Osmotic stress at the barley root affects expression of circadian clock genes in the shoot

The circadian clock is an important timing system that controls physiological responses to abiotic stresses in plants. However, there is little information on the effects of the clock on stress adaptation in important crops, like barley. In addition, we do not know how osmotic stress perceived at the roots affect the shoot circadian clock. Barley genotypes, carrying natural variation at the photoperiod response and clock genes Ppd‐H1 and HvELF3, were grown under control and osmotic stress conditions to record changes in the diurnal expression of clock and stress‐response genes and in physiological traits. Variation at HvELF3 affected the expression phase and shape of clock and stress‐response genes, while variation at Ppd‐H1 only affected the expression levels of stress genes. Osmotic stress up‐regulated expression of clock and stress‐response genes and advanced their expression peaks. Clock genes controlled the expression of stress‐response genes, but had minor effects on gas exchange and leaf transpiration. This study demonstrated that osmotic stress at the barley root altered clock gene expression in the shoot and acted as a spatial input signal into the clock. Unlike in Arabidopsis, barley primary assimilation was less controlled by the clock and more responsive to environmental perturbations, such as osmotic stress.     

Silencing barley cystatins HvCPI‐2 and HvCPI‐4 specifically modifies leaf responses to drought stress

Protein breakdown and mobilization are some of the major metabolic features associated with abiotic stresses, essential for nutrient recycling and plant survival. Genetic manipulation of protease and/or protease inhibitors may contribute to modulate proteolytic processes and plant responses. The expression analysis of the whole cystatin family, inhibitors of C1A cysteine proteases, after water deprivation in barley leaves highlighted the involvement of Icy‐2 and Icy‐4 cystatin genes. Artificial microRNA lines independently silencing the two drought‐induced cystatins were generated to assess their function in planta. Phenotype alterations at the final stages of the plant life cycle are represented by the stay‐green phenotype of silenced cystatin 2 lines. Besides, the enhanced tolerance to drought and differential responses to water deprivation at the initial growing stages are observed. The mutual compensating expression of Icy‐2 and Icy‐4 genes in the silencing lines pointed to their cooperative role. Proteolytic patterns by silencing these cystatins were concomitant with modifications in the expression of potential target proteases, in particular, HvPap‐1, HvPap‐12, and HvPap‐16 C1A proteases. Metabolomics analysis lines also revealed specific modifications in the accumulation of several metabolites. These findings support the use of plants with altered proteolytic regulation in crop improvement in the face of climate change.     

Effects of Russian wheat aphid infestation on barley plant response to drought stress

The influence of Russian wheat aphid ( Diuraphis noxia Mordvilko) infestation on the response of barley ( Hordeum vulgare L. ev Hazen) plants to drought stress was investigated. Fourteen-day-old plants were infested with eight apterous adult aphids, which were removed 7 days later with systemic insecticide. Leaves previously infested with aphids had lower relative water content, reduced stomatal conductance, more negative water potential, lower levels of chlorophyll and higher levels of amino-N, proline and glycinebetaine than corresponding leaves from uninfested plants. When water was withheld for a period of 7 days after aphids were removed, the relative water content of previously infested plants dropped steadily from 0.89 to 0.60, while the relative water content of uninfested plants remained at about 0.94 for the first 4 days of the drought stress period followed by a steady drop to about 0.77 by the end of the drought stress period. Leaf water potentials dropped steadily during the drought stress period in both previously infested (-1.14 to -1.91 MPa) and unin-fested (-0.54 to -1.52 MPa) plants. Analysis of glycinebetaine and proline levels at the end of the drought stress period indicated that leaves of previously infested plants accumulated lower levels of these solutes than leaves from uninfested plants. Upon alleviation of drought stress, plants previously infested with aphids showed little increase in dry weight while younger leaves and tillers from uninfested plants showed large increases. It is concluded that Russian wheat aphids cause drought-stress symptoms in leaves of infested plants even in the presence of ample root moisture. The observations of low levels of glycinebetaine and proline present in leaves after water was withheld from roots and lack of leaf growth upon alleviation of drought stress in previously-infested plants, suggest that aphid infestation limits the capacity of barley plants to adjust successfully to drought stress.     

Mild phosphorus stress in barley and a related low-phosphorus-adapted barleygrass: Phosphorus fractions and phosphate absorption in relation to growth

Barleygrass ( Hordeum leporinum ) from Australian low-P (phosphorus) soils and commercial barley ( H. vulgare ) with high fertilizer requirements were grown in solution culture at 3 levels of P supply. The high-P-adapted barley produced more biomass at all levels of P supply and was more responsive to added P in terms of rate of tillering, rate of leaf production, final leaf size, and therefore total shoot weight compared to barleygrass. In both species root: shoot ratio decreased in response to improved tissue P status, even at P levels where total biomass did not respond to P supply. Removal of endosperm reserves of barley reduced total biomass to a greater extent than it altered phosphate absorption rate, thus increasing tissue P status and making plants less responsive to added P. Similarly, barleygrass had a slower growth rate but a comparable P absorption rate to that of barley. Thus barleygrass also accumulated tissue P and was unresponsive to added P. All phosphorus chemical fractions increased in response to improved tissue P status, but to differing extents (inorganic-P > nucleic acid-P > lipid-P > ester-P), suggesting that all P fractions (particularly inorganic P) serve, in part, a storage function. Both barleygrass and barley without endosperm had higher concentrations of all P fractions (particularly inorganic P) than did unaltered barley, but this was due entirely to their higher P status (due to slow growth) rather than to any major difference in P metabolism between species. We conclude that slow growth is more important than interspecific differences in P metabolism, P absorption, or efficiency of P utilization in explaining the success of barleygrass and other low-P-adapted species on infertile soils.     

Analyses of abiotic stress and brassinosteroid-related some genes in barley roots grown under salinity stress and HBR treatments: Expression profiles and phylogeny

We aimed to investigate abiotic stress (WAK, HvPIP1.1, HvPIP1.2, HvPIP1.3, HvPIP1.5, CYCD3, DREB2) and brassinosteroid-related gene (DWARF4) expressions in barley (Hordeum vulgare L. cv. “Hilal”) roots grown under different salt (150 and 250 mM), HBR (0.5 and 1 μM), and salt + HBR applications during 48 and 72 h at dark with their controls. Phylogenetic trees were also constructed to observe relationships among genes found in other plants. The expression of HvPIP1.2 and WAK reduced after salt treatment while HvPIP1.3, DREB2 and DWARF4 expressions increased. HvPIP1.1, HvPIP1.2, HvPIP1.3, HvPIP1.5 and DWARF4 expressions were upregulated under only HBR applications. Salt + HBR treatments increased HvPIP1.1, DREB2 and DWARF4 but decreased HvPIP1.2. Phylogenetic analyses indicated that Oryza sativa L. shared similar sequences with HvPIP1.5. CYCD3 could diverge relatively earlier from cyclin genes during evolution as it segregates in a distinct clade. Sorghum bicolor showed sequence homology with DREB2. Oryza australiensis L. and DWARF4 were found in the same clade. To our knowledge, this is the first detailed report related to salt stress and HBR applications in terms of the expression of different genes in barley, providing a valuable information for molecular breeding improvement of stress-related traits.     

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.     

Probing the responses of barley cultivars (Hordeum vulgare L.) by chlorophyll a fluorescence OLKJIP under drought stress and re-watering

The main objective of this study was to evaluate the effects of drought and re-watering on 10 varieties of barley (Hordeum vulgare L.) originating from Morocco. Five varieties obtained from the National Institute of Agricultural Research (INRA) of Morocco and five landraces (local varieties defined by high stress tolerance, high yield stability, an intermediate yield and low-input demand) collected at five localities in the south of Morocco were used in the present study. After 2 weeks of growth, drought stress was initiated by withholding water for 2 weeks followed by 1 week of re-watering. The polyphasic OJIP fluorescence transient was used to evaluate photosystem II (PSII) criteria at the end of the first week of drought stress (moderate drought), at the end of the second week (severe drought) and the end of the recovery phase. Drought and re-watering had little effect on the maximum quantum yield of primary photochemistry φ_Po(=F_V/F_M). The photosynthetic performance index (PI) is the product of an antenna, reaction center and electron transport dependent parameter. It revealed differences between varieties as a function of drought and re-watering. For the screening for drought stress tolerance, changes in the PI during a 2-week drought stress treatment were analysed and a new parameter was defined: the drought factor index (DFI) = log(PI_week 1/PI_control) + 2 log(PI_week 2/PI_control). The DFI of the tested varieties correlated with their drought tolerance. Another parameter that was analysed was the relative water content. It decreased during the drought stress treatment varying between 61% and 78.2% at the end of the drought period. During the subsequent recovery period, it increased in a species-dependent manner (65.1–94.1%). A third parameter studied were changes in the initial fluorescence rise. The fluorescence rise during the first 300 μs (L-band) can give information on the energetic connectivity between PSII units whereas changes in the rise during the first 2 ms (K-band) offer information on developing limitations on the donor side of PSII. Changes in respectively the L and K-bands of the fluorescence transients OJIP were shown to have predictive value with respect to the vitality of leaves and the tolerance of the varieties to drought stress.     

The effect of genotype and environment on the fatty acid content of barley (Hordeum vulgare L.) grains

Abstract A comparison was made of the content of total and some individual fatty acids in grains of nine barley varieties grown at six sites in Belgium. The varieties represented six- and two-rowed winter types and two-rowed spring types. The results showed that the winter types contain more linolenic acid (C18 : 3) than spring types and that six-rowed barleys have less total fatty acids than two-rowed barleys, due mainly to a low concentration of palmitic (C16:0), oleic (CI8 : 1) and linoleic (C18 : 2) acids. Analysis of variance showed that fatty acid content is affected by both the genotype and the environment and multiple regression analysis suggested that weather conditions before and after flowering affected lipid composition.     

Circadian rhythmicity of nitrate reductase activity in barley leaves

Nitrate reductase (EC 1.6.6.1) activity showed circadian rhythmicity in the first leaf of 8–11 days old barley ( Hordeum vulgare L. cv. Herta) plants. Circadian rhythms were found using both the in vitro and in vivo method for testing the enzyme activity. When the light intensity was reduced from 65 to 20 W m⁻², the amplitude was smaller and the oscillations were damped sooner. In continuous darkness nitrate reductase activity decreased in a two step process. Three different light qualities were tested which all gave the same results.     

Differential expression of phenylalanine ammonia-lyase genes in barley induced by fungal infection or elicitors

Gene-specific probes were used to assess the expression patterns of four different phenylalanine ammonia-lyase ( pal ) genes in infected or elicitor-treated leaves and suspension-cultured cells of barley. Genes corresponding to hpal2 , hpal3 , hpal4 , and hpal6 were all induced by mercuric chloride and fungal infection by Bipolaris sorokiniana Sacc. (Shoem.) in barley ( Hordeum vulgare L. cv. Pokko) leaves, but with considerable variation in their expression level and timing. The expression patterns of hpal2 and hpal6 were similar, both showing a rapid, strong induction after treatment with mercuric chloride and a slower induction after fungal inoculation, whereas the more divergent hpal3 was induced at a later time and at a lower level after both treatments. Hpal4 was expressed with timing like that of hpal2 and hpal6 in infected or mercuric chloride-treated leaves but its expression was much weaker. Hpal2 and hpal4 were induced in elicitor-treated, suspension-cultured barley cells, whereas the expression of hpal3 was nearly undetectable, and hpal6 was strongly and constitutively present. All pal genes except hpal4 were developmentally regulated, but differentially expressed in various barley tissues. The results suggest that the four pal genes studied here might be responsible for the activation of different branches in the phenylpropanoid biosynthesis of barley.     

Diurnal variations of nitrate reductase activity and stability in barley leaves

Diurnal variations of nitrate reductase (NR) activity and stability have been studied in leaves of barley seedlings ( Hordeum vulgare L. cv. Herta) grown in an 8 h light/16 h darkness regime. Stability (decay) of NR was tested both in the extracts and in the plants. In the morning, when the plants were transferred to light, NR activity increased rapidly during the first hour and then remained constant. After the photoperiod, activity decreased rapidly during the first hour of darkness and then remained fairly constant during the rest of the dark period. The high NR activity during the photoperiod was associated with low NR stability both in the extracts and in the plants. On the other hand the low NR activity during the dark period was associated with high stability in the extracts and in the plants.