Miguel A. Blázquez

Trehalose-6-phosphate, a new regulator of yeast glycolysis that inhibits hexokinases

Trehalose‐6‐phosphate (P) competitively inhibited the hexokinases from Saccharomyces cerevisiae. The strongest inhibition was observed upon hexokinase II, with a K i, of 40 μM, while in the case of hexokinase I the K i was 200 μM. Glucokinase was not inhibited by trehalose‐6‐P up to 5 mM. This inhibition appears to have physiological significance, since the intracellular levels of trehalose‐6‐P were about 0.2 mM. Hexokinases from other organisms were also inhibited, while glucokinases were unaffected. The hexokinase from the yeast, Yarrowia lipolytica, was particularly sensitive to the inhibition by trehalose‐6‐P: when assayed with 2 mM fructose an apparent K i, of 5 μM was calculated. Two S. cerevisiae mutants with abnormal levels of trehalose‐6‐P exhibited defects in glucose metabolism. It is concluded that trehalose‐6‐P plays an important role in the regulation of the first steps of yeast glycolysis, mainly through the inhibition of hexokinase II.

Polarization of PIN3-dependent auxin transport for hypocotyl gravitropic response in Arabidopsis thaliana

Gravitropism aligns plant growth with gravity. It involves gravity perception and the asymmetric distribution of the phytohormone auxin. Here we provide insights into the mechanism for hypocotyl gravitropic growth. We show that the Arabidopsis thaliana PIN3 auxin transporter is required for the asymmetric auxin distribution for the gravitropic response. Gravistimulation polarizes PIN3 to the bottom side of hypocotyl endodermal cells, which correlates with an increased auxin response at the lower hypocotyl side. Both PIN3 polarization and hypocotyl bending require the activity of the trafficking regulator GNOM and the protein kinase PINOID. Our data suggest that gravity-induced PIN3 polarization diverts the auxin flow to mediate the asymmetric distribution of auxin for gravitropic shoot bending.

Role of polyamines in plant vascular development.

Several pieces of evidence suggest a role for polyamines in the regulation of plant vascular development. For instance, polyamine oxidase gene expression has been shown to be associated with lignification, and downregulation of S-adenosylmethionine decarboxylase causes dwarfism and enlargement of the vasculature. Recent evidence from Arabidopsis thaliana also suggests that the active polyamine in the regulation of vascular development is the tetraamine thermospermine. Thermospermine biosynthesis is catalyzed by the aminopropyl transferase encoded by ACAULIS5, which is specifically expressed in xylem vessel elements. Both genetic and molecular evidence support a fundamental role for thermospermine in preventing premature maturation and death of the xylem vessel elements. This safeguard action of thermospermine has significant impact on xylem cell morphology, cell wall patterning and cell death as well as on plant growth in general. This manuscript reviews recent reports on polyamine function and places polyamines in the context of the known regulatory mechanisms that govern vascular development.

Hierarchy of hormone action controlling apical hook development in Arabidopsis

The apical hook develops in the upper part of the hypocotyl when seeds buried in the soil germinate, and serves to protect cotyledons and the shoot apical meristem from possible damage caused by pushing through the soil. The curvature is formed through differential cell growth that occurs at the two opposite sides of the hypocotyl, and it is established by a gradient of auxin activity and refined by the coordinated action of auxin and ethylene. Here we show that gibberellins (GAs) promote hook development through the transcriptional regulation of several genes of the ethylene and auxin pathways in Arabidopsis. The level of GA activity determines the speed of hook formation and the extent of the curvature during the formation phase independently of ethylene, probably by modulating auxin transport and response through HLS1, PIN3, and PIN7. Moreover, GAs cooperate with ethylene in preventing hook opening, in part through the induction of ethylene production mediated by ACS5/ETO2 and ACS8.

Large-Scale Identification of Gibberellin-Related Transcription Factors Defines Group VII ETHYLENE RESPONSE FACTORS as Functional DELLA Partners

Transcription factors of the APETALA2 superfamily are regulated by DELLAs which represents a cross regulatory node for gibberellins and ethylene to control apical hook opening. DELLA proteins are the master negative regulators in gibberellin (GA) signaling acting in the nucleus as transcriptional regulators. The current view of DELLA action indicates that their activity relies on the physical interaction with transcription factors (TFs). Therefore, the identification of TFs through which DELLAs regulate GA responses is key to understanding these responses from a mechanistic point of view. Here, we have determined the TF interactome of the Arabidopsis (Arabidopsis thaliana) DELLA protein GIBBERELLIN INSENSITIVE and screened a collection of conditional TF overexpressors in search of those that alter GA sensitivity. As a result, we have found RELATED TO APETALA2.3, an ethylene-induced TF belonging to the group VII ETHYLENE RESPONSE FACTOR of the APETALA2/ethylene responsive element binding protein superfamily, as a DELLA interactor with physiological relevance in the context of apical hook development. The combination of transactivation assays and chromatin immunoprecipitation indicates that the interaction with GIBBERELLIN INSENSITIVE impairs the activity of RELATED TO APETALA2.3 on the target promoters. This mechanism represents a unique node in the cross regulation between the GA and ethylene signaling pathways controlling differential growth during apical hook development.

Differential growth at the apical hook: all roads lead to auxin

The apical hook is a developmentally regulated structure that appears in dicotyledonous seedlings when seeds germinate buried in the soil. It protects the shoot apical meristem and cotyledons from damage while the seedling is pushing upwards seeking for light, and it is formed by differential cell expansion between both sides of the upper part of the hypocotyl. Its apparent simplicity and the fact that it is dispensable when seedlings are grown in vitro have converted the apical hook in one of the favorite experimental models to study the regulation of differential growth. The involvement of hormones –especially auxin—in this process was manifested already in the early studies. Remarkably, a gradient of this hormone across the hook curvature is instrumental to complete its development, similar to what has been proposed for other processes involving the bending of an organ, such as tropic responses. In agreement with this, other hormones—mainly gibberellins and ethylene—and the light, regulate in a timely and interconnected manner the auxin gradient to promote hook development and its opening, respectively. Here, we review the latest findings obtained mainly with the apical hook of Arabidopsis thaliana, paying special attention to the molecular mechanisms for the cross-regulation between the different hormone signaling pathways that underlie this developmental process.

Genomic analysis of DELLA protein activity.

Changes in gene expression are the main outcome of hormone signaling cascades that widely control plant physiology. In the case of the hormones gibberellins, the transcriptional control is exerted through the activity of the DELLA proteins, which act as negative regulators in the signaling pathway. This review focuses on recent transcriptomic approaches in the context of gibberellin signaling, which have provided useful information on new processes regulated by these hormones such as the regulation of photosynthesis and gravitropism. Moreover, the enrichment of specific cis-elements among DELLA primary targets has also helped extend the view that DELLA proteins regulate gene expression through the interaction with multiple transcription factors from different families.

SCHIZOSACCHAROMYCES POMBE POSSESSES AN UNUSUAL AND A CONVENTIONAL HEXOKINASE : BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF BOTH HEXOKINASES

Two hexokinases were characterized in Schizosaccharomyces pombe: hexokinase 1, with a low phosphorylation coefficient on glucose (Km 8.5 mM) and hexokinase 2, a kinetically conventional hexokinase. Genes hxk1 + and hxk2 + encoding these enzymes were cloned and sequenced. Disruption of hxk1 + had no effect on growth but disruption of hxk2 + doubled the generation time in glucose. Spores carrying the double disruption hxk1 + hxk2 + did not grow on glucose or fructose after one week. Expression of hxk1 + increased strongly during growth in fructose or glycerol. Expression of hxk2 + was highest during growth in glycerol. A NADP‐dependent glucose dehydrogenase was detected, but not a glucokinase.

A mutation affecting carbon catabolite repression suppresses growth defects in pyruvate carboxylase mutants from Saccharomyces cerevisiae

Yeasts with disruptions in the genes PYC1 and PYC2 encoding the isoenzymes of pyruvate carboxylase cannot grow in a glucose‐ammonium medium (Stucka et al. (1991) Mol. Gen. Genet. 229, 307–315). We have isolated a dominant mutation, BPC1‐1, that allows growth in this medium of yeasts with interrupted PYC1 and PYC2 genes. The BPC1‐1 mutation abolishes catabolite repression of a series of genes and allows expression of the enzymes of the glyoxylate cycle during growth in glucose. A functional glyoxylate cyle is necessary for suppression as a disruption of gene ICL1 encoding isocitrate lyase abolished the phenotypic effect of BPC1‐1 on growth in glucose‐ammonium. Concurrent expression from constitutive promoters of genes ICL1 and MLS1 (encoding malate synthase) also suppressed the growth phenotype of pyc1 pyc2 mutants. The mutation BPC1‐1 is either allelic or closely linked to the mutation DGT1‐1.

Identification of extragenic suppressors of the cif1 mutation in Saccharomyces cerevisiae

The cif1 mutation of Saccharomyces cerevisiae causes inability to grow on glucose and related fermentable carbon sources. We have isolated two different suppressor mutations that allow growth on glucose of yeasts carrying the cif1 mutation. One of them, sci1-1, is recessive and caused inability to grow on non-fermentable carbon sources and to de-repress fructose-1,6-bisphosphatase. The other suppressor mutation, SCI2-1, is dominant and diminished the capacity to phosphorylate glucose or fructose. The SCI2-1 mutation decreased sporulation efficiency by 70% in heterozygosis and by more than 90% in homozygosis. In a CIF1 background, cells carrying the mutation SCI2-1 accumulated trehalose during the logarithmic phase of growth and hyperaccumulated it during the stationary phase. Genetic tests showed that SCI2 was either allelic, or else closely linked, to HXK2. The concentrations of the glycolytic metabolites measured during growth on glucose in cells carrying the cif1 mutation and any of the suppressor mutations were similar to those of a wild-type. Both types of suppressor mutations restored the transient cAMP response to glucose to cif1 mutants.