Prevalence of gene expression additivity in genetically stable wheat allohexaploids.

The reprogramming of gene expression appears as the major trend in synthetic and natural allopolyploids where expression of an important proportion of genes was shown to deviate from that of the parents or the average of the parents. In this study, we analyzed gene expression changes in previously reported, highly stable synthetic wheat allohexaploids that combine the D genome of Aegilops tauschii and the AB genome extracted from the natural hexaploid wheat Triticum aestivum. A comprehensive genome-wide analysis of transcriptional changes using the Affymetrix GeneChip Wheat Genome Array was conducted. Prevalence of gene expression additivity was observed where expression does not deviate from the average of the parents for 99.3% of 34,820 expressed transcripts. Moreover, nearly similar expression was observed (for 99.5% of genes) when comparing these synthetic and natural wheat allohexaploids. Such near-complete additivity has never been reported for other allopolyploids and, more interestingly, for other synthetic wheat allohexaploids that differ from the ones studied here by having the natural tetraploid Triticum turgidum as the AB genome progenitor. Our study gave insights into the dynamics of additive gene expression in the highly stable wheat allohexaploids.

Alteration in expression of hormone-related genes in wild emmer wheat roots associated with drought adaptation mechanisms.

Transcriptomic and metabolomic profiles were used to unravel drought adaptation mechanisms in wild emmer wheat (Triticum turgidum ssp. dicoccoides), the progenitor of cultivated wheat, by comparing the response to drought stress in roots of genotypes contrasting in drought tolerance. The differences between the drought resistant (R) and drought susceptible (S) genotypes were characterized mainly by shifts in expression of hormone-related genes (e.g., gibberellins, abscisic acid (ABA) and auxin), including biosynthesis, signalling and response; RNA binding; calcium (calmodulin, caleosin and annexin) and phosphatidylinositol signalling, in the R genotype. ABA content in the roots of the R genotype was higher in the well-watered treatment and increased in response to drought, while in the S genotype ABA was invariant. The metabolomic profiling revealed in the R genotype a higher accumulation of tricarboxylic acid cycle intermediates and drought-related metabolites, including glucose, trehalose, proline and glycine. The integration of transcriptomics and metabolomics results indicated that adaptation to drought included efficient regulation and signalling pathways leading to effective bio-energetic processes, carbon metabolism and cell homeostasis. In conclusion, mechanisms of drought tolerance were identified in roots of wild emmer wheat, supporting our previous studies on the potential of this genepool as a valuable source for novel candidate genes to improve drought tolerance in cultivated wheat.

Multilevel regulation and signalling processes associated with adaptation to terminal drought in wild emmer wheat.

Low water availability is the major environmental factor limiting crop productivity. Transcriptome analysis was used to study terminal drought response in wild emmer wheat, Triticum dicoccoides, genotypes contrasting in their productivity and yield stability under drought stress. A total of 5,892 differentially regulated transcripts were identified between drought and well-watered control and/or between drought resistant (R) and drought susceptible (S) genotypes. Functional enrichment analyses revealed that multilevel regulatory and signalling processes were significantly enriched among the drought-induced transcripts, in particular in the R genotype. Therefore, further analyses were focused on selected 221 uniquely expressed or highly abundant transcripts in the R genotype, as potential candidates for drought resistance genes. Annotation of the 221 genes revealed that 26% of them are involved in multilevel regulation, including: transcriptional regulation, RNA binding, kinase activity and calcium and abscisic acid signalling implicated in stomatal closure. Differential expression patterns were also identified in genes known to be involved in drought adaptation pathways, such as: cell wall adjustment, cuticular wax deposition, lignification, osmoregulation, redox homeostasis, dehydration protection and drought-induced senescence. These results demonstrate the potential of wild emmer wheat as a source for candidate genes for improving drought resistance.

Newly synthesized wheat allohexaploids display progenitor-dependent meiotic stability and aneuploidy but structural genomic additivity.

To understand key mechanisms leading to stabilized allopolyploid species, we characterized the meiotic behaviour of wheat allohexaploids in relation to structural and genetic changes. For that purpose, we analysed first generations of synthetic allohexaploids obtained through interspecific hybridization, followed by spontaneous chromosome doubling, between several genotypes of Triticum turgidum and Aegilops tauschii wheat species, donors of AB and D genomes, respectively. As expected for these Ph1 (Pairing homoeologous 1) gene-carrying allopolyploids, chromosome pairing at metaphase I of meiosis essentially occurs between homologous chromosomes. However, the different synthetic allohexaploids exhibited progenitor-dependent meiotic irregularities, such as incomplete homologous pairing, resulting in univalent formation and leading to aneuploidy in the subsequent generation. Stability of the synthetic allohexaploids was shown to depend on the considered genotypes of both AB and D genome progenitors, where few combinations compare to the natural wheat allohexaploid in terms of regularity of meiosis and euploidy. Aneuploidy represents the only structural change observed in these synthetic allohexaploids, as no apparent DNA sequence elimination or rearrangement was observed when analysing euploid plants with molecular markers, developed from expressed sequence tags (ESTs) as well as simple sequence repeat (SSR) and transposable element sequences.

Genome-wide gene expression changes in genetically stable synthetic and natural wheat allohexaploids.

*The present study aims to understand regulation of gene expression in synthetic and natural wheat (Triticum aestivum) allohexaploids, that combines the AB genome of Triticum turgidum and the D genome of Aegilops tauschii; and which we have recently characterized as genetically stable. *We conducted a comprehensive genome-wide analysis of gene expression that allowed characterization of the effect of variability of the D genome progenitor, the intergenerational stability as well as the comparison with natural wheat allohexaploid. We used the Affymetrix GeneChip Wheat Genome Array, on which 55 049 transcripts are represented. *Additive expression was shown to represent the majority of expression regulation in the synthetic allohexaploids, where expression for more than c. 93% of transcripts was equal to the mid-parent value measured from a mixture of parental RNA. This leaves c. 2000 (c. 7%) transcripts, in which expression was nonadditive. No global gene expression bias or dominance towards any of the progenitor genomes was observed whereas high intergenerational stability and low effect of the D genome progenitor variability were revealed. *Our study suggests that gene expression regulation in wheat allohexaploids is established early upon allohexaploidization and highly conserved over generations, as demonstrated by the high similarity of expression with natural wheat allohexaploids.

CgOpt1, a putative oligopeptide transporter from Colletotrichum gloeosporioides that is involved in responses to auxin and pathogenicity.

BACKGROUND: The fungus Colletotrichum gloeosporioides f. sp. aeschynomene produces high levels of indole-3-acetic acid (IAA) in axenic cultures and during plant infection. We generated a suppression subtractive hybridization library enriched for IAA-induced genes and identified a clone, which was highly expressed in IAA-containing medium. RESULTS: The corresponding gene showed similarity to oligopeptide transporters of the OPT family and was therefore named CgOPT1. Expression of CgOPT1 in mycelia was low, and was enhanced by external application of IAA. cgopt1-silenced mutants produced less spores, had reduced pigmentation, and were less pathogenic to plants than the wild-type strain. IAA enhanced spore formation and caused changes in colony morphology in the wild-type strain, but had no effect on spore formation or colony morphology of the cgopt1-silenced mutants. CONCLUSION: Our results show that IAA induces developmental changes in C. gloeosporioides. These changes are blocked in cgopt1-silenced mutants, suggesting that this protein is involved in regulation of fungal response to IAA. CgOPT1 is also necessary for full virulence, but it is unclear whether this phenotype is related to auxin.

New insights into the origin of the B genome of hexaploid wheat: evolutionary relationships at the SPA genomic region with the S genome of the diploid relative Aegilops speltoides.

BACKGROUND: Several studies suggested that the diploid ancestor of the B genome of tetraploid and hexaploid wheat species belongs to the Sitopsis section, having Aegilops speltoides (SS, 2n = 14) as the closest identified relative. However molecular relationships based on genomic sequence comparison, including both coding and non-coding DNA, have never been investigated. In an attempt to clarify these relationships, we compared, in this study, sequences of the Storage Protein Activator (SPA) locus region of the S genome of Ae. speltoides (2n = 14) to that of the A, B and D genomes co-resident in the hexaploid wheat species (Triticum aestivum, AABBDD, 2n = 42). RESULTS: Four BAC clones, spanning the SPA locus of respectively the A, B, D and S genomes, were isolated and sequenced. Orthologous genomic regions were identified as delimited by shared non-transposable elements and non-coding sequences surrounding the SPA gene and correspond to 35,268, 22,739, 43,397 and 53,919 bp for the A, B, D and S genomes, respectively. Sequence length discrepancies within and outside the SPA orthologous regions are the result of non-shared transposable elements (TE) insertions, all of which inserted after the progenitors of the four genomes divergence. CONCLUSION: On the basis of conserved sequence length as well as identity of the shared non-TE regions and the SPA coding sequence, Ae speltoides appears to be more evolutionary related to the B genome of T. aestivum than the A and D genomes. However, the differential insertions of TEs, none of which are conserved between the two genomes led to the conclusion that the S genome of Ae. speltoides has diverged very early from the progenitor of the B genome which remains to be identified.

Ethylene sensing and gene activation in Botrytis cinerea: a missing link in ethylene regulation of fungus-plant interactions?

Ethylene production by infected plants is an early resistance response leading to activation of plant defense pathways. However, plant pathogens also are capable of producing ethylene, and ethylene might have an effect not only on the plant but on the pathogen as well. Therefore, ethylene may play a dual role in fungus-plant interactions by affecting the plant as well as the pathogen. To address this question, we studied the effects of ethylene on the gray mold fungus Botrytis cinerea and the disease it causes on Nicotiana benthamiana plants. Exposure of B. cinerea to ethylene inhibited mycelium growth in vitro and caused transcriptional changes in a large number of fungal genes. A screen of fungal signaling mutants revealed a Galpha null mutant (deltabcg1) which was ethylene insensitive, overproduced ethylene in vitro, and showed considerable transcriptional changes in response to ethylene compared with the wild type. Aminoethoxyvinylglycine (AVG)-treated, ethylene-nonproducing N. benthamiana plants developed much larger necroses than ethylene-producing plants, whereas addition of ethylene to AVG-treated leaves restricted disease spreading. Ethylene also affected fungal gene expression in planta. Expression of a putative pathogenicity fungal gene, bcspl1, was enhanced 24 h after inoculation in ethylene-producing plants but only 48 h after inoculation in ethylene-nonproducing plants. Our results show that the responses of B. cinerea to ethylene are partly mediated by a G protein signaling pathway, and that ethylene-induced plant resistance might involve effects of plant ethylene on both the plant and the fungus.

Functional characterization of LePT4: a phosphate transporter in tomato with mycorrhiza-enhanced expression.

Many plant roots acquire inorganic phosphate (Pi) from soils directly through the root-soil interface via high-affinity Pi transporters and/or through symbiotic associations between the cortical cells and arbuscular mycorrhizal fungi. In tomato, three phosphate transporters (LePT3, LePT4, and LePT5) are up-regulated upon colonization by arbuscular mycorrhizal fungi. In this study, the role of LePT4 in tomato is elucidated by molecular and physiological characterizations of a loss-of-function mutant lept4. In the absence of mycorrhizal infection and under solution-Pi concentrations (Cp) of 0.05 mM and 0.5 mM, the mutant exhibited severe Pi-deficiency symptoms which were associated with significantly lower Pi uptake as compared with that of the wild type. However, at a Cp of 5 mM, lept4 grew better than the wild type. Mycorrhizal infection at a Cp of 0.05 mM resulted in a significant increase in the transcripts of LePT4 in the wild type and a concomitant 2-fold increase in Pi uptake. Although upon mycorrhizal infection, lept4 also exhibited an increased Pi uptake, it was significantly lower than that of the wild type. Under a Cp of 1 mM and in the absence of mycorrhizal infection, LePT4 expression was suppressed in the wild type and a mutation in this gene resulted in a slight reduction in total Pi uptake. These data highlight the pivotal role of LePT4 in mycorrhizal-mediated Pi uptake in tomato, and show that this function may not be fully compensated by other members of the family. Characterization of the mycorrhiza-associated Pi transporter lept4 mutant, along with expression analysis of LePT3, provides evidence for the different routes of mycorrhiza-mediated Pi uptake in plants.

The characterization of novel mycorrhiza-specific phosphate transporters from Lycopersicon esculentum and Solanum tuberosum uncovers functional redundancy in symbiotic phosphate transport in solanaceous species.

Solanaceous species are among the >200 000 plant species worldwide forming a mycorrhiza, that is, a root living in symbiosis with soil-borne arbuscular-mycorrhizal (AM) fungi. An important parameter of this symbiosis, which is vital for ecosystem productivity, agriculture, and horticulture, is the transfer of phosphate (Pi) from the AM fungus to the plant, facilitated by plasma membrane-spanning Pi transporter proteins. The first mycorrhiza-specific plant Pi transporter to be identified, was StPT3 from potato [Nature414 (2004) 462]. Here, we describe novel Pi transporters from the solanaceous species tomato, LePT4, and its orthologue StPT4 from potato, both being members of the Pht1 family of plant Pi transporters. Phylogenetic tree analysis demonstrates clustering of both LePT4 and StPT4 with the mycorrhiza-specific Pi transporter from Medicago truncatula [Plant Cell, 14 (2002) 2413] and rice [Proc. Natl Acad. Sci. USA, 99 (2002) 13324], respectively, but not with StPT3, indicating that two non-orthologous mycorrhiza-responsive genes encoding Pi transporters are co-expressed in the Solanaceae. The cloned promoter regions from both genes, LePT4 and StPT4, exhibit a high degree of sequence identity and were shown to direct expression exclusively in colonized cells when fused to the GUS reporter gene, in accordance with the abundance of LePT4 and StPT4 transcripts in mycorrhized roots. Furthermore, extensive sequencing of StPT4-like clones and subsequent expression analysis in potato and tomato revealed the presence of a close paralogue of StPT4 and LePT4, named StPT5 and LePT5, respectively, representing a third Pi transport system in solanaceous species which is upregulated upon AM fungal colonization of roots. Knock out of LePT4 in the tomato cv. MicroTom indicated considerable redundancy between LePT4 and other Pi transporters in tomato.

Ethylene biosynthesis in Botrytis cinerea.

Ethylene is often released during plant pathogenesis. Enhanced ethylene biosynthesis by the attacked plant, and formation of ethylene by the attacking pathogen may be involved. We defined the biosynthetic pathway of ethylene in the pathogenic fungus Botrytis cinerea, and characterized the conditions that affect ethylene production in vitro. During the first 48 h of culture the fungus uses methionine to produce alpha-keto gamma-methylthiobutyric acid (KMBA) and secretes it to the medium. In darkness, KMBA accumulates in the medium. In light KMBA is photo-oxidized and ethylene is released. The photo-oxidation reaction is spontaneous and does not involve any enzymatic activity. Low levels of ethylene are produced in darkness between 48 and 96 h of culture. Adding peroxidase to dark-grown cultures induced ethylene formation. The results suggest that formation and secretion of KMBA by B. cinerea may affect ethylene levels during plant infection.