Despite the fact that fructans are the main constituent of water-soluble carbohydrates in forage grasses and cereal crops of temperate climates, little knowledge is available on the regulation of the ...enzymes involved in fructan metabolism. The analysis of enzyme activities involved in this process has been hampered by the low affinity of the fructan enzymes for sucrose and fructans used as fructosyl donor. Further, the analysis of fructan composition and enzyme activities is restricted to specialized labs with access to suited HPLC equipment and appropriate fructan standards. The degradation of fructan polymers with high degree of polymerization (DP) by fructan exohydrolases (FEHs) to fructosyloligomers is important to liberate energy in the form of fructan, but also under conditions where the generation of low DP polymers is required. Based on published protocols employing enzyme coupled endpoint reactions in single cuvettes, we developed a simple and fast kinetic 1-FEH assay. This assay can be performed in multi-well plate format using plate readers to determine the activity of 1-FEH against 1-kestotriose, resulting in a significant time reduction. Kinetic assays allow an optimal and more precise determination of enzyme activities compared to endpoint assays, and enable to check the quality of any reaction with respect to linearity of the assay. The enzyme coupled kinetic 1-FEH assay was validated in a case study showing the expected increase in 1-FEH activity during cold treatment. This assay is cost effective and could be performed by any lab with access to a plate reader suited for kinetic measurements and readings at 340 nm, and is highly suited to assess temporal changes and relative differences in 1-FEH activities. Thus, this enzyme coupled kinetic 1-FEH assay is of high importance both to the field of basic fructan research and plant breeding.
Purine nucleotides are essential components to sustain plant growth and development. In plants they are either synthesized "de novo" during the process of purine biosynthesis or are recycled from ...purine bases and purine nucleosides throughout the salvage pathway. Comparison between animals, microorganisms and Arabidopsis, the first plant species with a completely sequenced genome, shows that plants principally use the same biochemical steps to synthesize purine nucleotides and possess all the essential genes and enzymes. Here we report on the cloning and molecular analysis of the complete purine biosynthesis pathway in plants, and the in planta functional analysis of PRPP (5-phosphoribosyl-1-pyrophoshate) amidotransferase (ATase), catalyzing the first committed step of the "de novo" purine biosynthesis. The cloning of the genes involved in the purine biosynthesis pathway was attained by a screening strategy with heterologous cDNA probes and by using S. cerevisiae mutants for complementation. Southern hybridization showed a complex genomic organization for these genes in solanaceous species and their organ- and developmental specific expression was analyzed by Northern hybridization. The specific role of ATase for plant growth and development was analyzed in transgenic tobacco plants exhibiting a reduced ATase activity and in an Arabidopsis T-DNA mutant (atd2) deficient for ATase2. The transgenic tobacco plants as well as the Arabidopsis mutant exhibit a specific and comparable phenotype, which is characterized by strong growth retardation and severe chlorosis in leaves. The formation of white leaves, but green cotyledons is a characteristic trait of the Arabidopsis atd2 mutant.
Phytoalexins are pathogen induced low molecular weight compounds with antimicrobial activities derived from secondary metabolism. Following their identification, phytoalexins were directly ...incorporated into the network of plant defense responses. Due to their heterogeneity, the metabolic pathways involved in phytoalexin formation and in particular the regulatory mechanisms remained elusive. Consequently, research focus shifted to the characterization of other components of plant immunity such as defense signaling and resistance mechanisms, including components of systemic acquired and induced systemic resistance, effector and pathogen-associated molecular pattern triggered immunity as well as R-gene resistance. Despite the obtained knowledge on these immunity mechanisms, genetic engineering employing these mechanisms and classical breeding reached too low improvements in crop protection, probably because classical breeding focused on yield performance and taste, rather than pathogen resistance. The increasing demand for disease resistant crop species and the aim to reduce pesticide application therefore requires alternative approaches. Recent advances in the understanding of phytoalexin function, biosynthesis and regulation, in combination with novel methods of molecular engineering and advances in instrumental analysis, returned attention to phytoalexins as a potent target for improving crop protection. Based on this, the advantages as well as potential bottlenecks for molecular approaches of modulating inducible phytoalexins to improve crop protection are discussed.
Phytohormones are known as essential regulators of plant defenses, with ethylene, jasmonic acid, and salicylic acid as the central immunity backbone, while other phytohormones have been demonstrated ...to interact with this. Only recently, a function of the classic phytohormone cytokinin in plant immunity has been described in Arabidopsis, rice, and tobacco. Although interactions of cytokinins with salicylic acid and auxin have been indicated, the complete network of cytokinin interactions with other immunity-relevant phytohormones is not yet understood. Therefore, we studied the interaction of kinetin and abscisic acid as a negative regulator of plant immunity to modulate resistance in tobacco against Pseudomonas syringae. By analyzing infection symptoms, pathogen proliferation, and accumulation of the phytoalexin scopoletin as a key mediator of kinetin-induced resistance in tobacco, antagonistic interaction of these phytohormones in plant immunity was identified. Kinetin reduced abscisic acid levels in tobacco, while increased abscisic acid levels by exogenous application or inhibition of abscisic acid catabolism by diniconazole neutralized kinetin-induced resistance. Based on these results, we conclude that reduction of abscisic acid levels by enhanced abscisic acid catabolism strongly contributes to cytokinin-mediated resistance effects. Thus, the identified cytokinin-abscisic acid antagonism is a novel regulatory mechanism in plant immunity.
Drought stress conditions modify source–sink relations, thereby influencing plant growth, adaptive responses, and consequently crop yield. Invertases are key metabolic enzymes regulating sink ...activity through the hydrolytic cleavage of sucrose into hexose monomers, thus playing a crucial role in plant growth and development. However, the physiological role of invertases during adaptation to abiotic stress conditions is not yet fully understood. Here it is shown that plant adaptation to drought stress can be markedly improved in tomato (Solanum lycopersicum L.) by overexpression of the cell wall invertase (cwInv) gene CIN1 from Chenopodium rubrum. CIN1 overexpression limited stomatal conductance under normal watering regimes, leading to reduced water consumption during the drought period, while photosynthetic activity was maintained. This caused a strong increase in water use efficiency (up to 50%), markedly improving water stress adaptation through an efficient physiological strategy of dehydration avoidance. Drought stress strongly reduced cwInv activity and induced its proteinaceous inhibitor in the leaves of the wild-type plants. However, the CIN1-overexpressing plants registered 3- to 6-fold higher cwInv activity in all analysed conditions. Surprisingly, the enhanced invertase activity did not result in increased hexose concentrations due to the activation of the metabolic carbohydrate fluxes, as reflected by the maintenance of the activity of key enzymes of primary metabolism and increased levels of sugar-phosphate intermediates under water deprivation. The induced sink metabolism in the leaves explained the maintenance of photosynthetic activity, delayed senescence, and increased source activity under drought stress. Moreover, CIN1 plants also presented a better control of production of reactive oxygen species and sustained membrane protection. Those metabolic changes conferred by CIN1 overexpression were accompanied by increases in the concentrations of the senescence-delaying hormone trans-zeatin and decreases in the senescence-inducing ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) in the leaves. Thus, cwInv critically functions at the integration point of metabolic, hormonal, and stress signals, providing a novel strategy to overcome drought-induced limitations to crop yield, without negatively affecting plant fitness under optimal growth conditions.
Dear Editor, Phytohormones have been described as essential regula- tors of various processes throughout plant life, forming a strong interactive network. Because of this important func- tion, they ...are central and integrative modulators form- ing a physiological key interface between plant responses and primary parameters such as genotype, environmental conditions, and developmental status. Consequently, the determination of the phytohormone signature as a key physiological parameter is necessary to understand the correlations between genotype and phenotype, as well as the influence of exogenous modulations on the phenotype （Yin et al., 2004）. Thus, evaluation of the phytohormone signature has to be considered for physiological phenotyp- ing, especially for the improvement of crops or developing strategies for plant protection. This includes the important trait plant immunity, which is determined also by distinct and fine-tuned modulations of phytohormones （Robert- Seilaniantz et al., 2011）.
Phytohormones are essential regulators of various processes in plant growth and development. Several phytohormones are also known to regulate plant responses to environmental stress and pathogens. ...Only recently, cytokinins have been demonstrated to play an important role in plant immunity. Increased levels of cytokinins such as trans-zeatin, which are considered highly active, induced resistance against mainly (hemi)biotrophic pathogens in different plant species. In contrast, cis-zeatin is commonly regarded as a cytokinin exhibiting low or no activity. Here we comparatively study the impact of both zeatin isomers on the infection of Nicotiana tabacum by the (hemi)biotrophic microbial pathogen Pseudomonas syringae. We demonstrate a biological effect of cis-zeatin and a differential effect of the two zeatin isomers on symptom development, defense responses and bacterial multiplication.
Background: Berberine bridge enzyme-like proteins are a multigene family in plants. Results: Members of the berberine bridge enzyme-like family were identified as monolignol oxidoreductases. ...Conclusion: Berberine bridge enzyme-like enzymes play a role in monolignol metabolism and lignin formation. Significance: Our results indicate a novel and unexpected role of berberine bridge enzyme-like enzymes in plant biochemistry and physiology. Plant genomes contain a large number of genes encoding for berberine bridge enzyme (BBE)-like enzymes. Despite the widespread occurrence and abundance of this protein family in the plant kingdom, the biochemical function remains largely unexplored. In this study, we have expressed two members of the BBE-like enzyme family from Arabidopsis thaliana in the host organism Komagataella pastoris. The two proteins, termed AtBBE-like 13 and AtBBE-like 15, were purified, and their catalytic properties were determined. In addition, AtBBE-like 15 was crystallized and structurally characterized by x-ray crystallography. Here, we show that the enzymes catalyze the oxidation of aromatic allylic alcohols, such as coumaryl, sinapyl, and coniferyl alcohol, to the corresponding aldehydes and that AtBBE-like 15 adopts the same fold as vanillyl alcohol oxidase as reported previously for berberine bridge enzyme and other FAD-dependent oxidoreductases. Further analysis of the substrate range identified coniferin, the glycosylated storage form of coniferyl alcohol, as a substrate of the enzymes, whereas other glycosylated monolignols were rather poor substrates. A detailed analysis of the motifs present in the active sites of the BBE-like enzymes in A. thaliana suggested that 14 out of 28 members of the family might catalyze similar reactions. Based on these findings, we propose a novel role of BBE-like enzymes in monolignol metabolism that was previously not recognized for this enzyme family.
In a screen for leaf developmental mutants we have isolated an activator T-DNA-tagged mutant that produces leaves without a petiole. In addition to that leafy petiole phenotype this lettuce (let) ...mutant shows aberrant inflorescence branching and silique shape. The LEAFY PETIOLE (LEP) gene is located close to the right border of the T-DNA insert linked with these dominant phenotypes and encodes a protein with a domain with similarity to the DNA binding domain of members of the AP2/EREBP family of transcription factors. Introduction of the activation-tagged LEP gene in wild-type plants conferred all the phenotypic aberrations mentioned above. The leafy petiole phenotype consists of a conversion of the proximal part of the leaf from petiole into leaf blade, which means that leaf development in let is disturbed along the proximodistal axis. Therefore, LEP is involved in either cell division activity in the marginal meristem or patterning along the proximodistal axis.
Transgenic Arabidopsis thaliana plants containingthe Agrobacterium tumefaciens cytokinin-biosynthesis geneipt were produced to study the effect of increasedcytokinin (CK) levels on the development of ...this rosette plant species. Inthreeindependently transformed lines (ipt-156, 158 and 161),Arabidopsis plants had smaller leaves, an underdevelopedroot system and decreased apical dominance in inflorescence stems. The smallertransgenic leaves were highly serrated along the margins, pale green and hadpointed leaf tips. In cross section, transgenic leaves had smaller cells andirregularly shaped epidermal cells. In the ipt-161 line,leaves and hypocotyls frequently exhibited purple color due to anthocyaninproduction. The most severe phenotype was observed in tissue cultureconditions,while growth in soil reduced or eliminated some phenotypic effects. Compared toC24 wild type plants, ipt-161 plants accumulated zeatinandzeatin riboside with an approximate 10-fold increase in the total pool of CKs.Astudy of the progeny resulting from crosses between theipt-161 transgenic line and the ethylene insensitivemutants ein1, ein2 andeti5 suggested that part of the altered developmentexhibited by the ipt transgenic plants was caused byincreased ethylene levels.PUBLICATION ABSTRACT
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