Differential expression of dehydrin genes in wild barley, Hordeum spontaneum, associated with resistance to water deficit

Dehydrin gene (Dhn) expression is associated with plant response to dehydration. The aim of the present study was to investigate the association of differential expression of Dhn genes (Dhn 1, 3, 5, 6, and 9) with drought tolerance found in wild barley (Hordeum spontaneum). Tolerant and sensitive genotypes were identified from Israeli (Tabigha microsite) and Jordanian (Jarash and Waddi Hassa) populations (based on scoring of water loss rate of 390 genotypes). The five Dhn genes were up‐regulated by dehydration in resistant and sensitive wild barley genotypes. Notably, differences between resistant and sensitive genotypes were detected, mainly in the expression of Dhn1 and Dhn6 genes, depending on the duration of dehydration stress. Dhn1 tended to react earlier (after 3 h) and higher (12 h and 24 h) in resistant compared to sensitive genotypes. The level of expression of Dhn6 was significantly higher in the resistant genotypes at the earlier stages after stress. However, after 12 and 24 h Dhn6 expression was relatively higher in sensitive genotypes. The present results may indicate that these genes have some functional role in the dehydration tolerance in wild barley. The authors suggest that the observed differences of Dhn expression in wild barley, originating from different micro‐ and macro ecogeographic locations, may be the result of adaptive edaphic and climatic selective pressures.     

Genetic analysis of the accumulation of COR14 proteins in wild (Hordeum spontaneum) and cultivated (Hordeum vulgare) barley

The cold-regulated (COR14) protein of 14 kDa is a polypeptide accumulated under low-temperature conditions in the chloroplasts of barley leaves. In H. vulgare the COR14 antibody cross-reacts with two proteins, with a slightly different relative molecular weight around the marker of 14.4 kDa, referred to as COR14a and COR14b (high and low relative molecular weight, respectively). In a collection of H. spontaneum genotypes a clear polymorphism was found for the corresponding COR proteins. While some accessions showed the same COR pattern as cultivated barley, in 38 out of 61 accessions examined the COR14 antibody cross-reacted with an additional coldregulated protein with a relative molecular weight of about 24 kDa (COR24). The accumulation of COR24 was often associated with the absence of COR14b; the relationship between the COR14b/COR24 polymorphism and the adaptation of H. spontaneum to different environments is discussed. By studying COR14 accumulation in cultivated barley we have found that the threshold induction-temperature of COR14a is associated with the loci controlling winter hardiness. This association was demonstrated by using either a set of 30 cultivars of different origin, or two sets of frost-tolerant and frost-sensitive F1 doubled-haploid lines derived from the cross Dicktoo (winter type) x Morex (spring type). These results suggest that the threshold induction-temperature of COR14a can be a potential biochemical marker for the identification of superior frostresistant barley genotypes.     

Longitudinal leaf gradients of UV-absorbing screening pigments in barley (Hordeum vulgare)

In vivo leaf characteristics were examined to describe longitudinal gradients of UV-absorbing screening pigments in barley. Chlorophyll fluorescence properties and in vivo absorption spectra (210–750 nm) of leaves were measured from the base to the tip. Barley leaves showed strong longitudinal gradients of chlorophyll, where chlorophyll concentration increased within the first 5–8 cm from the leaf base, and did not significantly change for the remaining part of the leaf. Fluorescence microscopy was used to localize cell wall bound screening pigments different from flavonoids, since flavonoids lack a blue-green fluorescence emission (Lichtenthaler and Schweiger 1998). Measurements of in vivo chlorophyll fluorescence indicated that the ratio of UV-absorbing screening pigments per leaf area increases from the leaf base to the tip. These gradients were confirmed by in vivo absorption spectra. It is demonstrated that leaves in the early stage of development are less protected against UV-radiation than fully developed mature leaf regions. The experiments show that measurements of in vivo chlorophyll fluorescence are ideally suited as a fast non-invasive tool to estimate the epidermal UV-transmittance in different leaf sections.     

Tocopherol and tocotrienol accumulation during development of caryopses from barley (Hordeum vulgare L.)

Tocotrienols are lipophilic antioxidants belonging to the tocochromanols, better known as vitamin E. Although present in cereal grains in high quantities not much is known about their function in plants. In a detailed study the temporal and spatial accumulation of tocotrienols and tocopherols during grain development in two barley cultivars was analyzed. Tocochromanols and lipids accumulated in parallel until 80% of the final dry weight of the kernels was reached. Later on the tocochromanol content did not change while the lipid content decreased. Generally, only about 13% of the tocochromanols were found in the germ fraction, whereas the pericarp fraction contained about 50% and the endosperm fraction about 37% of the tocochromanols. Altogether, about 85% of the tocochromanols were tocotrienols in both cultivars. In case of the tocopherols about 80% were found in the germ fraction and the remaining 20% in the pericarp fraction. Tocotrienols were almost equally present in the pericarp and the endosperm fraction. Individual forms of tocopherols and tocotrienols accumulated with different kinetics during barley grain development. The differences in distribution and accumulation indicate different functions of the individual tocochromanols during grain development.     

Differential effect of ammonium on the induction of nitrate and nitrite reductase activities in roots of barley (Hordeum vulgare) seedlings

The effect of exogenous NH₄⁺ on the induction of nitrate reductase activity (NRA; EC 1.6.6.1) and nitrite reductase activity (NiRA; EC 1.7.7.1) in roots of 8-day-old intact barley (Hordeum vulgare L.) seedlings was studied. Enzyme activities were induced with 0.1, 1 or 10 mM NO₃⁺ in the presence of 0, 1 or 10 mM NH₄⁺, Exogenous NH₄⁺ partially inhibited the induction of NRA when roots were exposed to 0.1 mM, but not to 1 or 10 mM NO₃⁺, In contrast, the induction of NiRA was inhibited by NH₄⁺ at all NO₃⁺ levels. Maximum inhibition of the enzyme activities occurred at 1.0 mM NH₄⁺ Pre-treatment with NH₄⁺ had no effect on the subsequent induction of NRA in the absence of additional NH₄⁺ whereas the induction of NiRA in NH₄⁺-pretreated roots was inhibited in the absence of NH₄⁺ At 10 mM NO₃⁺ L-methionine sulfoximine stimulated the induction of NRA whether or not exogenous NH₄⁺ was present. In contrast, the induction of NiRA was inhibited by L-methionine sulfoximine irrespective of NH₄⁺ supply. During the postinduction phase, exogenous NH₄⁺ decreased NRA in roots supplied with 0.1 mM but not with 1mM NH₃⁺ whereas, NiRA was unaffected by NH₄⁺ at either substrate concentration. The results indicate that exogenous NH₄⁺ regulates the induction of NRA in roots by limiting the availability of NO₃⁺. Conversely, it has a direct effect, independent of the availability of NO₃⁺, on the induction of NiRA. The lack of an NH₄⁺ effect on NiRA during the postinduction phase is apparently due to a slower turnover rate of that enzyme.     

The regulation of leaf elongation and xyloglucan endotransglycosylase by gibberellin in 'Himalaya' barley (Hordeum vulgare L.)

The potential role of xyloglucan endotransglycosylase (XET)in GA-stimulated cell elongation was investigated during leafexpansion in barley (Hordeum vulgare L.). XET activity in aqueousextracts of leaves was detected in all segments along the elongatingblade of leaf 1 of seedlings, but was at highest levels in basalsegments. Leaf 1 elongation rates of gibberellin (GA)-responsivedwarf mutants were lower than the wild type, and accompaniedby reduced levels of XET activity. Leaf elongation rates ofthe dwarfs increased following treatment with gibberellic acid(GA₃) associated with higher levels of XET activity. The slendermutant, crossed into a dwarfing background, exhibited high ratesof leaf 1 elongation and high levels of XET activity withoutadded GA₃. The elongation of leaf 3 in a GA-responsive dwarfmutant was also studied. Treatment with GA₃ resulted in bladeand sheath lengths being 5-fold and 7-fold (respectively) thelengths of controls, and again there were increases in bladeand sheath XET activities. To investigate the basis for changesin XET activity levels two XET-related cDNA clones were isolated.RNAs detected by the two clones occurred at the highest levelsin basal segments of rapidly elongating leaves, but they haddifferent distribution patterns along the leaf. Overall, thedata indicate that an XET-like activity is detectable in barleyleaves, that the activity level and related. Key words: Gibberellin (GA), leaf elongation, Hordeum vulgare, xyloglucan endotransglycosylase (XET)     

HvPIP1;6, a barley (Hordeum vulgare L.) plasma membrane water channel particularly expressed in growing compared with non-growing leaf tissues

The aim of the present study was to identify water channel(s) which are expressed specifically in the growth zone of grass leaves and may facilitate growth-associated water uptake into cells. Previously, a gene had been described (HvEmip) which encodes a membrane intrinsic protein (MIP) and which is particularly expressed in the base 1 cm of barley primary leaves. The functionality of the encoding protein was not known. In the present study on leaf 3 of barley (Hordeum vulgare L.), a clone was isolated, termed HvPIP1;6, which has 99% amino acid sequence identity to HvEmip and belongs to the family of plasma membrane intrinsic proteins (PIPs). Expression of HvPIP1;6 was highest in the elongation zone, where it accounted for >85% of expression of known barley PIP1s. Within the elongation zone, faster grower regions showed higher expression than slower growing regions. Expression of HvPIP1;6 was confined to the epidermis, with some expression in neighboring mesophyll cells. Expression of HvPIP1;6 in Xenopus laevis oocytes increased osmotic water permeability 4- to 6-fold. Water channel activity was inhibited by pre-incubation of oocytes with 50 microM HgCl(2) and increased following incubation with the phosphatase inhibitor okadaic acid or the plant hormone ABA. Plasma membrane preparations were analyzed by Western blots using an antibody that recognized PIP1s. Levels of PIP1s were highest in the elongation and adjacent non-elongation zone. The developmental expression profile of HvPIP2;1, the only known barley water channel belonging to the PIP2 subgroup, was opposite to that of HvPIP1;6.     

Light-independent accumulation of chlorophyll a and b and protochlorophyllide in green barley (Hordeum vulgare)

Barley ( Hordeum vulgare L. cvs Clipper, Procter, Astrix) seedlings were transferred from daylight to darkness and changes in chlorophyll a , chlorophyll b , protochlorophyllide and chlorophyllide (μ leaf⁻¹) in either the first or second leaf determined spectrophotometrically after separating the esterified from unesterified pigments by partitioning between ammoniacal acetone and light petroleum ether. Chlorophyll a and b as well as protochlorophyllide accumulated in the dark. The ratio of chlorophyll to protochlorophyllide formed in the absence of light was 18:1. 5-aminolevulinic acid (10 m M ) promoted the synthesis of chlorophyll a and b and protochlorophyllide. Pigment synthesis and response to 5-aminolevulinic acid addition was related to tissue age. Mature tissue in the apical third of the leaf accumulated most chlorophyll, but per μg chlorophyll present at the time of transfer to darkness, was less efficient than immature tissue towards the base of the leaf. Immature tissue was also most responsive to added 5-aminolevulinic acid. Chlorophyll synthesis in the dark was accompanied by chloroplast development. Chloroplasts in immature leaf tissue increased in size and extent of thylakoid development when transferred from daylight to darkness. The results indicate that chlorophyll synthesis and chloroplast membrane development in light-grown barley continue into the dark phase of the diurnal cycle. A light-independent protochlorophyllide reductase in light-grown barley seedlings is postulated.     

Hordein-gene expression during development of the barley (Hordeum vulgare) endosperm.

A previous study [Rahman, Shewry & Miflin (1982) J. Exp. Bot. 33, 717-728] showed differential accumulation of the major storage proteins (called B and C hordeins) in developing endosperms of barley (Hordeum vulgare). To determine how this accumulation is regulated, we have studied mRNA fractions prepared from similar endosperms. Hordein-related mRNA species were detected some days before the deposition of hordeins in vivo. The translation products in vivo directed by polyribosomes, polysomal RNA and total cellular RNA showed similar changes in the proportions of the hordein products to those observed in the accumulations of the proteins in vivo. There was a relative increase in one of the subfamilies of B hordeins (called B1 hordein) and a decrease in the second subfamily of B hordeins (B3 hordein) and in C hordeins. The populations of RNA species related to these three groups of hordeins were studied by 'dot hybridization', with specific complementary-DNA probes for B1-, B3- and C-hordein-related sequences. This showed a 10-15-fold increase in sequences related to the B1 hordein during endosperm development, but only a 4-fold increase in sequences related to B3 and C hordeins. These results indicate that the rates of synthesis of hordeins are related to the abundance of their respective mRNA species. The different results observed for the two subfamilies of B hordeins are of interest, since they indicate differential expression of two subfamilies of genes present at a single multigenic locus.     

Enhancing the performance of ethephon with mepiquat chloride on barley (Hordeum distichon cV. Panda) using an adjuvant comprising acidified soyal phospholipid

Rates of ethephon with mepiquat chloride (as ‘Terpal’) were foliar applied to winter barley during early flag leaf emergence with or without soyal lecithin acidified with proprionic acid included as an adjuvant (‘LI700’). In both the glasshouse and field increasing the rate of ‘Terpal’ decreased the rate of stem extension exponentially, which in turn led to progressively shorter stands. As crop height extended above 0.9 m both the proportion of crop area lodged and the severity of lodging (mean angle of culms to the vertical) increased. As lodging progressed, mean grain weight was depressed and therefore ethephon with mepiquat chloride had the potential to increase yield. However, in the absence of lodging, applications of ‘Terpal’ reduced yield in direct proportion to increasing the dose rate. The influence of ‘Terpal’ on yield becomes a complex interaction between phytotoxicity, probably resulting from exogenous ethylene applications and benefits accruing from the control of lodging. Adding ‘LI700’ as an adjuvant enhanced the performance of ‘Terpal’ in reducing both rate of stem extension and mean grain weight in the absence of lodging.     

Differential localization of two acid proteinases in germinating barley (Hordeum vulgare) seed

A cathepsin D-like aspartic proteinase (EC 3.4.23) is abundant in ungerminated barley ( Hordeum vulgare ) seed while a 30 kDa cysteine endoproteinase (EC 3.4.22) is one of the proteinases synthesized de novo in the germinating seed. In this work, the localization of these two acid proteinases was studied at both the tissue and subcellular levels by immunomicroscopy. The results confirm that they have completely different functions. The aspartic proteinase was present in the ungerminated seed and, during germination, it appeared in all the living tissues of the grain, including the shoot and root. Contrary to previous suggestions, it was not observed in the starchy endosperm. By immunoblotting, the high molecular mass form of the enzyme (32 + 16 kDa) was found in all the living tissues, whereas the low molecular mass form (29 + 11 kDa) was not present in the shoot or root, indicating that the two enzyme forms have different physiological roles. The aspartic proteinase was localized first in the scutellar protein bodies of germinating seed, and later in the vacuoles which are formed by fusion of the protein bodies. In contrast to the aspartic proteinase, the expression of the 30 kDa cysteine proteinase began during the first germination day, and it was secreted into the starchy endosperm; first from the scutellum and later from the aleurone layer. It was not found in either shoots or roots. The 30 kDa cysteine proteinase was detected in the Golgi apparatus and in the putative secretory vesicles of the scutellar epithelium. These results suggest that the aspartic proteinase functions only in the living tissues of the grain, as opposed to the 30 kDa cysteine proteinase which is apparently one of the proteases initiating the hydrolysis of storage proteins in the starchy endosperm.     

Overexpression of HVA1 gene from barley generates tolerance to salinity and water stress in transgenic mulberry (Morus indica)

Late embryogenesis abundant (LEA) proteins are members of a large group of hydrophilic proteins found primarily in plants. The barley hva1 gene encodes a group 3 LEA protein and is induced by ABA and water deficit conditions. We report here the over expression of hva1 in mulberry under a constitutive promoter via Agrobacterium-mediated transformation. Molecular analysis of the transgenic plants revealed the stable integration and expression of the transgene in the transformants. Transgenic plants were subjected to simulated salinity and drought stress conditions to study the role of hva1 in conferring tolerance. The transgenic plants showed better cellular membrane stability (CMS), photosynthetic yield, less photo-oxidative damage and better water use efficiency as compared to the non-transgenic plants under both salinity and drought stress. Under salinity stress, transgenic plants show many fold increase in proline concentration than the non-transgenic plants and under water deficit conditions proline is accumulated only in the non-transgenic plants. Results also indicate that the production of HVA1 proteins helps in better performance of transgenic mulberry by protecting membrane stability of plasma membrane as well as chloroplastic membranes from injury under abiotic stress. Interestingly, it was observed that hva1 conferred different degrees of tolerance to the transgenic plants towards various stress conditions. Amongst the lines analysed for stress tolerance transgenic line ST8 was relatively more salt tolerant, ST30, ST31 more drought tolerant, and lines ST11 and ST6 responded well under both salinity and drought stress conditions as compared to the non-transgenic plants. Thus hva1 appears to confer a broad spectrum of tolerance under abiotic stress in mulberry.