CsAGA1 and CsAGA2 Mediate RFO Hydrolysis in Partially Distinct Manner in Cucumber Fruits

A Raffinose family oligosaccharides (RFOs) is one of the major translocated sugars in the vascular bundle of cucumber, but little RFOs can be detected in fruits. Alpha-galactosidases (α-Gals) catalyze the first catabolism step of RFOs. Six α-Gal genes exist in a cucumber genome, but their spatial functions in fruits remain unclear. Here, we found that RFOs were highly accumulated in vascular tissues. In phloem sap, the stachyose and raffinose content was gradually decreased, whereas the content of sucrose, glucose and fructose was increased from pedicel to fruit top. Three alkaline forms instead of acid forms of α-Gals were preferentially expressed in fruit vascular tissues and alkaline forms have stronger RFO-hydrolysing activity than acid forms. By inducible gene silencing of three alkaline forms of α-Gals, stachyose was highly accumulated in RNAi-CsAGA2 plants, while raffinose and stachyose were highly accumulated in RNAi-CsAGA1 plants. The content of sucrose, glucose and fructose was decreased in both RNAi-CsAGA1 and RNAi-CsAGA2 plants after β-estradiol treatment. In addition, the fresh- and dry-weight of fruits were significantly decreased in RNAi-CsAGA1 and RNAi-CsAGA2 plants. In cucurbitaceous plants, the non-sweet motif within the promoter of ClAGA2 is widely distributed in the promoter of its homologous genes. Taken together, we found RFOs hydrolysis occurred in the vascular tissues of fruits. CsAGA1 and CsAGA2 played key but partly distinct roles in the hydrolysis of RFOs.

TORC1 signaling modulates Cdk8-dependent GAL gene expression in Saccharomyces cerevisiae

Cdk8 of the RNA Polymerase II mediator kinase complex regulates gene expression by phosphorylating sequence-specific transcription factors. This function is conserved amongst eukaryotes, but the signals and mechanisms regulating Cdk8 activity and phosphorylation of its substrates are unknown. Full induction of the GAL genes in yeast requires phosphorylation of the transcriptional activator Gal4 by Cdk8. We used a screen to identify regulators of the Cdk8-dependent phosphorylation on Gal4, from which we identified multiple mutants with defects in TORC1 signaling. One mutant, designated gal four throttle 1 (gft1) was identified as a recessive allele of hom3, encoding aspartokinase, and mutations in hom3 caused effects typical of inhibition of TORC1, including rapamycin sensitivity and enhanced nuclear localization of the TORC1-responsive transcription factor Gat1. Mutations in hom3 also inhibit phosphorylation of Gal4 in vivo at the Cdk8-dependent site on Gal4, as did mutations of tor1, but these mutations did not affect activity of Cdk8 assayed in vitro. Disruption of cdc55, encoding a regulatory subunit of the TORC1-regulated protein phosphatase PP2A, suppressed the effect of hom3 and tor1 mutations on GAL expression, and also restored phosphorylation of Gal4 at the Cdk8-dependent site in vivo. These observations demonstrate that TORC1 signaling regulates GAL induction through the activity of PP2A/Cdc55, and suggest that Cdk8-dependent phosphorylation of Gal4 is opposed by PP2A/Cdc55 dephosphorylation. These results provide insight into how induction of transcription by a specific inducer can be modulated by global nutritional signals through regulation of Cdk8-dependent phosphorylation.

A glimpse at an early stage of microbe domestication revealed in the variable genome of Torulaspora delbrueckii, an emergent industrial yeast

The yeast Torulaspora delbrueckii is gaining importance for biotechnology due to its ability to increase wine sensorial complexity and for enhancing pre-frozen bread dough leavening. However, little is known about its population structure, variation in gene content, or possible domestication routes. Here, we address these issues and update the delimitation of T. delbrueckii along five major clades. Among the three European clades, a basal lineage is associated with the wild arboreal niche, while the two other lineages are linked with anthropic environments, one to wine fermentations and the other to diverse sources including dairy products and bread dough (Mix- Anthropic clade). Using 62 genomes we identified 5629 genes in the pangenome of T. delbrueckii and 270 genes in the cloud genome. A pangenome tree analysis showed that wine strains have a genome composition more similar to European wild arboreal strains than to those of the Mix Anthropic clade, in contradiction with the phylogenetic analysis. An association of gene content and ecology gave further support to the hypothesis that the Mix - Anthropic clade has the most specialized genome content and indicated that some of the exclusive genes were implicated in galactose and maltose utilization. More detailed analyses traced the acquisition of a cluster of GAL genes in strains associated with dairy products and the expansion and functional diversification of MAL genes in strains isolated from bread dough. Contrary to S. cerevisiae, domestication in T. delbrueckii is not primed by alcoholic fermentation and appears to be a recent event.

Chromosomal localization patterns of beta-galactosidase genes in streptomycetes

Aim. The aim is to identify trends of localization of genes encoding beta-galactosidase (β-gal genes) in the genomes of streptomycetes. Methods. Genetic maps of 94 strains of streptomycetes from the NCBI server database were analyzed. Results. Molecular sizes of genomes from the strains sample were from 6.84 Mbp up to 12.7 Mbp. Most of genomes (43.7 %) in the sample have molecular sizes 7.0 Mbp – 8.5 Mbp. As determined, the number of β-gal genes on genetic maps ranged from 0 to 13. 61.3 % of the strains contained 1 - 3 β-gal genes in their genomes. It has been established that these genes are located in the genomes of streptomycetes, as a rule, in terminal regions. 79 % of the studied β-gal genes had a molecular size from 1.5 kb. up to 2.1 kb. Conclusions. The following patterns of localization of β-gal genes in the genomes of streptomycetes were established: 1) Tendency of localization of the overwhelming majority of β-gal genes in the terminal regions of streptomycete genomes; 2) Trend has been established for the localization in the core region of individual β-gal genes, both in genomes of large molecular size and containing more than 8 genes; 3) The streptomycetes genomes contain the β-gal genes mainly of the size from 1.5 kb up to 2.1 kb regardless of molecular sizes of their genomes and the number of β-gal genes in them. Keywords: β-gal gene, genome, Streptomyces, molecular size.

The mRNA degradation factor Xrn1 regulates transcription elongation in parallel to Ccr4

Co-transcriptional imprinting of mRNA by Rpb4 and Rpb7 subunits of RNA polymerase II (RNAPII) and by the Ccr4–Not complex conditions its post-transcriptional fate. In turn, mRNA degradation factors like Xrn1 are able to influence RNAPII-dependent transcription, making a feedback loop that contributes to mRNA homeostasis. In this work, we have used repressible yeast GAL genes to perform accurate measurements of transcription and mRNA degradation in a set of mutants. This genetic analysis uncovered a link from mRNA decay to transcription elongation. We combined this experimental approach with computational multi-agent modelling and tested different possibilities of Xrn1 and Ccr4 action in gene transcription. This double strategy brought us to conclude that both Xrn1–decaysome and Ccr4–Not regulate RNAPII elongation, and that they do it in parallel. We validated this conclusion measuring TFIIS genome-wide recruitment to elongating RNAPII. We found that xrn1Δ and ccr4Δ exhibited very different patterns of TFIIS versus RNAPII occupancy, which confirmed their distinct role in controlling transcription elongation. We also found that the relative influence of Xrn1 and Ccr4 is different in the genes encoding ribosomal proteins as compared to the rest of the genome.

Polymorphisms in the yeast galactose sensor underlie a natural continuum of nutrient-decision phenotypes

In nature, microbes often need to “decide” which of several available nutrients to utilize, a choice that depends on a cell’s inherent preference and external nutrient levels. While natural environments can have mixtures of different nutrients, phenotypic variation in microbes’ decisions of which nutrient to utilize is poorly studied. Here, we quantified differences in the concentration of glucose and galactose required to induce galactose-responsive (GAL) genes across 36 wildS. cerevisiaestrains. Using bulk segregant analysis, we found that a locus containing the galactose sensorGAL3was associated with differences in GAL signaling in eight different crosses. Using allele replacements, we confirmed thatGAL3is the major driver of GAL induction variation, and thatGAL3allelic variation alone can explain as much as 90% of the variation in GAL induction in a cross. TheGAL3variants we found modulate the diauxic lag, a selectable trait. These results suggest that ecological constraints on the galactose pathway may have led to variation in a single protein, allowing cells to quantitatively tune their response to nutrient changes in the environment.Author summaryIn nature, microbes often need to decide which of many potential nutrients to consume. This decision making process is complex, involving both intracellular constraints and the organism’s perception of the environment. To begin to mimic the complexity of natural environments, we grew cells in mixtures of two sugars, glucose and galactose. We find that in mixed environments, the sugar concentration at which cells decides to induce galactose-utilizing (GAL) genes is highly variable in natural isolates of yeast. By analyzing crosses of phenotypically different strains, we identified a locus containing the galactose sensor, a gene that in theory could allow cells to tune their perception of the environment. We confirmed that the galactose sensor can explain upwards of 90% of the variation in the decision to induce GAL genes. Finally, we show that the variation in the galactose sensor can modulate the time required for cells to switch from utilizing glucose to galactose. Our results suggest that signaling pathways can be highly variable across strains and thereby might allow for rapid adaption in fluctuating environments.

Subnuclear positioning and interchromosomal clustering of the GAL1-10 locus are controlled by separable, interdependent mechanisms

On activation, the GAL genes in yeast are targeted to the nuclear periphery through interaction with the nuclear pore complex. Here we identify two cis-acting “DNA zip codes” from the GAL1-10 promoter that are necessary and sufficient to induce repositioning to the nuclear periphery. One of these zip codes, GRS4, is also necessary and sufficient to promote clustering of GAL1-10 alleles. GRS4, and to a lesser extent GRS5, contribute to stronger expression of GAL1 and GAL10 by increasing the fraction of cells that respond to the inducer. The molecular mechanism controlling targeting to the NPC is distinct from the molecular mechanism controlling interchromosomal clustering. Targeting to the nuclear periphery and interaction with the nuclear pore complex are prerequisites for gene clustering. However, once formed, clustering can be maintained in the nucleoplasm, requires distinct nuclear pore proteins, and is regulated differently through the cell cycle. In addition, whereas targeting of genes to the NPC is independent of transcription, interchromosomal clustering requires transcription. These results argue that zip code–dependent gene positioning at the nuclear periphery and interchromosomal clustering represent interdependent phenomena with distinct molecular mechanisms.