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

<p>Abstract</p> <p>Background</p> <p>Usually 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 <it>in silico </it>method based on the data publicly available in the wheat databases Unigene and TIGR.</p> <p>Results</p> <p>The 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.</p> <p>Conclusion</p> <p>The 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.</p

The PDI (Protein Disulfide Isomerase) gene family in wheat.

The PDI (Protein Disulfide Isomerase) gene family includes several members whose products are responsible for diversified metabolic functions. PDI and PDI-like proteins differ for number and position of thioredoxin-like (TRX-like) active (a type) and inactive (b type) domains, for presence/absence of other domains and of the KDEL signal of retention in the endoplasmic reticulum (ER). The phylogenetic analysis of typical PDI and PDI-like protein sequences resolved them into 10 groups (1), 5 of them (I-V) had 2 TRX-like active domains, whereas the remaining ones owned only a single TRX-like active domain (VI-VIII, QSOX and APRL). In particular, QRX and APRL were not included in this study due to their putative non-isomerase enzymatic activities encoded by an additional domain. The aim of the present research was the study of the complexity and diversity of the PDI gene family in wheat, with particular focus on the genes encoding PDIlike proteins structurally similar to TaPDIL1-1 (group I), the first identified and best characterized member of the PDI family, also named typical PDI. The most important function of typical PDI is the formation and isomerization of disulfide bonds during protein folding, which are accomplished by its two active TRX-like sites sharing the characteristic tetrapeptide –CGHC-. Several studies of molecular characterization, expression analysis and cell localisation in rice and maize have suggested the involvement of typical PDI in the assembly and deposition of storage proteins in these species (2, 3, 4). The characterization and chromosome location of the three homoeologous gene sequences encoding typical PDI and of their promoter sequences have been reported previously (5)

The Protein Disulfide Isomerase gene family in bread wheat (T. aestivum L.)

<p>Abstract</p> <p>Background</p> <p>The Protein Disulfide Isomerase (PDI) gene family encodes several PDI and PDI-like proteins containing thioredoxin domains and controlling diversified metabolic functions, including disulfide bond formation and isomerisation during protein folding. Genomic, cDNA and promoter sequences of the three homoeologous wheat genes encoding the "typical" PDI had been cloned and characterized in a previous work. The purpose of present research was the cloning and characterization of the complete set of genes encoding PDI and PDI like proteins in bread wheat (<it>Triticum aestivum </it>cv Chinese Spring) and the comparison of their sequence, structure and expression with homologous genes from other plant species.</p> <p>Results</p> <p>Eight new non-homoeologous wheat genes were cloned and characterized. The nine PDI and PDI-like sequences of wheat were located in chromosome regions syntenic to those in rice and assigned to eight plant phylogenetic groups. The nine wheat genes differed in their sequences, genomic organization as well as in the domain composition and architecture of their deduced proteins; conversely each of them showed high structural conservation with genes from other plant species in the same phylogenetic group. The extensive quantitative RT-PCR analysis of the nine genes in a set of 23 wheat samples, including tissues and developmental stages, showed their constitutive, even though highly variable expression.</p> <p>Conclusions</p> <p>The nine wheat genes showed high diversity, while the members of each phylogenetic group were highly conserved even between taxonomically distant plant species like the moss <it>Physcomitrella patens</it>. Although constitutively expressed the nine wheat genes were characterized by different expression profiles reflecting their different genomic organization, protein domain architecture and probably promoter sequences; the high conservation among species indicated the ancient origin and diversification of the still evolving gene family. The comprehensive structural and expression characterization of the complete set of <it>PDI </it>and <it>PDI</it>-like wheat genes represents a basis for the functional characterization of this gene family in the hexaploid context of bread wheat.</p

Phloem cytochemical modification and gene expression following the recovery of apple plants from apple proliferation

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 Ca2+ ions in the phloem of all the three plant groups, Ca2+ 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 upregulated 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 Ca2+-dependent processes, the present results suggest that an inward flux of Ca2+ across the phloem plasma membrane could act as a signal for activating defense reactions leading to recovery in phytoplasma-infected apple trees.L'articolo é disponibile sul sito dell'editore: http://www.apsjournals.apsnet.or