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.

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.

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.