Expression of cold-inducible genes and frost hardiness in the crown meristem of young barley (Hordeum vulgare L. cv. Igri) plants grown in different environments

The purpose of this work was to examine environmental control of expression, at the mRNA level, of cold-inducible genes and to test the relationship of the expression of the genes to cold acclimation. Barley plants (Hordeum vulgare L. cv. Igri) at the three- to four-leaf stage were (a) grown in different temperature environments between 20/15°C and +4/-4°C or (b) transferred between 20/15°C and 6/2°C or (c) grown under drought or nutrient stress conditions. Frost hardiness (using a regrowth method) and mRNA levels for three cold-induced genes, blt4-9, blt14 and blt101, from meristematic crown tissue (vegetative shoot meristem plus subtending stem and associated root initials) were measured. Hardiness and levels of blt4-9, blt14 and blt101 mRNAs increased with lower growth temperatures, below a maximum inductive temperature. Prior temperature environment and plant age affected the rate of change in mRNA levels of these genes in response to a change of temperature environment. Hardiness was strongly correlated with mRNA levels of these genes in plants grown in different temperature environments. This correlation did not extend to plants exposed to drought or nutrient stresses. Implications are drawn for plant responses to a warmer climate.     

Molecular Characterization of a Barley Gene Induced by Cold Treatment

A cDNA library was made from low positive temperature (6 ?C/2?C) grown barley shoot meristems. Several genes which are differentiallyexpressed, as measured by mRNA abundance, were selected fromthe library using a differential screen. This paper reportsan analysis of in vivo expression in several cultivars, theDNA sequence, copy number and chromosomal location of one gene(BLT14). In addition, genomic restriction fragment length polymorphismfor this gene in the 10 most widely UK-grown spring and 9 mostwidely UK-grown winter barley cultivars is analysed. Key words: Hordeum vulgare, low temperature, RFLP, differential expression, cDNA sequence     

Diversification of the barley and wheat <Emphasis Type="Italic">blt101</Emphasis>/<Emphasis Type="Italic">wpi6</Emphasis> promoters by the <Emphasis Type="Italic">Xumet</Emphasis> element without affecting stress responsiveness

BLT101-family plasma membrane proteins are found in a wide range of organisms from bacteria to nematodes and are involved in the regulation of cellular cation concentration under stress conditions. A comparison of the promoter regions of barley blt101 and its wheat ortholog, wpi6, revealed highly conserved nucleotide sequences between both genes and a unique insertion of a Xumet element in the blt101 promoter. The Xumet insertion occurred between a putative abscisic acid-responsive element (ABRE) and the dehydration-responsive element/c-repeat (DRE/CRT) within the blt101 promoter. However, blt101 and wpi6 were induced similarly in response to ABA, drought and low temperature, suggesting that the insertion does not affect promoter functions. The Xumet insertion in the blt101/wpi6 promoter region was detected in five barley cultivars, but absent in two wheat cultivars tested, suggesting that the insertion is barley-specific. Genomic Southern blot analysis revealed a large number of Xumet sequences interspersed in the barley genome, whereas only one or very few copies are present in the wheat genome. The data suggested that an expansion in copy number of Xumet elements occurred in the barley genome through evolution.     

Low Temperature Treatment of Barley Plants causes Altered Gene Expression in Shoot Meristems

We have examined whether acclimation to cold may involve alteredgene expression by testing for the appearance of new mRNA, capableof in vitro translation, in shoot meristems of the temperatecereal, barley, grown at 6 ?C day/2? C night. New mRNA is foundafter only 2 d in the cold, when acclimation is detectable butnot complete. The altered pattern of gene expression persistsduring subsequent growth in the cold and is associated witha number of new mRNA molecules including a major mRNA whichencodes a polypeptide Mt 77000. Key words: Hordeum vulgare cv. Igri (barley), low temperature, acclimation, gene expression     

Localization of Expression of Three Cold-Induced Genes, blt101, blt4.9, and blt14, in Different Tissues of the Crown and Developing Leaves of Cold-Acclimated Cultivated Barley

Tissues expressing mRNAs of three cold-induced genes, blt101, blt14, and blt4.9, and a control gene, elongation factor 1α, were identified in the crown and immature leaves of cultivated barley (Hordeum vulgare L. cv Igri). Hardiness and tissue damage were assessed. blt101 and blt4.9 mRNAs were not detected in control plants; blt14 was expressed in control plants but only in the inner layers of the crown cortex. blt101 was expressed in many tissues of cold-acclimated plants but most strongly in the vascular-transition zone of the crown; blt14 was expressed only in the inner layers of the cortex and in cell layers partly surrounding vascular bundles in the vascular-transition zone; expression of blt4.9, which codes for a nonspecific lipid-transfer protein, was confined to the epidermis of the leaf and to the epidermis of the older parts of the crown. None of the cold-induced genes was expressed in the tunica, although the control gene was most strongly expressed there. Thus, the molecular aspects of acclimation differed markedly between tissues. Damage in the vascular-transition zone of the crown correlated closely with plant survival. Therefore, the strong expression of blt101 and blt14 in this zone may indicate a direct role in freezing tolerance of the crown.     

Daily temperature gradients and processes of organogenesis in apical meristem of <Emphasis Type="Italic">Cucumis sativus</Emphasis> L.

We studied the influence of daily temperature gradients on organogenesis in apical and axil shoot meristems at different developmental stages in Cucumis sativus L. The level of organogenic activity of meristems was determined according to the number of leaf primordia on the main and lateral shoots, number of 2nd order shoots, and rudiments of flowers of different levels of development. At the studied ontogenetic stages (mesotrophic seedling or juvenile state), plants were grown under the controlled conditions: photoperiod 12 h, light intensity 100 Wt/m², range of mean daily temperatures 20…30°C, and daily temperature gradients ?20…+20°C. After the temperature treatment, some plants were returned to the optimal, for growth and development, conditions for two weeks (aftereffect). Three types of organogenic activity of meristems in response to the influence of variable daily temperatures were described: stimulation, inhibition, or absence of effect. The phenomenon of stimulation includes two subtypes: optimization, when a maximum effect, observed at other constant temperatures, was attained under the influence of variable temperatures and maximization, when maximum values markedly exceeded those at constant temperatures. The patterns described are preserved on the whole in the aftereffect of daily temperatures.     

Analysis of Organ Specificity of a Low Temperature Responsive Gene Family in Rye (Secale cereale L.)

The barley (Hordeum vulgare L.) low temperature responsive geneblt14 was used as a probe, to isolate two different cognateclones (rlt1412; rlt1421) from a rye (Secale cereale L.) cDNAlibrary prepared from low temperature-treated (6°C day/2°C night) shoot meristems of the cultivar, Puma. Northernblot analysis revealed that low temperature expression of rlt1412is highest in root tissues whereas, rlt1421 shows greatest mRNAaccumulation in mature leaf tissues. There is a relationshipbetween the steady-state levels of these mRNA species and thefrost hardiness of Puma (North American cultivar) and Rhayader(UK cultivar) such that the expression ofboth genes is higherin the more frost hardy cultivar, Puma, compared with Rhayader. DNA and predicted amino acid sequence analysis indicated thatthe rye and barley clones encode small proteins with consensusN-terminal signal sequences whose biological function is atpresent unknown. The relevant sequences are lodged in the EMBL data base. Key words: Rye, cold, cDNA, organ specificity, low temperature genes     

Differential gene expression in cucumber plants in response to brief daily cold treatments

The mechanisms of plant responses to short-term cold treatments applied daily in the period of active growth remain unknown. Cucumber (Cucumis sativus L.) plants were subjected to brief drops of temperature (2 h, 12°C) at the end of each night over a 6-day period (DROP treatment) and to prolonged (6 days) cooling at 12°C (permanent low-temperature treatment, PLT). The plants exposed to cold treatments and control plants grown at 20°C were compared in terms of cold resistance and changes in gene expression. Cold resistance of plants was determined on the basis of LT₅₀ temperature. The response of cucumber genetic machinery was assessed by means of a differential display method based on polymerase chain reaction (PCR). The changes in mRNA pool in cells of cucumber plants subjected to permanent and periodic chilling were assessed after comparing the populations of PCR fragments of cDNA. In both types of chilling protocols, the cold resistance started to increase from the 2nd day of low temperature treatment. At the end of the experiment (on the 6th day), the increment in cold resistance was three times larger for DROP compared to PLT treatment. Analysis of mRNA pool showed that the numbers of amplified fragments were nearly identical in both types of low-temperature treatment. The higher level of cold resistance under DROP conditions was assumed to depend on features of metabolism.     

Low-temperature conditioning of the ice nucleation active bacterium,Erwinia herbicola

Rapid “low-temperature conditioning” and “solute conditioning” of the ice nucleation active bacterium Erwinia herbicola No. 26 are described. Conditioning is the process by which the ability to initiate ice at high temperatures is gained in these bacteria. The cumulative ice nucleator concentration, N[T], was used to measure the number of ice nucleators present in the bacterial systems. N[T] was determined at temperatures from −2 ° to −10 °C and was measured under varying conditioning temperature, time, and solute regimes. Values of N[T] increased rapidly on cooling samples from 30 to 5 °C. The optimum low temperature for conditioning was 5 °C. The conditioning process followed first-order reaction kinetics and time constants (1/rate constant) were between 43 and 62 min at 5 °C. Individual ice nucleators were isolated in droplets and were stable for at least 2 hr. Low-temperature conditioning did not occur when protein synthesis was inhibited by eliminating amino acids in the low-temperature conditioning media or by using the protein synthesis inhibitors chloramphenicol and streptomycin. Analysis of low-temperature conditioning, using heterogeneous ice nucleation theory predicted that ice nucleators are large and have diameters ranging from 80 Å (active at −8 °C) to 300 Å (active at −3 °C). In conclusion, it was predicted that conditioning resulted from growth of the nucleator from about 80 to 300 Å, from a change in the surface properties of 300 Å nucleator making it more similar to ice, or from a combination of these.     

Mediation of high-temperature injury by roots and shoots during reproductive growth of wheat

Abstract Previous studies suggest that high temperature stress on wheat (Triticum aestivum L.) involves root processes and acceleration of monocarpic senescence. Physiological changes in wheat roots and shoots were investigated to elucidate their relationship to injury from elevated temperatures after anthesis. Plants were grown under uniform conditions until 10 d after anthesis, when shoot/root regimes of 25°C/25°C, 25°C/35°C, 35°C/25°C and 35°C/35°C were imposed. Growth and senescence of shoots and grain were influenced more by root temperatures than by shoot temperatures. High root temperatures increased activities of protease and RNasc enzymes, and loss of chlorophyll, protein and RNA from shoots, whereas low root temperatures had opposite effects. High root temperatures appeared to induce shoot senescence directly. High shoot temperatures probably disrupted root processes, including export of cytokinins, and induced high leaf protease activity, senescence and cessation of grain development. The authors concluded that responses of wheat to high temperatures, whether of roots or shoots, are manifested as acceleration of senescence and may be mediated by roots during grain development.     

Produktion von Ochratoxin A und B durch<Emphasis Type="Italic">Penicillium verrucosum</Emphasis> auf Gerste in Abhängigkeit der Parameter Wasseraktivität,Wassergehalt und Temperatur

The ochratoxin A and B (OTA, OTB) production by a toxigenic isolate ofPenicillium verrucosum grown on brewing barley up to six weeks was studied at a storage temperature of 25 °C and different moisture and water activity conditions. Sorption isothermes for barley were prepared at temperatures of 10°C, 15°C and 25°C. OTA was produced after 2 weeks of storage at moisture contents of ≥19%, which is equivalent to water activities (a_w) of 0.83 (adsorptive) and 0.82 (desorptive) at 25 °C. Increased OTA concentrations (5.8-fold and 16.1-fold) were noticed when the moisture contents were adjusted to 20% (a_{w [ads]} 25 °C=0.86) and 21% (a_{w [ads]} [ 25 °C=0.88), respectively. An increase was also shown during storage of 4 and 6 weeks (1.2-fold and 2.4-fold, respectively). Production of OTB was shown to occur at moisture contents ≥18% (a_{w [ads]} 25 °C=0.81). The findings document that OTA and OTB are not produced byP. verrucosum grown on barley stored below 18% moisture content.     

Nitrogen utilization by Typha latifolia L. as affected by temperature and rate of nitrogen application

Greenhouse and growth chamber studies were conducted to evaluate growth and N utilization by Typha latifolia L. in flooded organic soil under varying temperatures and rates of N additions. Elevation of temperature from 10 to 25°C increased shoot biomass yields by 275%. Root biomass yields were lowest at 10°C and increased linearly as a function of temperature. Shoot/root ratios were low (0.72–0.82) at lower temperatures (10–15°C) and ratios increased by about three times at higher temperatures (20–30°C). Biomass yields were increased by addition of N fertilizers, while the shoot/root ratios were directly related to plant-available N present in the soil.Fertilizer ¹⁵N uptake (expressed as % of applied N) by the whole plant was 5.3% at 10°C, 37.5% at 20°C and at 30°C decreased to 20.8%. Fertilizer N accumulation in shoots was 2.1–29.8% of applied N, while roots accumulated 3.2–7.7%. Under greenhouse conditions, N uptake by T. latifolia was found to increase with increased rate of N application. Fertilizer N uptake by both shoots and roots was in the range of 61–77%. Plants cultured in growth chambers were affected by low light conditions resulting in poor growth and low fertilizer ¹⁵N uptake, as compared to plants grown under greenhouse conditions. Added fertilizer N was the major source of N during the early part of the growing season, while soil organic N was the major and perhaps the sole source of N during the latter part of the growing season.     

An Optimized Protocol to Increase Virus-Induced Gene Silencing Efficiency and Minimize Viral Symptoms in Petunia

Virus-induced gene silencing (VIGS) is used to down-regulate endogenous plant genes. VIGS efficiency depends on viral proliferation and systemic movement throughout the plant. Although tobacco rattle virus (TRV)-based VIGS has been successfully used in petunia (Petunia?×?hybrida), the protocol has not been thoroughly optimized for efficient and uniform gene down-regulation in this species. Therefore, we evaluated six parameters that improved VIGS in petunia. Inoculation of mechanically wounded shoot apical meristems induced the most effective and consistent silencing compared to other methods of inoculation. From an evaluation of ten cultivars, a compact petunia variety, 'Picobella Blue', exhibited a 1.8-fold higher CHS silencing efficiency in corollas. We determined that 20 °C day/18 °C night temperatures induced stronger gene silencing than 23 °C/18 °C or 26 °C/18 °C. The development of silencing was more pronounced in plants that were inoculated at 3–4 versus 5 weeks after sowing. While petunias inoculated with pTRV2-NbPDS or pTRV2-PhCHS showed very minimal viral symptoms, plants inoculated with the pTRV2 empty vector (often used as a control) were stunted and developed severe necrosis, which often led to plant death. Viral symptoms were eliminated by developing a control construct containing a fragment of the green fluorescent protein (pTRV2-sGFP). These optimization steps increased the area of chalcone synthase (CHS) silencing by 69 % and phytoene desaturase (PDS) silencing by 28 %. This improved VIGS protocol, including the use of the pTRV2-sGFP control plants, provides stronger down-regulation for high-throughput analyses of gene function in petunia.     

Molecular characterization of a cold-induced plasma membrane protein gene from wheat

As a means to study the function of plasma membrane proteins during cold acclimation, we have isolated a cDNA clone for wpi6 which encodes a putative plasma membrane protein from cold-acclimated winter wheat. The wpi6 gene encodes a putative 5.9 kDa polypeptide with two predicted membrane-spanning domains, the sequence of which shows high sequence similarity with BLT101-family proteins from plants and yeast. Strong induction of wpi6 mRNA was observed during an early stage of cold acclimation in root and shoot tissues of both winter and spring wheat cultivars. In contrast to blt101 in barley, wpi6 mRNA was also induced by drought and salinity stresses, and exogenous application of ABA. Expression of wpi6 in a Δpmp3 mutant of Saccharomyces cerevisiae, which is disturbed in plasma membrane potential due to the lack of a BLT101-family protein, partially complemented NaCl sensitivity of the mutant. Transient expression analysis of a WPI6::GFP fusion protein in onion epidermal cells revealed that WPI6 is localized in the plasma membrane. Taken together, these data suggested that WPI6 may have a protective role in maintaining plasma membrane function during cold acclimation in wheat. The nucleotide sequence data for wpi6 have been recorded in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers AB030210 (cDNA) and AB221353 (genomic DNA).     

The importance of hardening and winter temperature for growth in mountain birch populations

Seedlings of five mountain birch populations (Betula pubescens Ehrh. ssp. czerepanovii) from Fennoscandia and Iceland were raised and grown at natural daylengths at Tromsø, Norway (69°N) and different temperatures during late summer and fall season, followed by winter temperature treatment at ambient and +4 °C above ambient temperatures at Bergen, Norway (60°N). The experiment took place during two seasons (2000/01 and 2001/02). The following summer shoot and biomass growth were reduced as a result of winter warming and subsequent premature dehardening in early flushing provenances and treatments. Biomass increased in plants grown at low hardening temperature when compared with high temperature treatment. As a conclusion, increased winter temperatures would tend to increase the risk of spring frost damage and reduce growth in birch seedlings, because the differences between the frost hardening and ambient temperatures are decreasing, and because the time from budbreak to dehardening is shortened. The results are discussed in relation to simultaneous experiments with frost hardiness in the same populations and treatments.