Jasmonate-induced changes in flavonoid metabolism in barley (Hordeum vulgare) leaves

The effects of jasmonic acid (JA) on secondary metabolism in barley (Hordeum vulgare L.) were investigated. A reversed-phase HPLC analysis revealed that the amount of a particular compound increased in excised barley leaf segments that had been treated with JA. This compound was purified and identified as 6'-feruloylsaponarin (1) by spectroscopic analyses and alkaline hydrolysis. A related compound, 6'-sinapoylsaponarin (2), was also found to accumulate in excised leaves independently of the JA treatment. The accumulation of these compounds was accompanied by a decrease in the saponarin (3) content. [8,9-(13)C]p-Coumaric acid and [2,3,4,5,6-(2)H]L-phenylalanine were effectively incorporated into the hydroxycinnamoyl moieties in 1 and 2, while the degree of incorporation of the labeled precursors into the saponarin part was small. These findings indicate that the hydroxycinnamoyl moieties of 1 and 2 are synthesized de novo from phenylalanine via the phenylpropanoid pathway, and that the saponarin part is mainly provided by the constitutive pool of 3.     

Fractionation and characterization of histones from barley (Hordeum vulgare) leaves

Histones were extracted from purified nuclei isolated from four cereals,viz. barley (Hordeum vulgare), wheat(Triticum aestivum), Aegilops squarrosa and corn (Zea mais), and from tobacco (Nicotiana tabacum). Analysis of the histones on SDS gels showed complex electrophoretic patterns with one species of both H3 and H4, one to three species of H1 and two to five species of H2. Judged from the electrophoretic patterns, the histones from barley, wheat and Aegilops are identical but different from those of corn with respect to H2. Like tobacco, corn showed two H2 components, whereas barley, wheat and Aegilops showed five H2 components. SDS gel electrophoresis of histones extracted from buds and roots of germinating seeds at different steps of germination and from different parts of ten-day-old leaves revealed that the existence in barley of multiple histone 2 variants is not restricted to any particular stage of differentiation of barley. Histones from barley leaves were resolved into four fractions by Biogel P-100 gel filtration and histones 2 were further fractionated by their differential solubility in HCl-ethanol. Each of these five fractions (H1, H3, H4, H2A and H2B, respectively) were characterized by electrophoresis on SDS or Triton-acid-urea gels and by their amino acid compositions as compared with the homologous histones of calf thymus and chicken erythrocytes. This revealed the following:

1. H3 and H4 are strictly analogous to their animal counterparts. However, H4 has an unexplained lower electrophoretic mobility in Triton-containing acid-urea gels.
2. H1 contains three components with lower electrophoretic mobilities than H1 from erythrocytes, contains more alanine than lysine and has a lower ratio of basic to acidic residues.
3. Both H2A and H2B contain at least four variants each, with higher molecular weights than in animals and higher lysine to arginine ratios. H2A variants comigrate in acid-urea-Triton gels with chicken erythrocytes H2A, whereas H2B migrate much slower.

It was concluded that the presence of multiple major variants of H2A and H2B is a frequent but not universal feature in cereals. The existence of these variants is not restricted to the embryonic stage as previously suggested for wheat (31).     

Genetical analysis of microspore derived plants of barley (Hordeum vulgare)

Summary From an F₁ hybrid between the two barley (Hordeum vulgare L.) cultivars Golden Promise and Mazurka a series of doubled haploid (DH) lines were generated both from microspores by anther culture and from immature zygotic embryos after hybridization withH. bulbosum. The DH lines from both sources were used to monitor the segregation of the five major genes, rachilla hair length, DDT susceptibility, height, C hordein polymorphism and mildew resistance. Whereas the microspore-derived samples showed significant departures from the expected 11 ratio for three of the five genes, theH. bulbosum lines showed deviation for only one gene. Analysis of linkage data also showed differences between the two series of DH lines. Cytogenetic analysis revealed a mean chiasma frequency in theH. bulbosum lines which was very similar to the F₁ hybrid. In contrast, four of the ten microspore derived lines examined showed a reduced chiasma frequency. One showed evidence of translocation heterozygosity.     

Chlorophyll synthesis in green leaves and isolated chloroplasts of barley (Hordeum vulgare)

The photoreduction of protochlorophyllide was studied in leaves and isolated chloroplasts of barley. Leaves of plants which had been preilluminated for varying lengths of time were incubated with [¹⁴C]-δ- aminolevulinic acid for 2 h in the dark. The subsequent photoreduction of [¹⁴C]-protochlorophyllide was analyzed by high performance liquid chromatography of pigments extracted from illuminated leaves and plastids. The plastids used in this study were isolated in the dark from leaves at the end of the 2 h labelling period. Three major results were obtained:

• 1

  The extent of protochlorophyllide reduction in vivo was rapidly reduced as a function of the preillumination period. In 24 h preilluminated plants only a small fraction of the radioactively labelled protochlorophyllide was reduced during the subsequent light period.
• 2

  The amount of NADPH-protochlorophyllide oxidoreductase (EC 1.6.99.-) present in plastids of fully-green plants was drastically reduced relative to levels in plastids of dark-grown plants as estimated by the methods of immunoblotting of plastid proteins and immunogold labelling of ultrathin sections of the leaf tissue.
• 3

  In etiolated plants light seemed to affect the reduction of protochlorophyllide directly through the excitation of protochlorophyllide. In fully green plants, however, light also affected chlorophyll formation indirectly by the supply of NADPH via photosynthetic electron transport.

     

Cuticular transpiration and epicuticular lipids of primary leaves of barley (Hordeum vulgare)

Twenty cultivars of barley and 15 eceriferum mutants from one of the cultivars have been analysed for cuticular transpiration and epicuticular lipids of their primary leaves. The relative cuticular transpiration rates of the cultivars ranged from 0.61 to 1.98. In spite of this variation in transpiration most of the cultivars had almost the same amount of epicuticular lipids per leaf area, about 16 μg cm⁻². The eceriferum mutants showed a wider range in amount of epicuticular lipids, from 5.0 to 15.5 μg cm⁻². Nevertheless, most of the mutants transpired almost at the same rate. Only a weak correlation was found between cuticular transpiration and total amount of epicuticular lipids. None of the analysed lipid components (alkanes, aldehydes, primary alcohols, esters or fatty acids) was better correlated to the cuticular transpiration than the total amount of lipids. When the cultivars were exposed to a mild water stress their cuticular transpiration rates decreased by about 11%. This reduction was not accompanied by any corresponding increase in total amount of epicuticular lipids. The most pronounced effect of the water stress treatment was a stimulation in the ester formation and a reduced formation of primary alcohols. This shift in lipid composition could not be correlated to the decreased cuticular transpiration rates of the individual cultivars. From this investigation it is concluded that the cuticular transpiration is poorly correlated to the amount or composition of the epicuticular lipids in this barley material. As a consequence it was not possible to use any characteristic of the epicuticular lipids as a selection criterion in breeding for drought resistance.     

The mis-identification of the major antioxidant flavonoids in young barley (Hordeum vulgare) leaves

Several papers have appeared in the literature since 1992 which refer to a major "isoflavonoid" antioxidant in young green barley leaves (Hordeum vulgare) as 2'-O-glucosylisovitexin. In the present paper the original NMR data supporting this structural assignment are examined and found to have been misinterpreted. HPLC and NMR data are used to prove that the major flavonoid antioxidants in young green barley leaves are in fact the flavone-C-glycosides, saponarin and lutonarin.     

Identification and characterization of novel senescence-associated genes from barley (Hordeum vulgare) primary leaves

Leaf senescence is the final developmental stage of a leaf. The progression of barley primary leaf senescence was followed by measuring the senescence-specific decrease in chlorophyll content and photosystem II efficiency. In order to isolate novel factors involved in leaf senescence, a differential display approach with mRNA populations from young and senescing primary barley leaves was applied. In this approach, 90 senescence up-regulated cDNAs were identified. Nine of these clones were, after sequence analyses, further characterized. The senescence-associated expression was confirmed by Northern analyses or quantitative RealTime-PCR. In addition, involvement of the phytohormones ethylene and abscisic acid in regulation of these nine novel senescence-induced cDNA fragments was investigated. Two cDNA clones showed homologies to genes with a putative regulatory function. Two clones possessed high homologies to barley retroelements, and five clones may be involved in degradation or transport processes. One of these genes was further analysed. It encodes an ADP ribosylation factor 1-like protein (HvARF1) and includes sequence motifs representing a myristoylation site and four typical and well conserved ARF-like protein domains. The localization of the protein was investigated by confocal laser scanning microscopy of onion epidermal cells after particle bombardment with chimeric HvARF1-GFP constructs. Possible physiological roles of these nine novel SAGs during barley leaf senescence are discussed.     

Primary callus as source of totipotent barley (Hordeum vulgare L.) protoplasts

Summary Primary callus of barley (Hordeum vulgare L.) derived from scutella (cv. Dissa) and anthers (cv. Igri) was used for protoplast isolation and plant regeneration. The protoplasts were embedded in agarose and cultured with nurse cells. The plating efficiency varied from 0.1% to 0.7%. Shoots regenerated from the developing callus. Plantlets were transferred to soil and cultivated in the greenhouse three to five months after protoplast isolation. All plants were normal in morphology, and most of them flowered and set seeds.     

Photosynthate partitioning in diploid and autotetraploid barley (Hordeum vulgare)

Net rates of carbon assimilation per unit leaf area by fully expanded, vegetative leaves of diploid (2x) and autotetraploid (4x) barley (Hordeum vulgare L. cultivars OAC-21 and Brant) were not significantly different (90% level) when measured under controlled environment conditions with air levels of CO₂ and either 2 or 20% O₂. Leaf thickness increased with ploidy so that net photosynthetic rates measured on single leaves were lower for 4x than 2x barley varieties when compared on a dry or fresh weight basis. Rates of ¹⁴CO₂ fixation by isolated mesophyll protoplasts prepared from seedlings were also lower for 4x than 2x varieties [about 108 and 125 μmol (mg ChI)^?1 h^?1, respectively]. Carbohydrate accumulation in leaves of 5-weekold plants averaged 28% (2x) and 47% (4x) of the total photosynthetic weight gain during the first 9 h of the light period. Estimated photoassimilate export from leaves was 15% (OAC-21) and 38% (Brant) lower for 4x compared to 2x isolines. The sucrose and oligofructan content of 4x compared to 2x leaves increased as a result of decreased photosynthate transport. Total tiller dry weight of plants raised in a glasshouse was greater for 4x than 2x barley varieties at ear emergence, but tiller height decreased with increasing ploidy. The nonstructural carbohydrate content of the inflorescence, leaves and lower stem organs was significantly (P≤ 0.01) higher in 4x than in 2x lines at this sampling. During the first 15 days of grain development total tiller dry weight increased by 46% (2x) compared to 8% (4x) when the results of both varieties were averaged together. The dry weight gain of the ear during this period was about 60 to 80% lower for 4x compared to 2x isolines. The nonstructural carbohydrate content of the inflorescence was also about 24% (Brant) and 51% (OAC-21) lower for 4x as compared to 2x plants 15 days post ear emergence. The above results suggested that photosynthate partitioning in autotetraploid barley was sink-limited.     

Aldehyde oxidase in roots, leaves and seeds of barley (Hordeum vulgare L.)

Aldehyde oxidase (AO, EC 1.2.3.1) proteins in leaves, roots and seeds of barley (Hordeum vulgare L.) plants were studied. Differences in substrate specificity and mobility in native PAGE between AO proteins extracted from roots, leaves and seeds have been observed. Four clear bands of AO reacting proteins were detected in barley plants capable of oxidizing a number of aliphatic and aromatic aldehydes such as indole-3-aldehyde, acetaldehyde, heptaldehyde, and benzaldehyde. Mouse polyclonal antibodies raised against purified maize AO cross-reacted with barley AO proteins extracted from roots, leaves and seeds. At least three different AO proteins were detected in roots on the basis of their mobility during PAGE after native Western blot analysis while in leaves and seeds only one polypeptide cross-reacted with the antibody. SDS-immunoblot analysis showed marked differences in molecular weight between subunits of the AO bands extracted from roots, leaves and seeds. Two distinct subunit bands were observed in roots, with relatively close molecular weights (160 kDa and 145 kDa), while a single subunit with a molecular weight of 150 kDa was observed in leaf and seed extracts.Menadione, a specific and potent inhibitor of animal AO did not affect barley AO proteins. Root and leaf AO differed in their thermostability and susceptibility to exogenous tungstate. The AO proteins in plants may be a group of enzymes with different substrate specificity, tissue distribution and presumably fulfilling different metabolic roles in each plant organ.     

Kernel texture and hordoindoline patterns in barley (Hordeum vulgare)

Hordoindolines, the tryptophan-rich polypeptides affecting grain hardness in barley, appeared as three pairs of polypeptides in the acidic polyacrylamide gel electrophoresis (A-PAGE) and two-dimensional A-PAGE?×?SDS-PAGE patterns of starch-granule proteins from 18 barley cultivars. On capillary RP-HPLC/nESI-MS/MS spectrometry, one pair of polypeptides was found to correspond to hordoindoline A (HINA), one to hordoindoline B1 (HINB1) and one to hordoindoline B2 (HINB2), the two polypeptides of each pair deriving from post-translational cleavage of a native hordoindoline at different positions at the N-terminus and/or C-terminus. Amongst the barley cultivars analyzed, cvs Hart and Sundance, which were claimed to be unique in lacking the Hina gene coding for HINA, revealed similar Hina coding sequences and accumulated hordoindoline HINA on their starch granules. The amount of total hordoindolines (HINA?+?HINB1?+?HINB2) on the starch granules, as quantified by densitometric scanning of A-PAGE gels, was comparable with that of puroindolines (PINA?+?PINB) in soft-textured wheat. By contrast, the amount of B-type hordoindolines (HINB1 and HINB2 combined) was 50?% lower than that of PINB, suggesting that the absence of barley cultivars with soft kernels is likely due to the reduced amount of B-type hordoindolines accumulated on the starch granules. Approximately 22 and 27?% of the phenotypic variation for kernel hardness in 56 barley cultivars analyzed by the Single Kernel Characterization System (SKCS) were explained by differences in kernel weight and B-type hordoindoline level, respectively. By contrast, the outer husk of barley grain showed no effect on the SKCS index.     

Latent manganese deficiency increases transpiration in barley (Hordeum vulgare)

To investigate if latent manganese (Mn) deficiency leads to increased transpiration, barley plants were grown for 10 weeks in hydroponics with daily additions of Mn in the low n M range. The Mn-starved plants did not exhibit visual leaf symptoms of Mn deficiency, but Chl a fluorescence measurements revealed that the quantum yield efficiency of PSII (F_v/F_m) was reduced from 0.83 in Mn-sufficient control plants to below 0.5 in Mn-starved plants. Leaf Mn concentrations declined from 30 to 7 μg Mn g⁻¹ dry weight in control and Mn-starved plants, respectively. Mn-starved plants had up to four-fold higher transpiration than control plants. Stomatal closure and opening upon light/dark transitions took place at the same rate in both Mn treatments, but the nocturnal leaf conductance for water vapour was still twice as high in Mn-starved plants compared with the control. The observed increase in transpiration was substantiated by ¹³C-isotope discrimination analysis and gravimetric measurement of the water consumption, showing significantly lower water use efficiency in Mn-starved plants. The extractable wax content of leaves of Mn-starved plants was approximately 40% lower than that in control plants, and it is concluded that the increased leaf conductance and higher transpirational water loss are correlated with a reduction in the epicuticular wax layer under Mn deficiency.     

Alkylresorcinols in barley (Hordeum vulgare L. distichon) grains

This study was carried out to compare grains of barley (Hordeum vulgare L. distichon) regarding contents and compositions of 5-n-alkylresorcinols. Mixtures of resorcinol homologues were isolated from acetone extracts from five barley cultivars. These polyketide metabolites were identified by chromatographic and spectroscopic means. The content and homologue patterns among different varieties were similar. The predominant compounds were 1,3-dihydroxy-5-n-heneicosylbenzene (C21:0), 1,3-dihydroxy-5-n-nonadecylbenzene (C19:0) and 1,3-dihydroxy-5-n-pentacosylbenzene (C25:0). The alkylresorcinol concentrations, in contrast to their compositions, depended on environmental and agricultural factors.     

Effects of nitrogen deficiency on accumulation of fructan and fructan metabolizing enzyme activities in sink and source leaves of barley (Hordeum vulgare)

Seedlings of barley ( Hordeum vulgare L. cv. Agneta) were grown hydroponically under continuous light, constant temperature and relative humidity. During the first two weeks, the relative growth rate (RGR) was kept at 25% by limiting only the supply of nitrogen. The cultures were then transferred to nitrogen-free media and the amounts of fructan, starch, sucrose, glucose and fructose in sink and source leaves were measured at 0, 12, 24, 48, 72, 120 and 156 h. The activities of two key enzymes in fructan metabolism, sucrose:sucrose fructosyltransferase (SST), fructan exohydrolase (FEH), as well as acid invertase were also measured in the two types of leaves.
The fructan and starch levels in both sink and source leaves increased during nitrogen deficiency. The highest increase in starch was 200% of the control while for fmctans a 700% increase was recorded. The activity of SST increased parallel to fructan accumulation in sink leaves. However the FEH activity was constant and not affected by nitrogen deficiency. The invertase activity both in sink and source leaves was reduced by nitrogen deficiency. More fructans as well as sucrose and fructose accumulated in source leaves compared to sink leaves both before and after nitrogen starvation. The results show that fructan is the major carbohydrate reserve accumulating under nitrogen deficiency both in sink and source leaves in barley plants. The induction of fructan accumulation in sink leaves caused by nitrogen deficiency is intimately connected with the regulation of SST     

Morphological and physiological analysis of cleistogamy in barley (Hordeum vulgare)

We previously reported that cleistogamy/chasmogamy (CL/CH) of barley ( Hordeum vulgare L.) is controlled by either two tightly linked genes or one gene with multiple alleles. To clarify the morphological and physiological mechanisms of barley CL, we analysed the lodicule size and auxin response of two cultivars whose CL/CH was controlled by two different genes, cly1 and Cly2 . In both cases, lodicules of the CL parent were smaller than those of the CH parent. Analyses of lodicule size and flowering phenotype of f ₁ plants and segregation analyses of the mapping population indicated that lodicule size co-segregated with the flowering phenotype. Indole-3-acetic acid (IAA) and other synthetic auxins, such as 2,4-dichlorophenoxyacetic acid, induced abnormal flowering in CH ears, in which florets remained open for a few days instead of the normal hour or so, but not in CL ears. This auxin effect also co-segregated with the flowering phenotype. Analyses of auxin-related compounds in the floret organs revealed that the anther contained high levels of IAA, whereas indole-3-carboxylic acid, a putative decarboxylated metabolite of IAA, accumulated only in lodicules of CH plants just at flowering. These results indicate that lodicule size and auxin response are pleiotropic effects of the CL gene, which may play a role in auxin response or metabolism.     

Discovery and assay of single-nucleotide polymorphisms in barley (Hordeum vulgare)

The least ambiguous genetic markers are those based on completely characterized DNA sequence polymorphisms. Unfortunately, assaying allele states by allele sequencing is slow and cumbersome. The most desirable type of genetic marker would be unambiguous, inexpensive to assay and would be assayable singly or in parallel with hundreds of other markers (multiplexable). In this report we sequenced alleles at 54 barley (Hordeum vulgare ssp. vulgare) loci, 38 of which contained single-nucleotide polymorphisms (SNPs). Many of these 38 loci contained multiple polymorphisms, and a total of 112 polymorphisms were scored in five barley genotypes. The polymorphism data set was analyzed both by using the individual mutations as cladistic characters and by reducing data for each locus to haplotypes. We compared the informativeness of these two approaches by consensus tree construction and bootstrap analysis. Both approaches provided similar results. Since some of the loci sequenced contained insertion/deletion events and multiple point mutations, we thought that these multiple-mutated loci might represent old alleles that predated the divergence of barley from H. spontaneum. We evaluated sequences from a sample of H. spontaneum accessions from the Eastern Mediterranean, and observed similar alleles present in both cultivated barley and H. spontaneum, suggesting either multiple domestication events or multiple transfers of genes between barley and its wild ancestor.     

Ring chromosomes in barley (Hordeum vulgare L.)

A plant with 2n = 14 + 1 ring chromosomes was obtained in the progeny of a primary trisomie for chromosome 7 of a two-rowed cultivar, Shin Ebisu 16. The morphological characteristics of the trisomic plants with an extra ring chromosome were similar to the primary trisomic for chromosome 7 (Semierect), which suggests that it originated from this chromosome. The ring chromosomes were not completely stable in mitotic cells because of abnormal behavior. Chromosome complements varied in different plants and in different roots within a plant. Root tip cells and spikes with 2n = 14 and 14 + 2 ring chromosomes were observed on plants with 14 + 1 ring chromosomes. Breakage-fusion-bridge cycle was inferred. The ring chromosome was associated with two normal homologues forming a trivalent in 17.6% sporocytes at metaphase I. The transmission of the extra ring chromosome was 23.1% in the progeny of the plant with 14 + 1 ring chromosomes. Trivalent formation may have been much higher at early prophase stages which were difficult to analyze in barley; only 4 of 120 sporocytes analyzed showed an isolated ring at pachytene. The ring chromosome moved to one pole without separation in 24.7% of the sporocytes at AI, and divided in 27.1% sporocytes giving rise to 8-8 separation. Only 10% of the sporocytes showed bridge formation at AI.     

Molecular organization of the barley (Hordeum vulgare) genome]

Studies in peculiarities of the DNA secondary structure in barley by means of thermal denaturation and renaturation shows that there are three types of the nucleotide sequences organization in DNA. More than 95% of the genome composition contain distributed repetitive sequences, in one part of the concentration of the repetitive sequences being higher as compared to bulk of them. About 3.5% of DNA is enriched with A-T pairs and contains no repetitive sequences. There is no "unique" part in the barley genome, which is natural for animals. Slowly renaturation sequences repeat 4 times.