from PeSBP1 to PeSBP14 based on their chromosomal distribution. The phylogenetic tree, gene structure, conserved motifs, collinearity analysis, and expression patterns of the identified SBP members were analyzed. We classified the PeSBP genes into eight groups (I to VIII) according to the phylogenetic tree, gene structure, and conserved motifs. Synteny analysis found that 5 homologous gene pairs existed in PeSBP genes and 11 orthologous gene pairs existed between passion fruit and Arabidopsis. Synonymous nucleotide substitution analysis showed that the PeSBP genes were under strong negative selection. The expression pattern of PeSBP genes in seed, root, leaf, and flower showed that nine of the PeSBP genes displayed high expression in the leaf and the flower. The expression patterns of PeSBP3/6/8/9/10 were further detected by qRT-PCR. In addition, differences in the expression levels occurred for each gene in the different flower organs and at the different developmental stages. There were large differences among SBPs based on transcriptional levels under cold, heat, salt, and osmotic stress conditions. Altogether, this study provides an overview of SBP genes in passion fruit and lays the foundation for further functional analysis.

Genome-wide identification and expression analysis of SBP-box gene family reveal their involvement in hormone response and abiotic stresses in Chrysanthemum nankingense.

SQUAMOSA promoter-binding-protein (SBP)-box family proteins are a class of plant-specific transcription factors, and widely regulate the development of floral and leaf morphology in plant growth and involve in environment and hormone signal response. In this study, we isolated and identified 21 non-redundant SBP-box genes in Chrysanthemum nankingense with bioinformatics analysis. Sequence alignments of 21 CnSBP proteins discovered a highly conserved SBP domain including two zinc finger-like structures and a nuclear localization signal region. According to the amino acid sequence alignments, 67 SBP-box genes from Arabidopsis thaliana, rice, Artemisia annua and C. nankingense were clustered into eight groups, and the motif and gene structure analysis also sustained this classification. The gene evolution analysis indicated the CnSBP genes experienced a duplication event about 10 million years ago (Mya), and the CnSBP and AtSPL genes occurred a divergence at 24 Mya. Transcriptome data provided valuable information for tissue-specific expression profiles of the CnSBPs, which highly expressed in floral tissues and differentially expressed in leaf, root and stem organs. Quantitative Real-time Polymerase Chain Reaction data showed expression patterns of the CnSBPs under exogenous hormone and abiotic stress treatments, separately abscisic acid, salicylic acid, gibberellin A3, methyl jasmonate and ethylene spraying as well as salt and drought stresses, indicating that the candidate CnSBP genes showed differentiated spatiotemporal expression patterns in response to hormone and abiotic stresses. Our study provides a systematic genome-wide analysis of the SBP-box gene family in C. nankingense. In general, it provides a fundamental theoretical basis that SBP-box genes may regulate the resistance of stress physiology in chrysanthemum via exogenous hormone pathways.

The miR156-Targeted SQUAMOSA PROMOTER BINDING PROTEIN (PmSBP) Transcription Factor Regulates the Flowering Time by Binding to the Promoter of SUPPRESSOR OF OVEREXPRESSION OF CO1 (PmSOC1) in Prunus mume.

Prunus mume, a famous perennial ornamental plant and fruit tree in Asia, blooms in winter or early spring in the Yangtze River area. The flowering time directly determines its ornamental and economic value, so it is of great significance to study the molecular mechanism of flowering time. SQUAMOSA PROMOTER BINDING PROTEIN (SBP), often regulated by miR156, is an important flowering regulator, although its function is unknown in P. mume. Here, 11 miR156 precursors were analyzed and located in five chromosomes of the P. mume genome. The expression pattern showed that PmSBP1/6 was negatively correlated with miR156. The promoters of PmSBP1/6 were specifically expressed in the apical meristem. Overexpression of PmSBP1/6 in tobacco promoted flowering and changed the length ratio of pistil and stamen. Moreover, PmSBP1 also affected the number and vitality of pollen and reduced the fertility of transgenic tobacco. Furthermore, ectopic expression of PmSBP1/6 caused up-regulated expression of endogenous SUPPRESSOR OF OVEREXPRESSION OF CO1 (NtSOC1). The yeast-one hybrid assay showed that PmSBP1 was bonded to the promoters of PmSOC1s. In conclusion, a miR156-PmSBP1-PmSOC1s pathway was formed to participate in the regulation of flowering time in P. mume, which provided references for the molecular mechanism of flowering time regulation and molecular breeding of P. mume.

Lipid turnover and SQUAMOSA promoter-binding proteins mediate variation in fatty acid desaturation under early nitrogen deprivation revealed by lipidomic and transcriptomic analyses in Chlorella pyrenoidosa.

Nitrogen deprivation induces variations in fatty acid desaturation in microalgae, which determines the performance of biodiesel and the nutritional value of bioproducts. However, the detailed scenario and the underlying regulatory mechanism remain unclear. In this study, we attempt to outline these scenario and mechanisms by performing biochemical, lipidomic, and transcriptomic analyses in Chlorella pyrenoidosa and functional characterization of transcription factors in Yarrowia lipolytica. We found that early nitrogen deprivation dramatically reduced fatty acid desaturation without increasing lipid content. The contents of palmitic acid (16:0) and oleic acid (18:1) dramatically increased to 2.14 and 2.87 times that of nitrogen repletion on the second day, respectively. Lipidomic analysis showed the transfer of polyunsaturated fatty acids from phospholipids and glycolipids to triacylglycerols, and an increase in lipid species with 16:0 or 18:1 under nitrogen deprivation conditions. Upregulated stearoyl-ACP desaturase and oleyl-ACP thioesterase promoted the synthesis of 18:1, but restricted acetyl-CoA supply revealed that it was the intensive lipid turnover instead of an attenuated Kennedy pathway that played an important role in the variation in fatty acid composition under early nitrogen deprivation. Finally, two differentially expressed SQUAMOSA promoter-binding proteins (SBPs) were heterologously expressed in Y. lipolytica, demonstrating their role in promoting the accumulation of total fatty acid and the reduction in fatty acid desaturation. These results revealed the crucial role of lipid turnover and SBPs in determining fatty acid desaturation under early nitrogen deprivation, opening new avenues for the metabolic engineering of fatty acid desaturation in microalgae.

Genome-Wide Analysis of the SPL Gene Family and Expression Analysis during Flowering Induction in Prunus × yedoensis 'Somei-yoshino'.

SQUAMOSA Promoter-Binding Protein-Like (SPL) genes encode plant-specific transcription factors which bind to the SQUAMOSA promoter of the MADS-box genes to regulate its expression. It plays important regulatory roles in floral induction and development, fertility, light signals and hormonal transduction, and stress response in plants. In this study, 32 PySPL genes with complete SBP (squamosa promoter binding protein) conserved domain were identified from the genome of Prunus × yedoensis ‘Somei-yoshino’ and analyzed by bioinformatics. 32 PySPLs were distributed on 13 chromosomes, encoding 32 PySPL proteins with different physical and chemical properties. The phylogenetic tree constructed with Arabidopsis thaliana and Oryza sativa can be divided into 10 subtribes, indicating PySPLs of different clusters have different biological functions. The conserved motif prediction showed that the number and distribution of motifs on each PySPL is varied. The gene structure analysis revealed that PySPLs harbored exons ranging from 2 to 10. The predictive analysis of acting elements showed that the promoter of PySPLs contain a large number of light-responsive elements, as well as response elements related to hormone response, growth and development and stress response. The analysis of the PySPLs expressions in flower induction and flower organs based on qRT-PCR showed that PySPL06/22 may be the key genes of flower development, PySPL01/06 and PySPL22 may play a role in the development of sepal and pistil, respectively. The results provide a foundation for the study of SPL transcription factors of Prunus × yedoensis ‘Somei-yoshino’ and provide more reference information of the function of SPL gene in flowering.

Genome wide identification of lncRNAs and circRNAs having regulatory role in fruit shelf life in health crop cucumber (Cucumis sativus L.).

Cucumber is an extremely perishable vegetable; however, under room conditions, the fruits become unfit for consumption 2–3 days after harvesting. One natural variant, DC-48 with an extended shelf-life was identified, fruits of which can be stored up to 10–15 days under room temperature. The genes involved in this economically important trait are regulated by non-coding RNAs. The study aims to identify the long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) by taking two contrasting genotypes, DC-48 and DC-83, at two different fruit developmental stages. The upper epidermis of the fruits was collected at 5 days and 10 days after pollination (DAP) for high throughput RNA sequencing. The differential expression analysis was performed to identify differentially expressed (DE) lncRNAs and circRNAs along with the network analysis of lncRNA, miRNA, circRNA, and mRNA interactions. A total of 97 DElncRNAs were identified where 18 were common under both the developmental stages (8 down regulated and 10 upregulated). Based on the back-spliced reads, 238 circRNAs were found to be distributed uniformly throughout the cucumber genomes with the highest numbers (71) in chromosome 4. The majority of the circRNAs (49%) were exonic in origin followed by inter-genic (47%) and intronic (4%) origin. The genes related to fruit firmness, namely, polygalacturonase, expansin, pectate lyase, and xyloglucan glycosyltransferase were present in the target sites and co-localized networks indicating the role of the lncRNA and circRNAs in their regulation. Genes related to fruit ripening, namely, trehalose-6-phosphate synthase, squamosa promoter binding protein, WRKY domain transcription factors, MADS box proteins, abscisic stress ripening inhibitors, and different classes of heat shock proteins (HSPs) were also found to be regulated by the identified lncRNA and circRNAs. Besides, ethylene biosynthesis and chlorophyll metabolisms were also found to be regulated by DElncRNAs and circRNAs. A total of 17 transcripts were also successfully validated through RT PCR data. These results would help the breeders to identify the complex molecular network and regulatory role of the lncRNAs and circRNAs in determining the shelf-life of cucumbers.

The spatiotemporal regulations of epicatechin biosynthesis under normal flowering and the continuous inflorescence removal treatment in Fagopyrum dibotrys

BackgroundFlowering is a critical physiological change that interferes with not only biomass yield but also secondary metabolism, such as the biosynthesis of flavonoids, in rhizome/root plants. The continuous inflorescence removal (CIR) treatment is frequently conducted to weaken this effect. Fagopyrum dibotrys (D.Don) H.Hara (Golden buckwheat) is a kind of rhizome medicinal plant rich in flavonoids and is widely used for the treatment of lung diseases. The CIR treatment is usually conducted in F. dibotrys because of its excessive reproductive growth. To uncover the molecular mechanisms, comprehensive analysis was performed using metabolome and transcriptome data obtained from normally bloomed and the CIR treated plants.ResultsMetabolome results demonstrated that in the rhizomes of F. dibotrys, its bioactive compound called epicatechin has higher amount than most of the detected precursors. Compared with the normally bloomed plants, the level of epicatechin in the rhizomes of the CIR group increased by 25% at the withering stage. Based on 96 samples of the control and the CIR groups at 4 flowering stages for 4 tissues, RNA-Seq results revealed a 3 ~ 5 times upregulations of all the key enzyme genes involved in the biosynthesis of epicatechin in both time (from the bud stage to the withering stage) and spatial dimensions (from the top of branch to rhizome) under the CIR treatment compared to normal flowering. Integrated analysis of LC–MS/MS and transcriptome revealed the key roles of several key enzyme genes besides anthocyanidin reductase (ANR). A total of 93 transcription factors were identified to co-expressed with the genes in epicatechin biosynthetic pathway. The flowering activator SQUAMOSA promoter-binding protein like (SPLs) exhibited opposite spatiotemporal expression patterns to that of the epicatechin pathway genes; SPL3 could significantly co-express with all the key enzyme genes rather than the flowering repressor DELLA. Weighted gene co-expression network analysis (WGCNA) further confirmed the correlations among chalcone synthases (CHSs), chalcone isomerases (CHIs), ANRs, SPLs and other transcription factors.ConclusionsSPL3 might dominantly mediate the effect of normal flowering and the CIR treatment on the biosynthesis of epicatechin in rhizomes mainly through the negative regulations of its key enzyme genes including CHS, CHI and ANR.

Genome-Wide Identification, Characterization, and Expression Profiling Analysis of SPL Gene Family during the Inflorescence Development in Trifolium repens.

Trifolium repens is the most widely cultivated perennial legume forage in temperate region around the world. It has rich nutritional value and good palatability, seasonal complementarity with grasses, and can improve the feed intake and digestibility of livestock. However, flowering time and inflorescence development directly affects the quality and yield of T. repens, as well as seed production. The Squa promoter binding protein-like (SPL) gene family is a plant specific transcription factor family, which has been proved to play a critical role in regulating plant formation time and development of flowers. In this study, a total of 37 TrSPL genes were identified from the whole genome of T. repens and were divided into nine clades based on phylogenetic tree. Seventeen TrSPL genes have potential target sites for miR156. The conserved motif of squamosa promoter binding protein (SBP) contains two zinc finger structures and one NLS structure. Gene structure analysis showed that all TrSPL genes contained SBP domain, while ankyrin repeat region was just distributed in part of genes. 37 TrSPL genes were relatively dispersedly distributed on 16 chromosomes, and 5 pairs of segmental repeat genes were found, which indicated that segmental duplication was the main way of gene expansion. Furthermore, the gene expression profiling showed that TrSPL11, TrSPL13, TrSPL22, and TrSPL26 were highly expressed only in the early stage of inflorescence development, while TrSPL1 and TrSPL6 are highly expressed only in the mature inflorescence. Significantly, the expression of TrSPL4 and TrSPL12 increased gradually with the development of inflorescences. The results of this study will provide valuable clues for candidate gene selection and elucidating the molecular mechanism of T. repens flowering regulation.