A Suite of Lotus japonicus Starch Mutants Reveals Both Conserved and Novel Features of Starch Metabolism

The metabolism of starch is of central importance for many aspects of plant growth and development. Information on leaf starch metabolism other than in Arabidopsis (Arabidopsis thaliana) is scarce. Furthermore, its importance in several agronomically important traits exemplified by legumes remains to be investigated. To address this issue, we have provided detailed information on the genes involved in starch metabolism in Lotus japonicus and have characterized a comprehensive collection of forward and TILLING (for Targeting Induced Local Lesions IN Genomes) reverse genetics mutants affecting five enzymes of starch synthesis and two enzymes of starch degradation. The mutants provide new insights into the structure-function relationships of ADP-glucose pyrophosphorylase and glucan, water dikinase1 in particular. Analyses of the mutant phenotypes indicate that the pathways of leaf starch metabolism in L. japonicus and Arabidopsis are largely conserved. However, the importance of these pathways for plant growth and development differs substantially between the two species. Whereas essentially starchless Arabidopsis plants lacking plastidial phosphoglucomutase grow slowly relative to wild-type plants, the equivalent mutant of L. japonicus grows normally even in a 12-h photoperiod. In contrast, the loss of GLUCAN, WATER DIKINASE1, required for starch degradation, has a far greater effect on plant growth and fertility in L. japonicus than in Arabidopsis. Moreover, we have also identified several mutants likely to be affected in new components or regulators of the pathways of starch metabolism. This suite of mutants provides a substantial new resource for further investigations of the partitioning of carbon and its importance for symbiotic nitrogen fixation, legume seed development, and perenniality and vegetative regrowth.Recent studies in Arabidopsis (Arabidopsis thaliana) have greatly enhanced our knowledge about pathways of transitory starch metabolism (Zeeman et al., 2007; Keeling and Myers, 2010; Kötting et al., 2010; Zeeman et al., 2010). The pathway of synthesis is well established for several species, but the degradative pathway is understood only in Arabidopsis. During synthesis, the plastidial isoforms of phosphoglucoisomerase (PGI1) and phosphoglucomutase (PGM1), together with ADP-Glc pyrophosphorylase (AGPase), catalyze the conversion of the Calvin cycle intermediate Fru 6-P to ADPGlc, the substrate for starch synthases (Supplemental Fig. S1). Leaves of mutants lacking any of these three enzymes either have strongly reduced starch contents or lack starch almost completely (Caspar et al., 1985; Hanson and McHale, 1988; Lin et al., 1988a, 1988b; Kruckeberg et al., 1989; Harrison et al., 1998; Yu et al., 2000; Streb et al., 2009). In contrast, the phenotypes of mutants lacking individual enzymes that convert ADPGlc into starch vary between species and are often much less pronounced (starch synthases [Delvallé et al., 2005; Zhang et al., 2005] and starch-branching enzymes [Tomlinson et al., 1997; Blauth et al., 2001; Dumez et al., 2006]).The degradation of the starch granule in Arabidopsis leaves is catalyzed primarily by β-amylases and isoamylase 3 (Wattebled et al., 2005; Delatte et al., 2006; Fulton et al., 2008). Normal rates of degradation require phosphorylation of the starch polymers by two glucan, water dikinases, GWD1 (Ritte et al., 2002) and GWD3 (or PWD, for phosphoglucan water, dikinase; Baunsgaard et al., 2005; Kötting et al., 2005), followed by dephosphorylation by a phosphoglucan phosphatase, STARCH EXCESS4 (SEX4; Kötting et al., 2009). Maltose produced by starch degradation is exported from the chloroplast by a maltose transporter and further metabolized to hexose phosphates in the cytosol (Zeeman et al., 2007; Supplemental Fig. S1). Mutations in numerous components of this pathway result in a starch-excess phenotype, in which the starch content of leaves at the end of the night is higher than that of wild-type plants.These studies have also revealed the importance of starch turnover for the productivity of the plant. Mutants of Arabidopsis that are essentially unable to synthesize transitory starch, or with reduced rates of starch degradation at night, have a reduced rate of growth and delayed flowering time relative to wild-type plants under most conditions (Caspar et al., 1985, 1991; Eimert et al., 1995; Corbesier et al., 1998; Smith and Stitt, 2007). However, it is not known whether information about the nature and importance of starch turnover in Arabidopsis is widely applicable. Plant species differ considerably in the extent to which starch is stored in leaves at night as well as in diurnal patterns of growth and metabolic demand. The function and regulation of starch metabolism in heterotrophic organs and its importance in major physiological and developmental processes such as perenniality, vegetative regrowth, symbiotic nitrogen fixation, and the accumulation of seed storage reserves cannot be studied easily in Arabidopsis and remain largely unknown. These processes represent traits of agronomic value in legumes (Fabaceae), a family that includes some of the most agriculturally important forage (e.g. alfalfa [Medicago sativa] and clover [Trifolium spp.]), grain (e.g. pea [Pisum sativum] and common bean [Phaseolus vulgaris]), and oilseed (e.g. soybean [Glycine max]) crops.Some information is already available about starch metabolism in pea and other legume crops (Martin and Smith, 1995; Wang et al., 1998b, and refs. therein). However, characteristics including large genome sizes and recalcitrant transformation and regeneration have limited progress on these species. There is insufficient information to allow either an overview of the nature and importance of starch metabolism in legumes or a meaningful comparison with the detailed picture emerging for Arabidopsis. The development of both Lotus japonicus and Medicago truncatula as legume model systems, and the wide range of genetic and genomic resources generated for them, offer the opportunity for a systematic analysis.To elucidate the pathway of starch synthesis and degradation in legumes and provide resources for future experimentation, we screened an ethyl methanesulfonate (EMS)-mutagenized population of L. japonicus (Perry et al., 2003) for mutants altered in transitory starch metabolism and carried out genetic mapping to identify the mutation responsible for their phenotype. We also used TILLING (for Targeting Induced Local Lesions IN Genomes; McCallum et al., 2000) to confirm that the mutations identified were indeed responsible for the mutant phenotype and to obtain additional mutations in genes known to affect leaf starch content in other species. We present the results of molecular and phenotypic analyses on the mutants that provide novel insights into the structure-function relationship of the AGPase and GWD1 enzymes. In addition, our analyses reveal new information on the nature and importance of starch metabolism for plant growth and development in L. japonicus. The importance of starch accumulation and degradation and a comparison with pathways in other plant species are also discussed.     

航天诱变百脉根结瘤突变体的筛选

通过返回式卫星搭载,利用太空环境对百脉根(Lotus japonicus)MG-20种子进行诱变。从种植的三代植株中,筛选到多种共生固氮根瘤的突变体,其中不结根瘤突变体18个株系,表现为接种根瘤菌两周后无根瘤形成;结无效根瘤突变体9个株系,表现为根瘤数目少且分布不均匀,根瘤呈白色,有些为半透明;花叶形态异常突变体1个株系,表现为除根瘤数目少外,植株矮小、托叶消失、花形态异常;纤细突变体1个株系,表现为除根瘤数目少外,植株变小、茎细叶小。     

Correction to: Mutants of Lotus japonicus deficient in flavonoid biosynthesis

Journal of Plant Research - The original article has been updated.     

Characterization of Mycorrhizas Formed by Glomus sp. on Roots of Hypernodulating Mutants of Lotus japonicus

Lotus japonicus hypernodulating mutants, Ljsym78-1 and Ljsym78-2, by the arbuscular mycorrhizal fungus Glomus sp. was characterized. The mutants are defective in systemic autoregulation of nodulation and nitrate inhibition, and form an excess of nodules and lateral roots. The percent root length colonized by the arbuscular mycorrhizal fungi was significantly higher for the mutant than wild-type roots. Detailed assessment of the colonization indicated that the percentage of colonization by arbuscules was increased, but that by external hyphae, internal hyphae and vesicles was decreased, in the mutant roots compared with the wild-type. The succinate dehydrogenase activity of arbuscules, external hyphae and internal hyphae showed similar trends. In addition, the majority of individual arbuscules that formed on the mutant roots had a well-developed and seemingly tough morphology. The results suggest that mutation at the Ljsym78 locus positively stimulates the growth and activity of arbuscules, but leads to reduced growth and activity of hyphae. We report the first identification of Lotus japonicus mutants that show significantly increased arbuscule formation and termed these mutants Arb⁺⁺. Received 8 August 2000/ Accepted in revised form 19 October 2000     

Genetic suppressors of the Lotus japonicus har1-1 hypernodulation phenotype

Lotus japonicus har1 mutants respond to inoculation with Mesorhizobium loti by forming an excessive number of nodules due to genetic lesions in the HAR1 autoregulatory receptor kinase gene. In order to expand the repertoire of mutants available for the genetic dissection of the root nodule symbiosis (RNS), a screen for suppressors of the L. japonicus har1-1 hypernodulation phenotype was performed. Of 150,000 M2 plants analyzed, 61 stable L. japonicus double-mutant lines were isolated. In the context of the har1-1 mutation, 26 mutant lines were unable to form RNS, whereas the remaining 35 mutant lines carried more subtle symbiotic phenotypes, either forming white ineffective nodules or showing reduced nodulation capacity. When challenged with Glomus intraradices, 18 of the 61 suppressor lines were unable to establish a symbiosis with this arbuscular mycorrhiza fungus. Using a combined approach of genetic mapping, targeting induced local lesions in genomics, and sequencing, all non-nodulating mutant lines were characterized and shown to represent new alleles of at least nine independent symbiotic loci. The class of mutants with reduced nodulation capacity was of particular interest because some of them may specify novel plant functions that regulate nodule development in L. japonicus. To facilitate mapping of the latter class of mutants, an introgression line, in which the har1-1 allele was introduced into a polymorphic background of L. japonicus ecotype MG20, was constructed.     

Enzymatic Defects in Three Genetic Classes of Serine-Requiring Mutants of Bacillus pumilus

Serine-requiring mutants of Bacillus pumilus NRRL B-3275 have been divided into three groups based on the position of the mutant loci on the linkage map of this organism. Representatives of each group were found deficient in enzymatic activities that constitute the phosphorylated pathway for serine biosynthesis. The evidence suggests that the genes coding for the enzymes of the phosphorylated pathway of serine biosynthesis are not clustered in B. pumilus.     

Reciprocal Interaction Matrix Reveals Complex Genetic and Dose-Dependent Specificity among Coinfecting Parasites

Abstract Understanding genetic specificity in factors determining the outcome of host-parasite interactions is especially important as it contributes to parasite epidemiology, virulence, and maintenance of genetic variation. Such specificity, however, is still generally poorly understood. We examined genetic specificity in interactions among coinfecting parasites. In natural populations, individual hosts are often simultaneously infected by multiple parasite species and genotypes that interact. Such interactions could maintain genetic variation in parasite populations if they are genetically specific so that the relative fitness of parasite genotypes varies across host individuals depending on (1) the presence/absence of coinfections and/or (2) the genetic composition of the coinfecting parasite community. We tested these predictions using clones of fish eye flukes Diplostomum pseudospathaceum and Diplostomum gasterostei. We found that interactions among parasites had a strong genetic basis and that this modified genetic variation in infection success of D. pseudospathaceum between single and multiple infections as well as across multiply infected host individuals depending on the genetic identity of the coinfecting D. gasterostei. The relative magnitude of these effects, however, depended on the exposure dose, suggesting that ecological factors can modify genetic interactions between parasites.     

百脉根BIO和豌豆突变位点ELE2的比较基因组定位(英文)

豆科两侧对称花的花瓣具有背腹(DV)的分化以及可变的器官内部(IN)非对称性,在大小与形状上显示出不同的发育特征;因而花瓣的发育为克隆决定植物器官的形状与大小的关键基因提供了很好的实验系统。本研究对百脉根中BIO基因进行研究。百脉根bio突变体具有多效性,既影响花器官内部的对称性也影响器官的大小和育性,豌豆ele突变体的表型与bio相似。定位结果表明BIO和ELE2位于豆科基因组的共线性区段,提示BIO和ELE2可能是同源基因突变所致。本研究利用比较基因组定位方法,将BIO和ELE2候选基因锚定在豆科模式植物百脉根和蒺藜苜蓿基因组含有11个同源基因的BAC重叠群上。BIO和ELE2基因的克隆将有助于揭示豆科花瓣形态和大小调控的分子机理,进而为豆科作物遗传改良提供分子理论基础。     

Fatty Acid Specificity in Lipase-Catalyzed Synthesis of Glucoside Esters

The fatty acid specificity of the B-lipase derived from Candida antarctica was investigated in the synthesis of esters of ethyl D-glucopyranoside. The specificity was almost identical with respect to straight-chain fatty acids with 10 to 18 carbon atoms. However, lower fatty acids such as hexanoic and octanoic acid and the unsaturated 9-cis-octadecenoic acid were found to be poor substrates of the enzyme. As a consequence of this selectivity, these fatty acids were accumulated in the unconverted fraction when ethyl D-glucopyranoside was esterified with an excess of a mixture of fatty acids. This accumulation can reduce the overall effectiveness of the process as the activity of the lipase was found to be reduced when exposed to high concentrations of short-chain fatty acids. Finally, using a simplified experimental set-up, the specificity of the C. antarctica B-lipase was compared to the specificity of lipases derived from C. rugosa, Mucor miehei, Humicola, and Pseudomonas. Apart from the C. rugosa lipase, which exhibited a very poor performance, all the enzymes showed a very similar specificity with respect to fatty acids longer than octanoic acid while only the C. antarctica B-lipase showed activity towards sort-chain fatty acids.     

Fatty Acid Specificity in Lipase-Catalyzed Synthesis of Glucoside Esters

The fatty acid specificity of the B-lipase derived from Candida antarctica was investigated in the synthesis of esters of ethyl D-glucopyranoside. The specificity was almost identical with respect to straight-chain fatty acids with 10 to 18 carbon atoms. However, lower fatty acids such as hexanoic and octanoic acid and the unsaturated 9-cis-octadecenoic acid were found to be poor substrates of the enzyme. As a consequence of this selectivity, these fatty acids were accumulated in the unconverted fraction when ethyl D-glucopyranoside was esterified with an excess of a mixture of fatty acids. This accumulation can reduce the overall effectiveness of the process as the activity of the lipase was found to be reduced when exposed to high concentrations of short-chain fatty acids. Finally, using a simplified experimental set-up, the specificity of the C. antarctica B-lipase was compared to the specificity of lipases derived from C. rugosa, Mucor miehei, Humicola, and Pseudomonas. Apart from the C. rugosa lipase, which exhibited a very poor performance, all the enzymes showed a very similar specificity with respect to fatty acids longer than octanoic acid while only the C. antarctica B-lipase showed activity towards sort-chain fatty acids.     

Genome and Chromosome Dimensions of Lotus japonicus

Lotus Japonicus , Miyakojima MG-20 and Gifu B-129. The genome sizes of Miyakojima and Gifu were determined as 472.1 and 442.8 Mbp, respectively. Both the accessions were diploid (2n=12) and six chromosomes were identified and characterized based on the condensation patterns and the locations of rDNA loci. The obvious polymorphism observed in the genome size and the chromosome morphology between the two accessions, revealed specific accumulation of heterochromatin in Miyakojima or elimination in Gifu. The chromosomes L. japonicus were numbered according to their length. A quantitative chromosome map was also developed by the imaging methods using the digital data of the condensation pattern. 45S rDNA loci were localized on chromosomes A and F, and 5S rDNA locus was localized on chromosome A by fluorescence in situ hybridization (FISH). Identification of the chromosome and genome sizes and development of the quantitative chromosome map represent significant contribution to the L. japonicus genome project as the basic information. Received 29 August 2000/ Accepted in revised form 17 October 2000     

TfoX-Based Genetic Mapping Identifies Vibrio fischeri Strain-Level Differences and Reveals a Common Lineage of Laboratory Strains

Bacterial strain variation exists in natural populations of bacteria and can be generated experimentally through directed or random mutation. The advent of rapid and cost-efficient whole-genome sequencing has facilitated strain-level genotyping. Even with modern tools, however, it often remains a challenge to map specific traits to individual genetic loci, especially for traits that cannot be selected under culture conditions (e.g., colonization level or pathogenicity). Using a combination of classical and modern approaches, we analyzed strain-level variation in Vibrio fischeri and identified the basis by which some strains lack the ability to utilize glycerol as a carbon source. We proceeded to reconstruct the lineage of the commonly used V. fischeri laboratory strains. Compared to the wild-type ES114 strain, we identify in ES114-L a 9.9-kb deletion with endpoints in tadB2 and glpF; restoration of the missing portion of glpF restores the wild-type phenotype. The widely used strains ESR1, JRM100, and JRM200 contain the same deletion, and ES114-L is likely a previously unrecognized intermediate strain in the construction of many ES114 derivatives. ES114-L does not exhibit a defect in competitive squid colonization but ESR1 does, demonstrating that glycerol utilization is not required for early squid colonization. Our genetic mapping approach capitalizes on the recently discovered chitin-based transformation pathway, which is conserved in the Vibrionaceae; therefore, the specific approach used is likely to be useful for mapping genetic traits in other Vibrio species.     

Enzymatic Biosynthesis of Novel Resveratrol Glucoside and Glycoside Derivatives

A UDP glucosyltransferase from Bacillus licheniformis was overexpressed, purified, and incubated with nucleotide diphosphate (NDP) d- and l-sugars to produce glucose, galactose, 2-deoxyglucose, viosamine, rhamnose, and fucose sugar-conjugated resveratrol glycosides. Significantly higher (90%) bioconversion of resveratrol was achieved with α-d-glucose as the sugar donor to produce four different glucosides of resveratrol: resveratrol 3-O-β-d-glucoside, resveratrol 4′-O-β-d-glucoside, resveratrol 3,5-O-β-d-diglucoside, and resveratrol 3,5,4′-O-β-d-triglucoside. The conversion rates and numbers of products formed were found to vary with the other NDP sugar donors. Resveratrol 3-O-β-d-2-deoxyglucoside and resveratrol 3,5-O-β-d-di-2-deoxyglucoside were found to be produced using TDP-2-deoxyglucose as a donor; however, the monoglycosides resveratrol 4′-O-β-d-galactoside, resveratrol 4′-O-β-d-viosaminoside, resveratrol 3-O-β-l-rhamnoside, and resveratrol 3-O-β-l-fucoside were produced from the respective sugar donors. Altogether, 10 diverse glycoside derivatives of the medically important resveratrol were generated, demonstrating the capacity of YjiC to produce structurally diverse resveratrol glycosides.     

A Genome-Wide Screen for Bacterial Envelope Biogenesis Mutants Identifies a Novel Factor Involved in Cell Wall Precursor Metabolism

The cell envelope of Gram-negative bacteria is a formidable barrier that is difficult for antimicrobial drugs to penetrate. Thus, the list of treatments effective against these organisms is small and with the rise of new resistance mechanisms is shrinking rapidly. New therapies to treat Gram-negative bacterial infections are therefore sorely needed. This goal will be greatly aided by a detailed mechanistic understanding of envelope assembly. Although excellent progress in the identification of essential envelope biogenesis systems has been made in recent years, many aspects of the process remain to be elucidated. We therefore developed a simple, quantitative, and high-throughput assay for mutants with envelope biogenesis defects and used it to screen an ordered single-gene deletion library of Escherichia coli. The screen was robust and correctly identified numerous mutants known to be involved in envelope assembly. Importantly, the screen also implicated 102 genes of unknown function as encoding factors that likely impact envelope biogenesis. As a proof of principle, one of these factors, ElyC (YcbC), was characterized further and shown to play a critical role in the metabolism of the essential lipid carrier used for the biogenesis of cell wall and other bacterial surface polysaccharides. Further analysis of the function of ElyC and other hits identified in our screen is likely to uncover a wealth of new information about the biogenesis of the Gram-negative envelope and the vulnerabilities in the system suitable for drug targeting. Moreover, the screening assay described here should be readily adaptable to other organisms to study the biogenesis of different envelope architectures.     

Characterization of shade avoidance responses in Lotus japonicus

Sessile plants must continuously adjust their growth and development to optimize photosynthetic activity under ever-fluctuating light conditions. Among such light responses in plants, one of the best-characterized events is the so-called shade avoidance, for which a low ratio of the red (R):far-red (FR) light intensities is the most prominent stimulus. Such shade avoidance responses enable plants to overtop their neighbors, thereby enhancing fitness and competitiveness in their natural habitat. Considerable progress has been achieved during the last decade in understanding the molecular mechanisms underlying the shade avoidance responses in the model rosette plant, Arabidopsis thaliana. We characterize here the fundamental aspects of the shade avoidance responses in the model legume, Lotus japonicus, based on the fact that its phyllotaxis (or morphological architecture) is quite different from that of A. thaliana. It was found that L. japonicus displays the characteristic shade avoidance syndrome (SAS) under defined laboratory conditions (a low R:FR ratio, low light intensity, and low blue light intensity) that mimic the natural canopy. In particular, the outgrowth of axillary buds (i.e., both aerial and cotyledonary shoot branching) was severely inhibited in L. japonicus grown in the shade. These results are discussed with special emphasis on the unique aspects of SAS observed with this legume.     

Characterization of Shade Avoidance Responses in Lotus japonicus

Sessile plants must continuously adjust their growth and development to optimize photosynthetic activity under ever-fluctuating light conditions. Among such light responses in plants, one of the best-characterized events is the so-called shade avoidance, for which a low ratio of the red (R):far-red (FR) light intensities is the most prominent stimulus. Such shade avoidance responses enable plants to overtop their neighbors, thereby enhancing fitness and competitiveness in their natural habitat. Considerable progress has been achieved during the last decade in understanding the molecular mechanisms underlying the shade avoidance responses in the model rosette plant, Arabidopsis thaliana. We characterize here the fundamental aspects of the shade avoidance responses in the model legume, Lotus japonicus, based on the fact that its phyllotaxis (or morphological architecture) is quite different from that of A. thaliana. It was found that L. japonicus displays the characteristic shade avoidance syndrome (SAS) under defined laboratory conditions (a low R:FR ratio, low light intensity, and low blue light intensity) that mimic the natural canopy. In particular, the outgrowth of axillary buds (i.e., both aerial and cotyledonary shoot branching) was severely inhibited in L. japonicus grown in the shade. These results are discussed with special emphasis on the unique aspects of SAS observed with this legume.