Analysis of ester-producing performance in high-yield ethyl hexanoate yeast and the effect on metabolites in bio- enhanced Daqu, the starter for Baijiu and other traditional fermented foods.

AIMS: Ethyl hexanoate, one of the key flavor compounds in strong-flavor Baijiu. To improve the content of ethyl hexanoate in strong-flavor Baijiu, a functional strain with high yield of ethyl hexanoate was screened and its ester-producing performance was studied. METHODS AND RESULTS: Upon identification, the strain was classified as Candida sp. and designated as ZY002. Under optimal fermentation conditions, the content of ethyl hexanoate synthesized by ZY002 can be as high as 170.56 mg L-1. A fermentation test was carried out using the ZY002 strain bioaugmented Daqu to verify the role of the strain applied to Baijiu brewing. It was found that strain ZY002 could not only improve the moisture and alcohol contents of fermented grains but also diminish the presence of reducing sugar and crude starch. Furthermore, it notably amplified the abundance of flavor compounds. CONCLUSION: In this study, Candida sp. ZY002 with a high yield of ethyl hexanoate provided high-quality strain resources for the actual industrial production of Baijiu.

Alfalfa MsATG13 Confers Cold Stress Tolerance to Plants by Promoting Autophagy.

Autophagy is a conserved cellular process that functions in the maintenance of physiological and metabolic balance. It has previously been demonstrated to improve plant tolerance to abiotic stress. Numerous autophagy-related genes (ATGs) that regulate abiotic stress have been identified, but there have been few functional studies showing how ATGs confer cold stress tolerance. The cold transcriptome data of the crown buds that experienced overwintering of the alfalfa (Medicago sativa L.) showed that MsATG13 is upregulated in response to cold stress. In the present study, we found that MsATG13 transgenic tobacco enhanced cold tolerance compared to wild-type (WT) plants. Transmission electron microscopy demonstrated that transgenic tobacco overexpressing MsATG13 formed more autophagosomes than WT plants in response to cold stress conditions. The transgenic tobacco increased autophagy levels due to upregulation of other ATGs that were necessary for autophagosome production under cold stress conditions. MsATG13 transgenic tobacco also increased the proline contents and antioxidant enzyme activities, enhancing the antioxidant defense capabilities under cold stress conditions. Furthermore, MsATG13 overexpression decreased levels of superoxide anion radicals and hydrogen peroxide under cold stress conditions. These findings demonstrate the role of MsATG13 in enhancing plant cold tolerance through modulation of autophagy and antioxidant levels.

Correlational Analysis of the Physicochemical Indexes, Volatile Flavor Components, and Microbial Communities of High-Temperature Daqu in the Northern Region of China.

Daqu is a microbial-rich baijiu fermentation starter. The high-temperature Daqu plays an essential role in the manufacturing of sauce-flavored baijiu. However, few studies have focused on three kinds of high-temperature Daqu (white, yellow, and black Daqu) in northern China. In this study, the physicochemical indexes, volatile flavor compounds, and microbial characteristics of the three different colors of high-temperature Daqu in northern China were comparatively analyzed to reveal their potential functions. White Daqu (WQ) exhibited the highest liquefying power and starch, and black Daqu (BQ) showed the highest saccharifying and esterifying powers. A total of 96 volatile components were identified in the three types of Daqu, and the contents of the volatile components of yellow Daqu (YQ) were the highest. The microbial community structure analysis showed that Bacillus and Byssochlamys were dominant in BQ, Kroppenstedtia and Thermoascus were dominant in WQ, and Virgibacillus and Thermomyces dominated the YQ. The RDA analysis revealed the correlation between the dominant microorganisms and different physicochemical indexes. The Spearman correlation analysis indicated that Oceanobacillus, Saccharopolyspora, Staphylococcus, Pseudogracilibacillus, Byssochlamys, and Thermomyces showed positive correlations with part of the majority of the key volatile flavor compounds. This work provides a scientific basis for the actual production of different colors of high-temperature Daqu in the northern region of China for sauce-flavored baijiu.

Dynamic Transcriptome Analysis Reveals Transcription Factors Involved in the Synthesis of Ethyl Acetate in Aroma-Producing Yeast.

Ethyl acetate is an important flavor element that is a vital component of baijiu. To date, the transcription factors that can help identify the molecular mechanisms involved in the synthesis of ethyl acetate have not been studied. In the present study, we sequenced and assembled the Wickerhamomyces anomalus strain YF1503 transcriptomes to identify transcription factors. We identified 307 transcription factors in YF1503 using high-throughput RNA sequencing. Some transcription factors, such as C2H2, bHLH, MYB, and bZIP, were up-regulated, and these might play a role in ethyl acetate synthesis. According to the trend of ethyl acetate content, heat map results and STEM, twelve genes were selected for verification of expression levels using quantitative real-time PCR. This dynamic transcriptome analysis presents fundamental information on the transcription factors and pathways that are involved in the synthesis of ethyl acetate in aroma-producing yeast. Of significant interest is the discovery of the roles of various transcription factor genes in the synthesis of ethyl acetate.

Proteomic analysis reveals responsive mechanisms for saline-alkali stress in alfalfa.

Saline-alkali stress is a major abiotic stress that limits plant growth, yield, and geographical distribution. Alfalfa is a perennial legume with the largest planting area in the world because of its high protein content, good palatability, and long utilization life. However, saline-alkali stress seriously affects alfalfa yield and quality. To better understand the saline-alkali stress response mechanisms of alfalfa, an isobaric tags proteomics method was used to compare and analyse alfalfa under saline-alkali stress for 0, 1, and 7 days, and 126 (1 vs. 0 days) and 1869 (7 vs. 0 days) differentially abundant proteins (DAPs) were found. Through integrative analysis with differentially expressed genes (DEGs), we found correlated DEGs-DAPs of RNA and protein with similar expression trends at the mRNA and protein levels; these were mainly involved in ABA and Ca2+ signal pathways, regulation of photosynthesis, ROS scavenging, secondary metabolism, and transcription factors (TFs) related to saline-alkali stress. Some genes not exhibiting such trends may have been regulated post-transcriptionally. Furthermore, through transgenic experiments, MsFTL was found to significantly improve the saline-alkali tolerance of plants. Overall, our findings provide important clues for understanding the molecular mechanisms underlying the response of alfalfa to saline-alkali stress.

Alfalfa (Medicago sativa L.) MsCML46 gene encoding calmodulin-like protein confers tolerance to abiotic stress in tobacco.

KEY MESSAGE: MsCML46 enhances tolerance to abiotic stresses through alleviating osmotic stress and oxidative damage by regulating the expression of stress-related genes to optimize osmolytes levels and antioxidant enzyme activity in transgenic tobacco. Abiotic stresses are major environmental factors that constraint crop productivity worldwide. Various stimuli regulate intracellular calcium levels and calcium-mediated signal transduction, and cellular responses. Ca2+ signals are perceived by different Ca2+ receptors. Calmodulin-like protein (CML) is one of the best-characterized Ca2+ sensors which shares sequence similarity with highly conserved calmodulin (CaM) ubiquitously expressed in plants. Currently, the molecular and physiological functions of CMLs are largely unknown. In this study, the MsCML46 was characterized in alfalfa (Medicago sativa cv. Zhaodong) under freezing stress. Results showed that MsCML46 was localized to the cytoplasm of Arabidopsis, and its expression was strongly elevated by cold, drought, salt, saline-alkali, and ABA treatments. Overexpressing MsCML46 in tobacco enhanced tolerance to freezing, drought, and salt stresses as evidenced by improved contents of osmotic regulatory solutes and antioxidant enzyme activity but decreased reactive oxygen species (ROS) accumulation. Furthermore, cold, drought, and salt stresses increased the expression of stress-related genes in transgenic tobacco. MsCML46 binds free Ca2+ to promote signal transduction and maintain higher K+/Na+ ratio. In this way, it protects intracellular homeostasis under sodium ion toxicity. These results suggest that MsCML46 plays a crucial role in resisting abiotic stresses and can be exploited in genetic engineering for crops.

Alfalfa MsCBL4 enhances calcium metabolism but not sodium transport in transgenic tobacco under salt and saline-alkali stress.

KEY MESSAGE: MsCBL4 expression in tobacco enhanced its salt and saline-alkali stress tolerance by regulating calcium accumulation in roots, indicating the important role of calcium metabolism in plant saline-alkali stress tolerance The calcineurin B-like (CBL) family of proteins play important roles in plant abiotic stress tolerance and signal transduction. CBL4 is known to participate in the Salt Overly Sensitive pathway; however, little is currently known regarding the mechanisms underlying the response of CBL4 to saline-alkali stress. In this study, we cloned and characterized the alfalfa MsCBL4 gene. We found that MsCBL4 showed the highest expression in root tissues and was induced by salt and saline-alkali stress, with the latter causing higher induction. Overexpression of MsCBL4 in tobacco enhanced salt and saline-alkali stress tolerance and reduced the Na+/K+ ratio in roots of transgenic lines. Salt (30 and 300 mM NaCl) and saline-alkali (30 mM NaHCO3) stress assays performed for MsCBL4 transgenic tobacco lines revealed a substantial influx of sodium ions in roots under saline-alkali stress and indicated that the expression of MsCBL4 had little influence on sodium ion content reduction. In contrast, in roots subjected to saline-alkali stress, calcium accumulation occurred and was significantly enhanced by the overexpression of MsCBL4. Physiological and biochemical analyses indicated that MsCBL4 plays an important role in saline-alkali stress tolerance via its influence on the regulation of calcium transport and accumulation. These results provide novel insights into the saline-alkali stress tolerance mechanisms of plants.

Genome-wide analysis of the TCP transcription factor genes in five legume genomes and their response to salt and drought stresses.

The teosinte branched1, cycloidea, and proliferating cell factor family (TCP) proteins, plant-specific transcription factors, are involved in the regulation of plant development; however, the TCP gene family of legumes has been based primarily on a single crop. Here, a total of 55, 22, 26, 21, and 25 genes containing the VQ motif were identified from the genomes of Glycine max, Cicer arietinum, Phaseolus vulgaris, Medicago truncatula, and Lotus japonicus, respectively. Based on the phylogenetic analysis, we divided these TCP genes into three distinct subfamilies: PCF, CYC/TB1, and CIN. The conserved domain analysis indicated that the TCP gene family members contain the bHLH and R domains. The TCP genes from the same evolutionary branches of legumes shared similar motifs and structures. The promoter analysis revealed that cis-elements were related to stress responses, phytohormone responses, and physical and reproductive growth regulation. In addition, the TCP genes presented different expression patterns in the five legumes. Most of them showed specific expression patterns during development. The results of qRT-PCR indicated that the TCP genes played regulatory roles in both salt and drought stresses. The present study provides novel and detailed information regarding the legume TCP gene family, which aids in functional characterisation of the TCP genes in other plants.

Overexpression of an alfalfa glutathione S-transferase gene improved the saline-alkali tolerance of transgenic tobacco.

Abiotic stresses restrict the productivity and quality of agricultural crops. Glutathione S-transferase (GST) utilizes glutathione to scavenge reactive oxygen species (ROS) that result from abiotic stresses. This study aimed to determine the expression pattern of the MsGSTU8 gene and its effects on saline-alkali tolerance. MsGSTU8, from alfalfa (Medicago sativa 'Zhaodong'), was transformed into transgenic tobacco (Nicotiana tabacum) and overexpressed to determine its effects on saline-alkali tolerance. The gene products in alfalfa localized to the cytoplasm and the transcript levels were higher in the leaves than the roots and stems. Expression was strongly induced by cold, drought, salt and saline-alkali stresses as well as abscisic acid (ABA) treatments. The transgenic tobacco lines had significantly higher transcription levels of the abiotic stress-related genes and higher GST activity than the wild types. Transgenic tobacco lines with saline-alkali treatments maintained their chlorophyll content, showed improved antioxidant enzyme activity and soluble sugar levels, reduced ion leakage, O2 .-, H2O2 accumulation and malondialdehyde content. Our results indicate that overexpression of MsGSTU8 could improve resistance to saline-alkali stresses by decreasing the accumulation of ROS and increasing the levels of antioxidant enzymes. Furthermore, they suggest that MsGSTU8 could be utilized for transgenic crop plant breeding.