参与木葡聚糖合成的糖基转移酶基因研究进展

半纤维素多糖木葡聚糖(XyG)存在于大多数植物的初生细胞壁中, 对细胞壁的结构组织和生长发育具有重要的调控作用。XyG在植物进化中存在结构的多样性。该文概述了参与XyG合成的糖基转移酶的最新研究进展, XyG合成需要多种糖基转移酶参与, 这些酶类很可能以蛋白酶复合体的形式存在并发挥作用, XyG的结构和组成的改变对植物的生长发育也产生影响。     

与木葡聚糖合成的糖基转移酶基因研究进展

半纤维素多糖木葡聚糖(XyG)存在于大多数植物的初生细胞壁中, 对细胞壁的结构组织和生长发育具有重要的调控作用。XyG在植物进化中存在结构的多样性。该文概述了参与XyG合成的糖基转移酶的最新研究进展, XyG合成需要多种糖基转移酶参与, 这些酶类很可能以蛋白酶复合体的形式存在并发挥作用, XyG的结构和组成的改变对植物的生长发育也产生影响。     

Xyloglucan Xylosyltransferases XXT1, XXT2, and XXT5 and the Glucan Synthase CSLC4 Form Golgi-Localized Multiprotein Complexes

Xyloglucan is the major hemicellulosic polysaccharide in the primary cell walls of most vascular dicotyledonous plants and has important structural and physiological functions in plant growth and development. In Arabidopsis (Arabidopsis thaliana), the 1,4-β-glucan synthase, Cellulose Synthase-Like C4 (CSLC4), and three xylosyltransferases, XXT1, XXT2, and XXT5, act in the Golgi to form the xylosylated glucan backbone during xyloglucan biosynthesis. However, the functional organization of these enzymes in the Golgi membrane is currently unknown. In this study, we used bimolecular fluorescence complementation and in vitro pull-down assays to investigate the supramolecular organization of the CSLC4, XXT1, XXT2, and XXT5 proteins in Arabidopsis protoplasts. Quantification of bimolecular fluorescence complementation fluorescence by flow cytometry allowed us to perform competition assays that demonstrated the high probability of protein-protein complex formation in vivo and revealed differences in the abilities of these proteins to form multiprotein complexes. Results of in vitro pull-down assays using recombinant proteins confirmed that the physical interactions among XXTs occur through their catalytic domains. Additionally, coimmunoprecipitation of XXT2YFP and XXT5HA proteins from Arabidopsis protoplasts indicated that while the formation of the XXT2-XXT2 homocomplex involves disulfide bonds, the formation of the XXT2-XXT5 heterocomplex does not involve covalent interactions. The combined data allow us to propose that the proteins involved in xyloglucan biosynthesis function in a multiprotein complex composed of at least two homocomplexes, CSLC4-CSLC4 and XXT2-XXT2, and three heterocomplexes, XXT2-XXT5, XXT1-XXT2, and XXT5-CSLC4.     

Xyloglucan Deficiency Disrupts Microtubule Stability and Cellulose Biosynthesis in Arabidopsis,Altering Cell Growth and Morphogenesis

Xyloglucan constitutes most of the hemicellulose in eudicot primary cell walls and functions in cell wall structure and mechanics. Although Arabidopsis (Arabidopsis thaliana) xxt1 xxt2 mutants lacking detectable xyloglucan are viable, they display growth defects that are suggestive of alterations in wall integrity. To probe the mechanisms underlying these defects, we analyzed cellulose arrangement, microtubule patterning and dynamics, microtubule- and wall-integrity-related gene expression, and cellulose biosynthesis in xxt1 xxt2 plants. We found that cellulose is highly aligned in xxt1 xxt2 cell walls, that its three-dimensional distribution is altered, and that microtubule patterning and stability are aberrant in etiolated xxt1 xxt2 hypocotyls. We also found that the expression levels of microtubule-associated genes, such as MAP70-5 and CLASP, and receptor genes, such as HERK1 and WAK1, were changed in xxt1 xxt2 plants and that cellulose synthase motility is reduced in xxt1 xxt2 cells, corresponding with a reduction in cellulose content. Our results indicate that loss of xyloglucan affects both the stability of the microtubule cytoskeleton and the production and patterning of cellulose in primary cell walls. These findings establish, to our knowledge, new links between wall integrity, cytoskeletal dynamics, and wall synthesis in the regulation of plant morphogenesis.The primary walls of growing plant cells are largely constructed of cellulose and noncellulosic matrix polysaccharides that include hemicelluloses and pectins (Carpita and Gibeaut, 1993; Somerville et al., 2004; Cosgrove, 2005). Xyloglucan (XyG) is the most abundant hemicellulose in the primary walls of eudicots and is composed of a β-1,4-glucan backbone with side chains containing Xyl, Gal, and Fuc (Park and Cosgrove, 2015). XyG is synthesized in the Golgi apparatus before being secreted to the apoplast, and its biosynthesis requires several glycosyltransferases, including β-1,4-glucosyltransferase, α-1,6-xylosyltransferase, β-1,2-galactosyltransferase, and α-1,2-fucosyltransferase activities (Zabotina, 2012). Arabidopsis (Arabidopsis thaliana) XYLOGLUCAN XYLOSYLTRANSFERASE1 (XXT1) and XXT2 display xylosyltransferase activity in vitro (Faik et al., 2002; Cavalier and Keegstra, 2006), and strikingly, no XyG is detectable in the walls of xxt1 xxt2 double mutants (Cavalier et al., 2008; Park and Cosgrove, 2012a), suggesting that the activity of XXT1 and XXT2 are required for XyG synthesis, delivery, and/or stability.Much attention has been paid to the interactions between cellulose and XyG over the past 40 years. Currently, there are several hypotheses concerning the nature of these interactions (Park and Cosgrove, 2015). One possibility is that XyGs bind directly to cellulose microfibrils (CMFs). Recent data indicating that crystalline cellulose cores are surrounded with hemicelluloses support this hypothesis (Dick-Pérez et al., 2011). It is also possible that XyG acts as a spacer-molecule to prevent CMFs from aggregating in cell walls (Anderson et al., 2010) or as an adapter to link cellulose with other cell wall components, such as pectin (Cosgrove, 2005; Cavalier et al., 2008). XyG can be covalently linked to pectin (Thompson and Fry, 2000; Popper and Fry, 2005, 2008), and NMR data demonstrate that pectins and cellulose might interact to a greater extent than XyG and cellulose in native walls (Dick-Pérez et al., 2011). Alternative models exist for how XyG-cellulose interactions influence primary wall architecture and mechanics. One such model posits that XyG chains act as load-bearing tethers that bind to CMFs in primary cell walls to form a cellulose-XyG network (Carpita and Gibeaut, 1993; Pauly et al., 1999; Somerville et al., 2004; Cosgrove, 2005). However, results have been accumulating against this tethered network model, leading to an alternative model in which CMFs make direct contact, in some cases mediated by a monolayer of xyloglucan, at limited cell wall sites dubbed “biomechanical hotspots,” which are envisioned as the key sites of cell wall loosening during cell growth (Park and Cosgrove, 2012a; Wang et al., 2013; Park and Cosgrove, 2015). Further molecular, biochemical, and microscopy experiments are required to help distinguish which aspects of the load-bearing, spacer/plasticizer, and/or hotspot models most accurately describe the functions of XyG in primary walls.Cortical microtubules (MTs) direct CMF deposition by guiding cellulose synthase complexes in the plasma membrane (Baskin et al., 2004; Paredez et al., 2006; Emons et al., 2007; Sánchez-Rodriguez et al., 2012), and the patterned deposition of cellulose in the wall in turn can help determine plant cell anisotropic growth and morphogenesis (Baskin, 2005). Disruption of cortical MTs by oryzalin, a MT-depolymerizing drug, alters the alignment of CMFs, suggesting that MTs contribute to CMF organization (Baskin et al., 2004). CELLULOSE SYNTHASE (CESA) genes, including CESA1, CESA3, and CESA6, are required for normal CMF synthesis in primary cell walls (Kohorn et al., 2006; Desprez et al., 2007), and accessory proteins such as COBRA function in cellulose production (Lally et al., 2001). Live-cell imaging from double-labeled YFP-CESA6; CFP-ALPHA-1 TUBULIN (TUA1) Arabidopsis seedlings provides direct evidence that cortical MTs determine the trajectories of cellulose synthesis complexes (CSCs) and patterns of cellulose deposition (Paredez et al., 2006). Additionally, MT organization affects the rotation of cellulose synthase trajectories in the epidermal cells of Arabidopsis hypocotyls (Chan et al., 2010). Recently, additional evidence for direct guidance of CSCs by MTs has been provided by the identification of CSI1/POM2, which binds to both MTs and CESAs (Bringmann et al., 2012; Li et al., 2012). MICROTUBULE ORGANIZATION1 (MOR1) is essential for cortical MT organization (Whittington et al., 2001), but disruption of cortical MTs in the mor1 mutant does not greatly affect CMF organization (Sugimoto et al., 2003), and oryzalin treatment does not abolish CSC motility (Paredez et al., 2006).Conversely, the organization of cortical MTs can be affected by cellulose synthesis. Treatment with isoxaben, a cellulose synthesis inhibitor, results in disorganized cortical MTs in tobacco cells, suggesting that inhibition of cellulose synthesis affects MT organization (Fisher and Cyr, 1998), and treatment with 2,6-dichlorobenzonitrile, another cellulose synthesis inhibitor, alters MT organization in mor1 plants (Himmelspach et al., 2003). Cortical MT orientation in Arabidopsis roots is also altered in two cellulose synthesis-deficient mutants, CESA6^52-isx and kor1-3, suggesting that CSC activity can affect MT arrays (Paredez et al., 2008). Together, these results point to a bidirectional relationship between cellulose synthesis/patterning and MT organization.MTs influence plant organ morphology, but the detailed mechanisms by which they do so are incompletely understood. The dynamics and stability of cortical MTs are also affected by MT-associated proteins (MAPs). MAP18 is a MT destabilizing protein that depolymerizes MTs (Wang et al., 2007), MAP65-1 functions as a MT crosslinker, and MAP70-1 functions in MT assembly (Korolev et al., 2005; Lucas et al., 2011). MAP70-5 stabilizes existing MTs to maintain their length, and its overexpression induces right-handed helical growth (Korolev et al., 2007); likewise, MAP20 overexpression results in helical cell twisting (Rajangam et al., 2008). CLASP promotes microtubule stability, and its mutant is hypersensitive to microtubule-destabilizing drug oryzalin (Ambrose et al., 2007). KATANIN1 (KTN1) is a MT-severing protein that can sever MTs into short fragments and promote the formation of thick MT bundles that ultimately depolymerize (Stoppin-Mellet et al., 2006), and loss of KTN1 function results in reduced responses to mechanical stress (Uyttewaal et al., 2012). In general, cortical MT orientation responds to mechanical signals and can be altered by applying force directly to the shoot apical meristem (Hamant et al., 2008). The application of external mechanical pressure to Arabidopsis leaves also triggers MT bundling (Jacques et al., 2013). Kinesins, including KINESIN-13A (KIN-13A) and FRAGILE FIBER1 (FRA1), have been implicated in cell wall synthesis (Cheung and Wu, 2011; Fujikura et al., 2014). The identification of cell wall receptors and sensors is beginning to reveal how plant cell walls sense and respond to external signals (Humphrey et al., 2007; Ringli, 2010); some of them, such as FEI1, FEI2, THESEUS1 (THE1), FERONIA (FER), HERCULES RECEPTOR KINASE1 (HERK1), WALL ASSOCIATED KINASE1 (WAK1), WAK2, and WAK4, have been characterized (Lally et al., 2001; Decreux and Messiaen, 2005; Kohorn et al., 2006; Xu et al., 2008; Guo et al., 2009; Cheung and Wu, 2011). However, the relationships between wall integrity, cytoskeletal dynamics, and wall synthesis have not yet been fully elucidated.In this study, we analyzed CMF patterning, MT patterning and dynamics, and cellulose biosynthesis in the Arabidopsis xxt1 xxt2 double mutant that lacks detectable XyG and displays altered growth (Cavalier et al., 2008; Park and Cosgrove, 2012a). To investigate whether and how XyG deficiency affects the organization of CMFs and cortical MTs, we observed CMF patterning in xxt1 xxt2 mutants and Col (wild-type) controls using atomic force microscopy (AFM), field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), and confocal microscopy (Hodick and Kutschera, 1992; Derbyshire et al., 2007; Anderson et al., 2010; Zhang et al., 2014). We also generated transgenic Col and xxt1 xxt2 lines expressing GFP-MAP4 (Marc et al., 1998) and GFP-CESA3 (Desprez et al., 2007), and analyzed MT arrays and cellulose synthesis using live-cell imaging. Our results show that the organization of CMFs is altered, that MTs in xxt1 xxt2 mutants are aberrantly organized and are more sensitive to external mechanical pressure and the MT-depolymerizing drug oryzalin, and that cellulose synthase motility and cellulose content are decreased in xxt1 xxt2 mutants. Furthermore, real-time quantitative RT-PCR measurements indicate that the enhanced sensitivity of cortical MTs to mechanical stress and oryzalin in xxt1 xxt2 plants might be due to altered expression of MT-stabilizing and wall receptor genes. Together, these data provide insights into the connections between the functions of XyG in wall assembly, the mechanical integrity of the cell wall, cytoskeleton-mediated cellular responses to deficiencies in wall biosynthesis, and cell and tissue morphogenesis.     

Multiple Genes for the Last Step of Proline Biosynthesis in Bacillus subtilis

The complete Bacillus subtilis genome contains four genes (proG, proH, proI, and comER) with the potential to encode Delta(1)-pyrroline-5-carboxylate reductase, a proline biosynthetic enzyme. Simultaneous defects in three of these genes (proG, proH, and proI) were required to confer proline auxotrophy, indicating that the products of these genes are mostly interchangeable with respect to the last step in proline biosynthesis.     

Processed Pseudogenes, Processed Genes, and Spontaneous Mutations in the Arabidopsis Genome

We identified 411 processed sequences in the Arabidopsis thaliana genome based on the fact that they have lost their intron(s) and have a length that is at least 95% of the length of the gene that gave rise to them. These sequences were generated by 230 different genes and clearly originated from retrotranspositons events because most of them (91%) have a poly(A)-tail. They are composed of 376 sequences with frame shifts and/or premature stop codons (processed pseudogenes) and 35 sequences without disablements (processed genes). Eleven of these processed genes are likely functional retrotransposed genes because they have low Ka/Ks ratios and high Ks values, and their sequences match numerous Arabidopsis ESTs. Processed sequences are mostly randomly distributed in the Arabidopsis genome and their rate of accumulation has steadily been decreasing since it peaked some 50 MYA. In contrast with the situation observed in mammals, the processed sequences found in the Arabidopsis genome originate from genes with high copy numbers and not from highly expressed genes. The patterns of spontaneous mutations in Arabidopsis are slightly different than those of mammals but are similar to those observed in Drosophila. This suggests that methylated cytosine deamination is less frequent in Arabidopsis than in mammals. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Juergen Brosius]     

Xyloglucan Endotransglucosylase-Hydrolase17 Interacts with Xyloglucan Endotransglucosylase-Hydrolase31 to Confer Xyloglucan Endotransglucosylase Action and Affect Aluminum Sensitivity in Arabidopsis

Previously, we reported that although the Arabidopsis (Arabidopsis thaliana) Xyloglucan Endotransglucosylase-Hydrolase31 (XTH31) has predominately xyloglucan endohydrolase activity in vitro, loss of XTH31 results in remarkably reduced in vivo xyloglucan endotransglucosylase (XET) action and enhanced Al resistance. Here, we report that XTH17, predicted to have XET activity, binds XTH31 in yeast (Saccharomyces cerevisiae) two-hybrid and coimmunoprecipitations assays and that this interaction may be required for XTH17 XET activity in planta. XTH17 and XTH31 may be colocalized in plant cells because tagged XTH17 fusion proteins, like XTH31 fusion proteins, appear to target to the plasma membrane. XTH17 expression, like that of XTH31, was substantially reduced in the presence of aluminum (Al), even at concentrations as low as 10 µm for 24 h or 25 µm for just 30 min. Agrobacterium tumefaciens-mediated transfer DNA insertion mutant of XTH17, xth17, showed low XET action and had moderately shorter roots than the wild type but was more Al resistant than the wild type. Similar to xth31, xth17 had low hemicellulose content and retained less Al in the cell wall. These data suggest a model whereby XTH17 and XTH31 may exist as a dimer at the plasma membrane to confer in vivo XET action, which modulates cell wall Al-binding capacity and thereby affects Al sensitivity in Arabidopsis.Soil acidity (pH < 5.5) affects about 40% of the world’s arable land (von Uexküll and Mutert, 1995) and more than 50% of land that is potentially arable, particularly in the tropics and subtropics (Kochian et al., 2004; Eticha et al., 2010). Al is the most growth-limiting factor for crop production on acid soils worldwide (Foy, 1988; Kochian, 1995), especially when the pH drops below 5 (Eswaran et al., 1997).To survive in an Al-toxic environment, Al-resistant plants have evolved two mechanisms to cope with Al toxicity. One is to restrict Al uptake from the root, while the other is to cope with internalized Al (Taylor, 1991; Kochian et al., 2004). The latter is usually employed by Al-accumulating species such as Hydrangea macrophylla (Ma et al., 1997a) and buckwheat (Fagopyrum esculentum; Ma et al., 1997b). In most cases, secretion of the organic acid anions is the most important Al exclusion mechanism (Kochian, 1995; Ryan et al., 2001; Ma and Furukawa, 2003), although it does not explain all the Al resistance in some plants such as signalgrass (Brachiaria decumbens Stapf cv Basilisk; Wenzl et al., 2001), maize (Zea mays; Piñeros et al., 2005), buckwheat (Zheng et al., 2005), rice (Oryza sativa; Ma et al., 2005; Yang et al., 2008), or Fagopyrum tataricum (Yang et al., 2011a). Therefore, it is possible that for some plant species (such as buckwheat), Al tolerance is a combination of mechanisms including organic anion efflux.Recently, evidence has accumulated that the cell wall, especially the hemicellulose component, may impact Al resistance. For example, Al induces significant changes in the hemicellulose fraction of wheat (Triticum aestivum; Tabuchi and Matsumoto, 2001), triticale (× Triticosecale Wittmack; Liu et al., 2008), rice (Yang et al., 2008), and Arabidopsis (Arabidopsis thaliana; Zhu et al., 2012), especially the Al-sensitive cultivars. Moreover, we found that Arabidopsis hemicellulose is not only very sensitive to Al stress (the content of hemicellulose increased quickly under Al stress), but is also the principal binding site for Al (Yang et al., 2011b). Furthermore, loss of Xyloglucan Endotransglucosylase-Hydrolase31 (XTH31) function resulted in lower xyloglucan content and cell wall Al-binding capacity in Arabidopsis (Zhu et al., 2012). Thus, xyloglucan may be a major Al-binding site in Arabidopsis, and any effects leading to xyloglucan modifications may regulate Al sensitivity.XTHs are a family of enzymes that play principal roles in the construction and restructuring of the load-bearing cross links among cellulose microfibrils (Osato et al., 2006) through catalyzing the molecular grafting or hydrolyzing of the xyloglucans to form the framework (Fry et al., 1992; Nishitani and Tominaga, 1992; Okazawa et al., 1993; Rose et al., 2002). There are 33 identified XTH genes in the Arabidopsis genome, and one-third occur as clusters resulting from genome duplication (Blanc et al., 2000; Yokoyama and Nishitani, 2001); XTH1-11 are classified in subgroup 1, XTH12-26 are in subgroup 2, and XTH27-33 are in subgroup 3 (Rose et al., 2002). Each member of the XTH gene family is likely regulated by specific cues and committed to cell wall dynamics specific to certain tissues or cell types (Nishitani, 2002; Becnel et al., 2006; Osato et al., 2006). For example, XTH27 is involved in the cell wall modification of tracheary elements at a specific stage of rosette leaf development and is essential for tertiary vein development (Matsui et al., 2005), whereas XTH31 is involved in cell wall modification and cell elongation through modulating xyloglucan endotransglucosylase (XET) action under Al stress (Zhu et al., 2012). However, XTH31 is an XTH for which xyloglucan endohydrolase (XEH) activity has been predicted (Baumann et al., 2007), and in our previous report, we demonstrated that XTH31 produced heterologously in Pichia pastoris has high XEH activity but low XET activity in vitro (Zhu et al., 2012), which is in accordance with Kaewthai et al. (2013), who reported that XTH31 is a predominant hydrolase using the in vitro activity assays and enzyme product analysis, as well as the use of a fluorogenic substrate in vivo. Unexpectedly, however, the xth31 mutant has very low XET action and activity (Zhu et al., 2012). One possible explanation for this result is that XTH31 may interact with and be required for activity of XET-active XTHs.In this study, we demonstrate that XTH17 can bind to XTH31 in vitro and in vivo and that a transfer DNA (T-DNA) insertional mutant of XTH17 has elevated Al resistance and exhibits a phenotype very similar to xth31. Together, these data are consistent with the interpretation that XTH17 and XTH31 may interact with each other and thereby contribute to Al-inhibited XET action in Arabidopsis.     

Biosynthesis of Xyloglucan in Suspension-Cultured Soybean Cells. Synthesis of Xyloglucan from UDP-Glucose and UDP-Xylose in the Cell-Free System

When UDP-[¹⁴C]glucose or UDP-[¹⁴C]xylose was incubated witha particulate fraction from soybean cells, radioactive polymerswere synthesized. On digestion with Aspergillus oryzae enzymes,these polymers gave ¹⁴C-monosaccharides and a ¹⁴C-disaccharidewith chromatographic and electrophoretic mobilities indistinguishablefrom those of authentic isoprimeverose (6-O--D-xylopyranosyl-D-glucopyranose).The disaccharide consisted of xylose and glucose, and the latterwas located at the reducing end. Evidence that the disaccharideis isoprimeverose was provided by methylation analysis. Hydrolysisof the methylated disaccharide yielded 2,3,4-tri-O-methyl-D-xyloseand 2,3,4-tri-O-methyl-D-glucose. Thus, incorporation of radioactivityinto isoprimeverose, the smallest structural unit of xyloglucan,suggests that xyloglucan is synthesized in vitro from UDP-glucoseand UDP-xylose. (Received November 20, 1980; Accepted February 14, 1981)     

Multiple Changes of Gene Expression and Function Reveal Genomic and Phenotypic Complexity in SLE-like Disease

The complexity of clinical manifestations commonly observed in autoimmune disorders poses a major challenge to genetic studies of such diseases. Systemic lupus erythematosus (SLE) affects humans as well as other mammals, and is characterized by the presence of antinuclear antibodies (ANA) in patients’ sera and multiple disparate clinical features. Here we present evidence that particular sub-phenotypes of canine SLE-related disease, based on homogenous (ANA^H) and speckled ANA (ANA^S) staining pattern, and also steroid-responsive meningitis-arteritis (SRMA) are associated with different but overlapping sets of genes. In addition to association to certain MHC alleles and haplotypes, we identified 11 genes (WFDC3, HOMER2, VRK1, PTPN3, WHAMM, BANK1, AP3B2, DAPP1, LAMTOR3, DDIT4L and PPP3CA) located on five chromosomes that contain multiple risk haplotypes correlated with gene expression and disease sub-phenotypes in an intricate manner. Intriguingly, the association of BANK1 with both human and canine SLE appears to lead to similar changes in gene expression levels in both species. Our results suggest that molecular definition may help unravel the mechanisms of different clinical features common between and specific to various autoimmune disease phenotypes in dogs and humans.     

Overexpression of Multiple Dehydrin Genes Enhances Tolerance to Freezing Stress in Arabidopsis

To elucidate the contribution of dehydrins (DHNs) to freezing stress tolerance in Arabidopsis, transgenic plants overexpressing multiple DHN genes were generated. Chimeric double constructs for expression of RAB18 and COR47 (pTP9) or LTI29 and LTI30 (pTP10) were made by fusing the coding sequences of the respective DHN genes to the cauliflower mosaic virus 35S promoter. Overexpression of the chimeric genes in Arabidopsis resulted in accumulation of the corresponding dehydrins to levels similar or higher than in cold-acclimated wild-type plants. Transgenic plants exhibited lower LT50 values and improved survival when exposed to freezing stress compared to the control plants. Post-embedding immuno electron microscopy of high-pressure frozen, freeze-substituted samples revealed partial intracellular translocation from cytosol to the vicinity of the membranes of the acidic dehydrin LTI29 during cold acclimation in transgenic plants. This study provides evidence that dehydrins contribute to freezing stress tolerance in plants and suggests that this could be partly due to their protective effect on membranes.     

Multiple New Genes That Determine Activity for the First Step of Leucine Biosynthesis in Saccharomyces Cerevisiae

The first step in the biosynthesis of leucine is catalyzed by α-isopropylmalate (α-IPM) synthase. In the yeast Saccharomyces cerevisiae, LEU4 encodes the isozyme responsible for the majority of α-IPM synthase activity. Yeast strains that bear disruption alleles of LEU4, however, are Leu(+) and exhibit a level of synthase activity that is 20% of the wild type. To identify the gene or genes that encode this remaining activity, a leu4 disruption strain was mutagenized. The mutations identified define three new complementation groups, designated leu6, leu7 and leu8. Each of these new mutations effect leucine auxotrophy only if a leu4 mutation is present and each results in loss of α-IPM synthase activity. Further analysis suggests that LEU7 and LEU8 are candidates for the gene or genes that encode an α-IPM synthase activity. The results demonstrate that multiple components determine the residual α-IPM synthase activity in leu4 gene disruption strains of S. cerevisiae.     

Gene and Enhancer Trap Transposable Elements Reveal Oxygen Deprivation-regulated Genes and their Complex Patterns of Expression in Arabidopsis

Transposon tagging with modified maize Ds–GUS constructswas used to isolate genes induced by oxygen deprivation in Arabidopsisthaliana. Seedlings of 800 gene-trap (DsG) and 600 enhancer-trap(DsE) lines were grown on vertically positioned plates for 1 week,oxygen deprived for up to 24 h and stained for GUS activity.Oxygen deprivation induced intricate patterns of gene expressionin seedlings of 65 lines. The insertion site and phenotypesof 15 lines were examined. Surprisingly, none of the insertionswere into genes that encode known anaerobic polypeptides. Insertionswere identified within or adjacent to genes encoding proteinsof regulatory, enzymatic, mitochondrial protein import and unknownfunction, as well as adjacent to genes encoding a putative receptor-likekinase and putative sensor-histidine kinase. Four lines hadsignificantly lower ADH activity after 24 h of oxygen deprivationand three of these showed reduced stress tolerance. Two lineswith wild-type levels of ADH were low-oxygen intolerant. Paradoxically,several lines had significantly higher ADH activity after 12 hof oxygen deprivation but reduced stress tolerance. Caffeinetreatment, which increased ADH specific activity in wild-typeseedlings under aerobic conditions, was sufficient to increaseGUS staining in seven of the 15 lines, providing evidence thatthese genes may be regulated by cytosolic calcium levels. Theseresults demonstrate the effectiveness of the Ds–GUS taggingsystem in the identification of genes that are regulated inresponse to oxygen deprivation and a calcium second messenger.