Functional Characterization Of Genes Research Articles

Comparative transcriptomic analyses of diploid and tetraploid citrus reveal how ploidy level influences salt stress tolerance.

Citrus is an important fruit crop for human health. The sensitivity of citrus trees to a wide range of abiotic stresses is a major challenge for their overall growth and productivity. Among these abiotic stresses, salinity results in a significant loss of global citrus yield. In order to find straightforward and sustainable solutions for the future and to ensure citrus productivity, it is of paramount importance to decipher the mechanisms responsible for salinity stress tolerance. Thisstudy aimed to investigate how ploidy levels influence salt stress tolerance in citrus by comparing the transcriptomic responses of diploid and tetraploid genotypes. In a previous article we investigated the physiological and biochemical response of four genotypes with different ploidy levels: diploid trifoliate orange (Poncirus trifoliata [L.] Raf.) (PO2x) and Cleopatra mandarin (Citrus reshni Hort. Ex Tan.) (CL2x) and their respective tetraploids (PO4x, CL4x). In this study, we useda multifactorial gene selection and gene clustering approach to finely dissect the influence of ploidy level on the salt stress response of each genotype. Following transcriptome sequencing, differentially expressed genes (DEGs) were identified in response to salt stress in leaves and roots of the different citrus genotypes. Gene expression profiles and functional characterization of genes involved in the response to salt stress, as a function of ploidy level and the interaction between stress response and ploidy level, have enabled us to highlight the mechanisms involved in the varieties tested. Saltstress induced overexpression of carbohydrate biosynthesis and cell wall remodelling- related genes specifically in CL4x Ploidy level enhanced oxidative stress response in PO and ion management capacity in both genotypes. Results further highlighted that under stress conditions, only the CL4x genotype up- regulated genes involved in sugar biosynthesis, transport management, cell wall remodelling, hormone signalling, enzyme regulation and antioxidant metabolism. These findings provide crucial insights that could inform breeding strategies for developing salt-tolerant citrus varieties.

Tissue-specific knockout in the Drosophila neuromuscular system reveals ESCRT's role in formation of synapse-derived extracellular vesicles.

Tissue-specific gene knockout by CRISPR/Cas9 is a powerful approach for characterizing gene functions during development. However, this approach has not been successfully applied to most Drosophila tissues, including the Drosophila neuromuscular junction (NMJ). To expand tissue-specific CRISPR to this powerful model system, here we present a CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) toolkit for knocking out genes in motoneurons, muscles, and glial cells. We validated the efficacy of CRISPR-TRiM by knocking out multiple genes in each tissue, demonstrated its orthogonal use with the Gal4/UAS binary expression system, and showed simultaneous knockout of multiple redundant genes. We used CRISPR-TRiM to discover an essential role for SNARE components in NMJ maintenance. Furthermore, we demonstrate that the canonical ESCRT pathway suppresses NMJ bouton growth by downregulating retrograde Gbb signaling. Lastly, we found that axon termini of motoneurons rely on ESCRT-mediated intra-axonal membrane trafficking to release extracellular vesicles at the NMJ. Thus, we have successfully developed an NMJ CRISPR mutagenesis approach which we used to reveal genes important for NMJ structural plasticity.

Genome-wide analysis of the CCT gene family and functional characterization of SlCCT6 in response to drought stress in tomato

CCT transcription factors are important for photoperiod and abiotic stress regulation in Arabidopsis and rice. However, the CCT gene family has not been reported in tomato. Here, we systematically analyzed this. Thirty-one SlCCT genes were identified and divided into five groups (CMF, TIFY, PRR, S8, and COL), with members unevenly distributed across 12 chromosomes and the third chromosome exhibiting the most distribution. SlCCT was found to interact with an interacting protein (SlGI), transcription factor (MYB), and non-coding RNA (sly-miR156-5p) to jointly regulate the tomato stress response. cis-Acting element analysis of the SlCCT promoter region indicated large stress- and hormone-response elements in this family. Real-time PCR results indicated that SlPRR subfamily genes respond to various abiotic stresses and hormones. Tissue expression analysis revealed that several PRR subfamily genes are highly expressed in flowers, and subcellular localization analysis indicated an SlCCT6 nuclear location. Notably, SlCCT6 expression was significantly induced by drought, and its silencing reduced drought stress tolerance. Moreover, SlCCT6 overexpression enhanced tomato drought resistance by increasing antioxidant enzyme activity and activating stress-related genes, whereas SlCCT6 knockout decreased drought resistance. In conclusion, this provides valuable insights for future research on SlCCT functions.

Agrobacterium-mediated transient expression in Torreya grandis cones: A simple and rapid tool for gene expression and functional gene assay

Agrobacterium-mediated transient gene expression is a powerful tool for characterizing gene function in plants, particularly in fruit trees with long juvenile periods. However, few studies have systematically explored transient expression in nut trees, such as T. grandis. In this study, we developed a rapid and easily manipulated transient transformation system using Agrobacterium-mediated injection infiltration in T. grandis cones. Cones at the enlargement stage (30 days after aril protrusion) were used, and the bacterial solution was gently injected into the kernel at a depth of 1 cm, filling nearly the entire cone, as indicated by a red dye. Using the green fluorescent protein (GFP) gene as a reporter for Agrobacterium-mediated transformation, we confirmed GFP expression in T. grandis cone successfully through confocal microscopy, western blot, and PCR. Transformation conditions, including bacterial density and days after agroinfiltration, were optimized for higher transformation efficiency, with the optimal conditions being an OD600 of 0.6 and an agroinfiltration period of 6 - 9 days. Transient overexpression of TgWRKY47, a cell size regulator, altered cell size in T. grandis cones, indicating that this Agrobacterium infiltration method can be used for gene functional analysis. Furthermore, confocal microscopy revealed that TgWRKY47-GFP was localized in the nucleus along with a nuclear marker, demonstrating its applicability for protein subcellular localization. In summary, this method presents a versatile tool for efficient and targeted transient gene functional analysis and subcellular localization in T. grandis cones, offering new insights into nut tree biology research.

High-throughput genetics enables identification of nutrient utilization and accessory energy metabolism genes in a model methanogen.

Archaea are widespread in the environment and play fundamental roles in diverse ecosystems; however, characterization of their unique biology requires advanced tools. This is particularly challenging when characterizing gene function. Here, we generate randomly barcoded transposon libraries in the model methanogenic archaeon Methanococcus maripaludis and use high-throughput growth methods to conduct fitness assays (RB-TnSeq) across over 100 unique growth conditions. Using our approach, we identified new genes involved in nutrient utilization and response to oxidative stress. We identified novel genes for the usage of diverse nitrogen sources in M. maripaludis including a putative regulator of alanine deamination and molybdate transporters important for nitrogen fixation. Furthermore, leveraging the fitness data, we inferred that M. maripaludis can utilize additional nitrogen sources including ʟ-glutamine, ᴅ-glucuronamide, and adenosine. Under autotrophic growth conditions, we identified a gene encoding a domain of unknown function (DUF166) that is important for fitness and hypothesize that it has an accessory role in carbon dioxide assimilation. Finally, comparing fitness costs of oxygen versus sulfite stress, we identified a previously uncharacterized class of dissimilatory sulfite reductase-like proteins (Dsr-LP; group IIId) that is important during growth in the presence of sulfite. When overexpressed, Dsr-LP conferred sulfite resistance and enabled use of sulfite as the sole sulfur source. The high-throughput approach employed here allowed for generation of a large-scale data set that can be used as a resource to further understand gene function and metabolism in the archaeal domain.IMPORTANCEArchaea are widespread in the environment, yet basic aspects of their biology remain underexplored. To address this, we apply randomly barcoded transposon libraries (RB-TnSeq) to the model archaeon Methanococcus maripaludis. RB-TnSeq coupled with high-throughput growth assays across over 100 unique conditions identified roles for previously uncharacterized genes, including several encoding proteins with domains of unknown function (DUFs). We also expand on our understanding of carbon and nitrogen metabolism and characterize a group IIId dissimilatory sulfite reductase-like protein as a functional sulfite reductase. This data set encompasses a wide range of additional conditions including stress, nitrogen fixation, amino acid supplementation, and autotrophy, thus providing an extensive data set for the archaeal community to mine for characterizing additional genes of unknown function.

Genome-Wide Identification of the Soybean AlkB Homologue Gene Family and Functional Characterization of GmALKBH10Bs as RNA m6A Demethylases and Expression Patterns under Abiotic Stress.

Soybean (Glycine max (L.) Merr) is one of the most important crops worldwide, but its yield is vulnerable to abiotic stresses. In Arabidopsis, the AlkB homologue (ALKBH) family genes plays a crucial role in plant development and stress response. However, the identification and functions of its homologous genes in soybean remain obscured. Here, we identified a total of 22 ALKBH genes in soybean and classified them into seven subfamilies according to phylogenetic analysis. Gene duplication events among the family members and gene structure, conserved domains, and motifs of all candidate genes were analyzed. By comparing the changes in the m6A levels on mRNA from hair roots between soybean seedlings harboring the empty vector and those harboring the GmALKBH10B protein, we demonstrated that all four GmALKBH10B proteins are bona fide m6A RNA demethylases in vivo. Subcellular localization and expression patterns of the GmALKBH10B revealed that they might be functionally redundant. Furthermore, an analysis of cis-elements coupled with gene expression data demonstrated that GmALKBH10B subfamily genes, including GmALKBH10B1, GmALKBH10B2, GmALKBH10B3, and GmALKBH10B4, are likely involved in the cis-elements' response to various environmental stimuli. In summary, our study is the first to report the genome-wide identification of GmALKBH family genes in soybean and to determine the function of GmALKBH10B proteins as m6A RNA demethylases, providing insights into GmALKBH10B genes in response to abiotic stresses.

Functional diversity of bacterial microbiota associated with the toxigenic benthic dinoflagellate Prorocentrum.

Interactions between bacterial microbiota and epibenthic species of the dinoflagellate Prorocentrum may define the onset and persistence of benthic harmful algal blooms (bHABs). Chemical ecological interactions within the dinoflagellate phycosphere potentially involve a complex variety of organic molecules, metabolites, and toxins, including undefined bioactive compounds. In this study, the bacterial diversity and core members of the dinoflagellate-associated microbiota were defined from 11 strains of three epibenthic Prorocentrum species, representing three geographically disjunct locations within Mexican coastal waters. Microbiota profiles in stable monoclonal Prorocentrum cultures were obtained by sequencing amplicons of the V3-V4 region of the 16S rRNA gene. Thirteen classes of bacteria were identified among dinoflagellate clones, where Alphaproteobacteria, Gammaproteobacteria, and Bacteroidia were consistently dominant. The bacterial community structure exhibited significantly different grouping by the location of origin of dinoflagellate clones. No significant diversity difference was found among free-living or unattached bacteria in the dinoflagellate culture medium (M) compared with those in closer association with the dinoflagellate host cells (H). Twelve taxa were defined as core members of the bacterial assemblage, representing the genera Algiphilus, Cohaesibacter, Labrenzia, Mameliella, Marinobacter, Marivita, Massilia, Muricauda, Roseitalea, and an unclassified member of the Rhodobacteraceae. The core members are inferred to significantly contribute to primary and secondary metabolic functions, but no direct correlation with dinoflagellate toxigenicity was apparent. Overall the bacterial profile and implied gene functionality indicated a suite of positive interactions, suggesting either mutualism or commensalism with the dinoflagellate. The further characterization and interpretation of specific gene functions and interactions between bacteria and dinoflagellates, such as epibenthic members of genus Prorocentrum, are key to understanding their role in toxigenesis and bHAB development.

Gene function characterization in Aspergillus niger using a dual resistance marker transformation system mediated by Agrobacterium tumefaciens

Aspergillus niger is a well-known workhorse for the industrial production of enzymes and organic acids. This fungus can also cause postharvest diseases in fruits. Although Agrobacterium tumefaciens-mediated transformation (ATMT) based on antibiotic resistance markers has been effectively exploited for inspecting functions of target genes in wild-type fungi, it still needs to be further improved in A. niger. In the present study, we re-examined the ATMT in the wild-type A. niger strains using the hygromycin resistance marker and introduced the nourseothricin resistance gene as a new selection marker for this fungus. Unexpectedly, our results revealed that the ATMT method using the resistance markers in A. niger led to numerous small colonies as false-positive transformants on transformation plates. Using the top agar overlay technique to restrict false positive colonies, a transformation efficiency of 87 ± 18 true transformants could be achieved for 106 conidia. With two different selection markers, we could perform both the deletion and complementation of a target gene in a single wild-type A. niger strain. Our results also indicated that two key regulatory genes (laeA and veA) of the velvet complex are required for A. niger to infect apple fruits. Notably, we demonstrated for the first time that a laeA homologous gene from the citrus postharvest pathogen Penicillium digitatum was able to restore the acidification ability and pathogenicity of the A. niger ΔlaeA mutant. The dual resistance marker ATMT system from our work represents an improved genetic tool for gene function characterization in A. niger.

Genome-Wide Identification of Sorghum Paclobutrazol-Resistance Gene Family and Functional Characterization of SbPRE4 in Response to Aphid Stress.

Sorghum (Sorghum bicolor), the fifth most important cereal crop globally, serves as a staple food, animal feed, and a bioenergy source. Paclobutrazol-Resistance (PRE) genes play a pivotal role in the response to environmental stress, yet the understanding of their involvement in pest resistance remains limited. In the present study, a total of seven SbPRE genes were found within the sorghum BTx623 genome. Subsequently, their genomic location was studied, and they were distributed on four chromosomes. An analysis of cis-acting elements in SbPRE promoters revealed that various elements were associated with hormones and stress responses. Expression pattern analysis showed differentially tissue-specific expression profiles among SbPRE genes. The expression of some SbPRE genes can be induced by abiotic stress and aphid treatments. Furthermore, through phytohormones and transgenic analyses, we demonstrated that SbPRE4 improves sorghum resistance to aphids by accumulating jasmonic acids (JAs) in transgenic Arabidopsis, giving insights into the molecular and biological function of atypical basic helix-loop-helix (bHLH) transcription factors in sorghum pest resistance.

Epigenetic control during root development and symbiosis.

The roots of plants play multiple functions that are essential for growth and development, including anchoring to the soil as well as water and nutrient acquisition. These underground organs exhibit the plasticity to modify their root system architecture in response to environmental cues, allowing adaptation to change in water and nutrient availability. In addition, roots enter in mutualistic interactions with soil microorganisms, for example, the root nodule symbiosis (RNS) established between a limited group of plants and nitrogen-fixing soil bacteria and the arbuscular mycorrhiza symbiosis involving most land plants and fungi of the Glomeromycetes phylum. In the past 20 years, genetic approaches allowed the identification and functional characterization of genes required for the specific programs of root development, root nodule, and arbuscular mycorrhiza symbioses. These genetic studies provided evidence that the program of the RNS recruited components of the arbuscular mycorrhiza symbiosis and the root developmental programs. The execution of these programs is strongly influenced by epigenetic changes-DNA methylation and histone post-translational modifications-that alter chromatin conformation modifying the expression of key genes. In this review, we summarize recent advances that highlight how DNA methylation and histone post-translational modifications, as well as chromatin remodeling factors and long noncoding RNAs, shape the root system architecture and allow the successful establishment of both root nodule and arbuscular mycorrhiza symbioses. We anticipate that the analysis of dynamic epigenetic changes and chromatin 3D structure in specific single cells or tissue types of root organs will illuminate our understanding of how root developmental and symbiotic programs are orchestrated, opening exciting questions and new perspectives to modulate agronomical and ecological traits linked to nutrient acquisition.

Enigmatic role of auxin response factors in plant growth and stress tolerance.

Abiotic and biotic stresses globally constrain plant growth and impede the optimization of crop productivity. The phytohormone auxin is involved in nearly every aspect of plant development. Auxin acts as a chemical messenger that influences gene expression through a short nuclear pathway, mediated by a family of specific DNA-binding transcription factors known as Auxin Response Factors (ARFs). ARFs thus act as effectors of auxin response and translate chemical signals into the regulation of auxin responsive genes. Since the initial discovery of the first ARF in Arabidopsis, advancements in genetics, biochemistry, genomics, and structural biology have facilitated the development of models elucidating ARF action and their contributions to generating specific auxin responses. Yet, significant gaps persist in our understanding of ARF transcription factors despite these endeavors. Unraveling the functional roles of ARFs in regulating stress response, alongside elucidating their genetic and molecular mechanisms, is still in its nascent phase. Here, we review recent research outcomes on ARFs, detailing their involvement in regulating leaf, flower, and root organogenesis and development, as well as stress responses and their corresponding regulatory mechanisms: including gene expression patterns, functional characterization, transcriptional, post-transcriptional and post- translational regulation across diverse stress conditions. Furthermore, we delineate unresolved questions and forthcoming challenges in ARF research.

Analysis of 14–3-3 Gene Family and Functional Characterization of DlGRF8/11 modulating flowering time in Ma bamboo (Dendrocalamus latiflorus Munro)

14–3–3 proteins, also known as general regulatory factors (GRFs), are common regulatory proteins that play a critical role in growth, development, and environmental responses in plants. However, there is limited understanding of the members and functions of 14–3–3 gene family in ma bamboo. In this study, 25 Dl14–3–3 genes were identified and designated as DlGRF1-DlGRF25, respectively. Bioinformatics analysis of DlGRF genes provided insights into the conservation and diversity during evolution in ma bamboo. It’s verified that the expression patterns of DlGRFs were discrepant in different flowering tissues. To further explore the floral transition, Y2H, BiFC, and LCI assays revealed that DlGRF8 interacted with DlFT1/DlTFL1 in the cytoplasm and with DlFT2/DlFD1 in the nucleus. Interestingly, DlGRF11 exclusively interacted with DlFD1 in the nucleus. Overexpressing experiments in rice indicated that DlGRF8 promoted flowering, while DlGRF11 delayed flowering. Moreover, they exhibited diverse expression patterns in response to drought and ABA treatments. These results provide preliminary insights into the role of DlGRF genes in floral transition, growth, and development.

Genome-wide investigation of HD-ZIP gene family and functional characterization of BnaHDZ149 and BnaHDZ22 in salt and drought response in Brassica napus L.

HD-ZIP proteins comprise a plant-specific transcription factor family, which play pivotal roles in plant development and adaptation to ever-changing environment. Although HD-ZIP family members have been identified in some plant species, so far our knowledge about HD-ZIP genes in rapeseed is still limited. In this study, 178 Brassica napus HD-ZIP (BnaHDZ) family members were identified in the rapeseed genome. The phylogenetic relationship, chromosomal locations, intron-exon structures, motif composition, and expression patterns of the BnaHDZ members were analyzed. The BnaHDZ family can be phylogenetically divided into four categories (Ⅰ, Ⅱ, Ⅲ and Ⅳ). Genome-wide transcriptome analysis revealed that most of the HD-ZIP I members respond to at least one abiotic stress. Two closely homologous stress-responsive HD-ZIP Ⅰ genes, BnaHDZ22 and BnaHDZ149, were identified to be involved in drought and salt responses, and selected for further functional characterization. Overexpressing BnaHDZ149 in rapeseed increased salt sensitivity of the transgenic plants, whereas overexpressing BnaHDZ22 increased sensitivity of the transgenic plants to polyethylene glycol (PEG)-simulated drought stress. This research provides not only a comprehensive landscape of BnaHDZ genes, but also a theoretical basis for elucidating the molecular mechanism of the abiotic stress responses of the HD-ZIP family in rapeseed.

Genome-wide evolutionary analysis of TKL_CTR1-DRK-2 gene family and functional characterization reveals that TaCTR1 positively regulates flowering time in wheat

BackgroundFlowering time has an important effect on regional adaptation and yields for crops. The tyrosine kinase-like (TKL) gene family is widely existed and participates in many biological processes in plants. Furthermore, only few TKLs have been characterized functions in controlling flowering time in wheat.ResultsHere, we report that TaCTR1, a tyrosine kinase-like (TKL) gene, regulates flowering time in wheat. Based on identification and evolutionary analysis of TKL_CTR1-DRK-2 subfamily in 15 plants, we proposed an evolutionary model for TaCTR1, suggesting that occurrence of some exon fusion events during evolution. The overexpression of TaCTR1 caused early flowering time in transgenic lines. Transcriptomics analysis enabled identification of mass differential expression genes including plant hormone (ET, ABA, IAA, BR) signaling, flavonoid biosynthesis, phenolamides and antioxidant, and flowering-related genes in TaCTR1 overexpression transgenic lines compared with WT plants. qRT–PCR results showed that the expression levels of ethylene (ET) signal-related genes (ETR, EIN, ERF) and flowering-related genes (FT, PPD1, CO, PRR, PHY) were altered in TaCTR1-overexpressing wheat compared with WT plants. Metabonomics analysis showed that flavonoid contents were altered.ConclusionsThus, the results show that TaCTR1 plays a positive role in controlling flowering time by activating various signaling pathways and regulating flowering-related genes, and will provide new insights on the mechanisms of wheat flowering regulation.

Genome-wide identification of the walnut MYC gene family and functional characterization of Xinjiang wild walnut under low-temperature stress.

Introduction: MYC transcription factors are the basic regulators of the jasmonic acid signaling pathway and play important roles in plant growth and development and the response to adverse stress. In recent years, severe winter freezing and late spring frost in the main planting area of walnut in Xinjiang have affected the growth and development of walnut, which has become a prominent problem restricting walnut production. Xinjiang wild walnut is the only remaining wild species of walnuts in China, which contains a lot of genes with excellent traits, and is important for the cultivation and breeding. Methods: In this paper, the physicochemical properties and bioinformatics of MYC transcription factor members in walnut were analyzed, and the nine MYC were screened from the transcriptome data under low temperature stress. At last, we study the subcellular localizations and the expression patterns of the nine MYC members in Xinjiang wild walnut. Results: The results revealed that 30 MYC members were identified from published walnut whole-genome data, and their evolutionary relationships with Arabidopsis and poplar were divided into six groups according to clustering analysis, among which JrMYC22 and JrMYC23 had high homology with PtrMYC2b, which is induced by jasmonic acid in response to low-temperature stress. Walnut MYC members are unevenly distributed on 12 chromosomes. The prediction of promoter cis-acting elements of walnut MYC transcription factor family members revealed that cis-acting elements related to jasmonic acid and lowtemperature stress were the ones with the greatest number of members, with 12. In addition, all nine MYC family members in Xinjiang wild walnut plants responding to low-temperature stress exhibited strong fluorescence responses in the nucleus. The expression levels of these members in response to low-temperature stress revealed that JrMYC28, JrMYC31, JrMYC33, JrMYC34, and JrMYC35 were highly expressed, and it was hypothesized that JrMYC28, JrMYC31, JrMYC33, JrMYC34, and JrMYC35 might play a key role in the response to lowtemperature stress. Discussion: The results of this study provide a theoretical basis for further research on the functional mechanisms of the MYC transcription factor family members in walnut.

Identification and targeting of metastatic biomarkers for hepatocellular carcinoma therapeutics using small molecules library of curcumin analogues.

The understanding of the molecular basis of complex diseases like hepatocellular carcinoma (HCC) needs large datasets of multiple genes and proteins involved in different phenomenon of its development. This study focuses on the molecular basis of HCC and the development of therapeutic strategies. We analyzed a dataset of 5475 genes (Homo sapiens) involved in HCC hallmarks, involving comprehensive data on multiple genes and frequently mutated genes. As HCC is characterized by metastasis, angiogenesis, and oxidative stress, exploration of genes associated with them has been targeted. Through gene ontology, functional characterization, and pathway enrichment analysis, we identified target proteins such as Lysyl oxidase, Survivin, Cofilin, and Cathepsin B. A library of curcumin analogs was used to target these proteins. Tetrahrydrocurcumin showed promising binding affinities for all four proteins, suggesting its potential as an inhibitor against these proteins for HCC therapy.

Genome-Wide Identification of the WRKY Gene Family and Functional Characterization of CpWRKY5 in Cucurbita pepo.

The WRKY gene family is crucial for regulating plant growth and development. However, the WRKY gene is rarely studied in naked kernel formation in hull-less Cucurbita pepo L. (HLCP), a natural mutant that lacks the seed coat. In this research, 76 WRKY genes were identified through bioinformatics-based methods in C. pepo, and their phylogenetics, conserved motifs, synteny, collinearity, and temporal expression during seed coat development were analyzed. The results showed that 76 CpWRKYs were identified and categorized into three main groups (I-III), with Group II further divided into five subgroups (IIa-IIe). Moreover, 31 segmental duplication events were identified in 49 CpWRKY genes. A synteny analysis revealed that C. pepo shared more collinear regions with cucumber than with melon. Furthermore, quantitative RT-PCR (qRT-PCR) results indicated the differential expression of CpWRKYs across different varieties, with notable variations in seed coat development between HLCP and CP being attributed to differences in CpWRKY5 expression. To investigate this further, CpWRKY5-overexpression tobacco plants were generated, resulting in increased lignin content and an upregulation of related genes, as confirmed by qRT-PCR. This study offers valuable insights for future functional investigations of CpWRKY genes and presents novel information for understanding the regulation mechanism of lignin synthesis.

Integrated Full-Length Transcriptomics and Metabolomics Reveal Glycosyltransferase Involved in the Biosynthesis of Flavonol Glycosides in Laportea bulbifera.

Many species of the Urticaceae family are important cultivated fiber plants that are known for their economic and industrial values. However, their secondary metabolite profiles and associated biosynthetic mechanisms have not been well-studied. Using Laportea bulbifera as a model, we conducted widely targeted metabolomics, which revealed 523 secondary metabolites, including a unique accumulation of flavonol glycosides in bulblet. Through full-length transcriptomic and RNA-seq analyses, the related genes in the flavonoid biosynthesis pathway were identified. Finally, weighted gene correlation network analysis and functional characterization revealed four LbUGTs, including LbUGT78AE1, LbUGT72CT1, LbUGT71BX1, and LbUGT71BX2, can catalyze the glycosylation of flavonol aglycones (kaempferol, myricetin, gossypetin, and quercetagetin) using UDP-Gal and UDP-Glu as the sugar donors. LbUGT78AE1 and LbUGT72CT1 showed substrate promiscuity, whereas LbUGT71BX1 and LbUGT71BX2 exhibited different substrate and sugar donor selectivity. These results provide a genetic resource for studying Laportea in the Urticaceae family, as well as key enzymes responsible for the metabolism of valuable flavonoid glycosides.

Allelic variation in the autotetraploid potato: genes involved in starch and steroidal glycoalkaloid metabolism as a case study

BackgroundTuber starch and steroidal glycoalkaloid (SGA)-related traits have been consistently prioritized in potato breeding, while allelic variation pattern of genes that underlie these traits is less explored.ResultsHere, we focused on the genes involved in two important metabolic pathways in the potato: starch metabolism and SGA biosynthesis. We identified 119 genes consisting of 81 involved in starch metabolism and 38 in the biosynthesis of steroidal glycoalkaloids, and discovered 96,166 allelic variants among 2,169 gene haplotypes in six autotetraploid potato genomes. Comparative analyses revealed an uneven distribution of allelic variants among gene haplotypes and that the vast majority of deleterious mutations in these genes are retained in heterozygous state in the autotetraploid potato genomes. Leveraging full-length cDNA sequencing data, we find that approximately 70% of haplotypes of the 119 genes are transcribable. Population genetic analyses identify starch and SGA biosynthetic genes that are potentially conserved or diverged between potato varieties with varying starch or SGA content.ConclusionsThese results deepen the understanding of haplotypic diversity within functionally important genes in autotetraploid genomes and may facilitate functional characterization of genes or haplotypes contributing to traits related to starch and SGA in potato.

A simple and effective VIGS system facilitates the control of citrus canker by silencing CsLOB1

Virus-induced gene silencing (VIGS) is a powerful technology for molecular characterization of gene functions in plants and has the potential to prevent and control plant diseases. Unfortunately, VIGS in many woody perennials such as citrus are severely hindered because they have a long juvenile period and are recalcitrant to infection compared to herbaceous plants. Here, we report the development of a simple and effective VIGS system based on citrus leaf blotch virus (CLBV), a virus endemic in most citrus-producing countries but showing no symptoms in most citrus varieties. By Agrobacterium-mediated vacuum-infiltration (AVI), pCLBV201-su245 and pCLBV201-pds391 triggered efficient gene silencing (85%) and silencing phenotypes began to appear at 30 days post infiltration/inoculation (dpi), a similar period for achieving that on herbaceous plants. Moreover, the silencing phenotypes could still be observed four years post inoculation. Further, pCLBV201-lob369 was constructed to silence the gene lateral organ boundaries1 (lob1), encoding a key factor for susceptibility to citrus canker that caused by Xanthomonas citri ssp. citri (Xcc). The resistance evaluation results showed that the lesion area, disease index, and bacterial content in the pCLBV201-lob369 treatment group were decreased by 64%, 14%, and 67%, respectively. This work provides a simple and effective VIGS system for citrus which has the potential to be used for diseases control.