Mycotoxin Determination in Peaches and Peach Products with a Modified QuEChERS Extraction Procedure Coupled with UPLC-MS/MS Analysis.

Peaches are the most significant temperate fruit crop worldwide. However, peach fruits are susceptible to fungal and mycotoxin contamination. Consequently, monitoring the residual levels of multiple mycotoxins in peaches and related products is essential. In this study, a novel method based on QuEChERS extraction, followed by ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) detection, was developed for analyzing 14 mycotoxins in peaches and peach products from China. Matrix-matched calibrations were employed to accurately quantify the mycotoxins and compensate for matrix effects. Recoveries for the target analytes ranged from 84.6% to 117.6%, with intra-day and inter-day precision below 20%. The limits of quantification were 2 or 5 µg/L for the 14 mycotoxins. This method was utilized to detect the presence of target mycotoxins in 109 fresh peaches, 100 diseased peaches, and 89 peach products from China. Six mycotoxins were identified in the rotten parts of the diseased peaches, with concentrations ranging from 5.2 to 1664.3 µg/kg. In the remaining parts of the diseased peach samples, only two toxins, alternariol (AOH) and alternariol monomethyl ether (AME), were quantified at levels of 15.3 µg/kg and 15.5 µg/kg, respectively. No mycotoxins were detected in fresh peaches. For peach products, all contamination levels were below the quantitative limits and significantly lower than the maximum legal limits established for the products.

Complete mitochondrial genome of Agrostis stolonifera: insights into structure, Codon usage, repeats, and RNA editing.

BACKGROUND: Plants possess mitochondrial genomes that are large and complex compared to animals. Despite their size, plant mitochondrial genomes do not contain significantly more genes than their animal counterparts. Studies into the sequence and structure of plant mitochondrial genomes heavily imply that the main mechanism driving replication of plant mtDNA, and offer valuable insights into plant evolution, energy production, and environmental adaptation. RESULTS: This study presents the first comprehensive analysis of Agrostis stolonifera's mitochondrial genome, characterized by a branched structure comprising three contiguous chromosomes, totaling 560,800 bp with a GC content of 44.07%. Annotations reveal 33 unique protein-coding genes (PCGs), 19 tRNA genes, and 3 rRNA genes. The predominant codons for alanine and glutamine are GCU and CAA, respectively, while cysteine and phenylalanine exhibit weaker codon usage biases. The mitogenome contains 73, 34, and 23 simple sequence repeats (SSRs) on chromosomes 1, 2, and 3, respectively. Chromosome 1 exhibits the most frequent A-repeat monomeric SSR, whereas chromosome 2 displays the most common U-repeat monomeric SSR. DNA transformation analysis identifies 48 homologous fragments between the mitogenome and chloroplast genome, representing 3.41% of the mitogenome's total length. The PREP suite detects 460 C-U RNA editing events across 33 mitochondrial PCGs, with the highest count in the ccmFn gene and the lowest in the rps7 gene. Phylogenetic analysis confirms A. stolonifera's placement within the Pooideae subfamily, showing a close relationship to Lolium perenne, consistent with the APG IV classification system. Numerous homologous co-linear blocks are observed in A. stolonifera's mitogenomes and those of related species, while certain regions lack homology. CONCLUSIONS: The unique features and complexities of the A. stolonifera mitochondrial genome, along with its similarities and differences to related species, provide valuable insights into plant evolution, energy production, and environmental adaptation. The findings from this study significantly contribute to the growing body of knowledge on plant mitochondrial genomes and their role in plant biology.

Uncovering early transcriptional regulation during adventitious root formation in Medicago sativa.

BACKGROUND: Alfalfa (Medicago sativa L.) as an important legume plant can quickly produce adventitious roots (ARs) to form new plants by cutting. But the regulatory mechanism of AR formation in alfalfa remains unclear. RESULTS: To better understand the rooting process of alfalfa cuttings, plant materials from four stages, including initial separation stage (C stage), induction stage (Y stage), AR primordium formation stage (P stage) and AR maturation stage (S stage) were collected and used for RNA-Seq. Meanwhile, three candidate genes (SAUR, VAN3 and EGLC) were selected to explore their roles in AR formation. The numbers of differentially expressed genes (DEGs) of Y-vs-C (9,724) and P-vs-Y groups (6,836) were larger than that of S-vs-P group (150), indicating highly active in the early AR formation during the complicated development process. Pathways related to cell wall and sugar metabolism, root development, cell cycle, stem cell, and protease were identified, indicating that these genes were involved in AR production. A large number of hormone-related genes associated with the formation of alfalfa ARs have also been identified, in which auxin, ABA and brassinosteroids are thought to play key regulatory roles. Comparing with TF database, it was found that AP2/ERF-ERF, bHLH, WRKY, NAC, MYB, C2H2, bZIP, GRAS played a major regulatory role in the production of ARs of alfalfa. Furthermore, three identified genes showed significant promotion effect on AR formation. CONCLUSIONS: Stimulation of stem basal cells in alfalfa by cutting induced AR production through the regulation of various hormones, transcription factors and kinases. This study provides new insights of AR formation in alfalfa and enriches gene resources in crop planting and cultivation.

Genome-wide identification, expression analysis, and transcriptome analysis of the IAA gene family in Zoysia japonica.

BACKGROUND: AUX/IAA is an essential signaling molecule and has great physiological importance in various plants, but its function in Zoysia japonica remains unknown. METHODS AND RESULTS: Genome-wide identification and analysis of AUX/IAA genes used bioinformatics methods to investigate the ZjIAA genes' expression of exogenous IAA hydroponics treatment for 2 h by qRT-PCR, control and exogenous IAA treated zoysia were subjected to transcriptome sequencing. ZjIAAs were distributed across the 13 subfamilies by phylogenetic analysis with Oryza sativa and Arabidopsis thaliana. Multiple sequence alignment revealed that the majority of genes were non-canonical ZjIAAs with incomplete domain. The optimal growth concentration of the IAA hormone was 0.05 mM, and the qRT-PCR analysis revealed that eight ZjIAAs were differentially expressed, with seven genes considerably upregulating and one gene significantly downregulating. The result of transcriptome sequencing revealed that 515 differentially expressed genes (DEGs) were identified, with 344 upregulated genes and 171 downregulated genes. A total of 18 genes were annotated as involved in the plant hormone signal transduction pathway. And 8 ZjIAAs exhibited distinct expressions, 7 upregulated, and only one downregulated, according to the qRT-PCR study. CONCLUSIONS: Genome-wide identification and analysis increased the understanding of the evolution and function of the IAA family in zoysia. DEGs of control and treatment with 0.05 mM exogenous IAA hormone were investigated by transcriptome sequencing. ZjIAAs had substantial variations in the expression of associated genes, with the majority of genes upregulated and 18 genes implicated in plant hormone signal transduction.

Overexpression of MsSAG113 gene promotes leaf senescence in alfalfa via participating in the hormone regulatory network.

Introduction: Alfalfa (Medicago sativa) is a kind of high quality leguminous forage species, which was widely cultivated in the world. Leaf senescence is an essential process in plant development and life cycle. Here, we reported the isolation and functional analysis of an alfalfa SENESCENCE-ASSOCIATED GENE113 (MsSAG113), which belongs to the PP2C family and mainly plays a role in promoting plant senescence. Methods: In the study, Agrobacterium-mediated, gene expression analysis, next generation sequencing, DNA pull-down, yeast single hybridization and transient expression were used to identify the function of MsSAG113 gene. Results: The MsSAG113 gene was isolated from alfalfa, and the transgenic plants were obtained by Agrobacterium-mediated method. Compared with the wildtype, transgenic plants showed premature senescence in leaves, especially when cultivated under dark conditions. Meanwhile, application of exogenous hormones ABA, SA, MeJA, obviously acclerated leaf senescence of transgenic plants. Furthermore, the detached leaves from transgenic plants turned yellow earlier with lower chlorophyll content. Transcriptome analysis identified a total of 1,392 differentially expressed genes (DEGs), involving 13 transcription factor families. Of which, 234 genes were related to phytohormone synthesis, metabolism and transduction. Pull-down assay and yeast one-hybrid assay confirmed that alfalfa zinc finger CCCH domain-containing protein 39 (MsC3H-39) could directly bind the upstream of MsSAG113 gene. In conclusion, the MsSAG113 gene plays a crucial role in promoting leaf senescence in alfalfa via participating in the hormone regulatory network. Discussion: This provides an essential basis for further analysis on the regulatory network involving senescence-associated genes in alfalfa.

Overexpression of abscisic acid-insensitive gene ABI4 from Medicago truncatula, which could interact with ABA2, improved plant cold tolerance mediated by ABA signaling.

ABI4 is considered an important transcription factor with multiple regulatory functions involved in many biological events. However, its role in abiotic stresses, especially low-temperature-induced stress, is poorly understood. In this study, the MtABI4 gene was derived from M. truncatula, a widely used forage grass. Analysis of subcellular localization indicated that ABI4 was localized in the nucleus. Identification of expression characteristics showed that ABI4 was involved in the regulatory mechanisms of multiple hormones and could be induced by the low temperature. IP-MS assay revealed that MtABI4 protein could interact with xanthoxin dehydrogenase protein (ABA2). The two-hybrid yeast assay and the biomolecular fluorescence complementarity assay further supported this finding. Expression analysis demonstrated that overexpression of MtABI4 induced an increase in ABA2 gene expression both in M. truncatula and Arabidopsis, which in turn increased the ABA level in transgenic plants. In addition, the transgenic lines with the overexpression of MtABI4 exhibited enhanced tolerance to low temperature, including lower malondialdehyde content, electrical conductivity, and cell membrane permeability, compared with the wide-type lines after being cultivated for 5 days in 4°C. Gene expression and enzyme activities of the antioxidant system assay revealed the increased activities of SOD, CAT, MDHAR, and GR, and higher ASA/DHA ratio and GSH/GSSG ratio in transgenic lines. Additionally, overexpression of ABI4 also induced the expression of members of the Inducer of CBF expression genes (ICEs)-C-repeat binding transcription factor genes(CBFs)-Cold regulated genes (CORs) low-temperature response module. In summary, under low-temperature conditions, overexpression of ABI4 could enhance the content of endogenous ABA in plants through interactions with ABA2, which in turn reduced low-temperature damage in plants. This provides a new perspective for further understanding the molecular regulatory mechanism of plant response to low temperature and the improvement of plant cold tolerance.

Uncovering a Phenomenon of Active Hormone Transcriptional Regulation during Early Somatic Embryogenesis in Medicago sativa.

Somatic embryogenesis (SE) is a developmental process in which somatic cells undergo dedifferentiation to become plant stem cells, and redifferentiation to become a whole embryo. SE is a prerequisite for molecular breeding and is an excellent platform to study cell development in the majority of plant species. However, the molecular mechanism involved in M. sativa somatic embryonic induction, embryonic and maturation is unclear. This study was designed to examine the differentially expressed genes (DEGs) and miRNA roles during somatic embryonic induction, embryonic and maturation. The cut cotyledon (ICE), non-embryogenic callus (NEC), embryogenic callus (EC) and cotyledon embryo (CE) were selected for transcriptome and small RNA sequencing. The results showed that 17,251 DEGs, and 177 known and 110 novel miRNAs families were involved in embryonic induction (ICE to NEC), embryonic (NEC to EC), and maturation (EC to CE). Expression patterns and functional classification analysis showed several novel genes and miRNAs involved in SE. Moreover, embryonic induction is an active process of molecular regulation, and hormonal signal transduction related to pathways involved in the whole SE. Finally, a miRNA-target interaction network was proposed during M. sativa SE. This study provides novel perspectives to comprehend the molecular mechanisms in M. sativa SE.

Expression of a Hydroxycinnamoyl-CoA Shikimate/Quinate Hydroxycinnamoyl Transferase 4 Gene from Zoysia japonica (ZjHCT4) Causes Excessive Elongation and Lignin Composition Changes in Agrostis stolonifera.

Hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) is considered to be an essential enzyme for regulating the biosynthesis and composition of lignin. To investigate the properties and function of ZjHCT4, the ZjHCT4 gene was cloned from Zoysia japonica with a completed coding sequence of 1284-bp in length, encoding 428 amino acids. The ZjHCT4 gene promoter has several methyl jasmonate (MeJA) response elements. According to analysis of expression patterns, it was up-regulated by MeJA, GA3 (Gibberellin), and SA (Salicylic acid), and down-regulated by ABA (Abscisic acid). Ectopic ZjHCT4 expression in creeping bentgrass causes excessive plant elongation. In addition, the content of G-lingnin and H-lingnin fell in transgenic plants, whereas the level of S-lingnin increased, resulting in a considerable rise in the S/G unit ratio. Analysis of the expression levels of lignin-related genes revealed that the ectopic expression of ZjHCT4 altered the expression levels of a number of genes involved in the lignin synthesis pathway. Simultaneously, MeJA, SA, GA3, IAA, BR (Brassinosteroid), and other hormones were dramatically enhanced in transgenic plants relative to control plants, whereas ABA concentration was significantly decreased. Expression of ZjHCT4 impacted lignin composition and plant growth via altering the phenylpropionic acid metabolic pathway and hormone response, as revealed by transcriptome analysis. HCTs may influence plant lignin composition and plant development by altering hormone content. These findings contributed to a deeper comprehension of the lignin synthesis pathway and set the stage for further investigation and application of the HCTs gene.

Expression of a Chlorophyll b Reductase Gene from Zoysia japonica Causes Changes in Leaf Color and Chlorophyll Morphology in Agrostis stolonifera.

The NYC-like (NOL) enzyme is considered as an essential enzyme for chlorophyll b degradation, which catalyzes the formation of 7-hydroxymethyl chlorophyll a from chlorophyll b. The ZjNOL gene was cloned from Zoysia japonica with a completed coding sequence of 981-bp in length, encoding 326 amino acids. ZjNOL was localized on the stroma side of the thylakoid membrane, and co-localized with ZjNYC in the chloroplasts. Multiple photoregulatory elements and hormone regulatory elements were identified in the promoter region of the ZjNOL gene, and the expression level of the ZjNOL gene was dramatically up-regulated in senescence leaves, which were regulated by a variety of plant hormones. ZjNOL's ectopic expression in creeping bentgrass produced yellow leaves, thicker cortex, and smaller vascular column cells. Additionally, transgenic plants exhibited morphological alterations in their chloroplast structure, and the number of grana and thylakoids per grana stack reduced dramatically. Transgenic plants also had a lower photosynthetic rate and Fm/Fv than the control. The transgenic plants displayed a decreased chlorophyll content and a greater rate of ion leakage. The properties and activities of ZjNOL will serve as a foundation for future research into gene functions and regulatory processes.

The complete chloroplast genome of Medicago arabica (Fabaceae).

Medicago arabica (Linnaeus, 1762) Huds. is an important annual legume forage that grows in a wide range of climates, from subtropical to temperate. This study aimed to sequence the chloroplast genome of M. arabica and compare it with other legumes. In this study, we sequenced the entire chloroplast genome of M. arabica, which has 125,056 base pairs. The total GC content of the chloroplast genome of M. arabica was 34.4%. From the 110 unique genes of the circular genome, 30 tRNA genes, four rRNA genes, and 76 protein-coding genes were successfully annotated. A maximum likelihood (ML) tree was constructed using the model species and 17 species of the Medicago genus. M. arabica was shown to be phylogenetically closely related to M. polymorpha. The nucleotide diversity of the chloroplast genome may provide valuable molecular markers to study chloroplast, genetic breeding, and plant molecular evolution. These findings provide a solid foundation for future research on the molecular biology of the chloroplast.

Expression of ZjPSY, a Phytoene Synthase Gene from Zoysia japonica Affects Plant Height and Photosynthetic Pigment Contents.

Phytoene synthase (PSY) is a key limiting enzyme in the carotenoid biosynthesis pathway for regulating phytoene synthesis. In this study, ZjPSY was isolated and identified from Zoysia japonica, an important lawn grass species. ZjPSY cDNA was 1230 bp in length, corresponding to 409 amino acids. ZjPSY showed higher expression in young leaves and was downregulated after GA3, ABA, SA, and MeJA treatments, exhibiting a sensitivity to plant hormones. Regulatory elements of light and plant hormone were found in the upstream of ZjPSY CDS. Expression of ZjPSY in Arabidopsis thaliana protein led to carotenoid accumulation and altered expression of genes involved in the carotenoid pathway. Under no-treatment condition, salt treatment, and drought treatment, transgenic plants exhibited yellowing, dwarfing phenotypes. The carotenoid content of transgenic plants was significantly higher than that of wild-type under salt stress and no-treatment condition. Yeast two-hybrid screening identified a novel interacting partner ZjJ2 (DNAJ homologue 2), which encodes heat-shock protein 40 (HSP40). Taken together, this study suggested that ZjPSY may affect plant height and play an important role in carotenoid synthesis. These results broadened the understanding of carotenoid synthesis pathways and laid a foundation for the exploration and utilization of the PSY gene.

Melatonin influences the early growth stage in Zoysia japonica Steud. by regulating plant oxidation and genes of hormones.

Zoysia japonica is a commonly used turfgrass species around the world. Seed germination is a crucial stage in the plant life cycle and is particularly important for turf establishment and management. Experiments have confirmed that melatonin can be a potential regulator signal in seeds. To determine the effect of exogenous melatonin administration and explore the its potential in regulating seed growth, we studied the concentrations of several hormones and performed a transcriptome analysis of zoysia seeds after the application of melatonin. The total antioxidant capacity determination results showed that melatonin treatment could significantly improve the antioxidant capacity of zoysia seeds. The transcriptome analysis indicated that several of the regulatory pathways were involved in antioxidant activity and hormone activity. The hormones concentrations determination results showed that melatonin treatment contributed to decreased levels of cytokinin, abscisic acid and gibberellin in seeds, but had no significant effect on the secretion of auxin in early stages. Melatonin is able to affect the expression of IAA (indoleacetic acid) response genes. In addition, melatonin influences the other hormones by its synergy with other hormones. Transcriptome research in zoysia is helpful for understanding the regulation of melatonin and mechanisms underlying melatonin-mediated developmental processes in zoysia seeds.

Expression of a NGATHA1 Gene from Medicago truncatula Delays Flowering Time and Enhances Stress Tolerance.

Shoot branching is one of the most variable determinants of crop yield, and the signaling pathways of plant branches have become a hot research topic. As an important transcription factor in the B3 family, NGATHA1 (NGA1), plays an important role in regulating plant lateral organ development and hormone synthesis and transport, but few studies of the role of this gene in the regulation of plant growth and stress tolerance have been reported. In this study, the NGA1 gene was isolated from Medicago truncatula (Mt) and its function was characterized. The cis-acting elements upstream of the 5' end of MtNGA1 and the expression pattern of MtNGA1 were analyzed, and the results indicated that the gene may act as a regulator of stress resistance. A plant expression vector was constructed and transgenic Arabidopsis plants were obtained. Transgenic Arabidopsis showed delayed flowering time and reduced branching phenotypes. Genes involved in the regulation of branching and flowering were differentially expressed in transgenic plants compared with wild-type plants. Furthermore, transgenic plants demonstrated strong tolerances to salt- and mannitol-induced stresses, which may be due to the upregulated expression of NCED3 (NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3) by the MtNGA1 gene. These results provide useful information for the exploration and genetic modification use of MtNGA1 in the future.

Global transcriptome analysis of alfalfa reveals six key biological processes of senescent leaves.

Leaf senescence is a complex organized developmental stage limiting the yield of crop plants, and alfalfa is an important forage crop worldwide. However, our understanding of the molecular mechanism of leaf senescence and its influence on biomass in alfalfa is still limited. In this study, RNA sequencing was utilized to identify differentially expressed genes (DEGs) in young, mature, and senescent leaves, and the functions of key genes related to leaf senescence. A total of 163,511 transcripts and 77,901 unigenes were identified from the transcriptome, and 5,133 unigenes were differentially expressed. KEGG enrichment analyses revealed that ribosome and phenylpropanoid biosynthesis pathways, and starch and sucrose metabolism pathways are involved in leaf development and senescence in alfalfa. GO enrichment analyses exhibited that six clusters of DEGs are involved in leaf morphogenesis, leaf development, leaf formation, regulation of leaf development, leaf senescence and negative regulation of the leaf senescence biological process. The WRKY and NAC families of genes mainly consist of transcription factors that are involved in the leaf senescence process. Our results offer a novel interpretation of the molecular mechanisms of leaf senescence in alfalfa.

PacBio single-molecule long-read sequencing shed new light on the transcripts and splice isoforms of the perennial ryegrass.

Perennial ryegrass (Lolium perenne), one of the most widely used forage and cool-season turfgrass worldwide, has a breeding history of more than 100 years. However, the current draft genome annotation and transcriptome characterization are incomplete mainly because of the enormous difficulty in obtaining full-length transcripts. To explore the complete structure of the mRNA and improve the current draft genome, we performed PacBio single-molecule long-read sequencing for full-length transcriptome sequencing in perennial ryegrass. We generated 29,175 high-confidence non-redundant transcripts from 15,893 genetic loci, among which more than 66.88% of transcripts and 24.99% of genetic loci were not previously annotated in the current reference genome. The re-annotated 18,327 transcripts enriched the reference transcriptome. Particularly, 6709 alternative splicing events and 23,789 alternative polyadenylation sites were detected, providing a comprehensive landscape of the post-transcriptional regulation network. Furthermore, we identified 218 long non-coding RNAs and 478 fusion genes. Finally, the transcriptional regulation mechanism of perennial ryegrass in response to drought stress based on the newly updated reference transcriptome sequences was explored, providing new information on the underlying transcriptional regulation network. Taken together, we analyzed the full-length transcriptome of perennial ryegrass by PacBio single-molecule long-read sequencing. These results improve our understanding of the perennial ryegrass transcriptomes and refined the annotation of the reference genome.

Altered Promoter and G-Box Binding Factor for 1-Deoxy-d-Xylulose-5-Phosphate Synthase Gene Grown from Poa pratensis Seeds after Spaceflight.

In plant cells, the nucleus DNA is considered the primary site of injury by the space environment, which could generate genetic alteration. As the part of genomic mutation, genetic variation in the promoter region could regulate gene expression. In the study, it is observed that there is a deletion in the upstream regulatory region of the 1-deoxy-d-xylulose-5-phosphate synthase 1 gene (PpDXS1) of Poa pratensis dwarf mutant and the PpDXS1 transcript abundance is lower in the dwarf mutant. It is indicated that the deletion in the promoter region between wild type and dwarf mutant could be responsible for the regulation of PpDXS1 gene expression. The PpDXS1 promoter of dwarf mutant shows a lower activity as determined by dual luciferase assay in Poa pratensis protoplast, as well as the GUS activity is lower in transgenic Poa pratensis plant. To further investigate the effect of the deletion in the promoter region on PpDXS1 transcript accumulation, the transient assay and yeast one-hybrid experiment demonstrate that the deletion comprises a motif which is a target of G-box binding factor (GBF1), and the motif correlates with an increase in transactivation by GBF1 protein. Taken together, these results indicate that the deletion in the promoter of PpDXS1 isolated from dwarf mutant is sufficient to account for the decrease in PpDXS1 transcript level and GBF1 can regulate the PpDXS1 gene expression, and subsequently affect accumulation of various isoprenoids throughout the plant.

Analysis of transcripts and splice isoforms in Medicago sativa L. by single-molecule long-read sequencing.

KEY MESSAGE: The full-length transcriptome of alfalfa was analyzed with PacBio single-molecule long-read sequencing technology. The transcriptome data provided full-length sequences and gene isoforms of transcripts in alfalfa, which will improve genome annotation and enhance our understanding of the gene structure of alfalfa. As an important forage, alfalfa (Medicago sativa L.) is world-wide planted. For its complexity of genome and unfinished whole genome sequencing, the sequences and complete structure of mRNA transcripts remain unclear in alfalfa. In this study, single-molecule long-read sequencing was applied to investigate the alfalfa transcriptome using the Pacific Biosciences platform, and a total of 113,321 transcripts were obtained from young, mature and senescent leaves. We identified 72,606 open reading frames including 46,616 full-length ORFs, 1670 transcription factors from 54 TF families and 44,040 simple sequence repeats from 30,797 sequences. A total of 7568 alternative splicing events was identified and the majority of alternative splicing events in alfalfa was intron retention. In addition, we identified 17,740 long non-coding RNAs. Our results show the feasibility of deep sequencing full-length RNA from alfalfa transcriptome on a single-molecule level.

Analysis of transcripts and splice isoforms in red clover (Trifolium pratense L.) by single-molecule long-read sequencing.

BACKGROUND: Red clover (Trifolium pratense L.) is an important cool-season legume plant, which is the most widely planted forage legume after alfalfa. Although a draft genome sequence was published already, the sequences and completed structure of mRNA transcripts remain unclear, which limit further explore on red clover. RESULTS: In this study, the red clover transcriptome was sequenced using single-molecule long-read sequencing to identify full-length splice isoforms, and 29,730 novel isoforms from known genes and 2194 novel isoforms from novel genes were identified. A total of 5492 alternative splicing events was identified and the majority of alter spliced events in red clover was corrected as intron retention. In addition, of the 15,229 genes detected by SMRT, 8719 including 186,517 transcripts have at least one poly(A) site. Furthermore, we identified 4333 long non-coding RNAs and 3762 fusion transcripts. CONCLUSIONS: We analyzed full-length transcriptome of red clover with PacBio SMRT. Those new findings provided important information for improving red clover draft genome annotation and fully characterization of red clover transcriptome.

Genome-wide analysis reveals four key transcription factors associated with cadmium stress in creeping bentgrass (Agrostis stolonifera L.).

Cadmium (Cd) toxicity seriously affects the growth and development of plants, so studies on uptake, translocation, and accumulation of Cd in plants are crucial for phytoremediation. However, the molecular mechanism of the plant response to Cd stress remains poorly understood. The main objective of this study was to reveal differentially expressed genes (DEGs) under lower (BT2_5) and higher (BT43) Cd concentration treatments in creeping bentgrass. A total of 463,184 unigenes were obtained from creeping bentgrass leaves using RNA sequencing technology. Observation of leaf tissue morphology showed that the higher Cd concentration damages leaf tissues. Four key transcription factor (TF) families, WRKY, bZIP, ERF, and MYB, are associated with Cd stress in creeping bentgrass. Our findings revealed that these four TFs play crucial roles during the creeping bentgrass response to Cd stress. This study is mainly focused on the molecular characteristics of DEGs under Cd stress using transcriptomic analysis in creeping bentgrass. These results provide novel insight into the regulatory mechanisms of respond to Cd stress and enrich information for phytoremediation.

Transcriptome analysis of leaf senescence in red clover (Trifolium pratense L.).

Red clover (Trifolium pratense L.) is an important cool-season legume plant, which is used as forage. Leaf senescence is a critical developmental process that negatively affects plant quality and yield. The regulatory mechanism of leaf senescence has been studied, and genes involved in leaf senescence have been cloned and characterized in many plants. However, those works mainly focused on model plants. Information about regulatory pathways and the genes involved in leaf senescence in red clover is very sparse. In this study, to better understand leaf senescence in red clover, transcriptome analysis of mature and senescent leaves was investigated using RNA-Seq. A total of about 35,067 genes were identified, and 481 genes were differentially expressed in mature and senescent leaves. Some identified differentially expressed genes showed similar expression patterns as those involved in leaf senescence in other species, such as Arabidopsis, Medicago truncatula and rice. Differentially expressed genes were confirmed by quantitative real-time PCR (qRT-PCR). Genes involved in signal transduction, transportation and metabolism of plant hormones, transcription factors and plant senescence were upregulated, while the downregulated genes were primarily involved in nutrient cycling, lipid/carbohydrate metabolism, hormone response and other processes. There were 64 differentially expressed transcription factor genes identified by RNA-Seq, including ERF, WRKY, bHLH, MYB and NAC. A total of 90 genes involved in biosynthesis, metabolism and transduction of plant hormones, including abscisic acid, jasmonic acid, cyokinin, brassinosteroid, salicylic acid and ethylene, were identified. Furthermore, 207 genes with direct roles in leaf senescence were demonstrated, such as senescence-associated genes. These genes were associated with senescence in other plants. Transcriptome analysis of mature and senescent leaves in red clover provides a large number of differentially expressed genes. Further analysis and identification of senescence-associated genes can provide new insight into the regulatory mechanisms of leaf development and senescence in legume plant and red clover.