Transcriptome analysis and characterization of genes associated to leaf tannin content in foxtail millet [Setaria italica (L.) P. Beauv.].

BACKGROUND: Chinese chestnut is an economically important tree species whose yield and quality are seriously affected by red spider attack. Tannins is one of the most important class secondary metabolites in plants, and is closely associated with plant defense mechanisms against insect and herbivory. In our previous studies, it was revealed that several low-tannin foxtail millet varieties growing under the Chinese chestnut trees could attract red spiders to feed on their leaves and protect the chestnut trees from the infestation of red spiders, meanwhile, the growth and yield of foxtail millet plants themselves were not greatly affected. RESULTS: To identify genes related to leaf tannin content and selection of foxtail millet germplasm resources with low tannin content for interplanting with Chinese chestnut and preventing the red spider attack, the leaves of 4 varieties with different levels of tannin content were harvested for comparative transcriptome analysis. In total, 335 differentially expressed genes (DEGs) were identified. For acquisition of gene functions and biological pathways they involved in, gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were performed, and several DEGs were found to possibly participate in the tannins biosynthesis pathway and transport processes of precursors. In addition, according to the PlantTFDB database, some transcription factors were predicted among the DEGs, suggesting their role in regulation of tannins biosynthesis pathway. CONCLUSION: Our results provide valuable gene resources for understanding the biosynthesis and regulation mechanisms of tannins in foxtail millet, and pave the way for speeding up the breeding of low-tannin varieties through marker-assisted selection, which could be utilized for interplanting with Chinese chestnut trees to confer protection against red spider attack.

Comparison of Metabolome and Transcriptome of Flavonoid Biosynthesis Pathway in a Purple-Leaf Tea Germplasm Jinmingzao and a Green-Leaf Tea Germplasm Huangdan reveals Their Relationship with Genetic Mechanisms of Color Formation.

Purple-leaf tea is a phenotype with unique color because of its high anthocyanin content. The special flavor of purple-leaf tea is highly different from that of green-leaf tea, and its main ingredient is also of economic value. To probe the genetic mechanism of the phenotypic characteristics of tea leaf color, we conducted widely targeted metabolic and transcriptomic profiling. The metabolites in the flavonoid biosynthetic pathway of purple- and green-leaf tea were compared, and results showed that phenolic compounds, including phenolic acids, flavonoids, and tannins, accumulated in purple-leaf tea. The high expression of genes related to flavonoid biosynthesis (e.g., PAL and LAR) exhibits the specific expression of biosynthesis and the accumulation of these metabolites. Our result also shows that two CsUFGTs were positively related to the accumulation of anthocyanin. Moreover, genes encoding transcription factors that regulate flavonoids were identified by coexpression analysis. These results may help to identify the metabolic factors that influence leaf color differentiation and provide reference for future research on leaf color biology and the genetic improvement of tea.

Influence of Genotype, Environment, and Gypsy Moth Herbivory on Local and Systemic Chemical Defenses in Trembling Aspen (Populus tremuloides).

Numerous studies have explored the impacts of intraspecific genetic variation and environment on the induction of plant chemical defenses by herbivory. Relatively few, however, have considered how those factors affect within-plant distribution of induced defenses. This work examined the impacts of plant genotype and soil nutrients on the local and systemic phytochemical responses of trembling aspen (Populus tremuloides) to defoliation by gypsy moth (Lymantria dispar). We deployed larvae onto foliage on individual tree branches for 15 days and then measured chemistry in leaves from: 1) branches receiving damage, 2) undamaged branches of insect-damaged trees, and 3) branches of undamaged control trees. The relationship between post-herbivory phytochemical variation and insect performance also was examined. Plant genotype, soil nutrients, and damage all influenced phytochemistry, with genotype and soil nutrients being stronger determinants than damage. Generally, insect damage decreased foliar nitrogen, increased levels of salicinoids and condensed tannins, but had little effect on levels of a Kunitz trypsin inhibitor, TI3. The largest damage-mediated tannin increases occurred in leaves on branches receiving damage, whereas the largest salicinoid increases occurred in leaves of adjacent, undamaged branches. Foliar nitrogen and the salicinoid tremulacin had the strongest positive and negative relationships, respectively, with insect growth. Overall, plant genetics and environment concomitantly influenced both local and systemic phytochemical responses to herbivory. These findings suggest that herbivory can contribute to phytochemical heterogeneity in aspen foliage, which may in turn influence future patterns of herbivory and nutrient cycling over larger spatial scales.

Presence of tannins in sorghum grains is conditioned by different natural alleles of Tannin1.

Sorghum, an ancient old-world cereal grass, is the dietary staple of over 500 million people in more than 30 countries in the tropics and semitropics. Its C4 photosynthesis, drought resistance, wide adaptation, and high nutritional value hold the promise to alleviate hunger in Africa. Not present in other major cereals, such as rice, wheat, and maize, condensed tannins (proanthocyanidins) in the pigmented testa of some sorghum cultivars have been implicated in reducing protein digestibility but recently have been shown to promote human health because of their high antioxidant capacity and ability to fight obesity through reduced digestion. Combining quantitative trait locus mapping, meta-quantitative trait locus fine-mapping, and association mapping, we showed that the nucleotide polymorphisms in the Tan1 gene, coding a WD40 protein, control the tannin biosynthesis in sorghum. A 1-bp G deletion in the coding region, causing a frame shift and a premature stop codon, led to a nonfunctional allele, tan1-a. Likewise, a different 10-bp insertion resulted in a second nonfunctional allele, tan1-b. Transforming the sorghum Tan1 ORF into a nontannin Arabidopsis mutant restored the tannin phenotype. In addition, reduction in nucleotide diversity from wild sorghum accessions to landraces and cultivars was found at the region that codes the highly conserved WD40 repeat domains and the C-terminal region of the protein. Genetic research in crops, coupled with nutritional and medical research, could open the possibility of producing different levels and combinations of phenolic compounds to promote human health.

Transgenic upregulation of the condensed tannin pathway in poplar leads to a dramatic shift in leaf palatability for two tree-feeding Lepidoptera.

Transgenic hybrid aspen (Populus tremula x tremuloides) overexpressing the MYB134 tannin regulatory gene show dramatically enhanced condensed tannin (proanthocyanidin) levels, as well as shifts in other phenolic metabolites. A series of insect bioassays with forest tent caterpillars (Malacosoma disstria) and gypsy moth (Lymantria dispar) caterpillars was carried out to determine how this metabolic shift affects food preference and performance of generalist tree-feeding lepidopterans. Both species showed a distinct preference for the high-tannin MYB134 overexpressor plants, and L. dispar performance was enhanced relative to controls. L. dispar reached greater pupal weight and showed reduced time to pupation when reared on the MYB134 overexpressing poplar. These results were unexpected since enhanced condensed tannin levels were predicted to act as feeding deterrents. However, the data may be explained by the observed decrease in the salicinoids (phenolic glycosides) salicortin and tremulacin that accompanied the upregulation of the condensed tannins in the transgenics. We conclude that for these two lepidopteran species, condensed tannin levels are unlikely to be a major determinant of caterpillar food preference or performance. However, our experiments show that overexpression of a single regulatory gene in transgenic aspen can have a significant impact on herbivorous insects.

Headspace volatile components of Canadian grown low-tannin faba bean (Vicia faba L.) genotypes.

BACKGROUND: As flavor is a prerequisite for palatability of new crop varieties, 13 Canadian-grown, low-tannin faba bean genotypes were evaluated for volatile components isolated with headspace solid-phase microextraction, and analyzed with gas chromatography-mass spectrometry. RESULTS: A total of 45 volatiles consisting of aldehydes, aromatic hydrocarbons, alcohols, ketones and alkanes represented on average 57, 15, 14, 5 and 4.5% of the total peak area, respectively. Total extractable volatiles were highly location dependent, whereas 10 headspace volatiles (1-pentanol, 1-hexanol, pentanal, (E)-2-heptenal, 2-ethylfuran, 2-pentylfuran, acetone, 2-butanone, 2-heptanone and 3-octen-2-one) were genotype specific and/or regulated by environmental conditions. Multivariate data analysis performed on the functional group of the volatiles using principal component analysis and cluster analysis demonstrates that the total extractable volatiles were the major factor segregating faba bean genotypes. CONCLUSION: The solid-phase microextraction technique proved to be a rapid and effective method for routine evaluation of faba bean volatile profile that may be applicable in a screening/plant breeding program.

Phenotypic variation in sexually and asexually recruited individuals of the Baltic Sea endemic macroalga Fucus radicans: in the field and after growth in a common-garden.

BACKGROUND: Most species of brown macroalgae recruit exclusively sexually. However, Fucus radicans, a dominant species in the northern Baltic Sea, recruits new attached thalli both sexually and asexually. The level of asexual recruitment varies among populations from complete sexual recruitment to almost (> 90%) monoclonal populations. If phenotypic traits have substantial inherited variation, low levels of sexual activity will decrease population variation in these traits, which may affect function and resilience of the species. We assessed the level of inherited variation in nine phenotypic traits by comparing variation within and among three monoclonal groups and one group of unique multilocus genotypes (MLGs) sampled in the wild. RESULTS: Of the nine phenotypic traits, recovery after freezing, recovery after desiccation, and phlorotannin content showed substantial inherited variation, that is, phenotypic variation in these traits were to a large extend genetically determined. In contrast, variation in six other phenotypic traits (growth rate, palatability to isopod grazers, thallus width, distance between dichotomies, water content after desiccation and photochemical yield under ambient conditions) did not show significant signals of genetic variation at the power of analyses used in the study. Averaged over all nine traits, phenotypic variation within monoclonal groups was only 68% of the variation within the group of different MLGs showing that genotype diversity does affect the overall level of phenotypic variation in this species. CONCLUSIONS: Our result indicates that, in general, phenotypic diversity in populations of Fucus radicans increases with increased multilocus genotype (MLG) diversity, but effects are specific for individual traits. In the light of Fucus radicans being a foundation species of the northern Baltic Sea, we propose that increased MLG diversity (leading to increased trait variation) will promote ecosystem function and resilience in areas where F. radicans is common, but this suggestion needs experimental support.

Integrated Approaches to Reveal Genes Crucial for Tannin Degradation in Aureobasidium melanogenum T9.

Tannins biodegradation by a microorganism is one of the most efficient ways to produce bioproducts of high value. However, the mechanism of tannins biodegradation by yeast has been little explored. In this study, Aureobasidium melanogenum T9 isolated from red wine starter showed the ability for tannins degradation and had its highest biomass when the initial tannic acid concentration was 20 g/L. Furthermore, the genes involved in the tannin degradation process were analyzed. Genes tan A, tan B and tan C encoding three different tannases respectively were identified in the A. melanogenum T9. Among these genes, tan A and tan B can be induced by tannin acid simultaneously at both gene transcription and protein expression levels. Our assay result showed that the deletion of tanA and tanB resulted in tannase activity decline with 51.3 ± 4.1 and 64.1 ± 1.9 U/mL, respectively, which is much lower than that of A. melanogenum T9 with 91.3 ± 5.8 U/mL. In addition, another gene coding gallic acid decarboxylase (gad) was knocked out to better clarify its function. Mutant Δgad completely lost gallic acid decarboxylase activity and no pyrogallic acid was seen during the entire cultivation process, confirming that there was a sole gene encoding decarboxylase in the A. melanogenum T9. These results demonstrated that tanA, tanB and gad were crucial for tannin degradation and provided new insights for the mechanism of tannins biodegradation by yeast. This finding showed that A. melanogenum has potential in the production of tannase and metabolites, such as gall acid and pyrogallol.

Genomic Selection for Antioxidant Production in a Panel of Sorghum bicolor and S. bicolor × S. halepense Lines.

The purpose of this work was to assess the performance of four genomic selection (GS) models (GBLUP, BRR, Bayesian LASSO and BayesB) in 4 sorghum grain antioxidant traits (phenols, flavonoids, total antioxidant capacity and condensed tannins) using whole-genome SNP markers in a novel diversity panel of Sorghum bicolor lines and landraces and S. bicolor × S. halepense recombinant inbred lines. One key breeding problem modelled was predicting the performance in the antioxidant production of new and unphenotyped sorghum genotypes (validation set). The population was weakly structured (analysis of molecular variance, AMOVA R2 = 9%), showed a significant genetic diversity and expressed antioxidant traits with a good level of variability and high correlation. The S. bicolor × S. halepense lines outperformed Sorghum bicolor populations for all the antioxidants. The four GS models implemented in this work performed comparably across traits, with accuracy ranging from 0.49 to 0.58, and are considered high enough to sustain sorghum breeding for antioxidants production and allow important genetic gains per unit of time and cost. The results presented in this work are expected to contribute to GS implementation and the genetic improvement of sorghum grain antioxidants for different purposes, including the manufacture of health-promoting and specialty foods.

Characterization and diagnostic marker for TTG1 regulating tannin and anthocyanin biosynthesis in faba bean.

Condensed tannins, found in coloured-flowering varieties of faba bean (Vicia faba L) are, after vicine and convicine, one of the major anti-nutritional factors for monogastric animals. The development of tannin-free cultivars is a key goal in breeding to broaden the use of this legume in the animal feed industry. Two recessive genes, zt-1 and zt-2, control the zero-tannin content and promote white-flowered plants. Previous studies exploiting synteny with the model Medicago truncatula reported a mutation in TTG1, a gene encoding a WD40 transcription factor located in chromosome II, as the responsible for the zt-1 phenotypes. Here a comprehensive analysis of VfTTG1 (including phylogenetic relationships, gene structure and gene expression) has been conducted to confirm the identity of the gene and to reveal structural changes that may result in different functional alleles. The results confirmed the identity of the candidate and revealed the existence of two different alleles responsible for the phenotype: ttg1-a, probably due to a mutation in the promoter region, and ttg1-b caused by a deletion at the 5'end of VfTTG1. Based on the sequencing results, an allele-specific diagnostic marker was designed that differentiate zt-1 from wild and zt-2 genotypes and facilitates its deployment in faba bean breeding programs.

Aspen phenylpropanoid genes' expression levels correlate with genets' tannin richness and vary both in responses to soil nitrogen and associations with phenolic profiles.

Condensed tannin (CT) contents of European aspen (Populus tremula L.) vary among genotypes, and increases in nitrogen (N) availability generally reduce plants' tannin production in favor of growth, through poorly understood mechanisms. We hypothesized that intrinsic tannin production rates may co-vary with gene expression responses to soil N and resource allocation within the phenylpropanoid pathway (PPP). Thus, we examined correlations between soil N levels and both expression patterns of eight PPP genes (measured by quantitative-reverse transcription PCR) and foliar phenolic compounds (measured by liquid chromatography-mass spectrometry) in young aspen genets with intrinsically extreme CT levels. Monitored phenolics included salicinoids, lignins, flavones, flavonols, CT precursors and CTs. The PPP genes were consistently expressed more strongly in high-CT trees. Low N supplements reduced expression of genes throughout the PPP in all genets, while high N doses restored expression of genes at the beginning and end of the pathway. These PPP changes were not reflected in pools of tannin precursors, but varying correlations between gene expression and foliar phenolic pools were detected in young and mature leaves, suggesting that processes linking gene expression and the resulting phenolics vary spatially and temporally. Precursor fluxes suggested that CT-related metabolic rate or sink controls are linked to intrinsic carbon allocation strategies associated with N responses. Overall, we found more negative correlations (indicative of allocation trade-offs) between PPP gene expression and phenolic products following N additions in low-CT plants than in high-CT plants. The tannin-related expression dynamics suggest that, in addition to defense, relative tannin levels may also be indicative of intraspecific variations in the way aspen genets respond to soil fertility.

Identification and Characterization of a 25 kDa Protein That Is Indispensable for the Efficient Saccharification of Eisenia bicyclis in the Digestive Fluid of Aplysia kurodai.

The digestive fluid of the sea hare Aplysia kurodai can liberate approximately 2.5 mg of glucose from 10 mg of dried Eisenia bicyclis powder. Although laminaran, a major storage polysaccharide in E. bicyclis, is easily digested to glucose by the synergistic action of the 110 and 210 kDa A. kurodai ß-glucosidases (BGLs), glucose is not liberated from E. bicyclis by direct incubation with these BGLs. To clarify this discrepancy, we searched for an Eisenia hydrolysis enhancing protein (EHEP) in the digestive fluid of A. kurodai. A novel 25 kDa protein that enhances E. bicyclis saccharification by ß-glucosidases was purified to a homogeneous state from the digestive fluid of A. kurodai, and its cDNA was cloned from total cDNAs reverse-transcribed from hepatopancreas total RNA. The E. bicyclis extract strongly inhibited BGLs, suggesting some compound within this brown alga functioned as a feeding deterrent. However, when E. bicyclis was incubated with BGLs in the presence of EHEP, glucose production was markedly increased. As E. bicyclis is rich in phlorotannin, which are only found in brown algae, our study suggested that these compounds are the main BGL inhibitors in E. bicyclis extract. EHEP protects BGLs from phlorotannin inhibition by binding to phlorotannins and forming an insoluble complex with phloroglucinol and phlorotannins. These findings indicated that EHEP plays a key role in the saccharification of brown seaweeds containing phlorotannins in the digestive fluid of A. kurodai. This is the first report of EHEP as a phlorotannin-binding protein that protects BGLs from inhibition.

Genetics and Biochemistry of Zero-Tannin Lentils.

The zero-tannin trait in lentil is controlled by a single recessive gene (tan) that results in a phenotype characterized by green stems, white flowers, and thin, transparent, or translucent seed coats. Genes that result in zero-tannin characteristics are useful for studies of seed coat pigmentation and biochemical characters because they have altered pigmentation. In this study, one of the major groups of plant pigments, phenolic compounds, was compared among zero-tannin and normal phenotypes and genotypes of lentil. Biochemical data were obtained by liquid chromatography-mass spectrometry (LC-MS). Genomic sequencing was used to identify a candidate gene for the tan locus. Phenolic compound profiling revealed that myricetin, dihydromyricetin, flavan-3-ols, and proanthocyanidins are only detected in normal lentil phenotypes and not in zero-tannin types. The molecular analysis showed that the tan gene encodes a bHLH transcription factor, homologous to the A gene in pea. The results of this study suggest that tan as a bHLH transcription factor interacts with the regulatory genes in the biochemical pathway of phenolic compounds starting from flavonoid-3',5'-hydroxylase (F3'5'H) and dihydroflavonol reductase (DFR).

Greek yogurt with added sorghum flours: antioxidant potential and sensory acceptance / Yogurt griego adicionado de harinas integrales de sorgo: potencial antioxidante y evaluación sensorial

ABSTRACT The aim of this study was to develop and evaluate the antioxidant characteristics and sensory acceptance of Greek yogurt with added whole sorghum flours (WSF) from genotypes BRS305 (tannin) and BR501 (tannin-free). Five formulations were elaborated: 1) Control (without WSF); 2) 2% BR501; 3) 4% BR501; 4) 2% BRS305 and 5) 4% BRS305 and evaluated for antioxidant capacity, total anthocyanins, total phenols, condensed tannins, sensory acceptance and purchase intention. The 4% BRS305 formulation presented higher antioxidant properties, but lower sensory acceptance. No significant difference was observed for: color, flavor and overall acceptability between the control and 2% BR501; nor for texture and overall acceptability between the control and 4% BR501. Principal component analysis explained that all variables associated with antioxidant properties were positively correlated with the first major component (PC1: 82.7%). The 2% and 4% BR501 formulations were more highly correlated with PC1 (92.7%), as well as the control in relation to sensorial characteristics. The addition of WSF BRS305 improved the antioxidant properties of yogurts and the addition of WSF BR501 did not interfere with the sensory acceptance of the formulations. The similarity of the sensory acceptance of the yogurt containing WSF from the BR 501 genotype to the control, opens perspectives for the insertion of sorghum in human food, using dairy products as suitable matrices, adding potential functionality to this type of product.

RESUMEN El trabajo tuvo como propósito desarrollar y evaluar las características antioxidantes y la aceptación sensorial de yogurt griego con adición de harinas integrales de sorgo (HIS) de los genotipos BRS305 (con taninos) y BR501 (sin taninos). Cinco formulaciones fueron elaboradas: 1) Control (sin HIS); 2) 2% BR501; 3) 4% BR501; 4) 2% BRS305 y 5) 4% BRS305. Las formulaciones fueron evaluadas en función de su actividad antioxidante, antocianinas totales, fenólicos totales, taninos condensados, aceptación sensorial e intención de compra. La formulación 4% BRS305 presentó más propiedades antioxidantes, pero menor aceptación sensorial que las otras formulaciones. No se observó diferencia significativa entre el control y el 2% BR501 para el color, sabor e impresión global, ni tampoco para textura e impresión global en relación al 4% BR501. El análisis de componente principal ha explicado que todas las variables asociadas a las propiedades antioxidantes han sido positivamente correlacionadas con el primer componente principal (PC1: 82,7%). Las formulaciones 2% y 4% BR501 se han correlacionado más con el PC1 (92,7%), así como el control con las características sensoriales. La adición de HIS BRS305 ha mejorado las propiedades antioxidantes de los yogurts y la adición de HIS BR501 no ha interferido en la aceptación sensorial de las formulaciones. La similitud de la aceptación sensorial del yogurt que contiene el HIS del genotipo BR501 con el control, abre perspectivas para la inserción del sorgo en la alimentación humana, utilizando productos lácteos como matrices adecuadas, agregando funcionalidad potencial a este tipo de producto.

Genotypic Tannin Levels in Populus tremula Impact the Way Nitrogen Enrichment Affects Growth and Allocation Responses for Some Traits and Not for Others.

Plant intraspecific variability has been proposed as a key mechanism by which plants adapt to environmental change. In boreal forests where nitrogen availability is strongly limited, nitrogen addition happens indirectly through atmospheric N deposition and directly through industrial forest fertilization. These anthropogenic inputs of N have numerous environmental consequences, including shifts in plant species composition and reductions in plant species diversity. However, we know less about how genetic differences within plant populations determine how species respond to eutrophication in boreal forests. According to plant defense theories, nitrogen addition will cause plants to shift carbon allocation more towards growth and less to chemical defense, potentially enhancing vulnerability to antagonists. Aspens are keystone species in boreal forests that produce condensed tannins to serve as chemical defense. We conducted an experiment using ten Populus tremula genotypes from the Swedish Aspen Collection that express extreme levels of baseline investment into foliar condensed tannins. We investigated whether investment into growth and phenolic defense compounds in young plants varied in response to two nitrogen addition levels, corresponding to atmospheric N deposition and industrial forest fertilization. Nitrogen addition generally caused growth to increase, and tannin levels to decrease; however, individualistic responses among genotypes were found for height growth, biomass of specific tissues, root:shoot ratios, and tissue lignin and N concentrations. A genotype's baseline ability to produce and store condensed tannins also influenced plant responses to N, although this effect was relatively minor. High-tannin genotypes tended to grow less biomass under low nitrogen levels and more at the highest fertilization level. Thus, the ability in aspen to produce foliar tannins is likely associated with a steeper reaction norm of growth responses, which suggests a higher plasticity to nitrogen addition, and potentially an advantage when adapting to higher concentrations of soil nitrogen.

Sorghum phytochemicals and their potential impact on human health.

Sorghum is a rich source of various phytochemicals including tannins, phenolic acids, anthocyanins, phytosterols and policosanols. These phytochemicals have potential to significantly impact human health. Sorghum fractions possess high antioxidant activity in vitro relative to other cereals or fruits. These fractions may offer similar health benefits commonly associated with fruits. Available epidemiological evidence suggests that sorghum consumption reduces the risk of certain types of cancer in humans compared to other cereals. The high concentration of phytochemicals in sorghum may be partly responsible. Sorghums containing tannins are widely reported to reduce caloric availability and hence weight gain in animals. This property is potentially useful in helping reduce obesity in humans. Sorghum phytochemicals also promote cardiovascular health in animals. Such properties have not been reported in humans and require investigation, since cardiovascular disease is currently the leading killer in the developed world. This paper reviews available information on sorghum phytochemicals, how the information relates to current phytonutrient research and how it has potential to combat common nutrition-related diseases including cancer, cardiovascular disease and obesity.

Light and an exogenous transcription factor qualitatively and quantitatively affect the biosynthetic pathway of condensed tannins in Lotus corniculatus leaves.

The effects of increasing light and of a heterologous bHLH transcription factor on the accumulation of condensed tannins (CT) were investigated in leaves of Lotus corniculatus, a model legume species which accumulates these secondary metabolites in leaves as well as reproductive tissues. Light and expression of the transgene increased the level of CT in a synergistic way. To monitor how the changes in accumulation of condensed tannins were achieved, the level of expression of four key genes in the flavonoid pathway was estimated by real-time RT-PCR analysis. Early genes of the pathway (PAL and CHS) were affected less in their expression and so appeared to be less involved in influencing the final level of CT than later genes in the pathway (DFR and ANS). Steady-state levels of DFR and ANS transcripts showed a strong positive correlation with CT and these genes might be considered the first rate-limiting steps in CT biosynthesis in Lotus leaves. However, additional factors mediated by light are limiting CT accumulation once these genes are up-regulated by the transgene. Therefore, the increment of the steady-state mRNA level for DFR and ANS might not be sufficient to up-regulate condensed tannins in leaves. The real-time RT-PCR approach adopted showed that members within the CHS and DFR gene families are differentially regulated by the exogenous bHLH gene and light. This finding is discussed in relation to the approaches for controlling CT biosynthesis and for studying the expression profile of multi-gene families.

Inheritance patterns of phenolics in F1, F2, and back-cross hybrids of willows: implications for herbivore responses to hybrid plants.

The aim of this study was to determine the inheritance pattern of phenolic secondary compounds in pure and hybrid willows and its consequences for plant resistance to leaf-feeding insects. F1, F2, and back-cross hybrids along with pure species were produced by hand pollination of pure, naturally-growing Salix caprea (L., Salicaceae) and S. repens (L.) plants. Leaf concentrations of condensed tannins and seven different phenolic glucosides were determined by using butanol-HCI and HPLC analyses. Insect herbivore leaf damage was measured on the same leaves as used for chemical analyses. We found hybrids to be approximately intermediate between the parental species: S. caprea with high levels of condensed tannins and no phenolic glucosides. and S. repens with low levels of condensed tannins and high levels of phenolic glucosides. We also found a negative correlation between concentrations of condensed tannins and phenolic glucosides, suggesting a trade-off in production of these two substances. F2 hybrids and the hybrid back-crossed to S. caprea were significantly more damaged by insect herbivores than the parental species and the F1 hybrid, indicating reduced resistance and possibly a selective disadvantage for these hybrid categories.