Background and aims: 4-Coumarate:CoA ligase (4CL) is a key enzyme in the phenylpropanoid pathway, directing metabolic flux toward lignin and flavonoid biosynthesis. This study aimed to identify and characterize the 4CL gene family (Ah4CLs) in Agave and to analyze their expression patterns during leaf development and under heavy metal and biotic stresses. Methods: Ah4CL genes were identified from transcriptome data. Phylogenetic analysis was performed using the Maximum Likelihood (ML) method with IQ-TREE. Expression patterns were examined by qPCR across three leaf developmental stages and under copper (Cu), lead (Pb), and Phytophthora nicotianae treatments. Results: Phylogenetic analysis classified the five genes into three groups: Group I (Ah4CL1, predicted lignin-related), Group II (Ah4CL2, predicted flavonoid-related), and Group III (Ah4CL3-5, 4CL-like). Expression profiling revealed that Ah4CL1, Ah4CL4, and Ah4CL5 were highly expressed in unexpanded leaves and were coordinately upregulated under Cu and Pb stress. Conversely, Ah4CL1 and Ah4CL2 were significantly induced by P. nicotianae infection. Conclusions: The Ah4CL gene family exhibits functional diversification. Ah4CL1, Ah4CL4, and Ah4CL5 are primarily associated with leaf development and heavy metal tolerance, while Ah4CL1 and Ah4CL2 play a significant role in biotic defense. These findings provide a foundation for the genetic improvement of stress resilience in Agave.
Background: The mitochondrial calcium uniporter (MCU) protein family mediates calcium uptake into mitochondria and plays a vital role in stress-related calcium signaling. Although extensively studied in animals, its functions in plants remain unclear. Aims: This study aimed to identify and characterize the MCU gene family in Brassica rapa (Br) L. and to investigate their expression profiles under abiotic stress conditions. Methods: Six genes (Br-MCU1 to Br-MCU6) were identified, each containing conserved MCU domains and two transmembrane helices, indicating structural similarity to animal MCUs. Phylogenetic analysis, gene structure, motif composition, and chromosomal localization were analyzed to elucidate their evolutionary and functional significance. Results: Quantitative real-time PCR (qPCR) was conducted to assess gene expression under water deficit (20% PEG-6000), heat (42°C), and salt (200 mM NaCl) stresses at different time intervals. The Br-MCU genes exhibited dynamic and stress-specific expression patterns. Notably, Br-MCU2 and Br-MCU5 showed significant induction under salt and water deficit stresses, suggesting their involvement in mitochondrial calcium signaling during stress adaptation. Conclusions: These findings provide a comprehensive foundation for future functional studies of MCU genes in B. rapa and support the development of stress-tolerant varieties through genetic or biotechnological approaches.
Background: Detection of mixed infections involving viruses with different genome types requires a diagnostic workflow that can amplify RNA and DNA targets in a single reaction, thereby avoiding separate nucleic-acid-specific procedures. Aims: This study aimed to develop a one-step PCR assay for the simultaneous detection of multiple viruses using a universal single template, total nucleic acid (TNA). Methods: Wheat and barley field isolates were subjected to extraction of TNA using potassium ethyl xanthogenate (PEX) buffer. Wheat dwarf virus (WDV) and barley yellow dwarf virus (BYDV) were then detected using a one-enzyme RTX-PCR-based assay. Conventional PCR and two-step RT-PCR were used to validate the TNA-RTX-PCR assay, and absolute quantification by qPCR was performed to determine WDV and BYDV titres. Results: TNA-RTX-PCR confirmed the simultaneous detection of WDV and BYDV, enabling the detection of viruses with different genome types in a single reaction. Conventional PCR and RT-PCR validated the presence of WDV and BYDV in the same isolates. Absolute quantification showed different WDV and BYDV titres range coherent with the PCR detection. Conclusions: This one-step TNA-RTX-PCR assay is an efficient approach for detecting viruses with different genome types in a single reaction and eliminates the need for separate reverse transcription and DNA-based PCR workflows.
Background and aims: Abscisic acid (ABA) is essential for plant stress adaptation and growth regulation, and clade A PP2Cs function as core negative regulators in ABA signal transduction. Nevertheless, the biological functions of clade A PP2C family genes remain largely uncharacterized in tuber mustard (Brassica juncea var. tumida). This study aimed to systematically characterize clade A BjuPP2C genes and explore their potential roles in abiotic stress response, stem swelling development, and resistance to Plasmodiophora brassicae. Methods: We profiled the expression patterns of clade A BjuPP2C genes under multiple abiotic stresses and P. brassicae inoculation, as well as across successive stem swelling developmental stages. Four candidate genes (BjuPP2C56_B1, BjuPP2C37_A1, BjuPP2C37_B1, and BjuPP2C24_O1) with prominent expression changes were selected for CDS cloning. Subcellular localization assays were performed to determine their intracellular distribution, and yeast two-hybrid (Y2H) analysis was used to verify physical interactions between these PP2C proteins and PYR1-Like ABA receptors (BjuPYLs). Results: Most clade A BjuPP2C genes were significantly induced by abiotic stresses and pathogen infection, and the transcript abundance of several clade A BjuPP2C members was upregulated during stem swelling development. In particular, BjuPP2C56_B1, BjuPP2C37_A1, BjuPP2C37_B1, and BjuPP2C24_O1 were markedly upregulated both at the early stage of P. brassicae infection and the initial phase of stem swelling. Subcellular localization demonstrated that BjuPP2C37_A1, BjuPP2C37_B1, and BjuPP2C24_O1 localized to nucleus and BjuPP2C56_B1 localized to nucleus and cytoplasm. Y2H results revealed that BjuPP2C56_B1, BjuPP2C37_A1, and BjuPP2C37_B1 interact with BjuPYLs in tuber mustard, whereas no interaction was detected between BjuPP2C24_O1 and BjuPYLs. Conclusions: The clade A BjuPP2C genes participate in ABA-mediated stress response, clubroot resistance, and stem swelling development in tuber mustard. Four key PP2C members differ in subcellular distribution and their capacity to bind ABA receptors, laying a foundation for further dissecting the ABA regulatory network governing stem swelling development and pathogen defense in tuber mustard.
Background: Cymbidium faberi Rolfe is an ornamental orchid of high economic value, however, an efficient genetic transformation system has not yet been reported for this species. Aims: This study aimed to establish an Agrobacterium tumefaciens-mediated genetic transformation system for C. faberi and to optimize the key factors affecting transformation performance. Methods: Rhizomes, calli, and inflorescences were used as explants. The pCAMBIA3301-EGFP vector was introduced using Agrobacterium strain GV3101. Explant type, pre-culture duration, bacterial density (OD600), infection time, co-cultivation period, and antibiotic selection were systematically optimized. Putative transformants were screened by green fluorescence and verified by polymerase chain reaction (PCR). Results: Calli pre-cultured for 3 d were the most suitable recipient material. The optimal transformation conditions were an Agrobacterium suspension of OD600 = 0.2, infection for 5 min, and co-cultivation for 2 d; under these conditions, explant survival reached 72.22%. Two independent transgenic plantlets with stable integration of the enhanced green fluorescent protein gene were obtained and confirmed by PCR. Conclusions: This study provides an Agrobacterium-mediated transformation protocol for C. faberi and offers a technical basis for molecular breeding and gene function studies in this orchid.
