Tolerant Yeast Research Articles

A novel metallothionein gene HcMT from halophyte shrub Halostachys caspica respond to cadmium and sodium stress

Cadmium (Cd) and sodium (Na) are two of the most phytotoxic metallic elements causing environmental and agricultural problems. Metallothioneins (MTs) play an important role in the adaptation to abiotic stress. We previously isolated a novel type 2 MT gene from Halostachys caspica (H. caspica), named HcMT, which responded to metal and salt stress. To understand the regulatory mechanisms controlling HcMT expression, we cloned the HcMT promoter and characterized its tissue-specific and spatiotemporal expression patterns. β-Glucuronidase (GUS) activity analysis showed that the HcMT promoter was responsive to CdCl2, CuSO4, ZnSO4 and NaCl stress. Therefore, we further investigated the function of HcMT under abiotic stress in yeast and Arabidopsis thaliana (Arabidopsis). In CdCl2, CuSO4 or ZnSO4 stress, HcMT significantly enhanced the metal ions tolerance and accumulation in yeast through function as a metal chelator. Moreover, the HcMT protein also protected yeast cells from NaCl, PEG and hydrogen peroxide (H2O2) toxicity with less effectiveness. However, transgenic Arabidopsis carrying HcMT gene only displayed tolerance to CdCl2 and NaCl, accompanying by higher content of Cd2+ or Na+ and lower H2O2, compared to wild-type (WT) plants. Next, we demonstrated that the recombinant HcMT protein has the ability to bind Cd2+ and the potential of scavenging ROS (reactive oxygen species) in vitro. This result further confirmed that the role of HcMT to influence plants to CdCl2 and NaCl stress may bind metal ions and scavenge ROS. Overall, we described the biological functions of HcMT and developed a metal- and salt-inducible promoter system for using in genetic engineering.

Adaptive responses of yeast strains tolerant to acidic pH, acetate, and supraoptimal temperature

Ethanol fermentations can be prematurely halted as Saccharomyces cerevisiae faces adverse conditions, such as acidic pH, presence of acetic acid, and supraoptimal temperatures. The knowledge on yeast responses to these conditions is essential to endowing a tolerant phenotype to another strain by targeted genetic manipulation. In this study, physiological and whole-genome analyses were conducted to obtain insights on molecular responses which potentially render yeast tolerant towards thermoacidic conditions. To this end, we used thermotolerant TTY23, acid tolerant AT22, and thermo-acid tolerant TAT12 strains previously generated by adaptive laboratory evolution (ALE) experiments. The results showed an increase in thermoacidic profiles in the tolerant strains. The whole-genome sequence revealed the importance of genes related to: H+, iron, and glycerol transport (i.e., PMA1, FRE1/2, JEN1, VMA2, VCX1, KHA1, AQY3, and ATO2); transcriptional regulation of stress responses to drugs, reactive oxygen species and heat-shock (i.e., HSF1, SKN7, BAS1, HFI1, and WAR1); and adjustments of fermentative growth and stress responses by glucose signaling pathways (i.e., ACS1, GPA1/2, RAS2, IRA2, and REG1). At 30 °C and pH 5.5, more than a thousand differentially expressed genes (DEGs) were identified in each strain. The integration of results revealed that evolved strains adjust their intracellular pH by H+ and acetic acid transport, modify their metabolism and stress responses via glucose signaling pathways, control of cellular ATP pools by regulating translation and de novo synthesis of nucleotides, and direct the synthesis, folding and rescue of proteins throughout the heat-shock stress response. Moreover, the motifs analysis in mutated transcription factors suggested a significant association of SFP1, YRR1, BAS1, HFI1, HSF1, and SKN7 TFs with DEGs found in thermoacidic tolerant yeast strains.Key points• All the evolved strains overexpressed the plasma membrane H+-ATPase PMA1 at optimal conditions• Tolerant strain TAT12 mutated genes encoding weak acid and heat response TFs HSF1, SKN7, and WAR1• TFs HSF1 and SKN7 likely controlled the transcription of metabolic genes associated to heat and acid tolerance

Trehalose metabolism targeting as a novel strategy to modulate acid tolerance of yeasts and its application in food industry

Some spoilage yeasts are able to develop resistance to commonly used weak-acid preservatives. We studied the trehalose metabolism and its regulation in Saccharomyces cerevisiae in response to propionic acid stress. We show interruption of trehalose synthetic pathway caused the mutant hypersensitive to the acid stress, while its overexpression conferred acid-tolerance to yeast. Interestingly, this acid-tolerance phenotype was largely independent of trehalose but relied on the trehalose synthetic pathway. We demonstrate trehalose metabolism played a vital role in regulation of glycolysis flux and Pi/ATP homeostasis in yeast during acid-adaptation, and the PKA and TOR signaling pathways were involved in regulating trehalose synthesis at transcriptional level. This work confirmed the regulatory function of trehalose metabolism and improved our understanding of molecular mechanism of acid-adaptation of yeast. By exemplifying trehalose metabolism interruption limited the growth of S. cerevisiae exposed to weak acids, and trehalose pathway overexpression conferring acid-resistance to Yarrowia lipolytica enhanced citric acid production, this work provides new insights into the development of efficient preservation strategies and robust organic acid producers.

Interaction Network Construction and Functional Analysis of the Plasma Membrane H+-ATPase in Bangia fuscopurpurea (Rhodophyta).

Salinity is a serious threat to most land plants. Although seaweeds adapt to salty environments, intertidal species experience wide fluctuations in external salinities, including hyper- and hypo-saline stress. Bangia fuscopurpurea is an economic intertidal seaweed with a strong tolerance to hypo-salinity. Until now, the salt stress tolerance mechanism has remained elusive. Our previous study showed that the expression of B. fuscopurpurea plasma membrane H+-ATPase (BfPMHA) genes were the most upregulated under hypo-salinity. In this study, we obtained the complete sequence of BfPMHA, traced the relative expression of this BfPMHA gene in B. fuscopurpurea under hypo-salinity, and analyzed the protein structure and properties based on the gene's sequence. The result showed that the expression of BfPMHA in B. fuscopurpurea increased significantly with varying hypo-salinity treatments, and the higher the degree of low salinity stress, the higher the expression level. This BfPMHA had typical PMHA structures with a Cation-N domain, an E1-E2 ATPase domain, a Hydrolase domain, and seven transmembrane domains. In addition, through the membrane system yeast two-hybrid library, three candidate proteins interacting with BfPMHA during hypo-saline stress were screened, fructose-bisphosphate aldolase (BfFBA), glyceraldehyde 3-phosphate dehydrogenase (NADP+) (phosphorylating) (BfGAPDH), and manganese superoxide dismutase (BfMnSOD). The three candidates and BfPMHA genes were successfully transferred and overexpressed in a BY4741 yeast strain. All of them significantly enhanced the yeast tolerance to NaCl stress, verifying the function of BfPMHA in salt stress response. This is the first study to report the structure and topological features of PMHA in B. fuscopurpurea and its candidate interaction proteins in response to salt stress.

Bioactive dipeptides enhance the tolerance of lager yeast to ethanol-oxidation cross-stress by regulating the multilevel defense system

Although high gravity brewing technology has been widely used for beer industries due to its economic benefits, yeast cells are subjected to multiple environmental stresses throughout the fermentation process. Eleven bioactive dipeptides (LH, HH, AY, LY, IY, AH, PW, TY, HL, VY, FC) were selected to evaluate their effects on cell proliferation, cell membrane defense system, antioxidant defense system and intracellular protective agents of lager yeast against ethanol-oxidation cross-stress. Results showed that the multiple stresses tolerance and fermentation performance of lager yeast were enhanced by bioactive dipeptides. Cell membrane integrity was improved by bioactive dipeptides through altering the structure of macromolecular compounds of the cell membrane. Intracellular reactive oxygen species (ROS) accumulation was significantly decreased by bioactive dipeptides, especially for FC, decreasing by 33.1%, compared with the control. The decrease of ROS was closely related to the increase of mitochondrial membrane potential, intracellular antioxidant enzyme activities including superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), and glycerol level. In addition, bioactive dipeptides could regulate the expression of key genes (GPD1, OLE1, SOD2, PEX11, CTT1, HSP12) to enhance the multilevel defense systems under ethanol-oxidation cross-stress. Therefore, bioactive dipeptides should be potentially efficient and feasible bioactive ingredients to improve the multiple stresses tolerance of lager yeast during high gravity fermentation.

Schwanniomyces etchellsii, acid-thermotolerant yeasts from urban city soil

Acid-tolerant yeasts are one of the important keys to producing ethanol from acidic substrates, especially from molasses and agricultural waste. In this study, selected cultivars of yeasts isolated from a variety of locations such as botanical gardens in Thailand urban areas, which are often found highly polluted in the air such as carbon dioxide which is a cause of acid rain. There is limited information about how tolerant yeasts, are or their functional properties related to the environment. Yeast species were determined by using the18S rDNAsequence guide. The level of acid tolerance was evaluated by adding to the culture medium lactic acid (300-900mM), acetic acid (100-400mM), and propionic acid (25-100mM).18S rDNAanalysis has shown a %similarity of the nucleotide sequence higher than 98.65% compared to the database.Schwanniomyces etchellsiistrains found in urban city soil were notable for their tolerance of lactic acid up to 100mM. There are two main types of yeasts in overall acid tolerance:S. etchellsii,which is recognized as an osmotic pressure-resistant species that is highly resistant to fermentation inhibitors and produces ethanol; andSchizosaccharomyces pombe, which cell wall has been reported to be characterized by accumulation of α-(1,3)-glucan and malic acid can be used in metabolic pathways. The results show thatS. pombe, isolated from rice paddy fields, can grow efficiently in acetic and propionic acid up to 400mM and 100mM, respectively. This species could be cultured in ethanol at a concentration of 12.5% (v/v). Moreover, it presented high ethanol and acetic acid production of 14.5-15.9g/L and 7-10g/L, respectively, with or without acidic conditions. In comparison,S. etchellsii, isolated from the botanical garden soil, which is grown in acetic, propionic, and lactic acid, was also indicated to be an organic acid-tolerant species.

Genome-Wide Analysis of the SNARE Family in Cultivated Peanut (Arachis hypogaea L.) Reveals That Some Members Are Involved in Stress Responses.

The superfamily of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediates membrane fusion during vesicular transport between endosomes and the plasma membrane in eukaryotic cells, playing a vital role in plant development and responses to biotic and abiotic stresses. Peanut (Arachis hypogaea L.) is a major oilseed crop worldwide that produces pods below ground, which is rare in flowering plants. To date, however, there has been no systematic study of SNARE family proteins in peanut. In this study, we identified 129 putative SNARE genes from cultivated peanut (A. hypogaea) and 127 from wild peanut (63 from Arachis duranensis, 64 from Arachis ipaensis). We sorted the encoded proteins into five subgroups (Qa-, Qb-, Qc-, Qb+c- and R-SNARE) based on their phylogenetic relationships with Arabidopsis SNAREs. The genes were unevenly distributed on all 20 chromosomes, exhibiting a high rate of homolog retention from their two ancestors. We identified cis-acting elements associated with development, biotic and abiotic stresses in the promoters of peanut SNARE genes. Transcriptomic data showed that expression of SNARE genes is tissue-specific and stress inducible. We hypothesize that AhVTI13b plays an important role in the storage of lipid proteins, while AhSYP122a, AhSNAP33a and AhVAMP721a might play an important role in development and stress responses. Furthermore, we showed that three AhSNARE genes (AhSYP122a, AhSNAP33a and AhVAMP721) enhance cold and NaCl tolerance in yeast (Saccharomyces cerevisiae), especially AhSNAP33a. This systematic study provides valuable information about the functional characteristics of AhSNARE genes in the development and regulation of abiotic stress responses in peanut.

Characterization of the NAC Transcription Factor in Passion Fruit (Passiflora edulis) and Functional Identification of PeNAC-19 in Cold Stress

The NAC (NAM, ATAF and CUC) gene family plays an important role in plant development and abiotic stress response. However, up to now, the identification and research of the NAC (PeNAC) family members of passion fruit are still lacking. In this study, 25 PeNACs were identified from the passion fruit genome, and their functions under abiotic stress and at different fruit-ripening stages were analyzed. Furthermore, we analyzed the transcriptome sequencing results of PeNACs under four various abiotic stresses (drought, salt, cold and high temperature) and three different fruit-ripening stages, and verified the expression results of some genes by qRT-PCR. Additionally, tissue-specific analysis showed that most PeNACs were mainly expressed in flowers. In particular, PeNAC-19 was induced by four various abiotic stresses. At present, low temperatures have seriously endangered the development of passion fruit cultivation. Therefore, PeNAC-19 was transformed into tobacco, yeast and Arabidopsis to study their function of resisting low temperature. The results show that PeNAC-19 responded to cold stress significantly in tobacco and Arabidopsis, and could improve the low temperature tolerance of yeast. This study not only improved the understanding of the PeNAC gene family characteristics and evolution, but also provided new insights into the regulation of the PeNAC gene at different stages of fruit maturation and abiotic stresses.

Heterologous expression of Sesuvium portulacastrum SOS-related genes confer salt tolerance in yeast

Heterologous expression of Sesuvium portulacastrum SOS-related genes confer salt tolerance in yeast

Ecology of Pollen Storage in Honey Bees: Sugar Tolerant Yeast and the Aerobic Social Microbiota

Honey bee colonies are resource rich and densely populated, generating a constant battle to control microbial growth. Honey is relatively sterile in comparison with beebread: a food storage medium comprising pollen mixed with honey and worker head-gland secretions. Within colonies, the microbes that dominate aerobic niches are abundant throughout social resource space including stored pollen, honey, royal jelly, and the anterior gut segments and mouthparts of both queens and workers. Here, we identify and discuss the microbial load in stored pollen associated with non-Nosema fungi (primarily yeast) and bacteria. We also measured abiotic changes associated with pollen storage and used culturing and qPCR of both fungi and bacteria to investigate changes in stored pollen microbiology by both storage time and season. Over the first week of pollen storage, pH and water availability decreased significantly. Following an initial drop in microbial abundance at day one, both yeasts and bacteria multiply rapidly during day two. Both types of microbes then decline at 3-7 days, but the highly osmotolerant yeasts persist longer than the bacteria. Based on measures of absolute abundance, bacteria and yeast are controlled by similar factors during pollen storage. This work contributes to our understanding of host-microbial interactions in the honey bee gut and colony and the effect of pollen storage on microbial growth, nutrition, and bee health.

Metallochaperone OsHIPP9 is involved in the retention of cadmium and copper in rice.

Metallochaperones are a unique class of proteins that play crucial roles in metal homoeostasis and detoxification. However, few metallochaperones have been functionally characterised in rice. Heterologous expression of Heavy metal-associated Isoprenylated Plant Protein 9 (OsHIPP9), a metallochaperone, altered yeast tolerance to cadmium (Cd) and copper (Cu). We investigated the physiological role of OsHIPP9 in rice. OsHIPP9 was primarily expressed in the root exodermis and xylem region of enlarged vascular bundles (EVB) at nodes. KO of OsHIPP9 increased the Cd concentrations of the upper nodes and panicle, but decreased Cd in expanded leaves. KO of OsHIPP9 decreased Cu uptake and accumulation in rice. Constitutive OX of OsHIPP9 increased Cd and Cu accumulation in aboveground tissues and brown rice. OsHIPP9 showed binding capacity for Cd and Cu. We propose that OsHIPP9 has dual metallochaperone roles, chelating Cd in the xylem region of EVB for Cd retention in the nodes and chelating Cu in rice roots to aid Cu uptake.

Tung tree stearoyl-acyl carrier protein Δ9 desaturase improves oil content and cold resistance of Arabidopsis and Saccharomyces cerevisiae.

The seed oil of tung tree is rich in a-eleostearic acid (ESA), which endows tung oil with the characteristic of an excellently dry oil. The stearoyl-acyl carrier protein δ9 desaturase (SAD) is a rate-limiting enzyme that converts the stearic acid to the oleic acid, the substrate for the production of the α-ESA. However, the function of the two predicted VfSAD1 and VfSAD2 genes in the tung tree has not been determined. In this study, quantitative real-time PCR (qRT-PCR) analysis showed that VfSAD1 and VfSAD2 were expressed in multiple organs of tung tree but were highly expressed in the seed during the oil rapid accumulation period. Heterologous expression of VfSAD1 and VfSAD2 could promote the production of oleic acid and its derivatives in Arabidopsis thaliana and yeast BY4741, indicating that VfSAD1 and VfSAD2 possess the stearoyl-ACP desaturases function. Furthermore, both VfSAD1 and VfSAD2 could significantly improve seed oil accumulation in Arabidopsis. VfSAD1 could also significantly promote the oil accumulation in the yeast BY4741 strain. In addition, overexpression of VfSAD1 and VfSAD2 enhanced the tolerance of yeast and Arabidopsis seedlings to low temperature stress. This study indicates that the two VfSAD genes play a vital role in the process of oil accumulation and fatty acid biosynthesis in the tung tree seed, and both of them could be used for molecular breeding in tung tree and other oil crops.

Xylose consumption and ethanol production by Pichia guilliermondii and Candida oleophila in the presence of furans, phenolic compounds, and organic acids commonly produced during the pre-treatment of plant biomass.

For 2G ethanol production, pentose fermentation and yeast tolerance to lignocellulosic hydrolyzate components are essential to improve biorefinery yields. Generally, physicochemical pre-treatment methodologies are used to facilitate access to cellulose and hemicellulose in plant material, which consequently can generate microbial growth inhibitory compounds, such as furans, weak acids, and phenolic compounds. Because of the unsatisfactory yield of wild-type Saccharomyces cerevisiae during pentose fermentation, the search for xylose-fermenting yeasts tolerant to microbial growth inhibitors has gained attention. In this study, we investigated the ability of the yeasts Pichia guilliermondii G1.2 and Candida oleophila G10.1 to produce ethanol from xylose and tolerate the inhibitors furfural, 5-hydroxymethylfurfural (HMF), acetic acid, formic acid, ferulic acid, and vanillin. We demonstrated that both yeasts were able to grow and consume xylose in the presence of all single inhibitors, with greater growth limitation in media containing furfural, acetic acid, and vanillin. In saline medium containing a mixture of these inhibitors (2.5-3.5 mM furfural and HMF, 1 mM ferulic acid, 1-1.5 mM vanillin, 10-13 mM acetic acid, and 5-7 mM formic acid), both yeasts were able to produce ethanol from xylose, similar to that detected in the control medium (without inhibitors). In future studies, the proteins involved in the transport of pentose and tolerance to these inhibitors need to be investigated.

The Na+/H+ antiporter GbSOS1 interacts with SIP5 and regulates salt tolerance in Gossypium barbadense

Cotton is a globally cultivated economic crop and is a major source of natural fiber and edible oil. However, cotton production is severely affected by salt stress. Although Salt Overly Sensitive 1 (SOS1) is a well-studied Na+/H+ antiporter in multiple plant species, little is known about its function and regulatory mechanism in cotton. Here, we cloned a salt-induced SOS1 from sea-island cotton. Real-time quantitative PCR analysis revealed that GbSOS1 was induced by multiple stresses and phytohormones. Silencing GbSOS1 through virus-induced gene silencing significantly reduced cotton resistance to high Na+ but mildly affected Li+ tolerance. On the other hand, overexpression of GbSOS1 enhanced salt tolerance in yeast, Arabidopsis, and cotton largely due to the ability to maintain Na+ homeostasis in protoplasts. Yeast-two-hybrid assays and bimolecular fluorescence complementation identified a novel protein interacting with GbSOS1 on the plasma membrane, which we named SOS Interaction Protein 5 (SIP5). We found that the SIP5 gene encoded an unknown protein localized on the cell membrane. Silencing SIP5 significantly increased cotton tolerance to salt, exhibited by less wilting and plant death under salt stress. Our results revealed that GbSOS1 is crucial for cotton survival in saline soil, and SIP5 is a potentially negative regulator of SOS1-mediated salt tolerance in cotton. Overall, this study provides a theoretical basis for elucidating the molecular mechanism of SOS1, and a candidate gene for breeding salt-tolerant crops.

Lactiplantibacillus argentoratensis and Candida tropicalis Isolated from the Gastrointestinal Tract of Fish Exhibited Inhibitory Effects against Pathogenic Bacteria of Nile Tilapia

Nile tilapia is one of the most consumed farmed fish in the world. The outbreak of pathogenic bacterial diseases causes high mortality rates and economic losses in Nile tilapia farming. Antibiotic administrations are commonly utilized to inhibit and prevent bacterial infections. However, antibiotics are expensive and cause serious concerns for antibiotic resistance in fish that can be potentially transferred to humans. As an alternative solution, probiotics can be used to prevent infection of pathogenic bacteria in fish. In this work, both bacteria and yeast were isolated from fish gastrointestinal tracts and their inhibitory activity against Nile tilapia pathogenic bacteria was evaluated, as well as other probiotic properties. In this study, 66 bacteria and 176 acid tolerant yeasts were isolated from fish gastrointestinal tracts. Of all isolated microorganisms, 39 bacterial and 15 yeast isolates with inhibitory effect against pathogens were then examined for their probiotic properties (acidic and bile salt resistance, adhesion potential, and biofilm formation), formation of antibacterial factor survival rate under simulated gastrointestinal fluid, and safety evaluation. AT8/5 bacterial isolate demonstrated probiotic properties and the highest inhibition against all 54 tested pathogens while YON3/2 yeast isolate outperformed the inhibitory effect among all yeast isolates. These two probiotic isolates were further identified by 16S rDNA and the D1/D2 domain of 26S rDNA sequence analysis for bacterial and yeast identification, respectively. AT8/5 and YON3/2 showed the highest similarity to Lactiplantibacillus argentoratensis and Candida tropicalis, respectively. This is the first report on isolated L. argentoratensis and C. tropicalis with antipathogenic bacteria of Nile tilapia properties. Collectively, AT8/5 and YON3/2 could be potentially used as promising alternatives to existing antibiotic methods to prevent pathogenic bacteria infection in Nile tilapia farming.

Gene Profiling of the Ascorbate Oxidase Family Genes under Osmotic and Cold Stress Reveals the Role of AnAO5 in Cold Adaptation in Ammopiptanthus nanus.

The uplift of the Qinghai Tibet Plateau has led to a drastic change in the climate in Central Asia, from warm and rainy, to dry and less rainfall. Ammopiptanthus nanus, a rare evergreen broad-leaved shrub distributed in the temperate desert region of Central Asia, has survived the drastic climate change in Central Asia caused by the uplift of the Qinghai-Tibet Plateau. Ascorbate oxidase (AO) regulates the redox status of the apoplast by catalyzing the oxidation of ascorbate acid to dehydroascorbic acid, and plays a key role in the adaptation of plants to environmental changes. Analyzing the evolution, environmental response, and biological functions of the AO family of A. nanus is helpful for understanding how plant genome evolution responds to climate change in Central Asia. A total of 16 AOs were identified in A. nanus, all of which contained the ascorbate oxidase domain, most of which contained transmembrane domain, and many were predicted to be localized in the apoplast. Segmental duplication and tandem duplication are the main factors driving the gene amplification of the AO gene family in A. nanus. Gene expression analysis based on transcriptome data and fluorescence quantitative PCR, as well as enzyme activity measurements, showed that the expression levels of AO genes and total enzyme activity decreased under short-term osmotic stress and low-temperature stress, but the expression of some AO genes (AnAO5, AnAO13, and AnAO16) and total enzyme activity increased under 7 days of cold stress. AnAO5 and AnAO11 are targeted by miR4415. Further functional studies on AnAO5 showed that AnAO5 protein was localized in the apoplast. The expression of AnAO5 in yeast cells and the transient expression in tobacco enhanced the tolerance of yeast and tobacco to low-temperature stress, and the overexpression of AnAO5 enhanced the tolerance of Arabidopsis seedlings to cold stress. Our research provides important data for understanding the role of AOs in plant adaptation to environmental change.

Dephosphorylation of CatC at Ser-18 improves salt and oxidative tolerance via promoting its tetramerization in rice

Catalase (CAT) is a vital antioxidant enzyme, while phosphorylation pivotally regulates its function. Many phosphosites have been identified in CAT, but their functions remained largely elusive. We functionally studied five phosphoserines (Ser-9, −10, −11, −18, and −205) of CatC in rice (Oryza sativa L.). Phospho-Ser-9 and − 11 and dephospho-Ser-18 promoted the enzymatic activity of CatC and enhanced oxidative and salt tolerance in yeast. Phosphorylation status of Ser-18 did not affect CatC peroxisomal targeting and stability, but dephospho-Ser-18 promoted CatC tetramerization to enhance its activity. Moreover, overexpression of dephospho-mimic form CatCS18A in rice significantly improved the tolerance to salt and oxidative stresses by inhibiting the H2O2 accumulation. Together, these results elucidate the mechanism underlying dephosphorylation at Ser-18 promotes CatC activity and salt tolerance in rice. Ser-18 is a promising candidate phosphosite of CatC for breeding highly salt-tolerant rice.

Association studies of genes in a Pb response-associated network in maize (Zea mays L.) reveal that ZmPIP2;5 is involved in Pb tolerance

Lead (Pb) in the soil affects the growth and development of plants and causes damages to the human body through the food chain. Here, we identified and cloned a Pb-tolerance gene ZmPIP2;5 based on a weighted gene co-expression network analysis and gene-based association studies. We showed that ZmPIP2;5 encodes a plasma membrane aquaporin and positively regulated Pb tolerance and accumulation in Arabidopsis and yeast. Overexpression of ZmPIP2;5 increased root length and fresh weight of Arabidopsis seedlings under Pb stress. Heterologous expression of ZmPIP2;5 in yeast caused the enhanced growth speed under Pb treatment and Pb accumulation in yeast cells. A (T/A) SNP in the ZmPIP2;5 promoter affected the expression abundance of ZmPIP2;5 and thereby led to the difference in Pb tolerance among different maize lines. Our study helps to understand the mechanism underlying plant tolerance to Pb stress and provides new ideas for breeding Pb-tolerance maize varieties via molecular marker-assisted selection.

Analysis of the chloroplast crotonylome of wheat seedling leaves reveals the roles of crotonylated proteins involved in salt-stress responses.

Lysine crotonylation (Kcr) is a novel post-translational modification and its function in plant salt-stress responses remains unclear. In this study, we performed the first comprehensive chloroplast crotonylome analysis of wheat seedling leaves to examine the potential functions of Kcr proteins in salt-stress responses. In a total of 471 chloroplast proteins, 1290 Kcr sites were identified as significantly regulated by salt stress, and the Kcr proteins were mainly involved in photosynthesis, protein folding, and ATP synthesis. The identified Kcr sites that responded to salt stress were concentrated within KcrK and KcrF motifs, with the conserved KcrF motif being identified in the Kcr proteins of wheat chloroplasts for the first time. Notably, 10 Kcr sites were identified in fructose-1,6-bisphosphate aldolase (TaFBA6), a key chloroplast metabolic enzyme involved in the Calvin-Benson cycle. Site-directed mutagenesis of TaFBA6 showed that the Kcr at K367 is critical in maintaining its enzymatic activity and in conferring salt tolerance in yeast. Further molecular dynamic simulations and analyses of surface electrostatic potential indicated that the Kcr at K367 could improve the structural stability of TaFBA6 by decreasing the distribution of positive charges on the protein surface to resist alkaline environments, thereby promoting both the activity of TaFBA6 and salt tolerance.

Molecular identification of yeasts from Turkish traditional cheeses: Extracellular enzyme activities and physiological properties important for dairy industry

Abstract. Gunay M, Genc TT. 2023. Molecular identification of yeasts from Turkish traditional cheeses: Extracellular enzyme activities and physiological properties important for dairy industry. Nusantara Bioscience 15: 1-11. The determination of yeast microbiota in cheeses and the physiological properties of yeasts are very important for the dairy industry. In addition, the physiological features, proteolytic and lipolytic activities, and stress tolerance of yeasts have a significant role in the selection of starter yeast species for cheese ripening. This study aimed to determine industrially important yeasts isolated from cheese samples. Molecular techniques identified the isolated yeast strains. The yeast strains’ extracellular enzyme activities, fermentation capacities, and thermotolerance and osmotolerance properties were also evaluated. A total of 81 yeast strains were isolated and characterized from three types of cheese samples. PCR-RFLP determined the isolated yeast strains and sequence analysis of ITS1-5.8S-ITS2 and 26S rDNA regions. A maximum parsimony tree was constructed by MEGA X software to evaluate the phylogenetic relationship of identified yeast strains. Candida intermedia, Candida parapsilosis, Clavispora lusitaniae, Debaryomyces hansenii, Kluyveromyces marxianus, Pichia kudriavzevii, and Wickerhamomyces anomalus yeast species were identified on cheese samples. The distribution of identified yeast species on cheese samples was determined as 48.1% for W. anomalus, 17.3% for K. marxianus, 14.8% for C. parapsilosis, 8.6% for D. hansenii, 4.9% for Cl. lusitaniae, 3.7% for C. intermedia and 2.5% for P. kudriavzevii. The W. anomalus yeast species was common in three cheese types. All strains of W. anomalus and P. kudriavzevii yeast species, three C. parapsilosis, and two Cl. lusitaniae yeast strains have important physiological properties for industrial applications. These yeast strains have the potential to be used in combination as starter cultures to improve cheese maturation in the future. This comprehensive study identifies yeast species by ITS1-5.8S-ITS2 and 26S rDNA regions and determines industrially important yeast species using multiple criteria (extracellular enzyme activity, stress tolerance, and fermentation capacity).