Keith D. Richards

A homozygous diploid subset of commercial wine yeast strains

Genetic analysis was performed on 45 commercial yeasts which are used in winemaking because of their superior fermentation properties. Genome sizes were estimated by propidium iodide fluorescence and flow cytometry. Forty strains had genome sizes consistent with their being diploid, while five had a range of aneuploid genome sizes that ranged from 1.2 to 1.8 times larger. The diploid strains are all Saccharomyces cerevisiae, based on genetic analysis of microsatellite and minisatellite markers and on DNA sequence analysis of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA of four strains. Four of the five aneuploid strains appeared to be interspecific hybrids between Saccharomyces kudriavzevii and Saccharomyces cerevisiae, with the fifth a hybrid between two S. cerevisiae strains. An identification fingerprint was constructed for the commercial yeast strains using 17 molecular markers. These included six published trinucleotide microsatellites, seven new dinucleotide microsatellites, and four published minisatellite markers. The markers provided unambiguous identification of the majority of strains; however, several had identical or similar patterns, and likely represent the same strain or mutants derived from it. The combined use of all 17 polymorphic loci allowed us to identify a set of eleven commercial wine yeast strains that appear to be genetically homozygous. These strains are presumed to have undergone inbreeding to maintain their homozygosity, a process referred to previously as ‘genome renewal’.

Cloning and Expression of a Wheat (Triticum aestivumL.) Phosphatidylserine Synthase cDNA OVEREXPRESSION IN PLANTS ALTERS THE COMPOSITION OF PHOSPHOLIPIDS

We describe the cloning of a wheat cDNA (TaPSS1) that encodes a phosphatidylserine synthase (PSS) and provides the first strong evidence for the existence of this enzyme in a higher eukaryotic cell. The cDNA was isolated on its ability to confer increased resistance to aluminum toxicity when expressed in yeast. The sequence of the predicted protein encoded byTaPSS1 shows homology to PSS from both yeast and bacteria but is distinct from the animal PSS enzymes that catalyze base-exchange reactions. In wheat, Southern blot analysis identified the presence of a small family of genes that cross-hybridized to TaPSS1,and Northern blots showed that aluminum induced TaPSS1expression in root apices. Expression of TaPSS1complemented the yeast cho1 mutant that lacks PSS activity and altered the phospholipid composition of wild type yeast, with the most marked effect being increased abundance of phosphatidylserine (PS). Arabidopsis thaliana leaves overexpressingTaPSS1 showed a marked enhancement in PSS activity, which was associated with increased biosynthesis of PS at the expense of both phosphatidylinositol and phosphatidylglycerol. Unlike mammalian cells where PS accumulation is tightly regulated even when the capacity for PS biosynthesis is increased, plant cells accumulated large amounts of PS when TaPSS1 was overexpressed. High levels ofTaPSS1 expression in Arabidopsis and tobacco (Nicotiana tabacum) led to the appearance of necrotic lesions on leaves, which may have resulted from the excessive accumulation of PS. The cloning of TaPSS1 now provides evidence that the yeast pathway for PS synthesis exists in some plant tissues and provides a tool for understanding the pathways of phospholipid biosynthesis and their regulation in plants.

The yeast IRC7 gene encodes a β-lyase responsible for production of the varietal thiol 4-mercapto-4-methylpentan-2-one in wine.

Three varietal thiols are key aroma compounds in Sauvignon Blanc wines: 4-mercapto-4-methylpentan-2-one (4MMP), 3-mercaptohexanol (3MH) and its acetylated derivative 3-mercaptohexyl acetate (3MHA). Screening of Saccharomyces cerevisiae strains identified a clinical isolate with elevated 4MMP production after fermentation. Bulked Segregant Analysis of a cross between this isolate and the laboratory strain revealed a single major locus for 4MMP production near the telomere of chromosome 6. Deletion of the IRC7 gene from this region in YJM450 reduced 4MMP production below detectable levels, but did not affect yields of 3MH, in Sauvignon Blanc wine. Sequencing revealed that the IRC7 gene in YJM450 had been introgressed from a strain of Saccharomyces paradoxus. Most strains of S. cerevisiae, including the laboratory strain S288C, have a 38-bp deletion that inactivates IRC7. Overexpression of a full-length S. cerevisiae allele of IRC7 in a wine yeast, Zymaflore F15, increased 4MMP production in Sauvignon Blanc wine from undetectable levels (<10 ng L(-1)) to concentrations of 1000 ng L(-1), and also increased 3MH and 3MHA. Biochemical analysis of soluble protein extracts showed that both the cerevisiae and paradoxus IRC7 proteins show β-lyase activity, with a substrate preference for cys-4MMP over cys-3MH.

A database of microsatellite genotypes for Saccharomyces cerevisiae

A system for genotyping Saccharomyces cerevisiae is described based on a multiplex of ten microsatellite loci and the MAT locus. A database of genotypes has been developed for 246 yeast strains, including a large set of commercial wine yeasts, as well as 35 sequenced natural isolates currently being sequenced. The latter allow us, for the first time, to make direct comparisons of the relationship between DNA sequence data and microsatellite-based genotypes. The genotyping system provides a rapid and valuable system for strain identification as well as studying population genetics of S. cerevisiae.

A new yeast genetic resource for analysis and breeding.

We made a library of Saccharomyces cerevisiae F1 hybrids from all possible crosses of 16 wild‐type strains, including two common laboratory strains and two commercial winemaking varieties. Fourteen of the starting strains have been sequenced. Thus, the sequences of both genomes are known in 182 novel hybrids, and the sequence of one genome is known in 56. All tested strains sporulated. Fertilities were in the range 0–100%. Hybrids showed no more variation than parental strains for ethanol production, ethanol tolerance or growth at temperature extremes, but some F1s appeared to display hybrid vigour (heterosis). We tested four tetrads from one hybrid for their ability to grow at low temperature or in the presence of an inhibitory concentration of ethanol. Only one F2 was as tolerant as the most tolerant F0 parent. A few showed intermediate tolerance, but most were less tolerant than either parent or the F1 hybrid, consistent with uncoupling of genes contributing to an optimized quantitative trait. The diversity and structure of the library should make it useful for analysis of genetic interactions among diverse strains, quantitative inheritance and heterosis, and for breeding. Copyright

Genetic characterization of strains of Saccharomyces uvarum from New Zealand wineries

We present a genetic characterization of 65 isolates of Saccharomyces uvarum isolated from wineries in New Zealand, along with the complete nucleotide sequence of a single sulfite-tolerant isolate. The genome of the New Zealand isolate averaged 99.85% nucleotide identity to CBS7001, the previously sequenced strain of S. uvarum. However, three genomic segments (37-87 kb) showed 10% nucleotide divergence from CBS7001 but 99% identity to Saccharomyces eubayanus. We conclude that these three segments appear to have been introgressed from that species. The nucleotide sequence of the internal transcribed spacer (ITS) region from other New Zealand isolates were also very similar to that of CBS7001, and hybrids showed complete genetic compatibility for some strains, with tetrads giving four viable progeny that showed 2:2 segregations of marker genes. Some strains showed high tolerance to sulfite, with genetic analysis indicating linkage of this trait to the transcription factor FZF1, but not to SSU1, the sulfite efflux pump that it regulates in order to confer sulfite tolerance in Saccharomyces cerevisiae. The fermentation characteristics of selected strains of S. uvarum showed exceptionally good cold fermentation characteristics, superior to the best commercially available strains of S. cerevisiae.

Protein profiles in root-tips of two wheat (Triticum aestivum L.) cultivars with differential tolerance to aluminium

The molecular basis for differential aluminium tolerance of two wheat cultivars was investigated using two-dimensional gel electrophoresis of root-tip proteins labelled in vivo. Five proteins were specific to the tolerant cultivar in the absence of Al. The same five were induced in response to Al in both cultivars.

The effect of aluminium treatment on wheat roots: expression of heat shock, histone and SHH genes

Abstract The molecular mechanism by which aluminium inhibits cell division in plant roots under acidic conditions is not understood. We have used Northern hybridisations to monitor changes in gene expression following Al treatment of two wheat cultivars: Warigal, an Al-sensitive variety, and Waalt, an AAl-tolerant variety. Steady state transcript levels of two heat shock genes, hsp17 and hsp70, were not increased in roots by the Al treatment. Levels of histone H3 and H4 transcripts were reduced by long-term exposure to inhibitory Al treatments in both wheat cultivars, consistent with prevous reports that histone genes are expressed primarily in dividing cells. In addition, we have isolated a wheat gene for S- adenosyl - l - homocysteine hydrolase (SHH), and demonstrated that its mRNA is downregulated by long-term exposure to inhibitory levels of Al.

Magnesium Transport and Aluminium Tolerance

Aluminium toxicity is a major factor limiting crop productivity internationally. Under acidic soil conditions, Al is converted from insoluble forms into soluble Al. The ion is toxic to plants and causes an immediate cessation of root growth. The mechanism of this inhibition has not been clearly established, despite a plethora of suggestions (reviewed by Kochian, 1995).