Integrating GC-MS and comparative transcriptome analysis reveals that TsERF66 promotes the biosynthesis of caryophyllene in Toona sinensis tender leaves.

Introduction: The strong aromatic characteristics of the tender leaves of Toona sinensis determine their quality and economic value. Methods and results: Here, GC-MS analysis revealed that caryophyllene is a key volatile compound in the tender leaves of two different T. sinensis varieties, however, the transcriptional mechanisms controlling its gene expression are unknown. Comparative transcriptome analysis revealed significant enrichment of terpenoid synthesis pathway genes, suggesting that the regulation of terpenoid synthesis-related gene expression is an important factor leading to differences in aroma between the two varieties. Further analysis of expression levels and genetic evolution revealed that TsTPS18 is a caryophyllene synthase, which was confirmed by transient overexpression in T. sinensis and Nicotiana benthamiana leaves. Furthermore, we screened an AP2/ERF transcriptional factor ERF-IX member, TsERF66, for the potential regulation of caryophyllene synthesis. The TsERF66 had a similar expression trend to that of TsTPS18 and was highly expressed in high-aroma varieties and tender leaves. Exogenous spraying of MeJA also induced the expression of TsERF66 and TsTPS18 and promoted the biosynthesis of caryophyllene. Transient overexpression of TsERF66 in T. sinensis significantly promoted TsTPS18 expression and caryophyllene biosynthesis. Discussion: Our results showed that TsERF66 promoted the expression of TsTPS18 and the biosynthesis of caryophyllene in T. sinensis leaves, providing a strategy for improving the aroma of tender leaves.

NnSUS1 encodes a sucrose synthase involved in sugar accumulation in lotus seed cotyledons.

Fresh lotus seeds are gaining favor with consumers for their crunchy texture and natural sweetness. However, the intricacies of sugar accumulation in lotus seeds remain elusive, which greatly hinders the quality improvement of fresh lotus seeds. This study endeavors to elucidate this mechanism by identifying and characterizing the sucrose synthase (SUS) gene family in lotus. Comprising five distinct members, namely NnSUS1 to NnSUS5, each gene within this family features a C-terminal glycosyl transferase1 (GT1) domain. Among them, NnSUS1 is the predominately expressed gene, showing high transcript abundance in the floral organs and cotyledons. NnSUS1 was continuously up-regulated from 6 to 18 days after pollination (DAP) in lotus cotyledons. Furthermore, NnSUS1 demonstrates co-expression relationships with numerous genes involved in starch and sucrose metabolism. To investigate the function of NnSUS1, a transient overexpression system was established in lotus cotyledons, which confirmed the gene's contribution to sugar accumulation. Specifically, transient overexpression of NnSUS1 in seed cotyledons leads to a significant increase in the levels of total soluble sugar, including sucrose and fructose. These findings provide valuable theoretical insights for improving sugar content in lotus seeds through molecular breeding methods.

Identification of transcription factor genes responsive to MeJA and characterization of a LaMYC2 transcription factor positively regulates lycorine biosynthesis in Lycoris aurea.

Jasmonates (JAs) are among the main phytohormones, regulating plant growth and development, stress responses, and secondary metabolism. As the major regulator of the JA signaling pathway, MYC2 also plays an important role in plant secondary metabolite synthesis and accumulation. In this study, we performed a comparative transcriptome analysis of Lycoris aurea seedlings subjected to methyl jasmonate (MeJA) at different treatment times. A total of 31,193 differentially expressed genes (DEGs) were identified by RNA sequencing. Among them, 732 differentially expressed transcription factors (TFs) comprising 51 TF families were characterized. The most abundant TF family was WRKY proteins (80), followed by AP2/ERF-EFR (67), MYB (59), bHLH (52), and NAC protein (49) families. Subsequently, by calculating the Pearson's correlation coefficient (PCC) between the expression level of TF DEGs and the lycorine contents, 41 potential TF genes (|PCC| >0.8) involved in lycorine accumulation were identified, including 36 positive regulators and 5 negative regulators. Moreover, a MeJA-inducible MYC2 gene (namely LaMYC2) was cloned on the basis of transcriptome sequencing. Bioinformatic analyses revealed that LaMYC2 proteins contain the bHLH-MYC_N domain and bHLH-AtAIB_like motif. LaMYC2 protein is localized in the cell nucleus, and can partly rescue the MYC2 mutant in Arabidopsis thaliana. LaMYC2 protein could interact with most LaJAZs (especially LaJAZ3 and LaJAZ4) identified previously. Transient overexpression of LaMYC2 increased lycorine contents in L. aurea petals, which might be associated with the activation of the transcript levels of tyrosine decarboxylase (TYDC) and phenylalanine ammonia lyase (PAL) genes. By isolating the 887-bp-length promoter fragment upstream of the start codon (ATG) of LaTYDC, we found several different types of E-box motifs (CANNTG) in the promoter of LaTYDC. Further study demonstrated that LaMYC2 was indeed able to bind the E-box (CACATG) present in the LaTYDC promoter, verifying that the pathway genes involved in lycorine biosynthesis could be regulated by LaMYC2, and that LaMYC2 has positive roles in the regulation of lycorine biosynthesis. These findings demonstrate that LaMYC2 is a positive regulator of lycorine biosynthesis and may facilitate further functional research of the LaMYC2 gene, especially its potential regulatory roles in Amaryllidaceae alkaloid accumulation in L. aurea.

Caffeic Acid O-Methyltransferase Gene Family in Mango (Mangifera indica L.) with Transcriptional Analysis under Biotic and Abiotic Stresses and the Role of MiCOMT1 in Salt Tolerance.

Caffeic acid O-methyltransferase (COMT) participates in various physiological activities in plants, such as positive responses to abiotic stresses and the signal transduction of phytohormones. In this study, 18 COMT genes were identified in the chromosome-level reference genome of mango, named MiCOMTs. A phylogenetic tree containing nine groups (I-IX) was constructed based on the amino acid sequences of the 71 COMT proteins from seven species. The phylogenetic tree indicated that the members of the MiCOMTs could be divided into four groups. Quantitative real-time PCR showed that all MiCOMT genes have particularly high expression levels during flowering. The expression levels of MiCOMTs were different under abiotic and biotic stresses, including salt and stimulated drought stresses, ABA and SA treatment, as well as Xanthomonas campestris pv. mangiferaeindicae and Colletotrichum gloeosporioides infection, respectively. Among them, the expression level of MiCOMT1 was significantly up-regulated at 6-72 h after salt and stimulated drought stresses. The results of gene function analysis via the transient overexpression of the MiCOMT1 gene in Nicotiana benthamiana showed that the MiCOMT1 gene can promote the accumulation of ABA and MeJA, and improve the salt tolerance of mango. These results are beneficial to future researchers aiming to understand the biological functions and molecular mechanisms of MiCOMT genes.

Expression Patterns and Functional Analysis of Three SmTAT Genes Encoding Tyrosine Aminotransferases in Salvia miltiorrhiza.

Tyrosine aminotransferase (TAT, E.C. 2.6.1.5) is a pyridoxal phosphate-dependent aminotransferase that is widely found in living organisms. It catalyzes the transfer of the amino group on tyrosine to α-ketoglutarate to produce 4-hydroxyphenylpyruvic acid (4-HPP) and is the first enzyme for tyrosine degradation. Three SmTATs have been identified in the genome of Salvia miltiorrhiza (a model medicinal plant), but their information is very limited. Here, the expression profiles of the three SmTAT genes (SmTAT1, SmTAT2, and SmTAT3) were studied. All three genes expressed in different tissues and responded to methyl jasmonate stimuli. SmTAT proteins are localized in the cytoplasm. The recombinant SmTATs were subjected to in vitro biochemical properties. All three recombinant enzymes had TAT activities and SmTAT1 had the highest catalytic activity for tyrosine, followed by SmTAT3. Also, SmTAT1 preferred the direction of tyrosine deamination to 4-HPP, while SmTAT2 preferred transamination of 4-HPP to tyrosine. In parallel, transient overexpression of SmTATs in tobacco leaves revealed that all three SmTAT proteins catalyzed tyrosine to 4-HPP in vivo, with SmTAT1 exhibiting the highest enzymatic activity. Overall, our results lay a foundation for the production of tyrosine-derived secondary metabolites via metabolic engineering or synthetic biology in the future.

The Role of MbEGS1 and MbEGS2 in Methyleugenol Biosynthesis by Melaleuca bracteata.

Many aromatic plant volatile compounds contain methyleugenol, which is an attractant for insect pollination and has antibacterial, antioxidant, and other properties. The essential oil of Melaleuca bracteata leaves contains 90.46% methyleugenol, which is an ideal material for studying the biosynthetic pathway of methyleugenol. Eugenol synthase (EGS) is one of the key enzymes involved in the synthesis of methyleugenol. We recently reported two eugenol synthase genes (MbEGS1 and MbEGS2) present in M. bracteata, where MbEGS1 and MbEGS2 were mainly expressed in flowers, followed by leaves, and had the lowest expression levels in stems. In this study, the functions of MbEGS1 and MbEGS2 in the biosynthesis of methyleugenol were investigated using transient gene expression technology and virus-induced gene silencing (VIGS) technology in M. bracteata. Here, in the MbEGSs genes overexpression group, the transcription levels of the MbEGS1 gene and MbEGS2 gene were increased 13.46 times and 12.47 times, respectively, while the methyleugenol levels increased 18.68% and 16.48%. We further verified the function of the MbEGSs genes by using VIGS, as the transcript levels of the MbEGS1 and MbEGS2 genes were downregulated by 79.48% and 90.35%, respectively, and the methyleugenol content in M. bracteata decreased by 28.04% and 19.45%, respectively. The results indicated that the MbEGS1 and MbEGS2 genes were involved in the biosynthesis of methyleugenol, and the transcript levels of the MbEGS1 and MbEGS2 genes correlated with the methyleugenol content in M. bracteata.

Fast Identification of In Vivo Protein Phosphorylation Events Using Transient Expression in Leaf Mesophyll Protoplasts and Phos-tagTM SDS-PAGE.

Phosphorylation/dephosphorylation is a key posttranslational mechanism for signal transduction and amplification. Several techniques exist for assessing protein phosphorylation status, but each has its own drawbacks. The fast, straightforward, and low-tech approach described here uses transient overexpression of peptide-tagged proteins in Arabidopsis leaf mesophyll protoplasts and immunoblotting with Phos-tag™ SDS-PAGE and commercial anti-tag antibodies. We illustrate this with two relevant examples related to the SnRK1 protein kinase, which mediates metabolic stress signaling: Arabidopsis thaliana SnRK1 activation by T-loop (auto-)phosphorylation and SnRK1 phosphorylation of the Arabidopsis RAV1 transcription factor, which is involved in seed germination and early seedling development.

Genome-wide identification of the TGA gene family in kiwifruit (Actinidia chinensis spp.) and revealing its roles in response to Pseudomonas syringae pv. actinidiae (Psa) infection.

Kiwifruit bacterial canker, caused by Pseudomonas syringae pv. actinidiae (Psa), is a destructive disease of kiwifruit worldwide. Functional genes in response to Psa infection are needed, as they could be utilized to control disease. TGACG-binding transcription factor (TGA), as an essential regulator, involved in the process of plant against pathogens. However, the function of TGA regulators has not been reported in kiwifruit. It is unclear that whether TGA genes play a role in response to Psa infection. Here, we performed genome-wide screening and identified 13 TGA genes in kiwifruit. Phylogenetic analysis showed that 13 members of the AcTGA gene family could be divided into five groups. AcTGA proteins were mainly located in the nucleus, and significant differences were identified in their 3D structures. Segmental duplications promoted the expansion of the AcTGA family. Additionally, RNA-Seq and qRT-PCR revealed that four genes (AcTGA01/06/07/09) were tissue-specific and responsive to hormones at different levels. Subcellular localization showed that four proteins located in the nucleus, and among them, three (AcTGA01/06/07) had transcriptional activation activity. Lastly, transient overexpression proved that these three genes (AcTGA01/06/07) potentially played a role in the resistance to kiwifruit canker. These results provided a theoretical basis for revealing TGA involved in kiwifruit regulation against Psa.

Characterization of silicon transporter gene family in Saccharum and functional analysis of the ShLsi6 gene in biotic stress.

Silicon, one of the most prevalent elements in the soil, is beneficial for plant growth and defense against different stresses. The silicon transporter gene (Lsi) plays an important role in the uptake and transport of silicon in higher plants. In this study, a total of 32 Lsi genes, including 20 SsLsi in sugarcane wild species Saccharum spontaneum, 5 ShLsi in Saccharum hybrid cultivar R570 and 7 SbLsi in sugarcane related species Sorghum bicolor, were identified and classified into three groups. Bioinformatics analysis showed that instability, hydrophobicity, localization of cell membranes and vacuoles were the main features of the Lsi proteins. Whole genome and segmental duplication contributed to the main expansion of Lsi gene family. Collinearity analysis of the Lsi genes showed that S. spontanum and R570 had a collinear relationship with monocotyledonous plants S. bicolor and Oryza sativa, but not with dicotyledonous plants Arabidopsis thaliana and Vitis vinifera. The replicated Lsi genes were mainly subjected to strong selection pressure for purification. The diverse cis-regulatory elements in the promoter of SsLsi, ShLsi and SbLsi genes suggested that they were widely involved in the response of plants to various stresses and the regulation of the growth and development. Transcriptome data and real time quantitative PCR analysis showed that the Lsi genes exhibited different expression profiles in sugarcane tissues and under Sporisorium scitamineum, drought and cold stresses. In addition, the cDNA and genomic DNA sequences of ShLsi6 that was homologous to SsLsi1b gene was cloned from Saccharum hybrid cultivar ROC22. Transient expression analysis showed that, compared with the control, Nicotiana benthamiana leaves which overexpressed the ShLsi6 gene showed a high sensitivity after inoculation with tobacco pathogens Ralstonia solanacearum and Fusarium solani var. coeruleum. This study provides important information for further functional analysis of Lsi genes and resistant breeding in sugarcane.

Genome-wide analysis of the CCT gene family in Chinese white pear (Pyrus bretschneideri Rehd.) and characterization of PbPRR2 in response to varying light signals.

BACKGROUND: Canopy architecture is critical in determining the light environment and subsequently the photosynthetic productivity of fruit crops. Numerous CCT domain-containing genes are crucial for plant adaptive responses to diverse environmental cues. Two CCT genes, the orthologues of AtPRR5 in pear, have been reported to be strongly correlated with photosynthetic performance under distinct canopy microclimates. However, knowledge concerning the specific expression patterns and roles of pear CCT family genes (PbCCTs) remains very limited. The key roles played by PbCCTs in the light response led us to examine this large gene family in more detail. RESULTS: Genome-wide sequence analysis identified 42 putative PbCCTs in the genome of pear (Pyrus bretschneideri Rehd.). Phylogenetic analysis indicated that these genes were divided into five subfamilies, namely, COL (14 members), PRR (8 members), ZIM (6 members), TCR1 (6 members) and ASML2 (8 members). Analysis of exon-intron structures and conserved domains provided support for the classification. Genome duplication analysis indicated that whole-genome duplication/segmental duplication events played a crucial role in the expansion of the CCT family in pear and that the CCT family evolved under the effect of purifying selection. Expression profiles exhibited diverse expression patterns of PbCCTs in various tissues and in response to varying light signals. Additionally, transient overexpression of PbPRR2 in tobacco leaves resulted in inhibition of photosynthetic performance, suggesting its possible involvement in the repression of photosynthesis. CONCLUSIONS: This study provides a comprehensive analysis of the CCT gene family in pear and will facilitate further functional investigations of PbCCTs to uncover their biological roles in the light response.

Characterization of the Role of the Neoxanthin Synthase Gene BoaNXS in Carotenoid Biosynthesis in Chinese Kale.

Chinese kale (Brassica oleracea var. alboglabra) is rich in carotenoids, and neoxanthin is one of the most important carotenoids in Chinese kale. In this study, the function of the neoxanthin synthase gene (BoaNXS) in Chinese kale was investigated. BoaNXS, which had a 699-bp coding sequence, was cloned from the white flower cultivar of Chinese kale and was expressed in all developmental stages and organs of Chinese kale; its expression was highest in young seeds. The subcellular localization indicated that BoaNXS was localized in the chloroplast. BoaNXS-overexpressed plants were obtained via Agrobacterium-mediated transient overexpression methodology, and the gene overexpression efficiencies ranged from 2.10- to 4.24-fold. The color in the leaves of BoaNXS-overexpressed plants changed from green to yellow-green; the content of total and individual carotenoids, such as neoxanthin, violaxanthin, and lutein, was significantly increased, and the expression levels of most carotenoid biosynthetic genes were notably increased. These findings indicated that BoaNXS is of vital importance in carotenoid biosynthesis in Chinese kale and could be used as a candidate gene for enriching the carotenoid accumulation and color of Chinese kale and other Brassica vegetables.

CsUGT78A15 catalyzes the anthocyanidin 3-O-galactoside biosynthesis in tea plants.

Anthocyanins are a group of natural water-soluble pigments in plants that contribute to the pink-purple color of a range of tissues. Because anthocyanins have various biological activities in human health, there is great research interest in the development of anthocyanin-rich foods and beverages, including purple shoot tea. Anthocyanidin 3-O-galactosides have been identified as one of the main anthocyanin components in purple shoot tea, but the enzyme responsible for their biosynthesis remains unclear. UDP-galactose anthocyanidin 3-O-galactosyltransferase (UA3GalT) is presumed to catalyze the galactosylation of anthocyanidin. Therefore, we assayed the UA3GalT activity in five tea samples with varying degrees of purple color and found that its activity was strongly positively correlated (r = 0.929, p < 0.05) with anthocyanin content. Phylogenetic analysis and sequence alignment suggested that CsUGT78A15 encoded a UA3GalT enzyme. Enzymatic assays indicated that rCsUGT78A15 could catalyze the synthesis of cyanidin 3-O-galactoside and delphinidin 3-O-galactoside using UDP-galactose as a sugar donor, and it showed higher catalytic efficiency towards delphinidin than cyanidin. These results indicate that CsUGT78A15 acts as a UA3GalT in vitro. Subcellular localization showed that CsUGT78A15 was located in the endoplasmic reticulum (ER) and nucleus, consistent with the location of anthocyanin synthesis. Transient overexpression of CsUGT78A15 in the fruit of mature 'Granny Smith' apples showed that the upregulation of CsUGT78A15 promoted cyanidin 3-O-galactoside accumulation in apple skins. These results suggested that CsUGT78A15 could catalyze galactosylation of anthocyanidins in planta. Our findings provide insight into the biosynthesis of anthocyanins in tea plants.

Expression Characteristics and Functional Analysis of the ScWRKY3 Gene from Sugarcane.

The plant-specific WRKY transcriptional regulatory factors have been proven to play vital roles in plant growth, development, and responses to biotic and abiotic stresses. However, there are few studies on the WRKY gene family in sugarcane (Saccharum spp.). In the present study, the characterization of a new subgroup, IIc WRKY protein ScWRKY3, from a Saccharum hybrid cultivar is reported. The ScWRKY3 protein was localized in the nucleus of Nicotiana benthamiana leaves and showed no transcriptional activation activity and no toxic effects on the yeast strain Y2HGold. An interaction between ScWRKY3 and a reported sugarcane protein ScWRKY4, was confirmed in the nucleus. The ScWRKY3 gene had the highest expression level in sugarcane stem pith. The transcript of ScWRKY3 was stable in the smut-resistant Saccharum hybrid cultivar Yacheng05-179, while it was down-regulated in the smut-susceptible Saccharum hybrid cultivar ROC22 during inoculation with the smut pathogen (Sporisorium scitamineum) at 0⁻72 h. ScWRKY3 was remarkably up-regulated by sodium chloride (NaCl), polyethylene glycol (PEG), and plant hormone abscisic acid (ABA), but it was down-regulated by salicylic acid (SA) and methyl jasmonate (MeJA). Moreover, transient overexpression of the ScWRKY3 gene in N. benthamiana indicated a negative regulation during challenges with the fungal pathogen Fusarium solani var. coeruleum or the bacterial pathogen Ralstonia solanacearum in N. benthamiana. The findings of the present study should accelerate future research on the identification and functional characterization of the WRKY family in sugarcane.

Transient Overexpression of HvSERK2 Improves Barley Resistance to Powdery Mildew.

Somatic embryogenesis receptor-like kinases (SERKs) play an essential role in plant response to pathogen infection. Here we identified three SERK genes (HvSERK1/2/3) from barley, and aimed to determine their implication in defense responses to barley powdery mildew (Bgh). Although HvSERK1/2/3 share the characteristic domains of the SERK family, only HvSERK2 was significantly induced in barley leaves during Bgh infection. The expression of HvSERK2 was rapidly induced by hydrogen peroxide (H2O2) treatment, but not by treatment with salicylic acid (SA), methyl jasmonate (MeJA), ethephon (ETH), or abscisic acid (ABA). Bioinformatics analysis of the cloned HvSERK2 promoter revealed that it contains several elements responsible for defense responses against pathogens. Promoter functional analysis showed that the HvSERK2 promoter was induced by Bgh and H2O2. Subcellular localization analysis of HvSERK2 indicated that it is mainly located on the plasma membrane. Transient overexpression of HvSERK2 in epidermal cells of the susceptible barley cultivar Hua 30 reduced the Bgh haustorium index from 58.6% to 43.2%. This study suggests that the HvSERK2 gene plays a positive role in the improvement of barley resistance to powdery mildew, and provides new insight into the function of SERK genes in the biotic stress response of plants.

A sugarcane pathogenesis-related protein, ScPR10, plays a positive role in defense responses under Sporisorium scitamineum, SrMV, SA, and MeJA stresses.

KEY MESSAGE: A pathogenesis-related gene, ScPR10 , was isolated from sugarcane and its bio-function was characterized, demonstrating that ScPR10 was involved in plant defense responses to Sporisorium scitamineum , SrMV, SA, and MeJA stresses. Plant fungal and viral diseases are the major concerns in sugarcane industry. Many anti-fungal and antivirus components, including pathogenesis-related (PR) proteins, have been identified. The pathogenesis-related protein 10 (PR10) is the dominant group in PR families, involved in the plant defense mechanism. In this study, ScPR10 (GenBank Acc. No. KT887884), a 701-bp-length PR10 gene with a 483 bp-length open reading frame, was isolated from sugarcane. Its transient expression in the leaves of Nicotiana benthamiana indicated that the function role of ScPR10 is likely in the nucleus, and it increased the level of H2O2 accumulation in leaf cells. Moreover, ScPR10 could also enhance the resistance of N. benthamiana leaves to infection by Pseudomonas solanacearum and Fusarium solani var. coeruleum. Quantitative real-time PCR analysis revealed that ScPR10 was not constitutively expressed in sugarcane tissues due to its high expression in the buds and scant presence in root tips. In addition, the transcript of ScPR10 could be induced by a pathogenic fungus (Sporisorium scitamineum) and a virus (Sorghum mosaic virus, SrMV) in the resistant sugarcane cultivars, while it was down-regulated in the susceptible ones. After exposure to salicylic acid (SA) and methyl jasmonate (MeJA), ScPR10 peaked at 6 and 12 h, respectively. These results suggest that ScPR10 can play a positive role in sugarcane defense responses to S. scitamineum, SrMV, SA, and MeJA stresses.

DkPK Genes Promote Natural Deastringency in C-PCNA Persimmon by Up-regulating DkPDC and DkADH Expression.

The astringency of Chinese pollination-constant non-astringent (C-PCNA) persimmon (Diospyros kaki Thunb.) can be naturally removed on the tree. This process is controlled by a single locus and is dominant against other types of persimmons; therefore, this variant is an important candidate for commercial cultivation and the breeding of PCNA cultivars. In our previous study, six full-length coding sequences (CDS) for pyruvate kinase genes (DkPK1-6) were isolated, and DkPK1 is thought to be involved in the natural deastringency of C-PCNA persimmon fruit. Here, we characterize the eight other DkPK genes (DkPK7-14) from C-PCNA persimmon fruit based on transcriptome data. The transcript changes in DkPK7-14 genes and correlations with the proanthocyanidin (PA) content were investigated during different fruit development stages in C-PCNA, J-PCNA, and non-PCNA persimmon; DkPK7 and DkPK8 exhibited up-regulation patterns during the last developmental stage in C-PCNA persimmon that was negatively correlated with the decrease in soluble PAs. Phylogenetic analysis and subcellular localization analysis revealed that DkPK7 and DkPK8 are cytosolic proteins. Notably, DkPK7 and DkPK8 were ubiquitously expressed in various persimmon organs and abundantly up-regulated in seeds. Furthermore, transient over-expression of DkPK7 and DkPK8 in persimmon leaves led to a significant decrease in the content of soluble PAs but a significant increase in the expression levels of the pyruvate decarboxylase (DkPDC) and alcohol dehydrogenase genes (DkADH), which are closely related to acetaldehyde metabolism. The accumulated acetaldehyde that results from the up-regulation of the DkPDC and DkADH genes can combine with soluble PAs to form insoluble PAs, resulting in the removal of astringency from persimmon fruit. Thus, we suggest that both DkPK7 and DkPK8 are likely to be involved in natural deastringency via the up-regulation of DkPDC and DkADH expression during the last developmental stage in C-PCNA persimmon.

A Novel L-ascorbate Peroxidase 6 Gene, ScAPX6, Plays an Important Role in the Regulation of Response to Biotic and Abiotic Stresses in Sugarcane.

The L-ascorbate peroxidase 6 gene (APX6) is one of the most important genes for scavenging H2O2 and plays a vital role in plant resistance to environmental stresses. In this study, a novel ScAPX6 gene (GenBank Accession No. KT907352) was obtained from a sugarcane variety (ROC22). Bioinformatics analysis showed that ScAPX6 has a cDNA length of 1,086 bp and encoded 333 amino acid residues. Subcellular localization confirmed that ScAPX6 was located in the chloroplast. Enhanced growth of Escherichia coli BL21 cells that expressed ScAPX6 showed high tolerance under copper (Cu) stress. Real-time quantitative PCR analysis revealed that ScAPX6 was constitutively expressed wherein with the highest expression levels in sugarcane pith and leaf and the lowest in the root. ScAPX6 was down-regulated by salicylic acid (SA), hydrogen peroxide (H2O2), polyethylene glycol (PEG) and sodium chloride (NaCl) stimuli. Interestingly, it was significantly up-regulated under the stresses of abscisic acid (ABA) and methyl jasmonate (MeJA) wherein with the highest inducible expression levels at 6 h at 6.0- and 70.0-times higher, respectively than that of control. Overexpression of ScAPX6 in Nicotiana benthamiana leaves enhanced the resistance to the infection of tobacco pathogens Pseudomonas solanacearum and Fusarium solani var. coeruleum. These results implied that ScAPX6 might positively respond to ABA, MeJA, and Cu, but might negatively respond to the stresses of SA, H2O2, PEG, and NaCl. Keeping in view the current investigation, ScAPX6 could be associated with the hypersensitive response (HR) or immunity of sugarcane, which will provide a baseline for the function identification of sugarcane ScAPX6.

Agrobacterium-mediated transient expression via root absorption in flowering Chinese cabbage.

BACKGROUND: Because most transient transformation techniques are inadequate for functional genomics research in roots, we aimed to develop a simple and efficient Agrobacterium-mediated transient transformation system that utilized root absorption for research in flowering Chinese cabbage. RESULTS: Both semi-quantitative and fluorescent quantitative RT-PCR confirmed that the target gene BcAMT1;3 was more highly expressed in plants that were infected with the transformed Agrobacterium strain (EHA105-p35S-BcAMT1;3) than in control plants that were infected with the control strain (EHA105-p35S). Furthermore, GUS staining analysis conformed the availability of this transient transformation system. In addition, we found that the highest transformation efficiency was achieved using an Agrobacterium cell density of OD600 = 0.3 for 3-6 h, without hyperosmotic pretreatment, and under these conditions, the peak transformation efficiency was observed at 2 and 4 d after infection. CONCLUSIONS: The transformation method developed by the present study is simple and convenient, since no special equipment is required, and since the method causes no damage, the plants can be used for subsequent experiments.

Isolation and Characterization of DkPK Genes Associated with Natural Deastringency in C-PCNA Persimmon.

Chinese pollination-constant non-astringent (C-PCNA) persimmon (Diospyros kaki Thunb.) is considered to be an important germplasm resource for the breeding of PCNA cultivars, though its molecular mechanisms of astringency removal remain to be elucidated. Previously, we showed that the abundance of pyruvate kinase gene transcripts increased rapidly during astringency removal in C-PCNA persimmon fruit. Here, we report the full-length coding sequences of six novel DkPK genes from C-PCNA persimmon fruit isolated based on a complementary DNA (cDNA) library and transcriptome data. The expression patterns of these six DkPK genes and correlations with the soluble proanthocyanidin (PA) content were analyzed during various fruit development stages in different types of persimmon, with DkPK1 showing an expression pattern during the last stage in C-PCNA persimmon that was positively correlated with a decrease in soluble PAs. Phylogenetic analysis revealed that DkPK1 belongs to cytosolic-1 subgroup, and subcellular localization analysis confirmed that DkPK1 is located in the cytosol. Notably, tissue expression profiling revealed ubiquitous DkPK1 expression in different persimmon organs, with the highest expression in seeds. Furthermore, transient over-expression of DkPK1 in persimmon leaves resulted in a significant decrease in the content of soluble PAs but a significant increase in the transcript levels of pyruvate decarboxylase genes (DkPDC1, -3, -4, -5), which catalyze the conversion of pyruvate to acetaldehyde. Thus, we propose that an acetaldehyde-based coagulation effect reduces the content of soluble PAs. Taken together, our results suggest that DkPK1 might be involved in the natural removal of astringency at the last developmental stage in C-PCNA persimmon. This is the first report to identify several novel full-length DkPK genes as well as their potential roles in the natural loss of astringency in C-PCNA persimmon.

Identification and Transcript Analysis of the TCP Transcription Factors in the Diploid Woodland Strawberry Fragaria vesca.

Plant-specific TEOSINTE BRANCHED 1, CYCLOIDEA, and PROLIFERATING CELL FACTORS (TCP) transcription factors play versatile functions in multiple processes of plant growth and development. However, no systematic study has been performed in strawberry. In this study, 19 FvTCP genes were identified in the diploid woodland strawberry (Fragaria vesca) accession Heilongjiang-3. Phylogenetic analysis suggested that the FvTCP genes were classified into two main classes, with the second class further divided into two subclasses, which was supported by the exon-intron organizations and the conserved motif structures. Promoter analysis revealed various cis-acting elements related to growth and development, hormone and/or stress responses. We analyzed FvTCP gene transcript accumulation patterns in different tissues and fruit developmental stages. Among them, 12 FvTCP genes exhibited distinct tissue-specific transcript accumulation patterns. Eleven FvTCP genes were down-regulated in different fruit developmental stages, while five FvTCP genes were up-regulated. Transcripts of FvTCP genes also varied with different subcultural propagation periods and were induced by hormone treatments and biotic and abiotic stresses. Subcellular localization analysis showed that six FvTCP-GFP fusion proteins showed distinct localizations in Arabidopsis mesophyll protoplasts. Notably, transient over-expression of FvTCP9 in strawberry fruits dramatically affected the expression of a series of genes implicated in fruit development and ripening. Taken together, the present study may provide the basis for functional studies to reveal the role of this gene family in strawberry growth and development.