A new carboxypeptidase from Aspergillus niger with good thermostability, pH stability and broad substrate specificity

A new serine carboxypeptidase gene, capA, was identified in Aspergillus niger CBS 513.88 by reading genomic information and performing sequence alignment, and the gene was cloned and expressed in Pichia pastoris GS115. In a shake flask, the enzyme activity of the recombinant strain GS115 (pPIC9K-capA) reached 209.3 U mg−1. The optimal temperature and pH for enzyme activity were determined to be 45 °C and 6.0, respectively. After incubation at 40–50 °C or at pH 4.0–8.0 for 1 h, the enzyme retained more than 80% or 60% of its initial activity. The presence of 1–10 mmol L−1 Mg2+ enhanced the activity of CapA, whereas 1–10 mmol L−1 Cu2+, Fe2+, or Co2+, 10 mmol L−1 Mn2+, or 1–10 mmol L−1 phenylmethylsulfonyl fluoride (PMSF) significantly inhibited its activity. CapA had a broad substrate specificity and preferred the hydrophobic amino acids Leu and Lys at the C terminus of proteins, and N-benzyloxycarbonyl-l-phenylalanyl-l-leucine (Cbz-Phe-Leu) was the optimal substrate, for which CapA exhibited Km 0.063 mmol L−1 and kcat/Km 186.35 mmol L−1 s−1. The good thermostability, pH stability and hydrolysis characteristics of CapA provide a solid foundation for application in the food and biotechnology fields.

BRS1 mediates plant redox regulation and cold responses

Background Brassinosteroid-insensitive 1 suppressor 1 (BRS1) is a serine carboxypeptidase that mediates brassinosteroid signaling and participates in multiple developmental processes in Arabidopsis. However, little is known about the precise role of BRS1 in this context. Results In this study, we analyzed transcriptional and proteomic profiles of Arabidopsis seedlings overexpressing BRS1 and found that this gene was involved in both cold stress responses and redox regulation. Further proteomic evidence showed that BRS1 regulated cell redox by indirectly interacting with cytosolic NADP + -dependent isocitrate dehydrogenase (cICDH). One novel alternative splice form of BRS1 was identified in over-expression mutants brs1-1D, which may confer a new role in plant development and stress responses. Conclusions This study highlights the role of BRS1 in plant redox regulation and stress responses, which extends our understanding of extracellular serine carboxypeptidases.

Cloning, Expression and Biochemical Characterization of a new Carboxypeptidase from Aspergillus Niger

A new serine carboxypeptidase gene, capA, was identified in Aspergillus niger CBS 513.88 by reading genomic information and performing sequence alignment, and then, the gene was cloned and expressed in Pichia pastoris. In a shake-flask, the enzyme activity of the recombinant strain GS115 (pPIC9K-capA) reached 109.7 U/mL. The optimum temperature and pH for enzyme activity were determined to be 45°C and 6.0, respectively. After incubation at 40°C-50°C or at pH 4.0–8.0 for 1 h, the enzyme retained more than 80% or 60% of its initial activity. Mg2+ enhanced the activity of CapA, whereas Cu2+, Fe2+, Co2+ and PMSF inhibited its activity. Among the six substrates tested, five could be hydrolyzed by CapA. CapA had a broad substrate specificity and preferred the hydrophobic amino acids Leu and Lys in the C-terminus of proteins, and CBZ-Phe-Leu was the preferred substrate for CapA. The good thermostability, pH stability and hydrolysis characteristics provide a solid foundation for the application of CapA in the food and biotechnology fields.

Genome-wide analysis of the serine carboxypeptidase-like protein family in Triticum aestivum reveals TaSCPL184-6D is involved in abiotic stress response

Background The serine carboxypeptidase-like protein (SCPL) family plays a vital role in stress response, growth, development and pathogen defense. However, the identification and functional analysis of SCPL gene family members have not yet been performed in wheat. Results In this study, we identified a total of 210 candidate genes encoding SCPL proteins in wheat. According to their structural characteristics, it is possible to divide these members into three subfamilies: CPI, CPII and CPIII. We uncovered a total of 209 TaSCPL genes unevenly distributed across 21 wheat chromosomes, of which 65.7% are present in triads. Gene duplication analysis showed that ~ 10.5% and ~ 64.8% of the TaSCPL genes are derived from tandem and segmental duplication events, respectively. Moreover, the Ka/Ks ratios between duplicated TaSCPL gene pairs were lower than 0.6, which suggests the action of strong purifying selection. Gene structure analysis showed that most of the TaSCPL genes contain multiple introns and that the motifs present in each subfamily are relatively conserved. Our analysis on cis-acting elements showed that the promoter sequences of TaSCPL genes are enriched in drought-, ABA- and MeJA-responsive elements. In addition, we studied the expression profiles of TaSCPL genes in different tissues at different developmental stages. We then evaluated the expression levels of four TaSCPL genes by qRT-PCR, and selected TaSCPL184-6D for further downstream analysis. The results showed an enhanced drought and salt tolerance among TaSCPL184-6D transgenic Arabidopsis plants, and that the overexpression of the gene increased proline and decreased malondialdehyde levels, which might help plants adapting to adverse environments. Our results provide comprehensive analyses of wheat SCPL genes that might work as a reference for future studies aimed at improving drought and salt tolerance in wheat. Conclusions We conducte a comprehensive bioinformatic analysis of the TaSCPL gene family in wheat, which revealing the potential roles of TaSCPL genes in abiotic stress. Our analysis also provides useful resources for improving the resistance of wheat.

Versatility in acyltransferase activity completes chicoric acid biosynthesis in purple coneflower

Purple coneflower (Echinacea purpurea (L.) Moench) is a popular native North American herbal plant. Its major bioactive compound, chicoric acid, is reported to have various potential physiological functions, but little is known about its biosynthesis. Here, taking an activity-guided approach, we identify two cytosolic BAHD acyltransferases that form two intermediates, caftaric acid and chlorogenic acid. Surprisingly, a unique serine carboxypeptidase-like acyltransferase uses chlorogenic acid as its acyl donor and caftaric acid as its acyl acceptor to produce chicoric acid in vacuoles, which has evolved its acyl donor specificity from the better-known 1-O-β-D-glucose esters typical for this specific type of acyltransferase to chlorogenic acid. This unusual pathway seems unique to Echinacea species suggesting convergent evolution of chicoric acid biosynthesis. Using these identified acyltransferases, we have reconstituted chicoric acid biosynthesis in tobacco. Our results emphasize the flexibility of acyltransferases and their roles in the evolution of specialized metabolism in plants.

Isolation and Functional Analysis of Genes Involved in Polyacylated Anthocyanin Biosynthesis in Blue Senecio cruentus

Polyacylated anthocyanins with multiple glycosyl and aromatic acyl groups tend to make flowers display bright and stable blue colours. However, there are few studies on the isolation and functional characterization of genes involved in the polyacylated anthocyanin biosynthesis mechanism, which limits the molecular breeding of truly blue flowers. Senecio cruentus is an important potted ornamental plant, and its blue flowers contain 3′,7-polyacylated delphinidin-type anthocyanins that are not reported in any other plants, suggesting that it harbours abundant gene resources for the molecular breeding of blue flowers. In this study, using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis of blue, carmine and white colours of cineraria cultivars “Venezia” (named VeB, VeC, and VeW, respectively), we found that 3′,7-polyacylated anthocyanin, cinerarin, was the main pigment component that determined the blue colour of ray florets of cineraria. Based on the transcriptome sequencing and differential gene expression (DEG) analysis combined with RT- and qRT-PCR, we found two genes encoding uridine diphosphate glycosyltransferase, named ScUGT1 and ScUGT4; two genes encoding acyl-glucoside-dependent glucosyltransferases which belong to glycoside hydrolase family 1 (GH1), named ScAGGT11 and ScAGGT12; one gene encoding serine carboxypeptidase-like acyltransferase ScSCPL2; and two MYB transcriptional factor genes ScMYB2 and ScMYB4, that were specifically highly expressed in the ray florets of VeB, which indicated that these genes may be involved in cinerarin biosynthesis. The function of ScSCPL2 was analysed by virus-induced gene silencing (VIGS) in cineraria leaves combined with HPLC-MS/MS. ScSCPL2 mainly participated in the 3′ and 7-position acylation of cinerarin. These results will provide new insight into the molecular basis of the polyacylated anthocyanin biosynthesis mechanism in higher plants and are of great significance for blue flower molecular breeding of ornamental plants.

Candidate Genes and Stable QTL for Grain Yield and Seed Size in Durum Wheat

Background: In wheat grain yield is expressed as the product of different components. Among these, thousand kernels weight (TKW) reflects the combination of several grain related traits including grain length (GL), grain width (GW) and area. Grain weight is also affected by phenological traits, such as heading time (HT) and plant height (PH). To detect stable QTL and candidate genes involved in phenotypic control of grain yield, a recombinant inbred line (RIL) population derived from two elite durum wheat cultivars (Liberdur and Anco Marzio) was evaluated for yield components and grain related traits for three growing seasons in southern Italy. The mapping population was genotyped with a 90K SNP array and a high-density genetic linkage map with 5134 markers was obtained.Results: A total of 30 QTL were detected on the durum RIL population including 9 stable QTL for TKW (2 QTL), GL, GW (2 QTL), AREA, HT and PH (2 QTL) distributed on 1B, 2A, 3A and 6B chromosomes. Interestingly, a QTL cluster mapped on 2A included a major QTL for HT explaining at least 70% of phenotypic variance and co-located with a QTL for YLD, TKW, GL and GW and AREA, respectively. In the physical position of this QTL cluster a photoperiod sensitivity gene (Ppd-A1) was found. Serine carboxypeptidase, Big Grain 1 and β-fructofuranosidase candidate genes were mapped in clusters containing stable QTL. Candidate genes involved in auxin metabolism were also found in QTL clusters in which a QTL for AREA was declared. Conclusions: This study showed that yield components and phenological traits had higher inheritances than grain yield, allowing an accurate stable QTL cluster detection. This was a powerful requisite to physically map QTL on the reference durum wheat genome and to identify candidate genes strongly affecting the genetic grain yield network.

BRS1 mediates plant redox regulation and cold responses

Background: Brassinosteroid-insensitive 1 suppressor 1 (BRS1), is a serine carboxypeptidase that mediates brassinosteroid signaling and participates in multiple developmental processes in Arabidopsis. However, little is known about the precise role of BRS1 in this context. Results: In this study, we analyzed transcriptional and proteomic profiles of Arabidopsis seedlings overexpressing BRS1 and found that this gene is involved in both cold stress responses and redox regulation. Further proteomic evidence shows that BRS1 regulates cell redox by indirectly interacting with cytosolic NADP+-dependent isocitrate dehydrogenase (cICDH). We identified two novel splice products of BRS1, which might play important roles in development and stress responses in plants. Conclusions: This study highlights the role of BRS1 in plant redox regulation and stress responses, which extends our understanding of extracellular serine carboxypeptidases.

Proteomics of Seed Nutrition-Associated Proteins in Germinated Brown Rice in Four Thai Rice Cultivars Analyzed by GeLC-MS/MS

Brown rice (BR) and germinated brown rice (GBR) of Oryza sativa L. are popularly consumed by Asians because of their important healthy diet components. They are known to contain bioactive compounds and nutrients, such as phenolics, vitamins, fatty acids, and sugars, which help to maintain good health and reduce the incidences of various chronic diseases. The objective of this study was to investigate the effects of germination on changes of nutrition-associated proteins in 4 rice cultivars. After germination for 24 h, the changes of seed nutrition-associated proteins were examined by shotgun proteomics. The total proteins from non-germinated seeds and 24 h germinated seeds of 4 rice cultivars were extracted and analyzed by in-gel digestion coupled with tandem mass spectrometry (GeLC-MS/MS). A total phenolic content was analyzed from the crude methanol extract of those grains after germination for 0, 24, and 48 h using Folin-Ciocalteu assay. The analysis showed that seed nutritional-associated proteins, especially phenolic-associated proteins, increased after germination according to the accumulation of the total phenolic content. The expressions of 6 phenolic-associated proteins, including phenylalanine ammonia-lyase, serine carboxypeptidase-like protein, isoflavone-7-O-methyltransferase, isoflavonoid glucosyltransferase, glycosyltransferase family 61 protein and UDP-glucose flavonoid 3-O-glucosyltransferase were increased by 2.20 - 15.90 folds after germination. This study provides evidence that rice germination for 24 h has essentially influenced the increased nutritional values of BR and the phenolic biosynthetic pathway.