Response of Winter Wheat to Cold: Production of Phenolic Compounds and L-Phenylalanine Ammonia Lyase Activity

The formation of soluble and polymeric (lignin) phenolic compounds, activity of L-phenylalanine ammonia lyase (PAL, EC 4.3.1.5), and content of free L-phenylalanine during cold hardening of winter wheat plants (Triticum aestivum L.) were studied. Cold treatment increased accumulation of soluble phenolic compounds in leaves while not affecting the content of lignin. The opposite was observed in tillering nodes. The activity of PAL was lower than in control plants in both tissues, and the content of free L-phenylalanine in tissues increased.     

Response of winter wheat to cold: production of phenolic compounds and L-phenylalanine ammonia-lyase activity

The formation of soluble and polymeric (lignin) phenolic compounds, activity of L-phenylalanine ammonia lyase (PAL, EC 4.3.1.5), and content of free L-phenylalanine during cold hardening of winter wheat plants (Triticum aestivum L.) were studied. Cold treatment increased accumulation of soluble phenolic compounds in leaves while not affecting the content of lignin. The opposite was observed in tillering nodes. The activity of PAL was lower than in control plants in both tissues, and the content of free L-phenylalanine in tissues increased.     

Altering expression of cinnamic acid 4-hydroxylase in transgenic plants provides evidence for a feedback loop at the entry point into the phenylpropanoid pathway

Pharmacological evidence implicates trans-cinnamic acid as a feedback modulator of the expression and enzymatic activity of the first enzyme in the phenylpropanoid pathway, L-phenylalanine ammonia-lyase (PAL). To test this hypothesis independently of methods that utilize potentially non-specific inhibitors, we generated transgenic tobacco lines with altered activity levels of the second enzyme of the pathway, cinnamic acid 4-hydroxylase (C4H), by sense or antisense expression of an alfalfa C4H cDNA. PAL activity and levels of phenylpropanoid compounds were reduced in leaves and stems of plants in which C4H activity had been genetically down-regulated. However, C4H activity was not reduced in plants in which PAL activity had been down-regulated by gene silencing. In crosses between a tobacco line over-expressing PAL from a bean PAL transgene and a C4H antisense line, progeny populations harboring both the bean PAL sense and C4H antisense transgenes had significantly lower extractable PAL activity than progeny populations harboring the PAL transgene alone. Our data provide genetic evidence for a feedback loop at the entry point into the phenylpropanoid pathway that had previously been inferred from potentially artifactual pharmacological experiments.     

Molecular analysis of herbivore-induced condensed tannin synthesis: cloning and expression of dihydroflavonol reductase from trembling aspen (Populus tremuloides)

In order to study condensed tannin synthesis and its induction by herbivory, a dihydroflavonol reductase (DFR) cDNA was isolated from trembling aspen (Populus tremuloides). Bacterial overexpression demonstrated that this cDNA encodes a functional DFR enzyme, and Southern analysis revealed that DFR likely is a single-copy gene in the aspen genome. Aspen plants that were mechanically wounded showed a dramatic increase in DFR expression after 24 h in both wounded leaves and unwounded leaves on wounded trees. Feeding by forest tent caterpillar (Malacosoma disstria) and satin moth (Leucoma salicis) larvae, and treatment with methyl jasmonate, all strongly induced DFR expression. DFR enzyme activity was also induced in wounded aspen leaves, and phytochemical assays revealed that condensed tannin concentrations significantly increased in wounded and systemic leaves. The expression of other genes involved in the phenylpropanoid pathway were also induced by wounding. Our findings suggest that the induction of condensed tannins, compounds known to be important for defense against herbivores, is mediated by increased expression of DFR and other phenylpropanoid genes.     

The potential of metabolite profiling as a selection tool for genotype discrimination in Populus

Differences between wild-type Populus tremulaxalba and two transgenic lines with modified lignin monomer composition, were interrogated using metabolic profiling. Analysis of metabolite abundance data by GC-MS, coupled with principal components analysis (PCA), successfully differentiated between lines that had distinct phenotypes, whether samples were taken from the cambial zone or non-lignifying suspension tissue cultures. Interestingly, the GC-MS analysis detected relatively few phenolic metabolites in cambial extracts, although a single metabolite associated with the differentiation between lines was directly related to the phenylpropanoid pathway or other down-stream aspects of lignin biosynthesis. In fact, carbohydrates, which have only an indirect relationship with the modified lignin monomer composition, featured strongly in the line-differentiating aspects of the statistical analysis. Traditional HPLC analysis was employed to verify the GC-MS data. These findings demonstrate that metabolic traits can be dissected reliably and accurately by metabolomic analyses, enabling the discrimination of individual genotypes of the same tree species that exhibit marked differences in industrially relevant wood traits. Furthermore, this validates the potential of using metabolite profiling techniques for marker generation in the context of plant/tree breeding for industrial applications.     

Reduced Lignin Content and Altered Lignin Composition in Transgenic Tobacco Down-Regulated in Expression of L-Phenylalanine Ammonia-Lyase or Cinnamate 4-Hydroxylase

We analyzed lignin content and composition in transgenic tobacco (Nicotiana tabacum) lines altered in the expression of the early phenylpropanoid biosynthetic enzymes L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase (C4H). The reduction of C4H activity by antisense expression or sense suppression resulted in reduced levels of Klason lignin, accompanied by a decreased syringyl/guaiacyl monomer ratio as determined by pyrolysis gas chromatography/mass spectrometry Similar reduction of lignin levels by down -regulation of L-phenylalanine ammonia-lyase, the enzyme preceding C4H in the central phenylpropanoid pathway, did not result in a decreased syringyl/guaiacyl ratio. Rather, analysis of lignin methoxyl content and pyrolysis suggested an increased syringyl/guaiacyl ratio. One possible explanation of these results is that monolignol biosynthesis from L-phenylalanine might occur by more than one route, even at the early stages of the core phenylpropanoid pathway, prior to the formation of specific monolignol precursors.     

Biochemical characterization of two differentially expressed polyphenol oxidases from hybrid poplar

Two polyphenol oxidase isoforms with distinct expression patterns were identified in hybrid poplar (Populus trichocarpaxP. deltoides). PPO-1, corresponding to the previously cloned PtdPPO (Constabel et al., Plant Physiol. 124: 285-295) was primarily leaf tissue-specific and detected only after wounding. PPO-2 was expressed constitutively in all tissue types tested except mature leaves, with highest expression in very young leaves and conducting tissues such as roots, stems and petioles. These two PPO isoforms were partially purified from hybrid poplar by ammonium sulfate fractionation followed by hydrophobic interaction chromatography. They were found to differ in stability, pH optimum, and activation by SDS. Tests with common phenolic substrates showed that PPO-1 had a broader substrate specificity than PPO-2. The distinct enzymatic properties and expression patterns of these two PPO isoforms suggest that they may have different physiological functions in hybrid poplar.     

Primary and secondary metabolism of winter wheat under cold hardening and treatment with antioxidants

The content of saccharides and phenolic compounds (including flavonoids and lignin) and the activity of L-phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) were determined in leaves and tillering nodes of winter wheat Triticum aestivum L.) cultivars Inna and Moskovskaya 39, differing in the level of frost resistance. These parameters were determined in three groups of plants--control, hardened, and treated with synthetic antioxidants (ambiol and amerol 2000). Cold hardening increased accumulation of primary and secondary metabolites in tissues but decreased the enzyme activity. Treatment with antioxidants also increased the content of saccharides and phenolic compounds (primarily flavonoids) and PAL activity. These changes were more pronounced in cultivar Inna, which is less frost resistant than Moskovskaya 39.     

Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees.

Because lignin limits the use of wood for fiber, chemical, and energy production, strategies for its downregulation are of considerable interest. We have produced transgenic aspen (Populus tremuloides Michx.) trees in which expression of a lignin biosynthetic pathway gene Pt4CL1 encoding 4-coumarate:coenzyme A ligase (4CL) has been downregulated by antisense inhibition. Trees with suppressed Pt4CL1 expression exhibited up to a 45% reduction of lignin, but this was compensated for by a 15% increase in cellulose. As a result, the total lignin-cellulose mass remained essentially unchanged. Leaf, root, and stem growth were substantially enhanced, and structural integrity was maintained both at the cellular and whole-plant levels in the transgenic lines. Our results indicate that lignin and cellulose deposition could be regulated in a compensatory fashion, which may contribute to metabolic flexibility and a growth advantage to sustain the long-term structural integrity of woody perennials.     

欧洲黑杨基因资源材性关联基因的SNP分析

以115个欧洲黑杨(Populus nigra L.)无性系为材料, 利用TaqMan技术分析了欧洲黑杨基因资源参与木质素和纤维素合成的酶(4CL、PAL和CesA2)的单核苷酸多态性, 并对分型的SNPs与木材材性性状(物理性状：基本密度、纤维长、纤维宽、微纤丝角; 化学性状: 木质素含量、纤维素含量、a 纤维素含量等)进行了相关分析。结果如下: (1)在对4CL、PAL和CesA2等3个基因进行检测时, 共获得27个SNPs标记, 对其中转换(A-G, C-T)有17个位点, 颠换(A-C, G-C, G-T, A-T等)有10个位点; (2)对其中的3个SNPs进行了分型, 分别记作SNP1、SNP2和SNP3; (2)对已经分型SNPs位点与材性性状进行方差分析, 结果显示, 3个SNPs中只有SNP1与4年生欧洲黑杨综纤维素含量显著相关, 表现为负效应, 贡献率为11.11%; (3)对欧洲黑杨4CL基因的SNP1标记的不同基因型所对应的材性性状进行方差分析, 结果显示基因型为CC和CT的欧洲黑杨相对于基因型为TT的欧洲黑杨有较高的纤维素含量。