M. Ciaffi

Identification and validation of reference genes for quantitative RT-PCR normalization in wheat

BackgroundUsually the reference genes used in gene expression analysis have been chosen for their known or suspected housekeeping roles, however the variation observed in most of them hinders their effective use. The assessed lack of validated reference genes emphasizes the importance of a systematic study for their identification. For selecting candidate reference genes we have developed a simple in silico method based on the data publicly available in the wheat databases Unigene and TIGR.ResultsThe expression stability of 32 genes was assessed by qRT-PCR using a set of cDNAs from 24 different plant samples, which included different tissues, developmental stages and temperature stresses. The selected sequences included 12 well-known HKGs representing different functional classes and 20 genes novel with reference to the normalization issue. The expression stability of the 32 candidate genes was tested by the computer programs geNorm and NormFinder using five different data-sets. Some discrepancies were detected in the ranking of the candidate reference genes, but there was substantial agreement between the groups of genes with the most and least stable expression. Three new identified reference genes appear more effective than the well-known and frequently used HKGs to normalize gene expression in wheat. Finally, the expression study of a gene encoding a PDI-like protein showed that its correct evaluation relies on the adoption of suitable normalization genes and can be negatively affected by the use of traditional HKGs with unstable expression, such as actin and α-tubulin.ConclusionThe present research represents the first wide screening aimed to the identification of reference genes and of the corresponding primer pairs specifically designed for gene expression studies in wheat, in particular for qRT-PCR analyses. Several of the new identified reference genes outperformed the traditional HKGs in terms of expression stability under all the tested conditions. The new reference genes will enable more accurate normalization and quantification of gene expression in wheat and will be helpful for designing primer pairs targeting orthologous genes in other plant species.

Storage-protein variation in wild emmer wheat (Triticum turgidum ssp.dicoccoides) from Jordan and Turkey. I. Electrophoretic characterization of genotypes.

Seed storage-protein variation at theGlu-A1,Glu-B1 andGli-B1/Glu-B3 loci in the tetraploid wild progenitor of wheat,T. dicoccoides, was studied electrophoretically in 315 individuals representing nine populations from Jordan and three from Turkey. A total of 44 different HMW-glutenin patterns were identified, resulting from the combination of 15 alleles in the A genome and 19 in the B genome. Twenty-seven new allelic variants, 12 at theGlu-A1 locus and 15 at theGlu-B1 locus, were identified by comparing the mobilities of their subunits to those previously found in bread and durum wheats. The novel variants include six alleles at theGlu-A1 locus showing both x and y subunits. The genes coding for the 1Bx and 1By subunits showed no or very little (3%) inactivity, the 1Ax gene showed a moderate degree (6.3%) of inactivity whereas the gene coding for lAy showed the highest degree of inactivity (84.8%). A high level of polymorphism was also present for the omega- and gamma-gliadins and LMW-glutenin subunits encoded by genes at the linkedGli-B1 andGlu-B3 loci (19 alleles). Some Jordanian accessions were found to contain omega-gliadin 35, gamma-gliadin 45, and LMW-2 also present in cultivated durum wheats and related to good gluten viscoelasticity. The newly-discovered alleles enhance the genetic variability available for improving the technological quality of wheats. Additionally some of them may facilitate basic research on the relationship between industrial properties and the number and functionality of HMW- and LMW-glutenin subunits.

Introgression of Dasypyrum villosum chromatin into common wheat improves grain protein quality

Dasypyrum villosum (L.) Candargy (DV) is adiploid (2n = 14, VV genomes), allogamous grass of theMediterranean region. It may be hybridized with wheatand is thus a gene resource for wheat improvement. Westudied grain protein concentration andSDS-sedimentation (SED) as indicators of end-usequality. The latter is a good predictor of glutenstrength. A-PAGE and SDS-PAGE were used to identifymonomeric and polymeric seed storage proteins,respectively, to relate proteins of DV to those foundin Chinese Spring (CS), Triticum aestivum L.,wheat. Two full-sib lines of DV had high grain protein(19.3 and 20.3%), but one had very low mean SED (69mm) and one had very high (118 mm) based on onegreenhouse and one field test. CS had very low grainprotein (12.0%) and weak gluten (33 mm). Single-DVchromosome addition and substitution lines and twoDV-wheat recombinant lines all had higher grainprotein than CS (range 13.9 to 16.7%). SED valuesshowed a different pattern. CS=4V and CS=6V hadlow SED, 63 and 44 mm, similar to CS, whereas CS=1Vand full sib DV 200 had very strong gluten, 118 mm, asdid substitution lines CSċ1V (1A) and CSċ1V (1B), 125and 131 mm, respectively. One hybrid-derived line withDV-wheat 1V recombinant chromosome had SED of 99 mmand one line with a 6V added chromosome had SED of 64mm. The large positive effects of quality in the wheathaving DV chromosome 1V are believed to be due to DValleles at the Glu-V1 and Gli-V1/Glu-V3loci. DV chromosomes 4V and 6V did not contribute toimproved quality probably due to Gli-V2 and Gli-V3 which, as the orthologous loci in wheat, donot enhance wheat quality. Based on the positiveeffects of alleles on DV chromosome 1V in a breadwheat background, we conclude that D. villosumis a source of allelic diversity that can beconsidered for improving end-use quality in breadwheat.

Molecular aspects of flower development in grasses

The grass family (Poaceae) of the monocotyledons includes about 10,000 species and represents one of the most important taxa among angiosperms. Their flower morphology is remarkably different from those of other monocotyledons and higher eudicots. The peculiar floral structure of grasses is the floret, which contains carpels and stamens, like eudicots, but lacks petals and sepals. The reproductive organs are surrounded by two lodicules, which correspond to eudicot petals, and by a palea and lemma, whose correspondence to eudicot organs remains controversial. The molecular and genetic analysis of floral morphogenesis and organ specification, primarily performed in eudicot model species, led to the ABCDE model of flower development. Several genes required for floral development in grasses correspond to class A, B, C, D, and E genes of eudicots, but others appear to have unique and diversified functions. In this paper, we outline the present knowledge on the evolution and diversification of grass genes encoding MIKC-type MADS-box transcription factors, based on information derived from studies in rice, maize, and wheat. Moreover, we review recent advances in studying the genes involved in the control of flower development and the extent of structural and functional conservation of these genes between grasses and eudicots.

Phloem Cytochemical Modification and Gene Expression Following the Recovery of Apple Plants from Apple Proliferation Disease

Recovery of apple trees from apple proliferation was studied by combining ultrastructural, cytochemical, and gene expression analyses to possibly reveal changes linked to recovery-associated resistance. When compared with either healthy or visibly diseased plants, recovered apple trees showed abnormal callose and phloem-protein accumulation in their leaf phloem. Although cytochemical localization detected Ca(2+) ions in the phloem of all the three plant groups, Ca(2+) concentration was remarkably higher in the phloem cytosol of recovered trees. The expression patterns of five genes encoding callose synthase and of four genes encoding phloem proteins were analyzed by quantitative real-time reverse transcription-polymerase chain reaction. In comparison to both healthy and diseased plants, four of the above nine genes were remarkably up-regulated in recovered trees. As in infected apple trees, phytoplasma disappear from the crown during winter, but persist in the roots, and it is suggested that callose synthesis/deposition and phloem-protein plugging of the sieve tubes would form physical barriers preventing the recolonization of the crown during the following spring. Since callose deposition and phloem-protein aggregation are both Ca(2+)-dependent processes, the present results suggest that an inward flux of Ca(2+) across the phloem plasma membrane could act as a signal for activating defense reactions leading to recovery in phytoplasma-infected apple trees.

Molecular and phylogenetic analysis of MADS-box genes of MIKC type and chromosome location of SEP-like genes in wheat (Triticum aestivum L.).

Transcription factors encoded by MIKC-type MADS-box genes control many important functions in plants, including flower development and morphogenesis. The cloning and characterization of 45 MIKC-type MADS-box full-length cDNA sequences of common wheat is reported in the present paper. Wheat EST databases were searched by known sequences of MIKC-type genes and primers were designed for cDNA cloning by RT-PCR. Full-length cDNAs were obtained by 5′ and 3′ RACE extension. Southern analysis showed that three copies of the MIKC sequences, corresponding to the three homoeologous genes, were present. This genome organization was further confirmed by aneuploid analysis of six SEP-like genes, each showing three copies located in different homoeologous chromosomes. Phylogenetic analysis included the wheat MIKC cDNAs into 11 of the 13 MIKC subclasses identified in plants and corresponding to most genes controlling the floral homeotic functions. The expression patterns of the cDNAs corresponding to different homeotic classes was analysed in 18 wheat tissues and floral organs by RT-PCR, real time RT-PCR and northern hybridisation. Potential functions of the genes corresponding to the cloned wheat cDNAs were predicted on the basis of sequence homology and comparable expression pattern with functionally characterized MADS-box genes from Arabidopsis and monocot species.

Transcriptional and physiological changes in the S assimilation pathway due to single or combined S and Fe deprivation in durum wheat (Triticum durum L.) seedlings

The effect of iron (Fe) and sulphur (S) deprivation on sulphate uptake and assimilation pathways was investigated in durum wheat by analysing the expression of genes coding for major transporters and enzymes involved in sulphate assimilation and reduction: high-affinity sulphate transporters (TdSultr1.1 and TdSultr1.3), ATP sulphurylase (TdATPSul1 and TdATPSul2), APS reductase (TdAPR), sulphite reductase (TdSiR), O-acetylserine(thiol)lyase (TdOASTL1 and TdOASTL2), and serine acetyltransferase (TdSAT1 and TdSAT2). Further experiments were carried out to detect changes in the activities of these enzymes, together with the evaluation of growth parameters (fresh biomass accumulation, leaf green values, and total S, thiol, and Fe concentrations). Fe shortage in wheat plants under adequate S nutrition resulted in an S deficiency-like response. Most of the genes of the S assimilatory pathway induced by S deprivation (TdATPSul1, TdAPR, TdSir, TdSAT1, and TdSAT2) were also significantly up-regulated after the imposition of the Fe limitation under S-sufficient conditions. However, the differential expression of genes encoding the two high-affinity transporters (TdSultr1.1 and TdSultr1.3) indicates that the mechanisms of sulphate uptake regulation under Fe and S deficiency are different in wheat. Moreover, it was observed that the mRNA level of genes encoding ATPS, APR, and OASTL and the corresponding enzyme activities were often uncoupled in response to Fe and S availability, indicating that most probably their regulation involves a complex interplay of transcriptional, translational, and/or post-translational mechanisms induced by S and/or Fe deficiency.

Molecular characterization of gene sequences coding for protein disulfide isomerase (PDI) in durum wheat (Triticum turgidum ssp. durum).

The organisation of the durum wheat genomic sequence (3.5 kb) coding for protein disulfide isomerase (PDI), deduced by comparison between genomic fragments and cDNA sequences (1.5 kb) isolated from immature caryopses, is described. The gene structure consists of ten exons and nine introns. The presence of consensus sequences involved in splicing, such as intron-exon junctions and branchpoint, has been observed and discussed. Although the deduced wheat PDI amino acid sequence exhibited an overall identity of only 31% to that of human PDI, their modular architecture in terms of number, size, location and secondary structure-propensities of the constituent domains are remarkably similar. The comparison of the amino acid sequences with the eight available plant PDI-like sequences showed a high identity with four of them and low with the remaining ones. Analyses of transcription levels showed that the PDI mRNA was present in all analysed tissues, with much higher expression in immature caryopses.

Iron deprivation results in a rapid but not sustained increase of the expression of genes involved in iron metabolism and sulfate uptake in tomato (Solanum lycopersicum L.) seedlings

Characterization of the relationship between sulfur and iron in both Strategy I and Strategy II plants, has proven that low sulfur availability often limits plant capability to cope with iron shortage. Here it was investigated whether the adaptation to iron deficiency in tomato (Solanum lycopersicum L.) plants was associated with an increased root sulfate uptake and translocation capacity, and modified dynamics of total sulfur and thiols accumulation between roots and shoots. Most of the tomato sulfate transporter genes belonging to Groups 1, 2, and 4 were significantly upregulated in iron-deficient roots, as it commonly occurs under S-deficient conditions. The upregulation of the two high affinity sulfate transporter genes, SlST1.1 and SlST1.2, by iron deprivation clearly suggests an increased root capability to take up sulfate. Furthermore, the upregulation of the two low affinity sulfate transporter genes SlST2.1 and SlST4.1 in iron-deficient roots, accompanied by a substantial accumulation of total sulfur and thiols in shoots of iron-starved plants, likely supports an increased root-to-shoot translocation of sulfate. Results suggest that tomato plants exposed to iron-deficiency are able to change sulfur metabolic balance mimicking sulfur starvation responses to meet the increased demand for methionine and its derivatives, allowing them to cope with this stress.

Restriction Fragment Length Polymorphism (RFLP) for protein disulfide isomerase (PDI) gene sequences in Triticum and Aegilops species

Abstract RFLP variation revealed by protein disulfide isomerase (PDI) coding gene sequences was assessed in 170 accessions belonging to 23 species of Triticum and Aegilops. PDI restriction fragments were highly conserved within each species and confirmed that plant PDI is encoded either by single-copy sequences or by small gene families. The wheat PDI probe hybridized to single EcoRI or HindIII fragments in different diploid species and to one or two fragments per genome in polyploids. Four Aegilops species in the Sitopsis section showed complex patterns and high levels of intraspecific variation, whereas Ae. searsii possessed single monomorphic fragments. T. urartu and Ae. squarrosa showed fragments with the same mobility as those in the A and D genomes of Triticum polyploid species, respectively, whereas differences were observed between the hybridization patterns of T. monococcum and T. boeoticum and that of the A genome. The single fragment detected in Ae. squarrosa was also conserved in most accessions of polyploid Aegilops species carrying the D genome. The five species of the Sitopsis section showed variation for the PDI hybridization fragments and differed from those of the B and G genomes of emmer and timopheevi groups of wheat, although one of the Ae. speltoidesEcoRI fragments was similar to those located on the 4B and 4G chromosomes. The similarity between the EcoRI fragment located on the 1B chromosome of common and emmer wheats and one with a lower hybridization intensity in Ae. longissima, Ae. bicornis and Ae. sharonensis support the hypothesis of a polyphyletic origin of the B genome.