Attila Fábián

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.

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.

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.

New phenotypes of the drought-tolerant cbp20 Arabidopsis thaliana mutant have changed epidermal morphology

This paper provides a detailed phenotypic analysis of the abscisic acid (ABA) hypersensitive Cap Binding Protein 20 (cbp20) mutant. Some hitherto undescribed changes were found in the tissue structure and epidermal morphology of this mutant. These include more and smaller cells in the epidermis, a thicker cuticle and more frequent occurrence of trichomes on leaf surfaces. Some of these traits may contribute to the physiological processes responsible for the water-saving behaviour of the mutant. Abnormal spatial patterns between stomatal pore complexes were also found on various organs of the mutant. All these observations indicate profoundly disturbed development of epidermal tissue in the cbp20 mutant, which has not previously been reported for this class of mutants. A potential connection between the new phenotypes and disturbed miRNA metabolism and mRNA splicing of the mutant is discussed.

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.

Comprehensive Analysis of DWARF14-LIKE2 (DLK2) Reveals Its Functional Divergence from Strigolactone-Related Paralogs

Strigolactones (SLs) and related butenolides, originally identified as active seed germination stimulants of parasitic weeds, play important roles in many aspects of plant development. Two members of the D14 α/β hydrolase protein family, DWARF14 (D14) and KARRIKIN INSENSITIVE2 (KAI2) are essential for SL/butenolide signaling. The third member of the family in Arabidopsis, DWARF 14-LIKE2 (DLK2) is structurally very similar to D14 and KAI2, but its function is unknown. We demonstrated that DLK2 does not bind nor hydrolyze natural (+)5-deoxystrigol [(+)5DS], and weakly hydrolyzes non-natural strigolactone (-)5DS. A detailed genetic analysis revealed that DLK2 does not affect SL responses and can regulate seedling photomorphogenesis. DLK2 is upregulated in the dark dependent upon KAI2 and PHYTOCHROME INTERACTING FACTORS (PIFs), indicating that DLK2 might function in light signaling pathways. In addition, unlike its paralog proteins, DLK2 is not subject to rac-GR24-induced degradation, suggesting that DLK2 acts independently of MORE AXILLARY GROWTH2 (MAX2); however, regulation of DLK2 transcription is mostly accomplished through MAX2. In conclusion, these data suggest that DLK2 represents a divergent member of the DWARF14 family.

Expression of a WIN/SHN-type regulator from wheat triggers disorganized proliferation in the Arabidopsis leaf cuticle

Based on information from the Arabidopsis model system, a putative transcriptional activator of cuticle formation (TaSHN1) was selected among the expressed sequence tags in wheat (Triticum aestivum L.). RT-PCR indicated the preferential expression of this gene in the basal, but not in the middle parts of wheat leaves. This leaf region is a likely site of cuticle formation in cereals. TaSHN1 was cloned and expressed in Arabidopsis, resulting in shiny leaf surfaces and the overproliferation of cuticular material as observed by electron microscopy. Unlike the Arabidopsis WAX INDUCER/SHINE1 (WIN/SHN1) gene, TaSHN1 triggered disorganized cuticular ultrastructure in the transgenic leaves, with the continuous layers replaced by large electrodense bodies embedded in amorphous lipid material. Toluidine blue staining and dark-adapted water release indicated increased cuticular permeability in TaSHN1-expressing Arabidopsis leaves. The expression of TaSHN1 resulted in a moderate decrease of the total number of stomata per unit leaf area in comparison with the wild type. Drought tolerance of Arabidopsis was unaffected by the transgene. The data indicate that this putative wheat orthologue of WIN/SHN transcription factors (TaSHN1) elicited both overlapping and new, distinctive phenotypes compared to other WIN/SHN-overexpressing plants. TaSHN1 transgenic Arabidopsis lines should provide a rich source of material for further comparative biochemical, physiological, and genetic studies.

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).

A single point mutation on the cucumber mosaic virus surface induces an unexpected and strong interaction with the F1 complex of the ATP synthase in Nicotiana clevelandii plants

A previous study showed that a single amino acid difference in the cucumber mosaic virus (CMV) capsid protein (CP) elicits unusual symptoms. The wild-type strain (CMV-R) induces green mosaic symptoms and malformation while the mutant strain (CMV-R3E79R) causes chlorotic lesions on inoculated leaves and strong stunting with necrosis on systemic leaves. Virion preparations of CMV-R and CMV-R3E79R were partially purified from Nicotiana clevelandii A. Gray and analysed by two-dimensional gel electrophoresis. Their separated protein patterns showed remarkable differences at the 50-75 kDa range, both in numbers and intensity of spots, with more protein spots for the mutant CMV. Mass spectrometry analysis demonstrated that the virion preparations contained host proteins identified as ATP synthase alpha and beta subunits as well as small and large Rubisco subunits, respectively. Virus overlay protein binding assay (VOPBA), immunogold electron microscopy and modified ELISA experiments were used to prove the direct interaction between the virus particle and the N. clevelandii ATP synthase F1 motor complex. Protein-protein docking study revealed that the electrostatic change in the mutant CMV can introduce stronger interactions with ATP synthase F1 complex. Based on our findings we suggest that the mutation present in the CP can have a direct effect on the long-distance movement and systemic symptoms. In molecular view the mutant CMV virion can lethally block the rotation of the ATP synthase F1 motor complex which may lead to cell apoptosis, and finally to plant death.

In Vivo DNA Affinity Purification and Histone Deacetylase Inhibitor Treatment Proves the Role of Histone Acetylation in the Expression Regulation of High-Molecular-Weight Glutenin Genes

High-molecular-weight glutenin subunit (HMW GS) proteins are major components of the gluten matrix, which is the physical basis of bread-making in wheat. Epigenetic and transcriptional regulations of HMW GS genes were studied both in silico and in wet lab to understand their tissue (endosperm) specific expression. Our co-expressional network analysis identified key transcription factor (TF) genes that regulate HMW GS genes. We also show here that HMW GS genes are inhibited in vegetative tissues by histone deacetylation as revealed by strong GUS expression in vascular tissues of transgenic barley seedlings harbouring HMW GS gene promoter::uidA-reporter gene fusions upon treatment with a histone deacetylase inhibitor. A novel method termed in vivo DNA affinity purification (IP) has been developed here for the isolation of histones and transcription factors binding to target DNA regions. The technique is based on the biolistic introduction of biotinylated PCR probes amplified from HMW GS gene promoters into wheat leaves. Twenty-four hours later, the probe is cross-linked with interacting factors and subsequently re-purified from plant nuclear extracts. Many proteins, ribosomal proteins and histones have so far been isolated. No lysine-acetylated histone protein fragments were found which further highlight the inhibiting effect of histone deacetylation on HMW GS gene expression.