Barley Mutant Research Articles

고-Lysine 보리 돌연변이 계통 M98 종실의 아미노산 조성 및 Proteome Profile 특성

본 연구는 찰쌀보리와 M98의 종실에 함유된 아미노산 조성 및 단백질 profile의 특성 차이를 분석하여 M98의 고-Lysine 및 함몰배유 형질에 관련된 생화학적 정보를 얻고자 수행하였으며, 그 결과를 요약하면 다음과 같다. 1. 찰쌀보리와 M98 종실의 총 16개 아미노산 조성을 분석한 결과, lysine을 비롯하여 총 6개 아미노산의 조성비가 M98에서 찰쌀보리에 비해 1.2~1.8배 높았다. Proline과 그것의 전구물질인 glutamic acid의 조성비는 M98에서 찰쌀보리에 비해 절반수준으로 낮았는데, glutamic acid를 전구물질로 하여 합성되는 arginine의 조성비는 M98에서 찰쌀보리보다 1.8배 높았다. 2. 찰쌀보리와 M98간의 종실 단백질을 이차원전기영동으로 분리한 결과, 총 70개 단백질 spot에서 발현양상의 차이가 확인되었다. 찰쌀보리에 비해 M98에서 발현양이 높은 단백질 spot은 44개였으며, 발현양이 낮은 단백질 spot은 18개였다. 나머지 8개 단백질 spot에서 7개의 발현은 찰쌀보리에서만 확인되었고, 1개의 발현은 M98에서만 확인되었다. 3. 찰쌀보리와 M98의 종실에서 발현양상 차이를 나타낸 총 53개의 단백질 spot을 선택하여 nESI-LC-MS/MS 방식으로 동정하였으며, 대부분의 단백질 spot은 종실의 다양한 생물학적 과정에 관여하는 단백질이었다. 특히, 28개 단백질 spot은 M98의 함몰배유 및 고-Lysine 형질과 연관된 단백질이었다. 4. 본 연구에서 밝혀진 M98 종실의 아미노산 조성 및 단백질 발현특성 정보는 향후 M98 유래 계통을 이용한 유전학적 검토 등을 통하여 고-Lysine 형질 연관 바이오마커 및 보리 신품종 개발에 유용한 정보로 활용될 수 있을 것으로 판단되었다. Lysine is the first limiting essential amino acid in cereals for humans and monogastric animals, although its content is generally low. A chemically induced high-lysine barley mutant, M98, has an agronomically undesirable shrunken endosperm trait. In order to obtain detailed insight into the atypical traits of M98 grains, we characterized amino acid composition and protein profiles of M98 and its parent cultivar Chalssalbori. Among a total of 16 amino acids, the percentage of each of the 7 amino acids, including lysine, was 1.2~1.8 times higher in M98, comparing to Chalssalbori. The percentage of proline and its precursor, glutamic acid, in M98 was about the half of that of the amino acids in Chalssalbori, but arginine synthesized from glutamic acid was 1.8 times higher in M98, compared that in the parent cultivar. Theses results indicated that the mutation in M98 grains might alter the proportion of amino acids linked to each other in a biosynthetic pathway. A comparison of grain proteome profiles between Chalssalbori and M98 revealed 70 differentially expressed protein spots, where 45 protein spots were up-regulated and 25 protein spots down-regulated in M98 compared to those in Chalssalbori. Of these changed protein spots, 53 were identified using nano-electrospray ionization liquid chromatography mass spectrometry. Most of these identified proteins were involved in various biological processes. In particular, 28 protein spots such as <TEX>${\beta}$</TEX>-amylase, serpins and B3-hordein were identified as proteins associated with the atypical traits of M98. It was thought that a genetic study on the unique protein profile of M98 would be needed to develop an agronomically feasible barley cultivar with high-lysine trait.

Characterization of Mutations in Barley fch2 Encoding Chlorophyllide a Oxygenase

The barley (Hordeum vulgare L.) mutants fch2 and clo-f2 comprise an allelic group of 14 Chl b-deficient lines. The genetic map position of fch2 corresponds to the physical map position of the gene encoding chlorophyllide a oxygenase. This enzyme converts chlorophyllide a to chlorophyllide b and it is essential for Chl b biosynthesis. The fch2 and clo-f2 barley lines were shown to be mutated in the gene for chlorophyllide a oxygenase. A five-base insertion was found in fch2 and base deletions in clo-f2.101, clo-f2.105, clo-f2.2800 and clo-f2.3613. In clo-f2.105 and clo-f2.108, nonsense base exchanges were discovered. All of these mutations led to a premature stop of translation and none of the mutants formed Chl b. The mutant clo-f2.2807 was transcript deficient and formed no Chl b. Missense mutations in clo-f2.102 (leading to the amino acid exchange D495N) and clo-f2.103 (G280D) resulted in a total lack of Chl b, whereas in the missense mutants clo-f2.107 (P419L), clo-f2.109 (A94T), clo-f2.122 (C320Y), clo-f2.123 (A94T), clo-f2.133 (A376V) and clo-f2.181 (L373F) intermediate contents of Chl b were determined. The missense mutations affect conserved residues, and their effect on chlorophyllide a oxygenase is discussed. The mutations in clo-f2.102, clo-f2.103, clo-f2.133 and clo-f2.181 may influence electron transfer as illustrated in the active site of a structural model protein. The changes in clo-f2.107, clo-f2.109, clo-f2.122 and clo-f2.123 may lead to Chlb deficiency by interfering with the regulation of chlorophyllide a oxygenase. The correlation of mutations and phenotypes strongly supports that the barley locus fch2 encodes chlorophyllide a oxygenase.

Significance of the structure of data in partial least squares regression predictions involving both natural and human experimental design

When predicting the chemical composition of food samples from near‐infrared spectroscopy using partial least squares regression, deep knowledge of the origin of the information is not present. We are aiming at opening a Pandora's box of how the prediction of protein proceeds in a unique set of chemically diverse barley mutant samples. An external validation of the sources of co‐variation in nature that are exploited by chemometric models would give a framework for manipulating the deciding information to make expensive calibration more economical. The barley samples were supplemented by two designed data sets: one mirroring the coarse composition of the barley samples by mixing six main chemical components and one set where the biological covariance between the six chemical components had been reduced.The three original data sets give remarkably comparable prediction models, albeit their regression coefficients are quite different. The origin of the prediction ability of the data is elucidated by splitting the natural barley samples into two parts: one based on simulated biology extracted from a set of chemical mixtures, and the residual after the chemistry has been removed from the raw data. As much as 98.1% of the spectral information in the natural barley data is explained through the simulated biology, leaving as little as 1.9% of the spectral information for the unexplained biological variation and noise. However, unexplained biological variation still gives a fair prediction of protein (RMSECV = 1.23 and r2 = 0.80, compared with RMSECV = 0.46 and r2 = 0.97 for the natural data), and it gives a clear principal component analysis separation of the three genotype classes. The results were interpreted by conducting spectral inspection on the origin of the unique covariate patterns appearing in self‐organised biological systems that should motivate researchers and industry to investigate the compressive effect that the model has on the essential deterministic biological data. Copyright © 2012 John Wiley &amp; Sons, Ltd.

Induced mutations in circadian clock regulator Mat-a facilitated short-season adaptation and range extension in cultivated barley

Time to flowering has an important impact on yield and has been a key trait in the domestication of crop plants and the spread of agriculture. In 1961, the cultivar Mari (mat-a.8) was the very first induced early barley (Hordeum vulgare L.) mutant to be released into commercial production. Mari extended the range of two-row spring barley cultivation as a result of its photoperiod insensitivity. Since its release, Mari or its derivatives have been used extensively across the world to facilitate short-season adaptation and further geographic range extension. By exploiting an extended historical collection of early-flowering mutants of barley, we identified Praematurum-a (Mat-a), the gene responsible for this key adaptive phenotype, as a homolog of the Arabidopsis thaliana circadian clock regulator Early Flowering 3 (Elf3). We characterized 87 induced mat-a mutant lines and identified >20 different mat-a alleles that had clear mutations leading to a defective putative ELF3 protein. Expression analysis of HvElf3 and Gigantea in mutant and wild-type plants demonstrated that mat-a mutations disturb the flowering pathway, leading to the early phenotype. Alleles of Mat-a therefore have important and demonstrated breeding value in barley but probably also in many other day-length-sensitive crop plants, where they may tune adaptation to different geographic regions and climatic conditions, a critical issue in times of global warming.

Barley mutants with low rates of endosperm starch synthesis have low grain dormancy and high susceptibility to preharvest sprouting

• Studies of embryo dormancy in relation to preharvest sprouting (PHS) in cereals have focused on ABA and other hormones. The relationship between these phenomena and the rate of grain filling has not been investigated. • A collection of barley mutants impaired in starch synthesis was assessed for preharvest sprouting in the field. In subsequent glasshouse experiments, developing grains were assayed for germination index, sugars, abscisic acid (ABA) and the effects of temperature and exogenous ABA on germination. • Mutant lines displayed greater preharvest sprouting in the field than parental lines. In the glasshouse, nondeep physiological dormancy was reduced in developing grains of five lines with mutations affecting proteins involved in endosperm starch synthesis. Inhibition of germination by exogenous ABA and elevated temperature was decreased in developing mutant grains. Sugar concentrations were high but embryo and endosperm ABA contents were unaltered. • We reveal a direct connection between grain filling and the extent of grain dormancy. Impaired endosperm starch synthesis directly influences the acquisition of embryo dormancy, perhaps because endosperm sugar concentrations modulate the ABA responsiveness of the embryo. Thus environmental or genetic factors that reduce grain filling are likely to reduce dormancy and enhance susceptibility to PHS.

Mutagenic treatment induces high transposon variation in barley (Hordeum vulgare L.)

With more than 2700 mutant-derived cultivars, mutation techniques belong to the most efficient breeding methods. Despite a relatively good understanding of mutagenesis there is no agreement about the range of genome changes in mutants. Visualizing the gain and the loss of transposon insertion sites in SSAP profiles, the present studies aimed to answer whether or not mutants can be regarded as near isogenic lines with respect to their parent cultivar. Activities of BARE-1 retrotransposon and Tpo1-like DNA transposon from the CACTA superfamily were analysed in ten barley mutants derived by mutagenic treatment of two cultivars, Brenda and Scarlett. A large number and proportion of mutations (on average 21.4 mutations encompassing 19.7% loci) confirm high efficiency of applied mutagens. However, differences exist among both cultivars and transposons. The lack of clear correlation between transposon activities and morphology reflects different mechanisms shaping the mutant architecture. With respect to a transposon type, BARE-1 and Tpo1-like were equally active in inducing mutations. However, their activity differs in that the former was mainly responsible for new insertions while the latter equally for insertions and deletions. An excess of new insertions over recombinational loss suggests a transposon burst as a response to stress caused by chemical mutagens. Low estimations of Nei’s similarities, well within the range of semispecies demonstrate the role of mutagenic factors in diversification of populations. This way mutagenic treatment not only provides rough breeding materials but also can be used as a model in evolutionary studies.

Ant28 gene for proanthocyanidin synthesis encoding the R2R3 MYB domain protein (Hvmyb10) highly affects grain dormancy in barley

A number of anthocyanin- and proanthocyanidin-free mutants (ant mutants) in barley were induced and selected because of breeding interest to reduce proanthocyanidins, which could cause haze and degrade the quality of beer. Ant loci, known as anthocyanin or proanthocyanidin synthesis genes, are classified into Ant1 to Ant30 through allelism tests. However, only the Ant18 gene has been molecularly shown to encode dihydroflavonol 4-reductase (DFR), which is involved in both anthocyanin and proanthocyanidin synthesis. In this study, an R2R3 MYB gene of barley was isolated by PCR and named Hvmyb10 due to its similarity to Tamyb10 of wheat, which is a candidate for the R-1 gene grain color regulator. The predicted amino acid sequences of Hvmyb10 showed high similarity not only to Tamyb10 but also to TT2, the proanthocyanidin regulator of Arabidopsis. Non-synonymous nucleotide substitutions in the Hvmyb10 gene were found in all six ant28 mutants tested. Mapping showed that a polymorphism in Hvmyb10 perfectly cosegregated with the ant 28 phenotype on the distal region of the long arm of chromosome 3H. These results demonstrate that ant28 encodes Hvmyb10, the R2R3 MYB domain protein that regulates proanthocyanidin accumulation in developing grains. The reduced grain dormancy of ant28 mutants compared with those of the respective wild types indicates that Hvmyb10 is a key factor in grain dormancy in barley.

Effects of chloroplast dysfunction on mitochondria: white sectors in variegated leaves have higher mitochondrial DNA levels and lower dark respiration rates than green sectors.

Co-ordination between plastids and mitochondria is so essential that there should be extensive inter-organellar crosstalk during development of plant cells. Indeed, chloroplast dysfunction in white leaves of plastid ribosome-deficient mutant barley, albostrians, is reportedly accompanied by increases in the levels of mitochondrial DNA and mitochondrial transcripts, suggesting that (i) developmental/physiological status of plastids (or heterotrophic growth condition of albino leaves) can affect the status of mitochondrial genome, and (ii) the function of the affected mitochondria may also be up-regulated accordingly. However, functional aspects of the mitochondria affected by chloroplast dysfunction have not yet been examined in detail. Here, we examined the effects of chloroplast dysfunction on mitochondrial DNA level and dark respiration rate, by comparing white and green sectors within individual variegated leaves, using 12 ornamental plants as experimental materials. The pattern of leaf variegation differed from species to species, suggesting that different mechanisms were involved in the formation of white sectors in different species. Quantitative hybridization analysis revealed that mitochondrial DNA levels were generally higher in white sectors than in green sectors. In spite of the elevated mitochondrial DNA levels, however, dark respiration rates in white sectors were generally lower than those in green sectors. Several possible mechanisms for elevation of mitochondrial DNA level and suppression of dark respiration rates in white sectors are discussed.Electronic supplementary materialThe online version of this article (doi:10.1007/s00709-011-0325-y) contains supplementary material, which is available to authorized users.

Mapping of Mutant Gene prbs Controlling Poly-Row-and-Branched Spike in Barley (Hordeum vulgare L.)

Abstract A row-type mutant of barley named poly-row-and-branched spike (prbs) was previously obtained from a two-rowed cultivar Pudamai-2 after treated by inflorescence soaking in maize total DNA solution. The mutant produces branched spikes with irregular multiple rows. Genetic analysis indicated that the mutant phenotype was caused by a recessive gene prbs, and the PRBS locus had a recessive epistatic effect on an independent locus (denoted as Vrsx) conferring the variation of two-rowed spike vs. six-rowed spike. This study aimed to map PRBS as well as Vrsx using simple sequence repeats (SSR) markers. We developed an F 2 population from a cross between the prbs mutant and a six-rowed cultivar Putianwudu for the gene mapping. As the two target loci interacted to result in a segregation ratio of two-rowed type:six-rowed type:prbs=9:3:4 in the population, we adopted a special strategy to map the two loci. PRBS was mapped between SSR markers HvLTPPB and Bmag0508A on the short arm of chromosome 3H, with distances of 24.7 and 14.3 cM to the two markers, respectively. Vrsx was mapped between SSR markers Bmag0125 and Bmag0378 on chromosome 2H, with distances of 6.9 and 15.3 cM to the two markers, respectively. This suggests that Vrsx should be the known locus Vrsl, which predominantly controls row-type variation in barley cultivars, and PRBS is a new locus related to the row type of spikes in barley.

Functional characterization of barley betaglucanless mutants demonstrates a unique role for CslF6 in (1,3;1,4)-β-D-glucan biosynthesis

(1,3;1,4)-β-D-glucans (mixed-linkage glucans) are found in tissues of members of the Poaceae (grasses), and are particularly high in barley (Hordeum vulgare) grains. The present study describes the isolation of three independent (1,3;1,4)-β-D-glucanless (betaglucanless; bgl) mutants of barley which completely lack (1,3;1,4)-β-D-glucan in all the tissues tested. The bgl phenotype cosegregates with the cellulose synthase like HvCslF6 gene on chromosome arm 7HL. Each of the bgl mutants has a single nucleotide substitution in the coding region of the HvCslF6 gene resulting in a change of a highly conserved amino acid residue of the HvCslF6 protein. Microsomal membranes isolated from developing endosperm of the bgl mutants lack detectable (1,3;1,4)-β-D-glucan synthase activity indicating that the HvCslF6 protein is inactive. This was confirmed by transient expression of the HvCslF6 cDNAs in Nicotiana benthamiana leaves. The wild-type HvCslF6 gene directed the synthesis of high levels of (1,3;1,4)-β-D-glucans, whereas the mutant HvCslF6 proteins completely lack the ability to synthesize (1,3;1,4)-β-D-glucans. The fine structure of the (1,3;1,4)-β-D-glucan produced in the tobacco leaf was also very different from that found in cereals having an extremely low DP3/DP4 ratio. These results demonstrate that, among the seven CslF and one CslH genes present in the barley genome, HvCslF6 has a unique role and is the key determinant controlling the biosynthesis of (1,3;1,4)-β-D-glucans. Natural allelic variation in the HvCslF6 gene was found predominantly within introns among 29 barley accessions studied. Genetic manipulation of the HvCslF6 gene could enable control of (1,3;1,4)-β-D-glucans in accordance with the purposes of use.

Lipid composition and deposition during grain filling in intact barley ( Hordeum vulgare) mutant grains as studied by 1H HR MAS NMR

Conventional barley has an excellent nutritional value, but a lower content of lipids than oat. Nevertheless, barley lipids deserve a focused interest due to their fatty acid composition and their vitamin E composition. In this study, 1H HR MAS NMR was used to monitor the deposition of lipids during grain filling of conventional barley, and of two barley endosperm mutants with increased lipid content. The lipids in the mutants are primarily synthesized between days 9 and 13, whereas the lipid biosynthesis in the conventional barley took place ten days later. GC analysis on barley flour lipids showed that the mutants and the control exhibit comparable relative levels of the major fatty acids: C16:0, C18:1 and C18:2 despite the higher content of lipids in the mutants. Differences in the minor fatty acid composition between control and mutants were also observed. The degree of unsaturation was found to be increasing during grain filling for both conventional barley and the two mutants.

Information recovery from near infrared data

In many practical situations, classical modelling protocols are either inappropriate or infeasible for the recovery of an estimate of some specific property of an object, such as the protein content of wheat, from available indirect (spectroscopic) measurements. In such situations, some form of calibration-and-prediction (machine learning) is a popular alternative. For a representative set of samples, inexpensive and rapidly available indirect (spectroscopic) encapsulations of the property are recorded for each sample along with an independent laboratory measurement of the value of the specified property. Because many more indirect measurement values are recorded for each sample than the number of samples tested, the resulting system is highly under-determined in the sense of performing the calibration step: the identification of a predictor which can be applied to the indirect measurements of a new sample to predict its value of the property. Various dimension reduction methodologies have been proposed for performing the calibration step, including principal component regression, partial least squares, independent component analysis and neural network analysis. Independently, because of the high accuracy with which near infrared spectra are recorded using computer controlled instrumentation, derivative spectroscopy techniques can be utilised to explore differences in the molecular structure of cereal grains. For the optimisation of the recovery of estimates of the property from the indirect measurements of new samples, two questions are explored in this article; one practical, the other theoretical: (i)~To what extent should preprocessing (such as fourth differentiation) be applied to the indirect measurements before the calibration step is performed? (ii)~Is there an algorithmic advantage in viewing partial least squares as an implementation of simultaneous minimisation? References B. Anderssen, F. de Hoog, and M. Hegland. A stable finite difference ansatz for higher order differentiation of non-exact data. Bull. Austral. Math. Soc., 58(2):223--232, 1998. R. S. Anderssen, E. Carter, B. G. Osborne, and I. J. Wesley. Joint inversion of multi-modal spectroscopic data of wheat flours. Appl. Spectro., 59(7):920--925, JUL 2005. R. S. Anderssen and F. R. de Hoog. Finite-difference methods for the numerical differentiation of non-exact data. Computing, 33(3--4):259--267, 1984. R. S. Anderssen and R. Haraszi. Characterizing and exploiting the rheology of wheat hardness. Euro. Food Res. Tech., 229(1):159--174, MAY 2009. doi:10.1007/s00217-009-1037-9 R. S. Anderssen and M. Hegland. Derivative Spectroscopy---An enhanced role for numerical differentiation. J. Integ. Eqn. Appl., 22(3):355--367, 2010. doi:10.1216/JIE-2010-22-3-355 R. S. Anderssen, B. G. Osborne, and I. J. Wesley. The application of localisation to near infrared calibration and prediction through partial least squares regression. JNIRS, 11(1):39--48, 2003. L. Elden. Partial least-squares vs. Lanczos bidiagonalization---I: analysis of a projection method for multiple regression. Comp. Stats and Data Anal., 46(1):11--31, 2004. doi:10.1016/S0167-9473(03)00138-5 I. E. Frank. Beyond linear least-squares regression. Trac-Trends in Anal. Chem., 6(10):271--275, 1987. I. E. Frank. Intermediate least-squares regression method. Chemo. Intel. Lab. Systems, 1(3):233--242, 1987. G. H. Golub and C. Reinsch. Singular value decomposition and least squares solutions. Numer. Math., 14(5):403--420, 1970. R. Gosselin, D. Rodrigue, and C. Duchesne. A Bootstrap-VIP approach for selecting wavelength intervals in spectral imaging applications. Chemo. Intell. Lab. Systems, 100(1):12--21, 2010. doi:10.1016/j.chemolab.2009.09.005 A. Hyvarinen and E. Oja. Independent component analysis: algorithms and applications. Neural Net., 13(4--5):411--430, {} 2000. T. Naes, T. Isaksson, T. Fearn, and T. Davies. A User-Friendly Guide to Multivariate Calibration and Classification. NIR Publications, Chichester, UK, 2002. B. G. Osborne, T. Fearn, and P. H. Hindle. Practical NIR Spectroscopy with Applications in Food and Beverage Analysis. Longman Scientific and Technical, Harlow, UK, 1993. McGraw-Hill Series in Higher Mathematics. I. J. Wesley, O. Larroque, B. G. Osborne, N. Azudin, H. Allen, and J. H. Skerritt. Measurement of gliadin and glutenin content of flour by NIR spectroscopy. J. Cereal Sci., 34(2):125--133, 2001. P. R. Wiley, G. J. Tanner, P. M. Chandler, and R. S. Anderssen. Molecular classification of barley mutants using derivative spectroscopic analysis of NIR spectra of their wholemeal flours. J. Agri. Food Chem, (2), 2009.

An ATP-binding cassette subfamily G full transporter is essential for the retention of leaf water in both wild barley and rice

Land plants have developed a cuticle preventing uncontrolled water loss. Here we report that an ATP-binding cassette (ABC) subfamily G (ABCG) full transporter is required for leaf water conservation in both wild barley and rice. A spontaneous mutation, eibi1.b, in wild barley has a low capacity to retain leaf water, a phenotype associated with reduced cutin deposition and a thin cuticle. Map-based cloning revealed that Eibi1 encodes an HvABCG31 full transporter. The gene was highly expressed in the elongation zone of a growing leaf (the site of cutin synthesis), and its gene product also was localized in developing, but not in mature tissue. A de novo wild barley mutant named "eibi1.c," along with two transposon insertion lines of rice mutated in the ortholog of HvABCG31 also were unable to restrict water loss from detached leaves. HvABCG31 is hypothesized to function as a transporter involved in cutin formation. Homologs of HvABCG31 were found in green algae, moss, and lycopods, indicating that this full transporter is highly conserved in the evolution of land plants.

A Second infA Plastid Gene Point Mutation Shows a Compensatory Effect on the Expression of the Cytoplasmic Line 2 (CL2) Syndrome in Barley

The IF1 protein is one of the factors controlling translation initiation in bacteria. This protein is encoded by the infA gene, which, in several higher plants, is located in the plastome. Cytoplasmic Line 2 (CL2), an alboviridis barley mutant, was the first to be proposed as an infA gene mutation (T 157 C) in higher plants. This mutant was isolated from a chloroplast mutator genotype (cpm/cpm) and was made genetically stable by backcrosses with a wild-type nuclear genotype. In the present work, genetically stable CL2 plants were backcrossed as females by cpm/cpm plants in order to regain the mutator activity. Interestingly, a seedling carrying a first leaf blade with a darker green stripe on a typical CL2-mutant background was observed in the F(4) generation. The T 157 C transition was confirmed in tissues from the CL2 background, whereas a second transition (A 178 G) was also found in the darker stripe. Two clearly different levels of CL2 syndrome were observed in the seedlings of the F(5) and F(6) progenies. Those of the greener group carried both transitions. These results suggest a compensatory effect of the second mutation and support the involvement of the infA plastid gene in CL2 syndrome, confirming CL2 as the first mutant of this gene reported in higher plants.

Identification of barley DWARF gene involved in brassinosteroid synthesis

Brassinosteroids (BR) are plant steroid hormones regulating various aspects of morphogenesis, such as seed development and germination, cell division and elongation, differentiation of tracheary elements, development during growth in darkness (skotomorphogenesis), photosynthesis and response to environmental stress. Brassinosteroid synthesis has been studied to a great extent in the dicot species, Arabidopsis thaliana, resulting in the identification of genes participating in this process. Much less is known about BR synthesis in crops, including the monocots. The purpose of this study was to identify and characterize barley coding sequence HvDWARF involved in brassinosteroid synthesis. This sequence, encoding brassinosteroid-6-oxidase, was identified on the basis of barley ESTs. This sequence was screened for nucleotide substitutions in semi-dwarf, chemically-induced barley mutants exhibiting changes in etiolation. The responsiveness of these genotypes to exogenous brassinosteroids was determined with the use of leaf-blade segment unrolling tests. The semi-dwarf phenotype of the BR-deficient mutants was rescued by the application of 10−5 M 24-epi-brassinolide. Two missense mutations were identified within the HvDWARF sequence in BR-deficient mutants 522DK and 527DK from variety ‘Delisa’. These substitutions cause changes of amino acid residues located within the conserved fragments of the encoded polypeptide. The transcription profile of HvDWARF and HvBAK1/SERK3, involved in BR signaling, was determined during the early stages of seedling development in BR-deficient and BR-insensitive mutants using real-time quantitative PCR. This analysis indicated that HvDWARF displays a uniformly low level of this process, whereas the transcription level of HvBAK1 proved to be spatially and temporally regulated.

Differential disease resistance response in the barley necrotic mutant nec1

BackgroundAlthough ion fluxes are considered to be an integral part of signal transduction during responses to pathogens, only a few ion channels are known to participate in the plant response to infection. CNGC4 is a disease resistance-related cyclic nucleotide-gated ion channel. Arabidopsis thaliana CNGC4 mutants hlm1 and dnd2 display an impaired hypersensitive response (HR), retarded growth, a constitutively active salicylic acid (SA)-mediated pathogenesis-related response and elevated resistance against bacterial pathogens. Barley CNGC4 shares 67% aa identity with AtCNGC4. The barley mutant nec1 comprising of a frame-shift mutation of CNGC4 displays a necrotic phenotype and constitutively over-expresses PR-1, yet it is not known what effect the nec1 mutation has on barley resistance against different types of pathogens.Resultsnec1 mutant accumulated high amount of SA and hydrogen peroxide compared to parental cv. Parkland. Experiments investigating nec1 disease resistance demonstrated positive effect of nec1 mutation on non-host resistance against Pseudomonas syringae pv. tomato (Pst) at high inoculum density, whereas at normal Pst inoculum concentration nec1 resistance did not differ from wt. In contrast to augmented P. syringae resistance, penetration resistance against biotrophic fungus Blumeria graminis f. sp. hordei (Bgh), the causal agent of powdery mildew, was not altered in nec1. The nec1 mutant significantly over-expressed race non-specific Bgh resistance-related genes BI-1 and MLO. Induction of BI-1 and MLO suggested putative involvement of nec1 in race non-specific Bgh resistance, therefore the effect of nec1on mlo-5-mediated Bgh resistance was assessed. The nec1/mlo-5 double mutant was as resistant to Bgh as Nec1/mlo-5 plants, suggesting that nec1 did not impair mlo-5 race non-specific Bgh resistance.ConclusionsTogether, the results suggest that nec1 mutation alters activation of systemic acquired resistance-related physiological markers and non-host resistance in barley, while not changing rapid localized response during compatible interaction with host pathogen. Increased resistance of nec1 against non-host pathogen Pst suggests that nec1 mutation may affect certain aspects of barley disease resistance, while it remains to be determined, if the effect on disease resistance is a direct response to changes in SA signaling.

Identification of root morphology mutants in barley

In this study, a forward-genetics analysis was performed on a portion of TILLMore, a chemically mutagenized population of barley cv. ‘Morex’ (http://www.distagenomics.unibo.it/TILLMore/), to identify root morphology alterations by comparison with ‘Morex’ wild-type. For this purpose, a simple paper-roll approach was performed to identify phenotypic variants at the seedling stage. The analysis of c. 1000 M4 families allowed us to identify c. 70 lines with altered root morphology. A more accurate phenotypic characterization of a portion of the mutant lines has been performed using stereomicroscopy and a scanning electron microscopy approach.

Responses of chlorophyll b-less chlorina 3613 mutant of barley to a prolonged decrease in illuminance: 2. Dynamics of carotenoids in leaf chloroplasts

Barley (Hordeum vulgare L.) mutant chlorina 3613 is notable for a lack of chlorophyll b (Chl b), low content of chlorophyll a (Chl a) and carotenoids in the chloroplasts, as well as reduction in the majority of components of LHCI and LHCII. Incompletely developed photosynthetic machinery of chlorina 3613 results in suppressed growth, lower biomass, and the declined rate of photosynthesis (as compared with the wild-type cv. Donaria). The lack of Chl b and greater part of peripheral antenna suggests that this mutant will have difficulties during acclimation to long-term shading because the light-harvesting role of Chl b-containing antenna becomes more important under the shortage of light. Earlier, our experiments with the mature chlorina 3613 plants shaded for one week at PAR photon flux density of 60 and 40% of that in full sunlight showed a stimulating effect of shading on growth, biomass accumulation, and Chl a synthesis in chlorina 3613 when biosynthesis of Chl b did not occur [1]. In this work, we investigated in more detail the changes in the content of carotenoids in chlorina 3613. We found that in Donaria at both investigated levels of illumination (60 and 40% of full sunlight) and in chlorina 3613 at 60% illumination, moderate reversible changes typical of shade-enduring plants occur in the content of carotenoids. In chlorina 3613 at 40% illumination, the content of β-carotene increased considerably (by 3 times) with simultaneous accumulation of Chl a. When full illumination was restored, the content of β-carotene decreased and remained on the level, which exceeded its initial content in the plants without shading by 38%; this level, was maintained by the end of vegetation. The changes in the contents of β-carotene and Chl a in chlorina 3613 were not accompanied by any accumulation of xanthophylls or changes in the relative content of active violaxanthin. The obtained results suggest that a long-term shading of the leaves of mature chlorina 3613 plants induced the formation of certain components of photosynthetic apparatus: reactive centers and core parts of photosystems’ antennae as well as proteins CP26 and CP29 and in this way contributed to partial restoration of photosynthetic activity and production process in the mutant lacking Chl b.

New allele of HvBRI1 gene encoding brassinosteroid receptor in barley

The aim of these studies was to characterize nucleotide substitutions leading to the phenotype of brassinosteroid-insensitive, semi-dwarf barley mutant 093AR. Two substitutions in the sequence of barley HvBRI1 gene, encoding leucine-rich repeats receptor kinase (LRR-RK), which participates in brassinosteroid (BR) signalling, were identified in this chemically-induced barley mutant of the cv. Aramir. The LRR-RK is a transmembrane protein phosphorylating downstream components. The identified substitutions CC>AA at positions 1760 and 1761 in the HvBRI1 gene of this mutant led to a missense mutation, causing the Thr-573 to Lys-573 replacement in the protein sequence. The threonine residue is situated in the distal part of a 70-amino acids island responsible for binding of BR molecules. As this residue is conserved among BRI1 protein homologs in Arabidopsis thaliana, Lycopersicon esculentum, Oryza sativa and Hordeum vulgare, it was postulated that this residue is crucial for the protein function. The genetic analyses indicated that the mutant 093AR was allelic to the spontaneous, semi-dwarf mutant uzu which carries A>G substitution at position 2612 of the HvBRI1 gene (GenBank acc. no. AB088206). A comparison of the genomic sequence of HvBRI1 in the mutants uzu, 093AR and in the cv. ‘Aramir’ confirmed the presence of the single-nucleotide A>G substitution at position 2612 in the sequence encoding kinase domain of HvBRI1 polypeptide in uzu, but not in 093AR mutant, indicating that a new allele of the HvBRI1 gene was identified.

A nonsense mutation in a putative sulphate transporter gene results in low phytic acid in barley

Low phytic acid grains can provide a solution to dietary micronutrient deficiency and environmental pollution. A low phytic acid 1-1 (lpa1-1) barley mutant was identified using forward genetics and the mutant gene was mapped to chromosome 2HL. Comparative genomic analysis revealed that the lpa1-1 gene was located in the syntenic region of the rice Os-lpa-MH86-1 gene on chromosome 4. The gene ortholog of rice Os-lpa-MH86-1 (designated as HvST) was isolated from barley using polymerase chain reaction and mapped to chromosome 2HL in a doubled haploid population of Clipper×Sahara. The results demonstrate the collinearity between the rice Os-lpa-MH86-1 gene and the barley lpa1-1 region. Sequence analysis of HvST revealed a single base pair substitution (C→T transition) in the last exon of the gene in lpa1-1 (M422), which resulted in a nonsense mutation. These results will facilitate our understanding of the molecular mechanisms controlling the low phytic acid phenotype and assist in the development of a diagnostic marker for the selection of the lpa1-1 gene in barley.