The potato R locus codes for dihydroflavonol 4-reductase

The potato R locus is required for the production of red pelargonidin-based anthocyanin pigments in potato (Solanum tuberosum L.). Red color also requires tissue-specific regulatory genes, such as D (for expression in tuber skin) and F (expression in flowers). A related locus, P, is required for production of blue/purple anthocyanins; P is epistatic to R. We have previously reported that the dihydroflavonol 4-reductase gene (dfr) co-segregates with R. To test directly whether R corresponds to dfr, we placed the allele of dfr associated with red color under the control of the CaMV 35S promoter and introduced it into the potato cultivar Prince Hairy (genotype dddd rrrr P-), which has white tubers and pale blue flowers. Transgenic Prince Hairy tubers remained white, but flower color changed to purple. Three independent transgenic lines, as well as a vector-transformed line, were then crossed with the red-skinned variety Chieftain (genotype D-R-pppp), to establish populations that segregated for D, R, P, and the dfr transgene or empty vector. Markers were used to genotype progeny at D and R. Progeny carrying the empty vector in the genetic background D-rrrr produced white or purple tubers, while progeny with the same genotype and the dfr transgene produced red or purple tubers. HPLC and LC–MS/MS analyses of anthocyanins present in Chieftain and in a red-skinned progeny clone with the dfr transgene in a D-rrrr background revealed no qualitative differences. Thus, dfr can fully complement R, both in terms of tuber color and anthocyanin composition

The potato developer (D) locus encodes an R2R3 MYB transcription factor that regulates expression of multiple anthocyanin structural genes in tuber skin

A dominant allele at the D locus (also known as I in diploid potato) is required for the synthesis of red and purple anthocyanin pigments in tuber skin. It has previously been reported that D maps to a region of chromosome 10 that harbors one or more homologs of Petuniaan2, an R2R3 MYB transcription factor that coordinately regulates the expression of multiple anthocyanin biosynthetic genes in the floral limb. To test whether D acts similarly in tuber skin, RT-PCR was used to evaluate the expression of flavanone 3-hydroxylase (f3h), dihydroflavonol 4-reductase (dfr) and flavonoid 3′,5′-hydroxylase (f3′5′h). All three genes were expressed in the periderm of red- and purple-skinned clones, while dfr and f3′5′h were not expressed, and f3h was only weakly expressed, in white-skinned clones. A potato cDNA clone with similarity to an2 was isolated from an expression library prepared from red tuber skin, and an assay developed to distinguish the two alleles of this gene in a diploid potato clone known to be heterozygous Dd. One allele was observed to cosegregate with pigmented skin in an F1 population of 136 individuals. This allele was expressed in tuber skin of red- and purple-colored progeny, but not in white tubers, while other parental alleles were not expressed in white or colored tubers. The allele was placed under the control of a doubled 35S promoter and transformed into the light red-colored cultivar Désirée, the white-skinned cultivar Bintje, and two white diploid clones known to lack the functional allele of D. Transformants accumulated pigment in tuber skin, as well as in other tissues, including young foliage, flower petals, and tuber flesh

Tomato Pathogenesis-related Protein Genes are Expressed in Response to Trialeurodes vaporariorum and Bemisia tabaci Biotype B Feeding

The temporal and spatial expression of tomato wound- and defense-response genes to Bemisia tabaci biotype B (the silverleaf whitefly) and Trialeurodes vaporariorum (the greenhouse whitefly) feeding were characterized. Both species of whiteflies evoked similar changes in tomato gene expression. The levels of RNAs for the methyl jasmonic acid (MeJA)- or ethylene-regulated genes that encode the basic β-1,3-glucanase (GluB), basic chitinase (Chi9), and Pathogenesis-related protein-1 (PR-1) were monitored. GluB and Chi9 RNAs were abundant in infested leaves from the time nymphs initiated feeding (day 5). In addition, GluB RNAs accumulated in apical non-infested leaves. PR-1 RNAs also accumulated after whitefly feeding. In contrast, the ethylene- and salicylic acid (SA)-regulated Chi3 and PR-4 genes had RNAs that accumulated at low levels and GluAC RNAs that were undetectable in whitefly-infested tomato leaves. The changes in Phenylalanine ammonia lyase5 (PAL5) were variable; in some, but not all infestations, PAL5 RNAs increased in response to whitefly feeding. PAL5 RNA levels increased in response to MeJA, ethylene, and abscisic acid, and declined in response to SA. Transcripts from the wound-response genes, leucine aminopeptidase (LapA1) and proteinase inhibitor 2 (pin2), were not detected following whitefly feeding. Furthermore, whitefly infestation of transgenic LapA1:GUS tomato plants showed that whitefly feeding did not activate the LapA1 promoter, although crushing of the leaf lamina increased GUS activity up to 40 fold. These studies indicate that tomato plants perceive B. tabaci and T. vaporariorum in a manner similar to baterical pathogens and distinct from tissue-damaging insects

Tutorial: Multivariate Classification for Vibrational Spectroscopy in Biological Samples

Vibrational spectroscopy techniques, such as Fourier-transform infrared (FTIR) and Raman spectroscopy, have been successful methods for studying the interaction of light with biological materials and facilitating novel cell biology analysis. Spectrochemical analysis is very attractive in disease screening and diagnosis, microbiological studies and forensic and environmental investigations because of its low cost, minimal sample preparation, non-destructive nature and substantially accurate results. However, there is now an urgent need for multivariate classification protocols allowing one to analyze biologically derived spectrochemical data to obtain accurate and reliable results. Multivariate classification comprises discriminant analysis and class-modeling techniques where multiple spectral variables are analyzed in conjunction to distinguish and assign unknown samples to pre-defined groups. The requirement for such protocols is demonstrated by the fact that applications of deep-learning algorithms of complex datasets are being increasingly recognized as critical for extracting important information and visualizing it in a readily interpretable form. Hereby, we have provided a tutorial for multivariate classification analysis of vibrational spectroscopy data (FTIR, Raman and near-IR) highlighting a series of critical steps, such as preprocessing, data selection, feature extraction, classification and model validation. This is an essential aspect toward the construction of a practical spectrochemical analysis model for biological analysis in real-world applications, where fast, accurate and reliable classification models are fundamental

Rapid determination of swiss cheese composition by Fourier transform infrared/attenuated total reflectance spectroscopy

WOS: 000236686700006PubMed ID: 16606712There is a need for rapid and simple techniques that can be used to predict the quality of cheese. The aim of this research was to develop a simple and rapid screening tool for monitoring Swiss cheese composition by using Fourier transform infrared spectroscopy. Twenty Swiss cheese samples from different manufacturers and degree of maturity were evaluated. Direct measurements of Swiss cheese slices (similar to 0.5g) were made using a MIRacle 3-reflection diamond attenuated total reflectance (ATR) accessory. Reference methods for moisture (vacuum oven), protein content ( Kjeldahl), and fat (Babcock) were used. Calibration models were developed based on a cross-validated (leave- one- out approach) partial least squares regression. The information-rich infrared spectral range for Swiss cheese samples was from 3,000 to 2,800 cm(-1) and 1,800 to 900 cm(-1). The performance statistics for cross-validated models gave estimates for standard error of cross-validation of 0.45, 0.25, and 0.21% for moisture, protein, and fat respectively, and correlation coefficients r > 0.96. Furthermore, the ATR infrared protocol allowed for the classification of cheeses according to manufacturer and aging based on unique spectral information, especially of carbonyl groups, probably due to their distinctive lipid composition. Attenuated total reflectance infrared spectroscopy allowed for the rapid (similar to 3- min analysis time) and accurate analysis of the composition of Swiss cheese. This technique could contribute to the development of simple and rapid protocols for monitoring complex biochemical changes, and predicting the final quality of the cheese