Genetic and molecular basis of grass cell wall biosynthesis and degradability. II. Lessons from brown-midrib mutants

The brown-midrib mutants of maize have a reddish-brown pigmentation of the leaf midrib and stalk pith, associated with lignified tissues. These mutants progressively became models for lignification genetics and biochemical studies in maize and grasses. Comparisons at silage maturity of bm1, bm2, bm3, bm4 plants highlighted their reduced lignin, but also illustrated the biochemical specificities of each mutant in p-coumarate, ferulate ester and etherified ferulate content, or syringyl/guaiacyl monomer ratio after thioacidolysis. Based on the current knowledge of the lignin pathway, and based on presently developed data and discussions, C3H and CCoAOMT activities are probably major hubs in controlling cell-wall lignification (and digestibility). It is also likely that ferulates arise via the CCoAOMT pathway.     

Nucleotide diversity of the <Emphasis Type="Italic">ZmPox3</Emphasis> maize peroxidase gene: Relationships between a MITE insertion in exon 2 and variation in forage maize digestibility

Background  

Polymorphisms were investigated within the ZmPox3 maize peroxidase gene, possibly involved in lignin biosynthesis because of its colocalization with a cluster of QTL related to lignin content and cell wall digestibility. The purpose of this study was to identify, on the basis of 37 maize lines chosen for their varying degrees of cell wall digestibility and representative of temperate regions germplasm, ZmPox3 haplotypes or individual polymorphisms possibly associated with digestibility.     

Genetic and molecular basis of grass cell-wall degradability. I. Lignin-cell wall matrix interactions

Lignification limits grass cell-wall digestion by herbivores. Lignification is spatially and temporally regulated, and lignin characteristics differ between cell walls, plant tissues, and plant parts. Grass lignins are anchored within walls by ferulate and diferulate cross-links, p-coumarate cyclodimers, and possibly benzyl ester and ether cross-links. Cell-wall degradability is regulated by lignin concentration, cross-linking, and hydrophobicity but not directly by most variations in lignin composition or structure. Genetic manipulation of lignification can improve grass cell-wall degradability, but the degree of success will depend on genetic background, plant modification techniques employed, and analytical methods used to characterize cell walls.     

Measurement of cell-wall digestibility in lucerne using the filter bag technique

A new filter bag technique was tested for the measurement of enzymatic digestibility of cell-wall in lucerne. Breeding for a higher cell-wall digestibility requires its measurement in in vitro conditions on small quantities of forage samples with a low cost, rapid method. Three methods of evaluation of cell-wall digestibility were tested on 60 whole plant samples and 60 stem samples of lucerne and compared. These three methods consisted in 1/ measurement of enzymatic digestibility of cell-wall by incubation of cell-wall residue in enzymatic solutions, 2/ calculation of the ratio between cell-wall content of the sample and cell-wall content of the residue of the enzymatic digestion, and 3/ calculation of the cell-wall digestibility through sample cell-wall content and sample digestibility, assuming a non cell-wall digestibility of 90%. The three evaluations were highly correlated and gave similar ranges of variation. The first method was the most precise but the most time-consuming. However, the third one was precise, and this method was the most rapid. NIRS prediction of all cell-wall digestibility estimates was accurate. The choice between the three techniques for breeding for an improved forage digestibility of lucerne needs further investigations on the genetic variation of this criterion measured by the three methods.     

Genetic diversity associated with variation in silage corn digestibility for three<Emphasis Type="Italic"> O</Emphasis>-methyltransferase genes involved in lignin biosynthesis

Polymorphisms within three candidate genes for lignin biosynthesis were investigated to identify alleles useful for the improvement of maize digestibility. The allelic diversity of two caffeoyl-CoA 3-O-methyltransferase genes, CCoAOMT2 and CCoAOMT1, as well as that of the aldehyde O-methyltransferase gene, AldOMT, was evaluated for 34 maize lines chosen for their varying degrees of cell wall digestibility. Frequency of nucleotide changes averaged one SNP every 35 bp. Ninety-one indels were identified in non-coding regions and only four in coding regions. Numerous distinct and highly diverse haplotypes were identified at each locus. Numerous sites were in linkage disequilibrium that declined rapidly within a few hundred bases. For F4, an early flint French line with high cell wall digestibility, the CCoAOMT2 first exon presented many non-synonymous polymorphisms. Notably we found an 18-bp indel, which resembled a microsatellite and was associated with cell wall digestibility variation. Additionally, the CCoAOMT2 gene co-localized with a QTL for cell wall digestibility and lignin content. Together, these results suggest that genetic diversity investigated on a broader genetic basis could contribute to the identification of favourable alleles to be used in the molecular breeding of elite maize germplasm.     

引进美国GEM材料的抗玉米青枯病和丝黑穗病种质资源筛选鉴定

近年来,青枯病、丝黑穗病上升为我国玉米生产中的主要病害,挖掘新的抗病资源,进行抗病育种是解决病害威胁的根本所在。本研究对引进的美国GEM材料进行了接种条件下的抗病性鉴定,并结合抗病材料的多年重复验证,筛选出一批新的抗病种质资源。这些材料的深入研究和广泛应用,对拓宽我国玉米青枯病、丝黑穗病抗性遗传基础,增加种质资源遗传多样性具有重要利用价值。     

Quantitative trait loci for cell-wall components in recombinant inbred lines of maize (Zea mays L.) I: stalk tissue

Maize silage is a significant energy source for animal production operations, and the efficiency of the conversion of forage into animal mass is an important consideration when selecting cultivars for use as feed. Fiber and lignin are negatively correlated with digestibility of feed, so the development of forage with reduced levels of these cell-wall components (CWCs) is desirable. While variability for fiber and lignin is present in maize germplasm, traditional selection has focused on the yield of the ear rather than the forage quality of the whole plant, and little information is available concerning the genetics of fiber and lignin. The objectives of this study were to map quantitative trait loci (QTLs) for fiber and lignin in the maize stalk and compare them with QTLs from other populations. Stalk samples were harvested from 191 recombinant inbred lines (RILs) of B73 (an inbred line with low-to-intermediate levels of CWCs) x De811 (an inbred line with high levels of CWCs) at two locations in 1998 and one in 1999 and assayed for neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL). The QTLs were detected on nine chromosomes, mostly clustered in concordance with the high genetic correlations between NDF and ADF. Adjustment of NDF for ADF and ADF for ADL revealed that most of the variability for CWCs in this population is in ADF. Many of the QTLs detected in this study have also been detected in other populations, and several are linked to candidate genes for cellulose or starch biosynthesis. The genetic information obtained in this study should be useful to breeding efforts aimed at improving the quality of maize silage.     

玉米种质资源抗矮花叶病鉴定

采用摩擦接种方法,鉴定了3995份玉米种质对玉米矮花叶病毒(SCMV-MDB)的抗病性。经重复鉴定,筛选出抗病优良自交系73份,抗病丰产杂交种80份。玉米不同类群种质,对SCMV-MDB的抗病性有明显差异,改良Reid、Lancaster、旅大红骨类群种质大部分表现感病;塘四平头、外杂先锋选系类群种质大多数表现抗病。在不同类型种质中,硬粒型、马齿型和半马齿型表现抗病的较多,糯质型较少,甜质型和爆裂型中尚未发现抗源。     

玉米重要自交系的肿囊腐霉茎腐病抗性鉴定与评价

由肿囊腐霉菌(Pythium inflatum Matthews)引起的玉米茎腐病是影响玉米产量的一种重要病害。为进一步拓展可利用的抗源,于2010-2011年在田间采用人工接种方法对287份重要的玉米自交系种质进行了玉米茎腐病的抗性鉴定评价。结果表明,287份鉴定材料中有171份自交系对茎腐病的抗性达到中抗以上水平,占鉴定材料的59.58%,其中高抗自交系共43份,占鉴定材料总数的14.98%;感病类型自交系共116份,占鉴定材料的40.42%,其中高感自交系共95份,占鉴定材料总数的33.10%。Lancaster、Reid及P群种质中具有丰富的茎腐病抗源,而塘四平头种质群中茎腐病抗源相对缺乏,多为感病类型。该研究结果可为今后我国玉米茎腐病抗性种质的引进和改良提供重要参考。     

Ultrastructural study of the maize epidermal root surface. I. Preservation and extent of the mucilage layer

Summary This study clarifies the relationships between the root mucilage and the epidermal cell-wall in axenically grown maize roots. Mucilage appears to overlay the epidermal cell-wall which has two distinct layers. This mucilage is well preserved after cationized ferritin fixation and PATAg staining. It extends all over the entire root epidermis.     

A High-Throughput Platform for Screening Milligram Quantities of Plant Biomass for Lignocellulose Digestibility

The development of a viable lignocellulosic ethanol industry requires multiple improvements in the process of converting biomass to ethanol. A key step is the improvement of the plants that are to be used as biomass feedstocks. To facilitate the identification and evaluation of feedstock plants, it would be useful to have a method to screen large numbers of individual plants for enhanced digestibility in response to combinations of specific pretreatments and enzymes. This paper describes a high-throughput digestibility platform (HTDP) for screening collections of germplasm for improved digestibility, which was developed under the auspices of the Department of Energy-Great Lakes Bioenergy Research Center (DOE-GLBRC). A key component of this platform is a custom-designed workstation that can grind and dispense 1–5 mg quantities of more than 250 different plant tissue samples in 16 h. The other steps in the processing (pretreatment, enzyme digestion, and sugar analysis) have also been largely automated and require 36 h. The process is adaptable to diverse acidic and basic, low-temperature pretreatments. Total throughput of the HTDP is 972 independent biomass samples per week. Validation of the platform was performed on brown midrib mutants of maize, which are known to have enhanced digestibility. Additional validation was performed by screening approximately 1,200 Arabidopsis mutant lines with T-DNA insertions in genes known or suspected to be involved in cell wall biosynthesis. Several lines showed highly significant (p?<?0.01) increases in glucose and xylose release (20–40% above the mean). The platform should be useful for screening populations of plants to identify superior germplasm for lignocellulosic ethanol applications and also for screening populations of mutant model plants to identify specific genes affecting digestibility.     

Nucleotide diversity and molecular evolution of the PSY1 gene in Zea mays compared to some other grass species

Phytoene synthase (PSY), which is encoded by the phytoene synthase 1 (PSY1) gene, is the first rate-limiting enzyme in the plant carotenoid biosynthetic pathway. In order to examine the genetic diversity and evolution pattern of PSY1 within the Andropogoneae, sequences of 76 accessions from 5 species (maize, teosinte, tripsacum, coix, and sorghum) of the Andropogoneae were tested, along with 4 accessions of rice (Oryza sativa L.) included as outliers. Both the number and the order of exons and introns were relatively conserved across the species tested. Three domains were identified in the coding sequence, including signal peptide (SP), PSY, and highly conserved squalene synthase (SQS) domain. Although no positive selection signal was detected at an overall coding level among all species tested, the SP domain and the region upstream of the SQS–PSY domain appear to have undergone rapid evolution, as evidenced by a high d _N/d _S ratio (>1.0). At the nucleotide level, positive selection and balancing selection were detected only among the yellow maize germplasm and the white maize germplasm, respectively. The phylogenetic tree based on full-length sequences of PSY1-like regions supported the monophyletic theory of the Andropogoneae and the closest relationship between Zea and Tripsacum among the Andropogoneae. Coix, which was theorized to have a closer relationship with maize due to similarities in morphology and chromosome number, has been shown in this study to have diverged relatively early from the other Andropogoneae, including maize.     

Influence of different yeast cell-wall mutants on performance and protection against pathogenic bacteria (Vibrio campbellii) in gnotobiotically-grown Artemia

A selection of isogenic yeast strains (with deletion for genes involved in cell-wall synthesis) was used to evaluate their nutritional and immunostimulatory characteristics for gnotobiotically-grown Artemia. In the first set of experiments the nutritional value of isogenic yeast strains (effected in mannoproteins, glucan, chitin and cell-wall bound protein synthesis) for gnotobiotically-grown Artemia was studied. Yeast cell-wall mutants were always better feed for Artemia than the isogenic wild type mainly because they supported a higher survival but not a stronger individual growth. The difference in Artemia performance between WT and mutants feeding was reduced when stationary-phase grown cells were used. These results suggest that any mutation affecting the yeast cell-wall make-up is sufficient to improve the digestibility in Artemia. The second set of experiments, investigates the use of a small amount of yeast cells in gnotobiotic Artemia to overcome pathogenicity of Vibrio campbellii (VC). Among all yeast cell strains used in this study, only mnn9 yeast (less cell-wall bound mannoproteins and more glucan and chitin) seems to completely protect Artemia against the pathogen. Incomplete protection against the pathogen was obtained by the gas1 and chs3 mutants, which are lacking the gene for a particular cell-wall protein and chitin synthesis, respectively, resulting in more glucan. The result with the chs3 mutant is of particular interest, as its nutritional value for Artemia is comparable to the wild type. Hence, only with the chs3 strain, in contrast to the gas1 or mnn9 strains, the temporary protection to VC is not concomitant with a better growth performance under non-challenged conditions, suggesting non-interference of general nutritional effects.     

Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells. 3. Interplay between limited cell-wall autolysis, pectin methyl esterase activity and electrostatic effects in soybean cell walls

Soybean cell walls display a process of autolysis which results in the release of reducing sugars from the walls. Loosening and autolysis of cell wall are involved in the cell-wall growth process, for autolysis is maximum during both cell extension and cell-wall synthesis. Autolysis goes to completion within about 50 h and is an enzymatic process that results from the activity of cell wall exo- and endo-glycosyltransferases. The optimum pH of autolysis is about 5. Increasing the ionic strength of the bulk phase where cell-wall fragments are suspended, results in a shift of the pH profile towards low pH. This is consistent with the view that at 'low' ionic strength, the local pH in the cell wall is lower than in the bulk phase. One of the main ideas of the model proposed in a preceding paper, is that pectin methyl esterase reaction, by building up a high fixed charge density, results in proton attraction in the wall. Low pH must then activate the wall loosening enzymes involved in autolysis and cell growth. This view may be directly confirmed experimentally. The pH of a cell-wall suspension, initially equal to 5, was brought to 8 for 20 min, then back to 5. Under these conditions, the rate of cell-wall autolysis was enhanced with respect to the rate of autolysis obtained with cell-wall fragments kept at pH 5. The pH response of the multienzyme plant cell-wall system basically relies on opposite pH sensitivities of the two types of enzymes involved in the growth process. Pectin methyl esterase, which generates the cell-wall Donnan potential, is inhibited by protons, whereas the wall-loosening enzymes involved in cell growth are activated by protons.     

Meta-analysis of QTL involved in silage quality of maize and comparison with the position of candidate genes

A meta-analysis of quantitative trait loci (QTL) associated with plant digestibility and cell wall composition in maize was carried out using results from 11 different mapping experiments. Statistical methods implemented in “MetaQTL” software were used to build a consensus map, project QTL positions and perform meta-analysis. Fifty-nine QTL for traits associated with digestibility and 150 QTL for traits associated with cell wall composition were included in the analysis. We identified 26 and 42 metaQTL for digestibility and cell wall composition traits, respectively. Fifteen metaQTL with confidence interval (CI) smaller than 10 cM were identified. As expected from trait correlations, 42% of metaQTL for digestibility displayed overlapping CIs with metaQTL for cell wall composition traits. Coincidences were particularly strong on chromosomes 1 and 3. In a second step, 356 genes selected from the MAIZEWALL database as candidates for the cell wall biosynthesis pathway were positioned on our consensus map. Colocalizations between candidate genes and metaQTL positions appeared globally significant based on χ² tests. This study contributed in identifying key chromosomal regions involved in silage quality and potentially associated genes for most of these regions. These genes deserve further investigation, in particular through association mapping.     

Digestibility of wet cage layer excreta and an excreta-supplemented diet by cattle as determined by two methods

Digestibility studies were carried out with cattle to determine coefficients of digestibility for residual nutrients in wet cage layer excreta (CLE), and the effect of incorporating CLE in a maize silage-based diet, on nutrient digestibility. Two methods were used and compared — total collection, and the use of the recently identified internal marker, acid insoluble ash (AIASH).Digestibility coefficients for cage layer excreta were: dry matter, 74.0; organic matter, 73.1; crude protein, 76.8; energy 71.3; acid detergent fibre, 71.8%. Substituting CLE for soya bean meal or urea in the maize silage-based diet did not adversely affect nutrient digestibility. Comparable digestion coefficients were obtained with both the total collection method and the AIASH marker technique, confirming the results of others that the AIASH marker gives satisfactory estimates of nutrient digestibility.     

Light-controlled growth of the maize seedling mesocotyl: Mechanical cell-wall changes in the elongation zone and related changes in lignification

Cell extension in the mesocotyl elongation zone (MEZ) of maize ( Zea mays L.) seedlings is inhibited by light. The growth inhibition by blue light in the MEZ was reversible upon transfer to darkness. This experimental system was used for investigating the modification of mechanical cell-wall properties and the role of cell-wall lignification in cell elongation. The occurrence of lignin in the cortex and vascular bundle tissues of the MEZ was demonstrated by the isolation of diagnostic monomers released after thioacidolysis of the cell walls. Concomitantly with the inhibition of growth, blue light induces an increase in cell-wall stiffness (tensile modulus) as well as an increase in extractable lignin in the outer MEZ tissues (cortex+epidermis). Both effects are reversed when growth is resumed in the MEZ in darkness after a period of growth inhibition induced by 3 h light. In the vascular bundle light produces no comparable change in lignin content. Appearance and disappearance of phenylpropanoid material in MEZ cell walls in the light, or in darkness following a brief light treatment, respectively, can be visualized under the fluorescence microscope by characteristic changes in autofluorescence of tissue sections upon excitation with UV radiation. It is concluded from these results that light-induced lignification of primary walls is involved in cell-wall stiffening and thus inhibition of elongation growth in the MEZ of maize seedlings. Resumption of growth upon redarkening may be initiated by wall loosening in the uppermost MEZ region which displaces the lignified cell walls towards the lower mesocotyl region.     

转基因技术在玉米遗传育种中的应用

玉米转基因育种是通过基因转移方法创造玉米新种质或新品种。将转基因技术与玉米常规育种技术相结合,能尽快地培育出符合育种目标的新品种,带来了育种水平的提高、创新和突破,加快了玉米育种进程。论述了转基因技术在玉米育种中的主要应用方法、研究进展及应用现状。随着转基因技术的飞速发展及其研究的日趋成熟,转基因技术在拓宽玉米种质资源、提高杂交种的抗逆性、抗病虫性、提高产量和品质等方面将发挥更大的作用,应用前景广阔。     

玉米种质资源对纹枯病的抗性鉴定与评价

在人工接种条件下,对国内常用的285份玉米种质资源进行抗纹枯病大田鉴定与评价,未发现免疫材料,抗病材料很少.高抗、抗和中抗材料的比例分别为1.0%、3.2%、13.7%;绝大多数材料属于感病或高感,分别占29.8%、52.3%.株高、主穗位高、主穗位高/株高之比值与病情指数呈负相关,病斑高、病斑高/株高、病斑高/主穗位高之比值与病情指数呈正相关,可以将病斑高度与主穗位高度的比值作为抗性鉴定与评价指标之一.采用带菌麦粒在拔节中后期定位接种,以病斑到达的叶鞘位作为病情和抗性评价体系是较为准确和规范的方法.