Morpho-Anatomical, Physiological and Biochemical Adjustments in Response to Heat and Drought Co-Stress in Winter Barley.

This study aimed to investigate the combined effect of high temperatures 10 °C above the optimum and water withholding during microgametogenesis on vegetative processes and determine the response of winter barley genotypes with contrasting tolerance. For this purpose, two barley varieties were analyzed to compare the effect of heat and drought co-stress on their phenology, morpho-anatomy, physiological and biochemical responses and yield constituents. Genotypic variation was observed in response to heat and drought co-stress, which was attributed to differences in anatomy, ultrastructure and physiological and metabolic processes. The co-stress-induced reduction in relative water content, total soluble protein and carbohydrate contents, photosynthetic pigment contents and photosynthetic efficiency of the sensitive Spinner variety was significantly greater than the tolerant Lambada genotype. Based on these observations, it has been concluded that the heat-and-drought stress-tolerance of the Lambada variety is related to the lower initial chlorophyll content of the leaves, the relative resistance of photosynthetic pigments towards stress-triggered degradation, retained photosynthetic parameters and better-preserved leaf ultrastructure. Understanding the key factors underlying heat and drought co-stress tolerance in barley may enable breeders to create barley varieties with improved yield stability under a changing climate.

Stigma Functionality and Fertility Are Reduced by Heat and Drought Co-stress in Wheat.

As a consequence of climate change, unpredictable extremely hot and dry periods are becoming more frequent during the early stages of reproductive development in wheat (Triticum aestivum L.). Pollen sterility has long been known as a major determinant of fertility loss under high temperature and water scarcity, but it will be demonstrated here that this is not the exclusive cause and that damage to female reproductive organs also contributes to losses of fertility and production. Changes in the phenology, morphology, and anatomy of female reproductive cells and organs, in the ROS and RNS generation of stigmatic papilla cells, and in fertility and yield components in response to simultaneous high temperature and drought at gametogenesis were studied in two wheat genotypes with contrasting stress responses. The combination of high temperature (32/24°C) and total water withdrawal for 5 days at gametogenesis altered the phenology of the plants, reduced pollen viability, modified the morphology and the anatomy of the pistils, enhanced the generation of ROS and RNS, intensified lipid peroxidation and decreased the NO production of stigmatic papilla cells, all leading to reduced fertility and to production loss in the sensitive genotype, depending on the position of the floret on the spike. Reduced functionality of female and male reproductive parts accounted for 34% and 66%, respectively, of the total generative cell- and organ-triggered fertility loss.

ImmunoFISH: Simultaneous Visualisation of Proteins and DNA Sequences Gives Insight Into Meiotic Processes in Nuclei of Grasses.

ImmunoFISH is a method combining immunolabelling (IL) with fluorescent in situ hybridisation (FISH) to simultaneously detect the nuclear distribution of proteins and specific DNA sequences within chromosomes. This approach is particularly important when analysing meiotic cell division where morphogenesis of individual proteins follows stage-specific changes and is accompanied by a noticeable chromatin dynamism. The method presented here is simple and provides reliable results of high quality signal, low background staining and can be completed within 2 days following preparation. Conventional widefield epifluorescent or laser scanning microscopy can be used for high resolution and three-dimensional analysis. Fixation and preparation techniques were optimised to best preserve nuclear morphology and protein epitopes without the need for any antigen retrieval. Preparation of plant material involved short cross-linking fixation of meiotic tissues with paraformaldehyde (PFA) followed by enzyme digestion and slide-mounting. In order to avoid rapid sample degradation typical of shortly fixed plant materials, and to be able to perform IL later, slides were snap-frozen and stored at -80°C. Ultra-freezing produced a remarkable degree of structural preservation for up to 12 months, whereby sample quality was similar to that of fresh material. Harsh chemicals and sample dehydration were avoided throughout the procedure and permeability was ensured by a 0.1-0.3% detergent treatment. The ImmunoFISH method was developed specifically for studying meiosis in Triticeae, but should also be applicable to other grass and plant species.

Coevolving MAPK and PID phosphosites indicate an ancient environmental control of PIN auxin transporters in land plants.

Plant growth flexibly adapts to environmental conditions, implying cross-talk between environmental signalling and developmental regulation. Here, we show that the PIN auxin efflux carrier family possesses three highly conserved putative mitogen-activated protein kinase (MAPK) sites adjacent to the phosphorylation sites of the well-characterised AGC kinase PINOID, which regulates the polar localisation of PINs and directional auxin transport, thereby underpinning organ growth. The conserved sites of PIN1 are phosphorylated in vitro by two environmentally activated MAPKs, MPK4 and MPK6. In contrast to AGC kinases, MAPK-mediated phosphorylation of PIN1 at adjacent sites leads to a partial loss of the plasma membrane localisation of PIN1. MAPK-mediated modulation of PIN trafficking may participate in environmental adjustment of plant growth.

Powdery Mildew Resistance in Wheat Cultivar Mv Hombár is Conferred by a New Gene, PmHo.

A new powdery mildew resistance gene designated as PmHo was identified in 'Mv Hombár' winter wheat, bred in Martonvásár, Hungary. It has exhibited a high level of resistance over the last two decades. Genetic mapping of recombinant inbred lines derived from the cross 'Ukrainka'/Mv Hombár located this gene on chromosome 2AL. The segregation ratio and consistent effect in all environments indicated that PmHo is a major dominant powdery mildew resistance gene. The race-specific nature of resistance in Mv Hombár was shown by the emergence of a single virulent pathotype designated as 51-Ho. This pathotype was, to some extent, able to infect Mv Hombár, developing visible symptoms with sporulating colonies. Microscopic studies revealed that, in incompatible interactions, posthaustorial hypersensitivity reaction was the most prevalent but not exclusive plant defense response in Mv Hombár, and fungal growth was mostly arrested during haustorium formation or in the early stages of colony development. The delayed fungal development of the virulent pathotype 51-Ho may be explained by additional effects of other loci that were also involved in the powdery mildew resistance of Mv Hombár.

New Insights into the Life Cycle of the Wheat Powdery Mildew: Direct Observation of Ascosporic Infection in Blumeria graminis f. sp. tritici.

Although Blumeria graminis is an intensively studied pathogen, an important part of its life cycle (namely, the way ascospores initiate primary infections on cereal leaves) has not yet been explored in detail. This study reports, for the first time, the direct observation of this process in B. graminis f. sp. tritici using light and confocal laser-scanning microscopy. All the germinated ascospores produced a single germ tube type both in vitro and on host plant surfaces; therefore, the ascosporic and conidial germination patterns are markedly different in this fungus, in contrast to other powdery mildews. Germinated ascospores penetrated the epidermal cells of wheat leaves and produced haustoria as known in the case of conidial infections. This work confirmed earlier studies reporting that B. graminis chasmothecia collected from the field do not contain mature ascospores, only asci filled with protoplasm; ascospore development is induced by moist conditions and is a fast process compared with other powdery mildews. Although ascosporic infections are frequent in B. graminis f. sp. tritici in the field, as shown by this study and other works as well, a recent analysis of the genomes of four isolates revealed the signs of clonal or near-clonal reproduction. Therefore, chasmothecia and ascospores are probably more important as oversummering structures than genetic recombination factors in the life cycle of this pathogen.

A morpho-physiological approach differentiates bread wheat cultivars of contrasting tolerance under cyclic water stress.

Leaf micromorphological traits and some physiological parameters with potential relevance to drought tolerance mechanisms were investigated in four selected winter wheat varieties. Plants were subjected to two cycles of drought treatment at anthesis. Yield components confirmed contrasting drought-sensitive and -tolerant behavior of the genotypes. Drought tolerance was associated with small flag leaf surfaces and less frequent occurrence of stomata. Substantial variation of leaf cuticular thickness was found among the cultivars. Thin cuticle coincided with drought sensitivity and correlated with a high rate of dark-adapted water loss from leaves. Unlike in Arabidopsis, thickening of the cuticular matrix in response to water deprivation did not occur. Water stress induced epicuticular wax crystal depositions preferentially on the abaxial leaf surfaces. According to microscopy and electrolyte leakage measurements from leaf tissues, membrane integrity was lost earlier or to a higher extent in sensitive than in tolerant genotypes. Cellular damage and a decline of relative water content of leaves in sensitive cultivars became distinctive during the second cycle of water deprivation. Our results indicate strong variation of traits with potential contribution to the complex phenotype of drought tolerance in wheat genotypes. The maintained membrane integrity and relative water content values during repeated water limited periods were found to correlate with drought tolerance in the selection of cultivars investigated.

High-frequency generation and characterization of intergeneric hybrids and haploids from new wheat-barley crosses.

KEY MESSAGE: Hybrid plants and a high frequency of maternal haploids were obtained using an efficient wheat-barley hybridization system (with new genotype combinations) and confirmed by several cytological and molecular tools. An efficient hybridization system between wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) is presented on the basis of three new genotype combinations. A particularly high, 14% frequency of plant regeneration per florets was achieved in the wheat-barley genotype combination of 'Sichuan' × 'Morex'. The genome composition in 42 of the 95 plants regenerated by embryo rescue was determined using ploidy analysis, genomic in situ hybridization and the application of chromosome arm-specific molecular markers (SSR and STS). A high overall frequency (76%) of maternal (wheat) haploids was observed in all the tests for all three cross combinations. A major implication of this observation is that this new hybridization system represents a useful tool to study the mechanism of uniparental chromosome elimination in cereals.

Phylogenetic diversity of bacterial communities inhabiting the sediment of Lake Hévíz - a comparison of cultivation and cloning.

Lake Hévíz is the largest natural warm water lake of Europe. The curative mud of the lake comprises volcanic and marsh components although their species composition is hardly known yet. The aim of the present study was to gain information about the distribution and species diversity of bacterial communities inhabiting the sediment of Lake Hévíz using cultivation-based and molecular cloning methods. Samples from two depths and locations were taken in 2004 and 2007. Representatives of the altogether 255 bacterial isolates were affiliated with the phyla Firmicutes, Actinobacteria, Proteobacteria and Bacteroidetes. The most abundant groups belonged to the genus Bacillus (Firmicutes). Many of Lake Hévíz isolates showed the highest sequence similarity to bacteria known to be plant associated or members of normal human microbiota as well as participating in decomposition of highly resistant organic materials. In the three clone libraries, phylotypes belonging to altogether different phyla (Firmicutes, Actinobacteria, Proteobacteria, Bacteroidetes, Cyanobacteria, Chlorobi, Chloroflexi, Deferribacteres, Nitrospirae, Spirochaetes and Verrucomicrobia) were revealed from which members of Gammaproteobacteria and Cyanobacteria proved to be the most abundant. Regardless of the sampling times and methodology used, high spatial heterogeneities of bacterial community structures were characteristic of the sediment of Lake Hévíz.

Identification of genes preferentially expressed in wheat egg cells and zygotes.

KEY MESSAGE : Wheat genes differentially expressed in the egg cell before and after fertilization were identified. The data support zygotic gene activation before the first cell division in wheat. To have an insight into fertilization-induced gene expression, cDNA libraries have been prepared from isolated wheat egg cells and one-celled zygotes. Two-hundred and twenty-six egg cell and 253 zygote-expressed EST sequences were determined. Most of the represented transcripts were detected in the wheat egg cell or zygote transcriptome at the first time. Expression analysis of fourteen of the identified genes and three controls was carried out by real-time quantitative PCR. The preferential expression of all investigated genes in the female gametophyte-derived samples (egg cells, zygotes, two-celled proembryos, and basal ovule parts with synergids) in comparison to the anthers, and the leaves were verified. Three genes with putative signaling/regulatory functions were expressed at a low level in the egg cell but exhibited increased (2-to-33-fold) relative expression in the zygote and the proembryo. Genes with high EST abundance in cDNA libraries exhibited strong expression in the egg cell and the zygote, while the ones coding for unknown or hypothetical proteins exhibited differential expression patterns with preferential transcript accumulation in egg cells and/or zygotes. The obtained data support the activation of the zygotic genome before the first cell division in wheat.

Embryo and endosperm development in wheat (Triticum aestivum L.) kernels subjected to drought stress.

The aim of the present work was to reveal the histological alterations triggered in developing wheat kernels by soil drought stress during early seed development resulting in yield losses at harvest. For this purpose, observations were made on the effect of drought stress, applied in a controlled environment from the 5th to the 9th day after pollination, on the kernel morphology, starch content and grain yield of the drought-sensitive Cappelle Desprez and drought-tolerant Plainsman V winter wheat (Triticum aestivum L.) varieties. As a consequence of water withdrawal, there was a decrease in the size of the embryos and the number of A-type starch granules deposited in the endosperm, while the development of aleurone cells and the degradation of the cell layers surrounding the ovule were significantly accelerated in both genotypes. In addition, the number of B-type starch granules per cell was significantly reduced. Drought stress affected the rate of grain filling shortened the grain-filling and ripening period and severely reduced the yield. With respect to the recovery of vegetative tissues, seed set and yield, the drought-tolerant Plainsman V responded significantly better to drought stress than Cappelle Desprez. The reduction in the size of the mature embryos was significantly greater in the sensitive genotype. Compared to Plainsman V, the endosperm cells of Cappelle Desprez accumulated significantly fewer B-type starch granules. In stressed kernels of the tolerant genotype, the accumulation of protein bodies occurred significantly earlier than in the sensitive variety.

Low and high ψ ways from post-transcriptional RNA regulation to drought tolerance.

Plants withstand adverse environmental effects by stress responses governed by a complex multilayer regulatory network. Besides well established transcriptional cascades posttranscriptional modifications give more plasticity to the plant's behavior under unfavorable circumstances. These modifications include various RNA alterations typically interlaced with transcriptional or translational regulation. Recent examples have been described in RNA splicing, processing, translation and degradation, some of which operate through effects of small non-coding RNAs. So far details of physiological output mechanisms affected by RNA regulation have been uncovered in a few cases only, some of those will be detailed in this review. In the well documented example of the nuclear cap binding complex (nCBC) mutants, molecular mechanisms of the regulatory switch and downstream events have been established in detail. New results directly link nCBC function to splicing, RNA processing and abscisic acid (ABA). Potential output mechanisms of this control point have also been implicated, both in fast stress responses and in developmental regulation. This latter aspect provides a new insight into how RNA regulation may contribute to acclimation by facilitating drought tolerant morphology. Recent results pinpoint the importance of cuticular structure in acclimation to drought stress at high water potential (ψ).

Histological and microarray analysis of the direct effect of water shortage alone or combined with heat on early grain development in wheat (Triticum aestivum).

Based on the in silico analysis of the representation of expressed sequence tags (ESTs) in wheat grain-related cDNA libraries, a specific 15k oligonucleotide microarray has been developed in order to monitor environmental stress-dependent gene expression changes in the wheat caryopses. Using this array, the effect of water withdrawal, with and without additional heat stress, has been investigated during the first five days of kernel development on two wheat cultivars differing in their drought sensitivity. Water shortage affected (more than twofold change) the expression of only 0.5% of the investigated genes. A parallel heat treatment increased the ratio of responding genes to 5-7% because of the temperature stress and/or the increased water deficit because of enhanced evaporation. It could be established that the two cultivars, differing in their long-term adaptation capabilities to drought, responded to the short and direct stress treatments on the same way. In response to the combined drought and heat treatment, the coordinately altered expression of genes coding for storage proteins, enzymes involved in sugar/starch metabolism, histone proteins, heat shock proteins, proteases, tonoplast aquaporins as well as several transcription factors has been observed. These gene expression changes were in agreement with histological data that demonstrated the accelerated development of the embryo as well as the endosperm.

The effect of drought and heat stress on reproductive processes in cereals.

As the result of intensive research and breeding efforts over the last 20 years, the yield potential and yield quality of cereals have been greatly improved. Nowadays, yield safety has gained more importance because of the forecasted climatic changes. Drought and high temperature are especially considered as key stress factors with high potential impact on crop yield. Yield safety can only be improved if future breeding attempts will be based on the valuable new knowledge acquired on the processes determining plant development and its responses to stress. Plant stress responses are very complex. Interactions between plant structure, function and the environment need to be investigated at various phases of plant development at the organismal, cellular as well as molecular levels in order to obtain a full picture. The results achieved so far in this field indicate that various plant organs, in a definite hierarchy and in interaction with each other, are involved in determining crop yield under stress. Here we attempt to summarize the currently available information on cereal reproduction under drought and heat stress and to give an outlook towards potential strategies to improve yield safety in cereals.