Formation of early-light-inducible-protein complexes and status of xanthophyll levels under high light and cold stress in barley (Hordeum vulgare L.)

In our previous work we found considerable accumulation of early light-inducible proteins (ELIPs) in barley during adaptation to combined high light and cold stress, an accumulation which occurred preferentially in the apical part of the leaves (M.-H. Montané et al., 1997, Planta 202: 293–302). Here we studied, under the same conditions, the effect of adaptation on the composition of thylakoid membrane proteins and pigments, particularly xanthophylls and chlorophyll, and their distribution within the barley leaf. It was observed that high light fluxes appeared to favour the trimerization of the light-harvesting complex of photosystem II (LHC II) whereas cold appeared to favour the monomers of LHC II. High light, cold or the combination of both factors had only a small effect on the protein composition of the thylakoid membranes except for the proteins of LHC II which were found to decrease under high light to a greater extent at 25 °C than at 5 °C. The total xanthophyll-cycle carotenoid content increased linearly with cellular development, the highest amount being observed in the apical part of the leaf. Cold and high light acted synergistically to induce less than a doubling in the amount of total xanthophylls, while chlorophylls a and b remained nearly constant. The fraction consisting of antheraxanthin plus zeaxanthin was up to 4- to 5-fold higher at 5 °C than at 25 °C. As determined previously (Montané et al. 1997), the same conditions caused a 15-fold increase in the accumulation of ELIPs. Consequently, neither the distribution of total xanthophylls nor that of antheraxanthin plus zeaxanthin along the leaf followed the same pattern as ELIP. Thus, the accumulation of xanthophylls cannot be stoichiometrically correlated with that of ELIPs. Using electrophoresis in the presence of decylmaltoside, we could demonstrate for the first time that ELIPs of 13.5 kDa are contained in high-molecular-mass complexes of >100 kDa, which are located in the unstacked stroma lamellar region of the thylakoid membranes. Received: 6 April 1998 / Accepted: 26 January 1999     

Cadmium leads to stimulated expression of the lipid transfer protein genes in barley: implications for the involvement of lipid transfer proteins in wax assembly

In order to investigate the nature of genes expressed in leaf epidermal cells of higher plants, we have identified the nucleotide sequence of a cDNA designated ltp 7a2b encoding a novel nonspecific lipid transfer protein of barley (Hordeum vulgare L. cv. Gerbel). The cDNA of 755 basepairs contains an open reading frame of 366 nucleotides coding for a 12.3-kDa polypeptide. The first 29 amino acids constitute the putative signal peptide, characteristic for targeting to the secretory pathway. Analysis of mRNA levels by Northern blotting indicated that ltp 7a2b is preferentially expressed in the leaf epidermis. Levels of mRNA decreased during ageing of leaf tissue. Expression of ltp 7a2b was stimulated by a factor of 2–3 when the seedlings were grown in the presence of cadmium (10–1600 μM). Concomitantly, the primary leaves of Cd-exposed seedlings contained elevated levels of abscisic acid and a thicker wax layer of the cuticle. At 100 μM Cd in the hydroponic medium, the wax cover was increased by 50%. The increase in abscisic acid content, ltp 7a2b mRNA and wax coverage was either not seen, or seen much less, in Ni- and Zn-stressed seedlings. The data add circumstantial evidence to the recently proposed hypothesis that nonspecific lipid transfer proteins function in transfer of cutin and/or wax monomers from the site of synthesis in the cell to the cuticle. Received: 1 April 1996 / Accepted: 11 February 1997     

The expression of the salt-responsive gene salT from rice is regulated by hormonal and developmental cues

The expression pattern of the salT gene was analyzed in different cell types and organs of rice (Oryza sativa L.) in response to saline and hormonal treatments to obtain detailed information on the physiological cues controlling gene expression. Gel blot analysis of RNA and in-situ hybridization performed on seedlings grown for 10 ds in the presence of 1% NaCl revealed that salT was expressed mainly in the younger tissues of the plant. In contrast, 6-week-old plants exhibited maximal salT mRNA accumulation in sheaths of older leaves. In addition, salT was normally expressed in rapidly dividing suspension-cultured cells, but not in quiescent ones. Altogether, these results may indicate that salT expression in each region of the plant is dependent on the metabolic activity of the cells as well as on whether or not they are stressed. The effects of two growth regulators, abscisic acid (ABA) and gibberellic acid, were investigated in combination with the effects of NaCl. Gibberellic acid had a synergistic effect on the induction of the salT gene when combined with 0.5% NaCl, but did not induce salT on its own. At 10 μM, ABA induced salT both in the absence of NaCl and in its presence. Whereas 1 μM ABA acted additively with NaCl to induce gene expression, 5 μM ABA with NaCl was only as effective as NaCl alone. This may indicate that the two stimuli act independently and possibly through antagonistic signal transduction pathways. Received: 26 March 1998 / Accepted: 11 July 1998     

Regulation of the expression of ferredoxin-glutamate synthase in barley

We have investigated the regulation of ferredoxin–glutamate synthase (Fd-GOGAT) in leaves of barley (Hordeum vulgare L. cv. Maris Mink) at the mRNA, protein and enzyme activity levels. Studies of the changes in Fd-GOGAT during plant development showed that the activity in shoots increases rapidly after germination to reach a maximum (on a fresh-weight basis) at day 10 and then declines markedly to less than 50% of the maximal activity by day 30, this decline being correlated with an equivalent loss of Fd-GOGAT protein. Growing the plants in darkness reduced the maximum activity attained in the shoots, but did not affect the overall pattern of the changes or their timing. The activity of Fd-GOGAT increased two- to three-fold within 48 h when etiolated leaves were exposed to light, and Northern blots indicated that the induction occurred at the mRNA level. However, whilst a carbon source could at least partially substitute for light in the induction of nitrate reductase activity, no induction of Fd-GOGAT activity was seen when etiolated leaves were treated with either sucrose or glucose. Interestingly, the levels of Fd-GOGAT mRNA and activity remained high up to a period of 16 h or 72 h darkness, respectively. Compared with plants grown in N-free medium, light-grown plants supplied with nitrate had almost two-fold higher Fd-GOGAT activities and increased Fd-GOGAT mRNA levels, but nitrate had no effect on the abundance of the enzyme or its mRNA in etiolated plants, indicating that light is required for nitrate induction of barley Fd-GOGAT. Received: 23 April 1997 / Accepted: 28 May 1997     

Differential stress responses of early salt-stress responding genes in common wheat

Four early salt-stress responding genes (WESR1-4) in common wheat (Triticum aestivum L.) were analyzed for their temporal accumulation of mRNA during salt stress, osmotic stress and abscisic acid (ABA) treatment. All genes showed transient stimulation by 0.15 M NaCl treatment. WESR1 and WESR2 were induced by both osmotic stress and exogenous ABA treatment. WESR3 responded to exogenous ABA, but not to osmotic stress. WESR4 did not show significant response to either osmotic stress or exogenous ABA treatment. These results suggest that wheat has at least two salt stress signal transduction pathways, an ABA-dependent and ABA-independent pathway.     

Cloning and expression of the UGA4 gene coding for the inducible GABA-specific transport protein of Saccharomyces cerevisiae

Japanese pear (Pyrus serotina Rehd.) exhibits gametophytic self-incompatibility. Following our previous findings that basic ribonucleases in the styles of Japanese pear are associated with self-incompatibility genes (S-RNases), stylar proteins with high pI values were analyzed by two-dimensional gel electrophoresis further to characterize S-RNases. A group of basic proteins of about 30 kDa associated with self-incompatibility genes were identified. These proteins contained sugar chains which reacted with concanavalin A and wheat germ agglutinin, and thus were designated as S-glycoproteins of Japanese pear. The fact that the S-glycoprotein was expressed at a much lower level in a self-compatible mutant than in the original variety suggested a role of S-glycoproteins in mediating self-incompatibility of Japanese pear. Immunoblot analysis indicated that S-glycoproteins are identical to previously identified S-RNases. The S-glycoproteins were predominantly expressed in the style, in the ovary in trace amounts, and not in leaf, pollen or germinated pollen. The N-terminal amino acid sequences of the S-glycoproteins showed homology not only with each other but also with those of the S-allele-associated proteins from plants of the family Solanaceae at levels of about 30–50%.     

A cotton mitogen-activated protein kinase （GhMPK6） is involved in ABA-induced CAT1 expression and H2O2 production

The mitogen-activated protein kinase (MAPK) cascade is one of the major and evolutionally conserved signaling pathways and plays a pivotal role in the regulation of stress and developmental signals in plants.Here,we identified one gene,GhMPK6,encoding an MAPK protein in cotton.GFP fluorescence assay demonstrated that GhMAPK6 is a cytoplasm localized protein.Quantitative RT-PCR analysis revealed that mRNA accumulation of GhMPK6 was significantly promoted by abscisic acid (ABA).Overexpression of GhMPK6 gene in the T-DNA insertion mutant atmkkl (SALK_015914) conferred a wild-type phenotype to the transgenic plants in response to ABA.Under ABA treatment,cotyledon greening/expansion in GhMPK6 transgenic lines and wild type was significantly inhibited,whereas the atmkkl mutant showed a relatively high cotyledon greening/expansion ratio.Furthermore,CAT1 expression and H2O2 levels in leaves of GhMPK6 transgenic lines and wild type were remarkably higher than those of atmkkl mutant with ABA treatment.Collectively,our results suggested that GhMPK6 may play an important role in ABA-induced CAT1 expression and H2O2 production.     

Photosynthetic light-harvesting function of carotenoids in higher-plant leaves exposed to high light irradiances

Exposure of barley (Hordeum vulgare L.) leaves to strong white light (1500 μmol photons · m⁻² · s⁻¹) decreased the quantum yield of photosynthetic oxygen evolution in green light preferentially absorbed by carotenoids (Φ-510) but not in red light exclusively absorbed by chlorophylls (Φ-650). This phenomenon was observed to be (i) rapidly induced (within a few minutes), (ii) slowly reversible in darkness (within about 1 h), (iii) insensitive to dithiothreitol and (iv) maximally induced by photon flux densities higher than about 1000 μmol · m⁻² · s⁻¹. Determination of the carotenoid composition of the major light-harvesting complex of PSII (LHCII) and analysis of the thylakoid membrane lipid fluidity before and after strong illumination of barley leaves in the presence or the absence of dithiothreitol showed that the light-induced decrease in the Φ-510/Φ-650 ratio did not require the physical detachment of carotenoids from the pigment antennae. Compared to barley plants grown under moderate light and temperature conditions, plants grown in sustained high irradiance at elevated temperature exhibited (i) a lower Φ-510/Φ-650 ratio, (ii) a reduced size of the functional PSII pigment antenna in green light (but not in red light) and (iii) a marked increase in the amount of free carotenoids found in non-denaturing Deriphat-containing electrophoretic gels of thylakoid membranes. Similarly, the Φ-510/Φ-650 ratio of the LHCII-deficient chlorina-f2 barley mutant was very low compared to the wild type. Separation and quantification of the cis/trans carotenoid isomers of barley leaves revealed that strong illumination did not induce pronounced cis-trans isomerization of xanthophylls. Taken together, the data suggest that the efficiency of energy transfer from carotenoids to chlorophylls varies with the light environment both in the short term and in the long term, with excess light energy noticeably inhibiting the photosynthetic light-harvesting function of carotenoids. The photoprotective significance of this carotenoid decoupling from the chlorophyll antennae is discussed. Received: 28 July 1997 / Accepted: 25 October 1997     

Differential expression of the Arabidopsis genes coding for Em-like proteins

Late embryogenesis abundant (lea) genes are a large and diverse group of genes highly expressed during late stages of seed development. Five major groups of LEA proteins have been described. Two Em genes (group I lea genes) are present in the genome of Arabidopsis thaliana L., AtEm1 and AtEm6. Both genes encode for very similar proteins which differ basically in the number of repetitions of a highly hydrophilic amino acid motif. The spatial patterns of expression of the two Arabidopsis Em genes have been studied using in situ hybridization and transgenic plants transformed with the promoters of the genes fused to the beta-glucuronidase reporter gene (uidA). In the embryo, AtEm1 is preferentially expressed in the pro-vascular tissues and in meristems. In contrast, AtEm6 is expressed throughout the embryo. The activity of both promoters disappears rapidly after germination, but is ABA-inducible in roots of young seedlings, although in different cells: the AtEm1 promoter is active in the internal tissues (vasculature and pericycle) whereas the AtEm6 promoter is active in the external tissues (cortex, epidermis and root hairs). The AtEm1 promoter, but not AtEm6, is also active in mature pollen grains and collapsed nectaries of young siliques. These data indicate that the two Em proteins could carry out at least slightly different functions and that the expression of AtEm1 and AtEm6 is controlled at, at least, three different levels: temporal, spatial and hormonal (ABA).     

Lectin and alliinase are the predominant proteins in nectar from leek (Allium porrum L.) flowers

Analysis of nectar from leek (Allium porrum) flowers by SDS-PAGE revealed the presence of two major polypeptide bands of 50 kDa and 13 kDa, respectively. Using a combination of agglutination tests, enzyme assays and N-terminal sequencing, the polypeptides have been identified as subunits of alliin lyase (alliinase, EC 4.4.1.4) and mannose-binding lectin, respectively. The latter protein is particularly abundant since it represents about 75% of the total nectar protein. Honey produced by bees foraging on flowering leek plants still contains biologically active lectin and alliinase. However, the levels of both proteins are strongly reduced as compared to those in the original nectar. It is evident, therefore, that the lectin as well as the alliinase are inactivated/degraded during the conversion of nectar into honey. Received: 24 May 1996 / Accepted: 19 August 1996     

Chromosomal location of genes coding for endosperm proteins of Hordeum chilense,determined by two-dimensional electrophoresis of wheat-H. chilense chromosome addition lines

The proteins of Hordeum chilense grain were resolved into 25 major components by two-dimensional electrophoresis. Their solubilities in aqueous alcohol solutions were determined to distinguish prolamin storage proteins from metabolic and structural proteins. The prolamins were divided into two groups, based on the presence or absence of intermolecular disulfide bonds determined by gel-filtration chromatography. Using an incomplete set of Chinese Spring wheat-H. chilense disomic addition lines, the structural genes of 21 of the 26 most dominant seed proteins were assigned to chromosomes. The great majority of the prolamin genes, including those coding for a high molecular weight (HMW) prolamin subunit, was present on chromosome 1H^ch. However, a small number of prolamin genes also occurred on chromosomes 5H^ch and 7H^ch. A minor protein, probably belonging to the nonstorage group of proteins, is coded by genes on 5H^ch. Various ditelosomic addition lines and ditelosomic and disomic substitution lines for chromosome 7H^ch were also analyzed by electrophoresis. This technique revealed that the genes for three major prolamins occur on the arm of chromosome 7H^ch and that a gene for a minor protein, also thought to be a prolamin, occurs on the arm. These results are discussed in relation to the evolution of prolamin genes in the Triticeae.     

Identification of sequence homology between the internal hydrophilic repeated motifs of Group 1 late-embryogenesis-abundant proteinsin plants and hydrophilic repeats of the general stress protein GsiB of Bacillus subtilis

Late embryogenesis abundant (LEA) proteins are speculated to protect against water stress in plants. Group 1 LEA proteins are hydrophilic and vary mainly in the numbers of an extremely hydrophilic internal 20-amino-acid motif. This motif is present up to four times in Arabidopsisthaliana and Hordeum vulgare Group 1 proteins and has been described in numerous plant species. However, no similarity has yet been described between Group 1 genes or gene products and those from non-plant species. We report here the striking similarity between the repeated internal motif of Group 1 LEA proteins and a repeated hydrophilic motif present in a stress-related protein (GsiB) from Bacillus subtilis. Received: 20 April 1998 / Accepted: 18 May 1998