We used chloroplast DNA sequences from matK and rbcL to infer the phylogeny for 101 of the approximately 111 species of Pinus (Pinaceae). At the level of subsection and above, the cpDNA tree is congruent with phylogenies based on nuclear DNA with one notable exception: cpDNA sequences from subsect. Contortae are sister to all other North American hard pines rather than occupying a more derived position in the same clade. We used the cpDNA tree plus evidence from nuclear ribosomal DNA and morphology to propose a new classification for the genus. The molecular phylogenies are symmetrical at the deepest branches of the genus, allowing for the delineation of two subgenera, each with two sections that form sister groups. Within sections, clades were slightly asymmetric and sometimes ambiguously resolved. To accomodate ambiguity in some interrelationships, avoid the creation of new ranks, and retain traditional names, we recognised up to three monophyletic subsections per section. Subgenus Pinus (the diploxylon, or hard pines) is divided into the predominantly Eurasian and Mediterranean section Pinus, composed of subsections Pinus and Pinaster, and the strictly North American section Trifoliae, composed of subsections Australes, Ponderosae, and Contortae. Subgenus Strobus (the haploxylon, or soft pines) is divided into the strictly North American section Parrya, composed of subsections Cembroides, Nelsoniae, and Balfourianae, and the Eurasian and North American section Quinquefoliae, composed of subsections Gerardianae, Krempfianae, and Strobus. Mapping of ten morphological and distributional characters indicates that two were diagnostic for infrageneric taxa: the number of vascular bundles per leaf distinguishes subgenus Pinus from subgenus Strobus, and a terminal­positioned umbo on the ovulate cone scale is diagnostic of subsect. Strobus.

Uncertainties in the age and phylogenetic position of Pinaceae fossils present significant obstacles to our understanding of the timing of diversification in the family. We demonstrate that simultaneous phylogenetic analyses of chloroplast DNA (matK and rbcL) and nonmolecular characters that include both extant genera and a limited number of fossil taxa provide useful hypotheses for calibrating molecular trees. Root placements varied for Pinaceae, with Bayesian analyses recovering mutually monophyletic subfamilies Pinoideae and Abietoideae and parsimony analyses recovering Abietoideae as paraphyletic by placing the root between Cedrus and the remaining genera. The inferred phylogenetic positions of fossil taxa Pityostrobus bernissartensis as the sister group to Pinus and Pseudolarix erensis as the sister group to extant Pseudolarix were used to guide divergencetime calibrations; these calibrations yielded an Early Cretaceous and an Early Jurassic age for crown-group Pinaceae, respectively. The older age estimates based on Pseudolarix erensis are supported by weaker evidence from the fossil record but are consistent with recent reports of Early Cretaceous leaf fossils that appear to coincide with extant genera. There remains a great need to characterize the anatomy of extant and fossil species and to code additional nonmolecular characters.

BackgroundGene duplication is a key evolutionary mechanism providing material for the generation of genes with new or modified functions. The fate of duplicated gene copies has been amply discussed and several models have been put forward to account for duplicate conservation. The specialization model considers that duplication of a bifunctional ancestral gene could result in the preservation of both copies through subfunctionalization, resulting in the distribution of the two ancestral functions between the gene duplicates. Here we investigate whether the presumed bifunctional character displayed by the single branched chain amino acid aminotransferase present in K. lactis has been distributed in the two paralogous genes present in S. cerevisiae, and whether this conservation has impacted S. cerevisiae metabolism.Principal FindingsOur results show that the KlBat1 orthologous BCAT is a bifunctional enzyme, which participates in the biosynthesis and catabolism of branched chain aminoacids (BCAAs). This dual role has been distributed in S. cerevisiae Bat1 and Bat2 paralogous proteins, supporting the specialization model posed to explain the evolution of gene duplications. BAT1 is highly expressed under biosynthetic conditions, while BAT2 expression is highest under catabolic conditions. Bat1 and Bat2 differential relocalization has favored their physiological function, since biosynthetic precursors are generated in the mitochondria (Bat1), while catabolic substrates are accumulated in the cytosol (Bat2). Under respiratory conditions, in the presence of ammonium and BCAAs the bat1Δ bat2Δ double mutant shows impaired growth, indicating that Bat1 and Bat2 could play redundant roles. In K. lactis wild type growth is independent of BCAA degradation, since a Klbat1Δ mutant grows under this condition.ConclusionsOur study shows that BAT1 and BAT2 differential expression and subcellular relocalization has resulted in the distribution of the biosynthetic and catabolic roles of the ancestral BCAT in two isozymes improving BCAAs metabolism and constituting an adaptation to facultative metabolism.

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harbors and paralogous genes that encode branched chain aminotransferases and have opposed expression profiles and physiological roles . Accordingly, in primary nitrogen sources such as glutamine, expression is induced, supporting Bat1-dependent valine-isoleucine-leucine (VIL) biosynthesis, while expression is repressed. Conversely, in the presence of VIL as the sole nitrogen source, expression is hindered while that of is activated, resulting in Bat2-dependent VIL catabolism. The presented results confirm that expression is determined by transcriptional activation through the action of the Leu3-α-isopropylmalate (α-IPM) active isoform, and uncovers the existence of a novel α-IPM biosynthetic pathway operating in aΔ mutant grown on VIL, through Bat2-Leu2-Leu1 consecutive action. The classic α-IPM biosynthetic route operates in glutamine through the action of the leucine-sensitive α-IPM synthases. The presented results also show that repression in glutamine can be alleviated in aΔ mutant or through Gcn4-dependent transcriptional activation. Thus, when is grown on glutamine, VIL biosynthesis is predominant and is preferentially achieved through; while on VIL as the sole nitrogen source, catabolism prevails and is mainly afforded by .

Aims:  For this study, we performed a genetic screen of S. cerevisiae’s deletion library for mutants sensitive to dehydration stress, with which we aimed to discover cell dehydration–tolerant genes. Methods and Results:  We used a yeast gene deletion set (YGDS) of 4850 viable mutant haploid strains to perform a genome‐wide screen for the identification of desiccation stress modifiers. SIP18 is among the genes identified as essential for cells surviving to drying/rehydration process. Deletion of SIP18 promotes the accumulation of reactive oxygen species and enhances apoptotic and necrotic cell death in response to dehydration/rehydration process. Conclusions:  SIP18p acts as an inhibitor of apoptosis in yeast under dehydration stress, as suggested by its antioxidative capacity through the ROS accumulation reduction after an H2O2 attack. Significance and Impact of the Study:  To our knowledge, this is the first systematic screen for the identification of putative genes essential to overcoming cell dehydration process. A broad range of identified genes could help to understand why some strains of high biotechnological interest cannot cope with the drying and rehydration treatments. Dehydration sensitivity makes these strains not suitable to be commercialized by yeast manufactures.

Production of ␣-isopropylmalate (␣-IPM) is critical for leucine biosynthesis and for the global control of metabolism. The budding yeast Saccharomyces cerevisiae has two paralogous genes, LEU4 and LEU9, that encode ␣-IPM synthase (␣-IPMS) isozymes.Little is known about the biochemical differences between these two ␣-IPMS isoenzymes. Here, we show that the Leu4 homodimer is a leucine-sensitive isoform, while the Leu9 homodimer is resistant to such feedback inhibition. The leu4⌬ mutant, which expresses only the feedback-resistant Leu9 homodimer, grows slowly with either glucose or ethanol and accumulates elevated pools of leucine; this phenotype is alleviated by the addition of leucine. Transformation of the leu4⌬ mutant with a centromeric plasmid carrying LEU4 restored the wild-type phenotype. Bimolecular fluorescent complementation analysis showed that Leu4-Leu9 heterodimeric isozymes are formed in vivo. Purification and kinetic analysis showed that the hetero-oligomeric isozyme has a distinct leucine sensitivity behavior. Determination of ␣-IPMS activity in ethanol-grown cultures showed that ␣-IPM biosynthesis and growth under these respiratory conditions depend on the feedback-sensitive Leu4 homodimer. We conclude that retention and further diversification of two yeast ␣-IPMSs have resulted in a specific regulatory system that controls the leucine-␣-IPM biosynthetic pathway by selective feedback sensitivity of homomeric and heterodimeric isoforms. The Leu4 and Leu9 ␣-isopropylmalate synthases (␣-IPMSs), paralogous isozymes from Saccharomyces cerevisiae, catalyze the first committed step of leucine biosynthesis: the synthesis of ␣-isopropylmalate (␣-IPM) from acetyl coenzyme A (acetylCoA) and ␣-ketoisovalerate (␣-KIV). This reaction is carried out in the mitochondria (1-8), and ␣-IPM is then transported from the mitochondria to the cytosol by the yeast oxaloacetate/sulfate carrier Oac1 (9). The concerted action of Leu1 and Leu2 converts ␣-IPM to ␣-ketoisocaproate, the immediate precursor of leucine; these reactions are performed in the cytoplasm (8). The last step in leucine biosynthesis is carried out on both the mitochondria and the cytoplasm through the action of the differentially localized Bat1 or Bat2 aminotransferase (10) (Fig. 1). Most of the ␣-IPMS activity in wild-type S. cerevisiae cells is provided by the mitochondrially localized LEU4-encoded isozyme and not by the Leu4 (Leu4 s) isoform, which naturally lacks the mitochondrial import sequence and is thus localized in the cytosol (1, 2).Leu4 enzymatic activity is inhibited by leucine and CoA, and the amino acid residues responsible for this property have been identified (7). Although no detailed biochemical characterization of the LEU9-encoded isozyme has been performed, it has been shown that it is less sensitive to leucine inhibition than Leu4 is (3).It is noteworthy that the leucine biosynthesis intermediate ␣-IPM plays a dual cellular role. On the one hand, it acts as an intermediate in leucine biosynthesis (5, 6), and on the other, it acts as t...

SummaryIn Saccharomyces cerevisiae, the first committed step in the lysine (Lys) biosynthetic pathway is catalysed by the Lys20 and Lys21 homocitrate synthase (HCS) isoforms. Overexpression of Lys20 resulted in eightfold increased Lys, as well as 2-oxoglutarate pools, which were not attained by overexpressing Lys21 or other pathway enzymes (Lys1, Lys9 or Lys12). A metabolic control analysis-based strategy, by gradually and individually manipulating the Lys20 and Lys21 activities demonstrated that the cooperative and strongly feedback-inhibited Lys21 isoform exerted low control of the pathway flux whereas most of the control resided on the non-cooperative and weakly feedback-inhibited Lys20 isoform. Therefore, the higher control of Lys20 over the Lys flux represents an exception to the dogma of higher pathway control by the strongest feedback-inhibited enzyme and points out to multi-site engineering (HCS isoforms and supply of precursors) to increase Lys synthesis.