Diploid Wheat Triticum Monococcum Research Articles

Control of flowering time and spike development in cereals: the earliness per se Eps-1 region in wheat, rice, and Brachypodium

The earliness per se gene Eps-A m 1 from diploid wheat Triticum monococcum affects heading time, spike development, and spikelet number. In this study, the Eps1 orthologous regions from rice, Aegilops tauschii, and Brachypodium distachyon were compared as part of current efforts to clone this gene. A single Brachypodium BAC clone spanned the Eps-A m 1 region, but a gap was detected in the A. tauschii physical map. Sequencing of the Brachypodium and A. tauschii BAC clones revealed three genes shared by the three species, which showed higher identity between wheat and Brachypodium than between them and rice. However, most of the structural changes were detected in the wheat lineage. These included an inversion encompassing the wg241-VatpC region and the presence of six unique genes. In contrast, only one unique gene (and one pseudogene) was found in Brachypodium and none in rice. Three genes were present in both Brachypodium and wheat but were absent in rice. Two of these genes, Mot1 and FtsH4, were completely linked to the earliness per se phenotype in the T. monococcum high-density genetic map and are candidates for Eps-A m 1. Both genes were expressed in apices and developing spikes, as expected for Eps-A m 1 candidates. The predicted MOT1 protein showed amino acid differences between the parental T. monococcum lines, but its effect is difficult to predict. Future steps to clone the Eps-A m 1 gene include the generation of mot1 and ftsh4 mutants and the completion of the T. monococcum physical map to test for the presence of additional candidate genes.Electronic supplementary materialThe online version of this article (doi:10.1007/s10142-009-0146-7) contains supplementary material, which is available to authorized users.

The CArG-Box Located Upstream from the Transcriptional Start of Wheat Vernalization Gene VRN1 Is Not Necessary for the Vernalization Response

In diploid wheat (Triticum monococcum), and likely in other Triticeae species, the VRN1 gene is essential for the initiation of the reproductive phase, and therefore, a detailed characterization of its regulatory regions is required to understand this process. A CArG-box (MADS-box-binding site) identified in the VRN1 promoter upstream from the transcription initiation site has been proposed as a critical regulatory element for the vernalization response. This hypothesis was supported by the genetic linkage between CArG-box natural deletions and dominant Vrn1 alleles for spring growth habit and by physical interactions with VRT2, a MADS-box protein proposed as a putative flowering repressor regulated by vernalization. Here, we describe a T. monococcum accession with a strong vernalization requirement and a 48-bp deletion encompassing the CArG-box in the VRN1 promoter. Genetic analyses of 2 segregating populations confirmed that this VRN1 allele is completely linked with a strong winter growth habit (vrn-A(m)1b). Transcript levels of the VRN1 allele with the 48-bp deletion were very low in unvernalized plants and increased during vernalization to levels similar to those detected in other wild-type vrn-A(m)1 alleles. Taken together, these results indicate that the CArG-box found upstream of the VRN1 transcription initiation site is not essential for the vernalization response.

Gene expression profiling and silencing reveal that monolignol biosynthesis plays a critical role in penetration defence in wheat against powdery mildew invasion.

Cell wall apposition (CWA) formation is one of the first lines of defence used by plants to halt invading fungi such as powdery mildew. Lignin is a complex polymer of hydroxylated and methoxylated phenylpropane units (monolignols) and lignification renders the cell wall more resistant to pathogen attack. The role of monolignol biosynthesis in CWA-mediated defence against powdery mildew penetration into cereals is demonstrated here using RNA interference (RNAi)-mediated gene silencing and enzyme-specific inhibitors. Thirteen cDNAs representing eight genes involved in monolignol biosynthesis were cloned from an expression sequence tag (EST) library derived from the epidermis of diploid wheat (Triticum monococcum) infected with Blumeria graminis f. sp. tritici (Bgt). Differential expression patterns were found for these genes in susceptible and resistant plants after infection. Transcripts of phenylalanine ammonia lyase (PAL), caffeic acid O-methyltransferase (CAOMT), ferulic acid hydroxylase (FAH), caffeoyl-CoA O-methyltransferase (CCoAMT), and cinnamyl alcohol dehydrogenase (CAD) were accumulated, particularly in the epidermis. RNAi-mediated transient gene silencing in the epidermis led to a higher penetration efficiency of Bgt than in the controls. Gene silencing also compromised penetration resistance to varying degrees with different genes against an inappropriate pathogen, B. graminis f. sp. hordei (Bgh). Co-silencing led to greater penetration of Bgt or Bgh than when the genes were silenced separately. Fluorescence emission spectra analyses revealed that gene silencing hampered host autofluorescence response at fungal contact sites. These results illustrate that monolignol biosynthesis is critically important for host defence against both appropriate and inappropriate pathogen invasion in wheat.

Genetic and Molecular Characterization of theVRN2Loci in Tetraploid Wheat

Winter wheat (Triticum spp.) varieties require long exposures to low temperatures to flower, a process called vernalization. The VRN2 locus includes two completely linked zinc finger-CCT domain genes (ZCCT1 and ZCCT2) that act as flowering repressors down-regulated during vernalization. Deletions or mutations in these two genes result in the elimination of the vernalization requirement in diploid wheat (Triticum monococcum). However, natural allelic variation in these genes has not been described so far in polyploid wheat (tetraploid Triticum turgidum and hexaploid Triticum aestivum). A tetraploid wheat population segregating for both VRN-A2 and VRN-B2 loci facilitated the characterization of different alleles. Comparisons between functional and nonfunctional alleles revealed that both ZCCT1 and ZCCT2 genes are able to confer vernalization requirement and that different ZCCT genes are functional in different genomes. ZCCT1 and ZCCT2 proteins from nonfunctional vrn2 alleles have mutations at arginine amino acids at position 16, 35, or 39 of the CCT domain. These positions are conserved between CCT and HEME ACTIVATOR PROTEIN2 (HAP2) proteins, supporting a model in which the action of CCT domains is mediated by their interactions with HAP2/HAP3/HAP5 complexes. This study also revealed natural variation in gene copy number, including a duplication of the functional ZCCT-B2 gene and deletions or duplications of the complete VRN-B2 locus. Allelic variation at the VRN-B2 locus was associated with a partially dominant effect, which suggests that variation in the number of functional ZCCT genes can be used to expand allelic diversity for heading time in polyploid wheat and, hopefully, improve its adaptation to different environments.

The chromosome region including the earliness per se locus Eps-Am1 affects the duration of early developmental phases and spikelet number in diploid wheat

Earliness per se genes are those that regulate flowering time independently of vernalization and photoperiod, and are important for the fine tuning of flowering time and for the wide adaptation of wheat to different environments. The earliness per se locus Eps-Am1 was recently mapped within a 0.8 cM interval on chromosome 1AmL of diploid wheat Triticum monococcum L., and it was shown that its effect was modulated by temperature. In this study, this precise mapping information was used to characterize the effect of the Eps-Am1 region on both duration of different developmental phases and spikelet number. Near isogenic lines (NILs) carrying the Eps-Am1-l allele from the cultivated accession DV92 had significantly longer vegetative and spike development phases (P <0.0001) than NILs carrying the Eps-Am1-e allele from the wild accession G3116. These differences were paralleled by a significant increase in the number of spikelets per spike, in both greenhouse and field experiments (P <0.0001). Significant interactions between temperature and Eps-Am1 alleles were detected for heading time (P <0.0001) but not for spikelet number (P=0.67). Experiments using NILs homozygous for chromosomes with recombination events within the 0.8 cM Eps-Am1 region showed that the differences in number of spikelets per spike were linked to the differences in heading time controlled by the Eps-Am1 locus. These results indicate that the differences in these two traits are either pleiotropic effects of a single gene or the effect of closely linked genes. A similar effect on spikelet number was detected in the distal region of chromosome 1AL in common wheat (T. aestivum L.).

Identification of candidate CBF genes for the frost tolerance locus Fr-A m 2 in Triticum monococcum

A cluster of eleven CBF genes was recently mapped to the Frost resistance-2 (Fr-Am2) locus on chromosome 5 of diploid wheat (Triticum monococcum) using a cross between frost tolerant accession G3116 and frost sensitive DV92. The Fr-Am2 locus was mapped at the peak of two overlapping quantitative trait loci (QTL), one for frost survival and the other for differential expression of the cold regulated gene COR14b. Seven lines with recombination events within the CBF cluster were used to identify CBF candidate genes for these QTL. The lines carrying the critical recombination events were tested for whole plant frost survival and for differential transcript levels of cold induced COR14b and DHN5 genes. The strongest effect for these traits was associated to the linked TmCBF12, TmCBF14 and TmCBF15 genes, with the G3116 allele conferring improved frost tolerance and higher levels of COR14b and DHN5 transcript at mild cold temperatures (12-15 degrees C) than the DV92 allele. Comparison of CBF protein sequences revealed that the DV92 TmCBF12 protein contains a deletion of five amino acids in the AP2 DNA binding domain. Electrophoretic Mobility Shift Assays (EMSA) confirmed that the protein encoded by this allele cannot bind to the CRT/DRE (C-repeat/ dehydration-responsive element) motif present in the promoters of several cold induced genes. A smaller effect on frost tolerance was mapped to the distal group of CBF genes including TmCBF16. Transcript levels of TmCBF16, as well as those of TmCBF12 and TmCBF15 were up-regulated at mild cold temperatures in G3116 but not in DV92. Higher threshold induction temperatures can result in earlier initiation of the cold acclimation process and better resistance to subsequent freezing temperatures. The non-functional TmCBF12 allele in DV92 can also contribute to its lower frost tolerance.

Micro-colinearity between rice, Brachypodium, and Triticum monococcum at the wheat domestication locus Q

Brachypodium, a wild temperate grass with a small genome, was recently proposed as a new model organism for the large-genome grasses. In this study, we evaluated gene content and microcolinearity between diploid wheat (Triticum monococcum), Brachypodium sylvaticum, and rice at a local genomic region harboring the major wheat domestication gene Q. Gene density was much lower in T. monococcum (one per 41 kb) because of gene duplication and an abundance of transposable elements within intergenic regions as compared to B. sylvaticum (one per 14 kb) and rice (one per 10 kb). For the Q gene region, microcolinearity was more conserved between wheat and rice than between wheat and Brachypodium because B. sylvaticum contained two genes apparently not present within the orthologous regions of T. monococcum and rice. However, phylogenetic analysis of Q and leukotriene A-4 hydrolase-like gene orthologs, which were colinear among the three species, showed that Brachypodium is more closely related to wheat than rice, which agrees with previous studies. We conclude that Brachypodium will be a useful tool for gene discovery, comparative genomics, and the study of evolutionary relationships among the grasses but will not preclude the need to conduct large-scale genomics experiments in the Triticeae.

Molecular Diversity at 18 Loci in 321 Wild and 92 Domesticate Lines Reveal No Reduction of Nucleotide Diversity during Triticum monococcum (Einkorn) Domestication: Implications for the Origin of Agriculture

The diploid wheat Triticum monococcum L. (einkorn) was among the first crops domesticated by humans in the Fertile Crescent 10,000 years ago. During the last 5,000 years, it was replaced by tetraploid and hexaploid wheats and largely forgotten by modern breeders. Einkorn germplasm is thus devoid of breeding bottlenecks and has therefore preserved in unfiltered form the full spectrum of genetic variation that was present during its domestication. We investigated haplotype variation among >12 million nucleotides sequenced at 18 loci across 321 wild and 92 domesticate T. monococcum lines. In contrast to previous studies of cereal domestication, we sampled hundreds of wild lines, rather than a few dozen. Unexpectedly, our broad sample of wild lines reveals that wild einkorn underwent a process of natural genetic differentiation, most likely an incipient speciation, prior to domestication. That natural differentiation was previously overlooked within wild einkorn, but it bears heavily upon inferences concerning the domestication process because it brought forth 3 genetically, and to some extent morphologically, distinct wild einkorn races that we designate here as alpha, beta, and gamma. Only one of those natural races, beta, was exploited by humans for domestication. Nucleotide diversity and haplotype diversity in domesticate einkorn is higher than in its wild sister group, the einkorn beta race, indicating that einkorn underwent no reduction of diversity during domestication. This is in contrast to findings from previous studies of domestication history among more intensely bred crop species. Taken together with archaeological findings from the Fertile Crescent, the data indicate that a specific wild einkorn race that arose without human intervention was subjected to multiple independent domestication events.

Transcriptional regulation of genes involved in the pathways of biosynthesis and supply of methyl units in response to powdery mildew attack and abiotic stresses in wheat

From a library of 3,000 expression sequence tags (ESTs), derived from the epidermis of a diploid wheat (Triticum monococcum) inoculated with Blumeria graminis f. sp. tritici (Bgt), we cloned 23 cDNAs representing 12 genes that are involved in the pathways of biosynthesis and supply of methyl units. We studied the transcription of these genes to investigate how the methyl units are generated and regulated in response to Bgt infection and abiotic stresses in wheat. Expression of 5, 10-methylene-tetrahydrofolate reductase, methionine synthase, S-adenosylmethionine synthetase, and S-adenosylhomocystein hydrolase transcripts were highly induced at an early stage of infection. This induction was specific to the epidermis and linked to host cell wall apposition (CWA) formation, suggesting that the pathways for generation of methyl units are transcriptionally activated for the host defense response. Levels of S-adenosylmethionine decarboxylase, caffeic acid 3-O-methyltransferase, 1-aminocyclopropane-1-carboxylate oxidase mRNA, but not phosphoethanolamine N-methyltransferase and nicotianamine synthase mRNA, were up-regulated after infection and showed similar expression patterns to genes involved in the pathways of generation of methyl units, revealing possible routes of methyl transfer towards polyamine, lignin and ethylene biosynthesis rather than glycine betaine and nicotianamine in response to Bgt attack. After imposing various abiotic stresses, genes involved in the pathways of generation and supply of methyl units were also up-regulated differentially, suggesting that the generation of sufficient methyl units at an early stage might be crucial to the mitigation of multiple stresses.

Reduction of a Triticum monococcum Chromosome Segment Carrying the Softness Genes Pina and Pinb Translocated to Bread Wheat

ABSTRACTEndosperm texture, i.e., the hardness or softness of the grain, is an important trait because it determines many end‐use properties of wheat (Triticum aestivum L.). It is primarily controlled by the puroindoline genes (Pina and Pinb) at the Hardness (Ha) locus, mapped on the short arm of chromosome 5D. The introgression of functional Pin genes from diploid wheat Triticum monococcum L. chromosome 5Am into hexaploid wheat resulted in softer grains, suggesting that this translocation might be useful for soft wheat breeders. However, the translocated segment includes a large portion of the 5Am short arm and may carry detrimental genes for agronomic performance. In this study we have generated a backcross (BC) population of 210 individuals where 5A‐5Am homeologous recombination was induced by the ph1b mutation to recover individuals with a reduced translocated segment. A map of this region was constructed using specific sequence tagged site (STS) markers for the three T. monococcum Ha–related genes, the completely linked BGGP gene, three wheat ESTs (BG606847, BF474606, and BQ168958), and two microsatellite markers. Eight plants with recombination events between XBggp and the closest proximal locus BG606847 were identified. Of these, four have the desired T. monococcum allele at the Ha locus. These plants carry a 6.3‐cM segment of T. monococcum chromatin proximal to the Ha locus. This germplasm, which will be publicly available, and the molecular markers developed in this study will be valuable tools for soft wheat breeding programs.

Wheat VIN3-like PHD finger genes are up-regulated by vernalization

The term 'vernalization' describes the acceleration of the transition between the vegetative and reproductive stages after exposing plants to an extended period of low temperature. In Arabidopsis, vernalization promotes flowering by silencing the flowering repressor gene FLOWERING LOCUS C (FLC). Mitotically stable repression of FLC is the result of chromatin modifications mediated by the Vernalization-INsensitive 3 (VIN3) and VIN3-Like (VIL) proteins. In this study, we identified and characterized three VIL genes in diploid wheat (Triticum monococcum L.), named TmVIL1, TmVIL2, and TmVIL3. Similar to Arabidopsis VIN3, all three wheat VIL proteins carry three conserved domains including a plant homeodomain finger motif (PHD), a fibronectin type III domain (FNIII), and a VIN3 interacting domain (VID). Genetic mapping placed TmVIL1, TmVIL2, and TmVIL3 loci in the centromeric regions of chromosome 5, 6, and 1, respectively. The chromosome location of TmVIL1 is close to that of the vernalization gene VRN-D5, but more precise mapping information is required to validate this relationship. Transcription of the wheat VIL genes was up-regulated by vernalization, with a peak after 4-6 weeks of cold treatment. When transferred back to warm conditions, transcript levels of the wheat VIL genes returned to pre-vernalization levels. In addition, the transcript levels of wheat VIL genes are affected by photoperiod. This study indicates that wheat VIL genes have retained a similar structure and transcriptional regulation as their Arabidopsis VIN3/VIL homologues, suggesting that they might have retained some of their functions.

Analysis of retrotransposon structural diversity uncovers properties and propensities in angiosperm genome evolution.

Analysis of LTR retrotransposon structures in five diploid angiosperm genomes uncovered very different relative levels of different types of genomic diversity. All species exhibited recent LTR retrotransposon mobility and also high rates of DNA removal by unequal homologous recombination and illegitimate recombination. The larger plant genomes contained many LTR retrotransposon families with >10,000 copies per haploid genome, whereas the smaller genomes contained few or no LTR retrotransposon families with >1,000 copies, suggesting that this differential potential for retroelement amplification is a primary factor in angiosperm genome size variation. The average ratios of transition to transversion mutations (Ts/Tv) in diverging LTRs were >1.5 for each species studied, suggesting that these elements are mostly 5-methylated at cytosines in an epigenetically silenced state. However, the diploid wheat Triticum monococcum and barley have unusually low Ts/Tv values (respectively, 1.9 and 1.6) compared with maize (3.9), medicago (3.6), and lotus (2.5), suggesting that this silencing is less complete in the two Triticeae. Such characteristics as the ratios of point mutations to indels (insertions and deletions) and the relative efficiencies of DNA removal by unequal homologous recombination compared with illegitimate recombination were highly variable between species. These latter variations did not correlate with genome size or phylogenetic relatedness, indicating that they frequently change during the evolutionary descent of plant lineages. In sum, the results indicate that the different sizes, contents, and structures of angiosperm genomes are outcomes of the same suite of mechanistic processes, but acting with different relative efficiencies in different plant lineages.

Vernalization Response in Perennial Ryegrass (Lolium perenne L.) Involves Orthologues of Diploid Wheat (Triticum monococcum) VRN1 and Rice (Oryza sativa) Hd1

Flowering time is important when adapting crop plants to different environments. While high feeding quality of forage grasses is facilitated by repression of flowering, flowering should also be inducible to facilitate grass seed production. Consequently, the identification and characterization of the genes controlling flowering time in forage grasses, including perennial ryegrass (Lolium perenne L.), is of great interest. In this study, three candidate genes for vernalization response genes in perennial ryegrass were identified based on DNA sequence homology to TmVRN1 and TmVRN2 of diploid wheat (Triticum monococcum), and Hd1 of rice (Oryza sativa). High sequence similarity between LpVRN1 and TmVRN1, co-localization of LpVRN1 with a major quantitative trait loci (QTL) for vernalization response in perennial ryegrass, synteny between map-positions of LpVRN1 and TmVRN1, mRNA expression analysis of LpVRN1 alleles during vernalization, and the correspondence between LpVRN1 mRNA expression levels and flowering time leads us to conclude that LpVRN1 is orthologous to TmVRN1 and that its function is conserved between diploid wheat and perennial ryegrass. Of the remaining two candidate genes, a putative Hd1 orthologue, LpCO, co-localized with a second QTL for vernlization response. LpCO has recently been shown to be involved in the photoperiodic regulation of flowering time. While epistasis, at the level of LpVRN1 transcription, was observed between the LpVRN1 and LpCO genomic regions, no differential expression of LpCO transcripts was observed during vernalization. While orthologous genes controlling flowering time can thus be identified, future allele sequencing efforts will reveal if causative polymorphisms are conserved across the grasses.

A cluster of 11 CBF transcription factors is located at the frost tolerance locus Fr-A m 2 in Triticum monococcum

Due to the adverse effects of cold temperatures on winter wheat, frost tolerance is an important trait for breeding programs in regions with severe winters. Frost tolerance locus Fr-A(m)2 was recently discovered in diploid wheat (Triticum monococcum L.). This locus was mapped as a QTL on chromosome 5A(m) in the same region as a QTL for the level of transcription of the cold-regulated gene COR14b at 15 degrees C. A CBF transcription factor was mapped in the center of these two overlapping QTLs. However, since the CBF gene family in wheat has numerous members, it was possible that multiple CBF genes were present at Fr-A(m)2. To investigate this possibility we initiated a systematic characterization of the CBF family in T. monococcum. Here we report the molecular characterization of thirteen TmCBF genes. Nine of them were numbered according to the closest barley HvCBF gene, and the other four that have no clear barley orthologues were assigned numbers TmCBF15 to TmCBF18. TmCBF5 and TmCBF18 were mapped on T. monococcum chromosomes 7A(m) and 6A(m), respectively, and are thus not candidates for the Fr-A(m)2 gene. The remaining eleven TmCBF genes are clustered at the Fr-A(m)2 locus within five different Bacterial Artificial Chromosome (BAC) clones. These BACs were mapped using a high-density map and recombination events were found between most BACs. Lines carrying these recombination events will be useful to identify which of the CBF genes is responsible for the differences in frost tolerance between the T. monococcum parental lines at the Fr-A(m)2 locus.

Differential regulation of wheat quinone reductases in response to powdery mildew infection

At least two types of quinone reductases are present in plants: (1) the zeta-crystallin-like quinone reductases (QR1, EC 1.6.5.5) that catalyze the univalent reduction of quinones to semiquinone radicals, and (2) the DT-diaphorase-like quinone reductases (QR2, EC 1.6.99.2) that catalyze the divalent reduction of quinones to hydroquinones. QR2s protect cells from oxidative stress by making the quinones available for conjugation, thereby releasing them from the superoxide-generating one electron redox cycling, catalyzed by QR1s. Two genes, putatively encoding a QR1 and a QR2, respectively, were isolated from an expressed sequence tag collection derived from the epidermis of a diploid wheat Triticum monococcum L. 24 h after inoculation with the powdery mildew fungus Blumeria graminis (DC) EO Speer f. sp. tritici Em. Marchal. Northern analysis and tissue-specific RT-PCR showed that TmQR1 was repressed while TmQR2 was induced in the epidermis during powdery mildew infection. Heterologous expression of TmQR2 in Escherichia coli confirmed that the gene encoded a functional, dicumarol-inhibitable QR2 that could use either NADH or NADPH as an electron donor. The localization of dicumarol-inhibitable QR2 activity around powdery mildew infection sites was accomplished using a histochemical technique, based on tetrazolium dye reduction.

Profiling of Wheat Class III Peroxidase Genes Derived from Powdery Mildew-Attacked Epidermis Reveals Distinct Sequence-Associated Expression Patterns

A cDNA library was constructed from leaf epidermis of diploid wheat (Triticum monococcum) infected with the powdery mildew fungus (Blumeria graminis f. sp. tritici) and was screened for genes encoding peroxidases. From 2,500 expressed sequence tags (ESTs), 36 cDNAs representing 10 peroxidase genes (designated TmPRX1 to TmPRX10) were isolated and further characterized. Alignment of the deduced amino acid sequences and phylogenetic clustering with peroxidases from other plant species demonstrated that these peroxidases fall into four distinct groups. Differential expression and tissue-specific localization among the members were observed during the B. graminis f. sp. tritici attack using Northern blots and reverse-transcriptase polymerase chain reaction analyses. Consistent with its abundance in the EST collection, TmPRX1 expression showed the highest induction during pathogen attack and fluctuated in response to the fungal parasitic stages. TmPRX1 to TmPRX6 were expressed predominantly in mesophyll cells, whereas TmPRX7 to TmPRX10, which feature a putative C-terminal propeptide, were detectable mainly in epidermal cells. Using TmPRX8 as a representative, we demonstrated that its C-terminal propeptide was sufficient to target a green fluorescent protein fusion protein to the vacuoles in onion cells. Finally, differential expression profiles of the TmPRXs after abiotic stresses and signal molecule treatments were used to dissect the potential role of these peroxidases in multiple stress and defense pathways.

Activity and gene expression of acid invertases in einkorn wheat (Triticum monococcum) infected with powdery mildew

Hexose released from sucrose by cell wall bound acid invertases is considered to be a major carbon source for powdery mildews. This aspect of pathogenesis was investigated in diploid wheat (Triticum monococcum) infected with powdery mildew [Blumeria graminis f. sp. tritici]. Quantitative assays revealed an increase in acid invertase activity following infection, and that soluble invertases, of vacuolar and (or) extracellular origin, accounted for over 80% of the overall increase. In situ detection showed activity of cell wall bound invertase in both uninfected and infected leaf tissues, but no increase in activity following infection and no activity at the infection sites. Two cDNAs of the putative genes Tm-exin1 and Tm-vin1 coding for a soluble extracellular invertase and a vacuolar invertase, respectively, were identified in a collection of expressed sequence tags derived from mildewed wheat leaf epidermis. Tm-exin1 was induced in infected leaves, and the enhanced expression coincided with conidiation and colonization of wheat leaf by the fungus. Conversely, Tm-vin1 expression remained relatively constant throughout infection. Collectively, these results support the view that invertases mediate hexose production during infection by the fungus causing powdery mildew and, furthermore, suggest that soluble extracellular invertase is responsible for providing the increased hexose required by the fungus.

Inhibition of α-Amylase Acting in Hexaploid Triticale Lines by Exogenous Abscisic Acid

Hexaploid triticale introgressive lines developed after recombination of A-genome with Am-genome of diploid wheat (Triticum monococcum) were analysed in respect of grains responsiveness to exogenous ABA treatment. This was assessed by in vivo bioassay as grain germination indices, and by α-amylase assay as quantity of synthesised α-amylase measured with the technique of radial diffusion in agarose gel. The results showed an important diminishing of seedling length caused by ABA (variable in different lines) as well as genotype dependant variability of α-amylase synthesis inhibition. The differences of ABA responsiveness were seen both in whole grains and in embryoless half-grains as a direct reaction of the aleurone layer. Variation of grain sensitivity to ABA treatment compared with two sprouting resistance indices showed a significant correlation with Falling Number values in grains, but not with a dormant grains germination in spikes. This is an evidence that in triticale precocious starch decompose in unripened and ungerminated grains is dependent on genotype ABA-responsiveness of the aleurone layer.

Diploid wheat — Triticum monococcum as a source of resistance genes to preharvest sprouting of triticale

Three accessions of diploid wheat Triticum monococcum var. monococcum (A m A m ) evaluated in respect of response of spikes to sprouting showed a markedly high values of sprouting resistance indices (number of sprouted kernels/spike, Hagbergs 1 Falling Number). The genome of T. monococcum var. monococcum (f. macedonicum Papag.) was incorporated into two triticale strains representing high and moderately level of susceptibility to sprouting. The appropriate two sets of introgressive triticale/T. monococcum lines were analysed in respect of variation of mature spikes sprouting (SR) and Falling Number (FN) values. The results showed different manner of inheritance of both characters. Full expression of Triticum monococcum genetic material responsible for resistance of mature spikes to sprouting was found and it was independent on hexaploid triticale recipient parent. In opposite to this, FN values higher than 200 sec. were originated on the basis of specific combination property of Triticum monococcum and triticale genes.

Metabolism, seedling growth and nucleolar activity in germinating diploid wheat Triticum monococcum ssp. monococcum cv. Einkorn

Seed germination, seedling growth, mitotic and nucleolar activity were investigated as indicators of metabolic activity in a diploid wheat, Triticum monococcum ssp. monococcum cv. Einkorn. Two developmental phases were identified, an initial lag phase during which reactivation of NOR's and the initiation of mitotic activity (10 h) took place prior to the appearance of the primary root (16 h). Dominance interactions were observed between the NOR's of chromosomes 1A and 5A with the NOR's of the one pair activated before the other and forming therefore larger nucleoli. The total number of nucleoli per 500 cells also varied according to the stages of the cell cycle over time. The mitotic activity had an initial exponential rise, but showed signs of flattening out after 10 h of mitosis with opposite levels of activity in the primary and first pair of lateral roots thereafter. Secondly, an exponential phase in root and shoot growth, mobilization of reserve food and respiration rate was observed during seedling growth. Comparisons were done on work from a previous investigation on two polyploid wheat accessions belonging toT. turgidum ssp. durum and T. aestivum species. T. monococcum displayed a higher embryo growth than the polyploids, that may be explained by having a lighter embryo and caryopsis weight that may influence the uptake of water and reactivation of metabolic activity. During the seedling growth period, the two polyploid species displayed a faster growth rate than the diploid probably due to the differences in the number of genomes, the hybrid nature of the polyploids and a more complex gene control in the polyploids.