Search Results (114)

The red plant phenotype of R1 cotton is a genetic marker produced by light-induced anthocyanin accumulation. GhPAP1D controls this trait. There are two 228 bp tandem repeats upstream of GhPAP1D in R1 cotton. In this study, GUS staining assays in transgenic Arabidopsis thaliana (L.) Heynh. demonstrated that tandem repeats in the GhPAP1D promoter-enhanced transcriptional activity. GhPAP1D is a homolog of A. thaliana AtPAP1. AtPAP1’s expression is regulated by photomorphogenesis-related transcription factors such as AtHY5 and AtBBXs. We identified the homologs of A. thaliana AtHY5, AtBBX21, and AtBBX24 in R1 cotton, designated as GhHY5, GhBBX21, and GhBBX24, respectively. Y1H assays confirmed that GhHY5, GhBBX21, and GhBBX24 each bound to the GhPAP1D promoter. Dual-luciferase reporter assays revealed that GhHY5 weakly activated the promoter activity of GhPAP1D. Heterologous expression assays in A. thaliana indicated that GhBBX21 promoted anthocyanin accumulation, whereas GhBBX24 had the opposite effect. Dual-luciferase assays showed GhBBX21 activated GhPAP1D transcription, while GhBBX24 repressed it. Further study indicated that GhHY5 did not enhance GhBBX21-mediated transcriptional activation of GhPAP1D but alleviates GhBBX24-induced repression. Together, our results demonstrate that GhBBX21 and GhBBX24 antagonistically regulate anthocyanin accumulation in R1 cotton under GhHY5 mediation, providing insights into light-responsive anthocyanin biosynthesis in cotton. Full article

Prolonged expression of gene-editing components in CRISPR-modified plants can interfere with phenotypic analysis of target traits, increase the risk of off-target mutations, and lead to unnecessary metabolic burden. To mitigate these issues in peanut (Arachis hypogaea L.), callus-specific promoters were screened to restrict Cas9 expression to the callus stage, minimizing its activity in regenerated plants. In this study, six callus-specific genes in peanut were identified by mining RNA sequencing datasets and validating their expression profiles using quantitative reverse transcriptase PCR. The promoters of Arahy.H0FE8D, Arahy.WT3AEF, Arahy.I20Q6X, Arahy.ELJ55T, and Arahy.N9CMH4 were cloned and assessed for their expression activity. Beta-glucuronidase (GUS) histochemical staining confirmed that all five promoters were functional in peanut callus. Further investigation revealed their ability to drive cytosine base editing via a deaminase-nCas9 fusion protein, with all promoters successfully inducing precise base substitutions in peanut. Notably, P_Ah-H0FE8D, P_Ah-WT3AEF, P_Ah-ELJ55T, and P_Ah-N9CMH4 exhibited comparable or higher editing efficiencies than the commonly used cauliflower mosaic virus 35S promoter. These findings provide valuable tools for improving the biosafety of CRISPR-based genome editing in peanut breeding programs. Full article

Soybean relies on symbiotic nitrogen fixation (SNF) to support sustainable agriculture. In this study, we conducted a comprehensive analysis of the GmMYB transcription factor subfamily 20, with a focus on GmMYB62a and GmMYB62b. Phylogenetic and structural analyses revealed that these genes are evolutionarily conserved among legumes and possess distinct domain architectures. Expression profiling and GUS staining showed that GmMYB62a and GmMYB62b are constitutively expressed in nodules. Functional analyses revealed that loss of GmMYB62s function significantly reduced nodule density, while overexpression promoted nodulation. Transcriptomic analysis (RNA-seq) further demonstrated that GmMYB62s regulate key pathways, including hormone signaling, immune responses, and cell wall metabolism, thereby coordinating symbiotic interactions. Collectively, our findings identify GmMYB62a and GmMYB62b as critical molecular regulators of nodulation in soybean, providing promising targets for improving symbiotic nitrogen fixation efficiency in legume crops. Full article

Wood is an important raw material for industrial applications. Its fiber-specific genetic modification provides an effective strategy to alter wood characteristics in tree breeding. Here, we performed a cross-analysis of previously reported single-cell RNA sequencing and the AspWood database during wood formation to identify potential xylem fiber-dominant expressing genes in poplar. As a result, 32 candidate genes were obtained, and subsequently, we further examined the expression of these genes in fibers and/or vessels of stem secondary xylem using the laser capture microdissection technique and RT-qPCR. Analysis identified nine candidate genes, including PtrFLA12-2, PtrIRX12, PtrFLA12-6, PtrMYB52, PtrMYB103, PtrMAP70, PtrLRR-1, PtrKIFC2-3, and PtrNAC12. Next, we cloned the promoter regions of the nine candidate genes and created promoter::GUS transgenic poplars. Histochemical GUS staining was used to investigate the tissue expression activities of these gene promoters in transgenic poplars. In one month, transgenic plantlets grown in medium showed intensive GUS staining signals that were visible in the leaves and apical buds, suggesting substantial expression activities of these gene promoters in plantlets predominantly undergoing primary growth. In contrast, for three-month-old transgenic poplars in the greenhouse with predominantly developed secondary stem tissues, the promoters of seven of nine candidate genes, including PtrMYB103, PtrIRX12, and PtrMAP70, showed secondary xylem fiber-dominant GUS signals with considerable spatial specificity. Overall, this study presents xylem fiber-dominant promoters that are well-suited for specifically expressing genes of interest in wood fibers for forest tree breeding. Full article

Orphan genes (OGs), which are unique to a specific taxon and have no detectable sequence homology to any known genes across other species, play a pivotal role in governing species-specific phenotypic traits and adaptive evolution. In this study, 20 OGs of Chinese cabbage (Brassica rapa OGs, BrOGs) were transferred into Arabidopsis thaliana by genetic transformation to construct an overexpression library in which 50% of the transgenic lines had a delayed flowering phenotype, 15% had an early flowering phenotype, and 35% showed no difference in flowering time compared to control plants. There were many other phenotypes attached to these transgenic lines, such as leaf color, number of rosette leaves, and silique length. To understand the impact of BrOGs on delayed flowering, BrOG142OE, which showed the most significantly delayed flowering phenotype, was chosen for further analysis, and BrOG142 was renamed BOLTING RESISTANCE 4 (BR4). In BR4OE, the expression of key flowering genes, including AtFT and AtSOC1, significantly decreased, and AtFLC and AtFRI expression increased. GUS staining revealed BR4 promoter activity mainly in the roots, flower buds and leaves. qRT-PCR showed that BR4 primarily functions in the flowers, flower buds, and leaves of Chinese cabbage. BR4 is a protein localized in the nucleus, cytoplasm, and cell membrane. The accelerated flowering time phenotype of BR4OE was observed under gibberellin and vernalization treatments, indicating that BR4 regulates flowering time in response to these treatments. These results provide a foundation for elucidating the mechanism by which OGs regulate delayed flowering and have significance for the further screening of bolting-resistant Chinese cabbage varieties. Full article

Dirigent proteins (DIRs) are pivotal regulators of lignin/lignan biosynthesis and play multifaceted roles in plant development and stress adaptation. Despite their functional significance, DIR genes remain unexplored in Rosa chinensis, a globally important woody ornamental species. This study identified 33 RcDIRs through whole-genome analysis, including their chromosomal distribution, phylogenetic relationships, collinearity, protein and gene structure, conserved motifs, and cis-acting element distribution, and classified them into three phylogenetically independent subgroups (DIR-a, DIR-b/d, and DIR-e). Notably, the DIR-e subgroup includes an exclusive tandem cluster comprising RcDIR7-RcDIR12, representing the largest lineage-specific RcDIR expansion in R. chinensis. Structural characterization revealed that most RcDIRs exhibit a conserved single-exon architecture. Promoter cis-element analysis uncovered abundant stress-/hormone-responsive elements and three pollen-specific motifs (AAATGA, POLLEN1LELAT52, GTGANTG10), with RcDIR12 from the DIR-e cluster showing high pollen-specific regulatory potential. Experimental validation included cloning the RcDIR12 promoter from R. chinensis ‘Old Blush’, constructing proRcDIR12::GUS vectors, and conducting histochemical GUS assays with pollen viability/DAPI staining in transgenic Arabidopsis. Histochemical assays demonstrated GUS activity localization in mature trinucleate pollen grains, marking the first experimental evidence of pollen-specific DIRs in rose. Our findings not only elucidate the DIR family’s genomic organization and evolutionary innovations in R. chinensis but also establish proRcDIR12 as a molecular tool for manipulating pollen development in plants. Full article

Apple (Malus domestica Borkh.) is an economically important fruit. The use of nitrate by plants plays a crucial role in their growth and development, and its absorption and dispersal are controlled by nitrate transport proteins (NRTs). In this study, we investigated the potential function of MdNRT2.4 under low-nitrogen (N) stress by overexpressing it in tobacco. Compared with plants treated with a normal nitrogen level (5 mM), the MdNRT2.4 overexpression lines under low-N stress (0.25 mM) exhibited significantly greater plant height and width, as well as larger leaves and a higher leaf density, than wild-type plants, suggesting that the overexpression of MdNRT2.4 enhances the low-N tolerance of tobacco. Enhanced antioxidant enzyme activities in the MdNRT2.4 overexpression plant lines promoted the scavenging of reactive oxygen species, which reduced damage to their cell membranes. GUS staining of pMdNRT2.4::GUS-transformed Arabidopsis thaliana lines showed that MdNRT2.4 was expressed in the roots, vascular bundles, seeds in fruit pods, and young anther sites, suggesting that MdNRT2.4 mediates the transport of nitrate to these tissues, indicating that MdNRT2.4 might promote nitrate utilization in apple and improve its tolerance to low-N stress. Experiments using yeast one-hybrid and dual-luciferase assays revealed that MdbHLH3 binds to the MdNRT2.4 promoter and activates its expression. MdbHLH3 belongs to the basic helix–loop–helix (bHLH) transcription factor (TF). It is speculated that MdbHLH3 may interact with the promoter of MdNRT2.4 to regulate N metabolism in plants and enhance their low-N tolerance. This study establishes a theoretical framework for investigating the regulatory mechanisms of low-N responsive molecules in apple, while simultaneously providing valuable genetic resources for molecular breeding programs targeting low-N tolerance. Full article

Background/Objectives: In crop genetic engineering, morphogenic genes have attracted increasing attention, given their ability to facilitate the transformation of a broad range of otherwise nontransformable cultivars. However, few callus-specific promoters have been identified to date that can be employed to avoid the adverse effects resulting from the ectopic expression of morphogenic genes on shoot regeneration and growth. Methods: A set of potential callus-specific genes were initially selected based on publicly available data. These genes were then screened using quantitative real-time polymerase chain reaction (qPCR), followed by promoter activity evaluation using a transgenic approach with the GUS gene serving as a reporter. Results: Of the 24 evaluated promoters, 12 were verified as being callus-specific using qPCR. Five genes (Os11g0295900, Os10g0207500, Os01g0300000, Os02g0252200, and Os04g0488100) were chosen, and their promoters were cloned. Based on GUS staining, the pOsTDL1B (Os10g0207500) promoter showed strong callus-specific expression, pOsEDC (Os01g0300000) was a medium-level callus-specific promoter, and pOsDLN53 (Os02g0252200) was strictly callus-specific, although its activity was low. Quantification of GUS activity indicated that all three pOsTDL1B:GUS transgenic lines exhibited strong callus specificity relative to the various tissues tested. Conclusions: A callus-specific promoter was identified that can be used to drive the expression of morphogenic genes in crop transformation. Full article

Phalaenopsis-type Dendrobium (Den-Phals) is a commercially valuable orchid, with floral color being key to its market appeal. Despite the significance of anthocyanin biosynthesis in color development, its transcriptional regulation in Den-Phals remains unclear. This study functionally characterized the promoters of DhMYB2 and DhbHLH1, two key transcription factors involved in anthocyanin biosynthesis. A 1864 bp DhMYB2 promoter and a 1995 bp DhbHLH1 promoter were isolated using genome walking. Bioinformatics analysis identified cis-acting elements associated with abiotic stress responses, phytohormone signaling, and floral-specific regulation. 5′-Deletion analysis in tobacco leaves identified core regulatory regions for the DhMYB2 promoter (−1864 to −937 bp) and DhbHLH1 promoter (−1995 to −924 bp). GUS staining and activity assays demonstrated that the activities of the DhMYB2 and DhbHLH1 promoters were significantly increased under treatments of long light, low temperature, drought, salicylic acid (SA), and abscisic acid (ABA), while the DhbHLH1 promoter was also induced by methyl jasmonate (MeJA) and indole-3-acetic acid (IAA). Furthermore, promoter activity of DhMYB2 and DhbHLH1 was detected only in transgenic Arabidopsis flowers, suggesting that these promoters exhibit floral-specific activity. This study provides the first functional analysis of Den-Phals anthocyanin promoters, revealing their stress-responsive nature and floral specificity, which will facilitate molecular breeding of novel orchid cultivars. Full article

Cannabis sativa produces pharmacologically valuable cannabinoids. In this study, we developed and optimized a transient transformation system using Cannabis sativa ‘Cheungsam’ to facilitate gene functional analysis. Various experimental conditions, including plant developmental stages, light conditions, Agrobacterium strains, tissue types, and physical treatments such as sonication and vacuum infiltration, were systematically evaluated using GUS histochemical staining and qPCR analysis. Among these, 7-day-old seedlings cultured under dark conditions and transformed with the GV3101 strain exhibited high transformation efficiency. Leaf tissue showed a higher GUS staining proportion and GUS staining area compared to hypocotyl and cotyledon tissues. The application of a combination of sonication and vacuum infiltration techniques resulted in the most intense GUS expression. Using the optimized protocol, we introduced a recombinant vector carrying CsCBDAS2, a key gene in cannabidiol (CBD) biosynthesis. qPCR analysis revealed that CsCBDAS2 overexpression led to significant upregulation of multiple upstream CBD biosynthetic genes (CsOAC, CsGOT, CsPT1, CsPT4, CsCBDAS1, and CsCBDAS2) and the transcription factor (TF) CsWRKY20, suggesting coordinated co-expression and potential involvement of a transcriptional feedback loop. These results demonstrate the effectiveness of our transient transformation system and provide insights into the regulatory mechanisms of cannabinoid biosynthesis in cannabis. Full article

Buckwheat is a promising crop with grains that are rich in nutrients and bioactive compounds. Genome sequence data for common and Tartary buckwheat have recently become available. Currently, there is a critical need for the development of a simple and reliable transient gene expression protocol, as well as a stable genetic transformation method, to facilitate metabolic engineering of bioactive compounds, functional analysis of genes, targeted editing, and, in a long-term perspective, to accelerate the breeding process in buckwheat. In this paper, we report optimized methods for Agrobacterium-mediated transient and stable transformation of Fagopyrum esculentum and F. tartaricum. Leaf and cotyledon tissues were infiltrated with an A. tumefaciens-bearing construct containing eGFP and GUS reporter genes. Histochemical staining and Western blotting were used to confirm the expression of reporter proteins. We also demonstrate the usefulness of the developed method for engineering the gramine biosynthetic pathway in buckwheat. HvAMIS and HvNMT genes were transiently expressed in buckwheat leaves, and the de novo production of gramine was confirmed by LC-MS. Moreover, in planta genetic transformation of common and Tartary buckwheat with a reporter gene (eGFP) and selectable marker gene (NptII) was achieved by Agrobacterium-mediated vacuum infiltration. Genomic integration of the construct was confirmed by polymerase chain reaction (PCR), whereas the production of eGFP was confirmed by fluorescence microscopy. Full article

Arabinogalactan proteins (AGPs) constitute a diverse class of hydroxyproline-rich glycoproteins implicated in various aspects of plant growth and development. However, their functional characterization in cotton (Gossypium spp.) remains limited. As a globally significant economic crop, cotton serves as the primary source of natural fiber, making it essential to understand the genetic mechanisms underlying its growth and development. This study aims to perform a comprehensive genome-wide identification and characterization of the AGP gene family in Gossypium spp., with a particular focus on elucidating their structural features, evolutionary relationships, and functional roles. A genome-wide analysis was conducted to identify AGP genes in Gossypium spp., followed by classification into distinct subfamilies based on sequence characteristics. Protein motif composition, gene structure, and phylogenetic relationships were examined to infer potential functional diversification. Subcellular localization of a key candidate gene, GhAGP50, was determined using fluorescent protein tagging, while gene expression patterns were assessed through β-glucuronidase (GUS) reporter assays. Additionally, hormonal regulation of GhAGP50 was investigated via treatments with methyl jasmonate (MeJA), abscisic acid (ABA), indole-3-acetic acid (IAA), and gibberellin (GA). A total of 220 AGP genes were identified in Gossypium spp., comprising 19 classical AGPs, 28 lysine-rich AGPs, 55 AG peptides, and 118 fasciclin-like AGPs (FLAs). Structural and functional analyses revealed significant variation in gene organization and conserved motifs across subfamilies. Functional characterization of GhAGP50, an ortholog of AGP18 in Arabidopsis thaliana, demonstrated its role in promoting epidermal hair formation in leaves and stalks. Subcellular localization studies indicated that GhAGP50 is targeted to the nucleus and plasma membrane. GUS staining assays revealed broad expression across multiple tissues, including leaves, inflorescences, roots, and stems. Furthermore, hormonal treatment experiments showed that GhAGP50 expression is modulated by MeJA, ABA, IAA, and GA, suggesting its involvement in hormone-mediated developmental processes. This study presents a comprehensive genome-wide analysis of the AGP gene family in cotton, providing new insights into their structural diversity and functional significance. The identification and characterization of GhAGP50 highlight its potential role in epidermal hair formation and hormonal regulation, contributing to a deeper understanding of AGP functions in cotton development. These findings offer a valuable genetic resource for future research aimed at improving cotton growth and fiber quality through targeted genetic manipulation. Full article

Calcium-dependent protein kinase (CPK), representing a group of typical Ca²⁺ sensors in plants, has been well characterized in plants. CPK is capable of binding to Ca²⁺, which sequentially activates CPK. CPK-related kinase (CRK) shows protein structures similar to CPK but only contains degenerative EF-hands, which likely makes the activation of CRK Ca²⁺ independent. Compared with CPK, CRK is barely functionally analyzed. In this study, we systematically investigated CRK genes in the Arabidopsis genome. We found that CRK appeared to emerge in land plants, suggesting CPK and CRK are divided at very early stages during plant evolution. In Arabidopsis, the detailed analysis of the calmodulin-like domain of CRK indicated the substitutions of key amino acid residues in its EF-hands result in disrupted Ca²⁺ association. Next, by using a YFP tag, we found that all Arabidopsis CRK proteins were localized at the plasma membrane. After cloning the promoters of all eight CRK genes, we found that CRKs were widely expressed at all stages of Arabidopsis by using GUS staining. Furthermore, the kinase activity of CRK was examined by using phospho-antibody and Pro-Q staining. CRK was shown to possess high autophosphorylation, which was not affected by the presence of Ca²⁺. Moreover, we analyzed the cis-elements of CRK promoters and discovered that stress signals potentially regulate the expression of CRK genes. Consistently, by using quantitative real-time PCR (qPCR), we found a number of CRK genes were regulated by a variety of biotic and abiotic treatments such as flg22, ABA, drought, salt, and high and low temperatures. Furthermore, by utilizing proteomic approaches, we identified more than 100 proteins that interacted with CRK5 in planta. Notably, RLK and channels/transporters were found in CRK5-containing complexes, suggesting they function upstream and downstream of CRK, respectively. Full article

Arabinogalactan proteins (AGPs) are complex proteoglycans present in plant cell walls across the kingdom. They play crucial roles in biological functions throughout the plant life cycle. In this study, we identified 43 gene members of the AG peptide (an AGP subfamily) within the rice genome, detailing their structure, protein-conserved domains, and motif compositions for the first time. We also examined the expression patterns of these genes across 18 tissues and organs, especially the different parts of the flower (anthers, pollen, pistil, sperm cells, and egg cells). Interestingly, the expression of some AG peptides is mainly present in the pollen grain. Transcription data and GUS staining confirmed that OsAGP6P—a member of the AG peptide gene family—is expressed in the stamen during pollen development stages 11–14, which are critical for maturation as microspores form after meiosis of pollen mother cells. It became noticeable from stage 11, when exine formation occurred—specifically at stage 12, when the intine began to develop. The overexpression of this gene in rice decreased the seed-setting rate (from 91.5% to 30.5%) and plant height (by 21.9%) but increased the tillering number (by 34.1%). These results indicate that AGP6P contributes to the development and fertility of pollen, making it a valuable gene target for future genetic manipulation of plant sterility through gene overexpression or editing. Full article

Soil lead (Pb) contamination is a severe environmental issue. Hydrangea, with high ornamental value, shows strong tolerance to the heavy metal Pb. Discovery of the gene(s) determining Pb resistance has been hindered by the lack of a stabilized and efficient genetic transformation system. Agrobacterium-mediated transient transformation overcomes the drawbacks of stabilized genetic transformation, such as long cycle, low efficiency, and high cost. In this study, an Agrobacterium-mediated method was adopted. The percentage of leaves that turned blue after GUS histochemical staining among the total number of infected leaves was used to represent the transient transformation efficiency. The effects of receptor material growth state (leaf age), Agrobacterium concentration, infection time, acetosyringone (Ace) concentration, negative pressure intensity, and co-culture time on the transient transformation efficiency of Hydrangea villosa Rehd. were investigated. Consequently, an efficient transient transformation system harboring the GUS reporter gene with a transient transformation efficiency as high as 100% was successfully established. Using this system, we successfully verified the Pb tolerance of HmPAT1, HmPIF1, and HmZAT7, proving the effectiveness of the transient transformation system. This transient transformation technology will help to discover new Pb-tolerant genes, provide new molecular targets for the development of Pb-resistant Hydrangea, and provide a potential phytoremediation strategy for the treatment of heavy metal pollution in soil. Full article