Histochemical Detection of Peroxidase and Laccase Activities in Populus Secondary Xylem.

Peroxidases (PRXs) and laccases (LACs) are enzymes involved in catalyzing the oxidation of the lignin monomers to facilitate lignin polymerization. However, due to the large number of genes composing these two families of enzymes, many details regarding their specific localization are only partially understood. Here, we present a fast and easy histochemical method that makes use of the artificial substrate 3,3',5,5'-tetramethylbenzidine (TMB) to visualize PRX and LAC activities in the hybrid aspen (Populus tremula x P. tremuloides) xylem tissue. In addition, we describe a protocol that allows the detection of the PRX substrate, H2O2, using the nonfluorescent dye 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) in woody tissues.

Monitoring of Crosstalk Between Jasmonate and Auxin in the Framework of Plant Stress Responses of Roots.

Over the last few years, it became more and more evident that plant hormone action is to great parts determined through their sophisticated crosstalk, rather than by their isolated activities. Thus, the parallel analysis of interconnected phytohormones in only very small amounts of tissue developed to an important issue in the field of plant sciences. In the following, a highly sensitive and accurate method is described for the quantitative analysis of the plant hormones jasmonic acid and indole-3-acetic acid in the model plant Arabidopsis thaliana. The described methodology is, however, not limited to the analysis of Arabidopsis samples but can also be applied to other plant species. The presented method is optimized for the working up of as little as 20-50 mg of plant tissue. Thus, it is well suited for the analysis of plant hormone contents in plant tissue of only little biomass, such as roots. The presented protocol facilitates the implementation of the method into other laboratories that have access to appropriate laboratory equipment and comparable state-of-the-art gas chromatography-mass spectrometry (GC-MS) technology.

Arabidopsis NITRILASE 1 Contributes to the Regulation of Root Growth and Development through Modulation of Auxin Biosynthesis in Seedlings.

Nitrilases consist of a group of enzymes that catalyze the hydrolysis of organic cyanides. They are found ubiquitously distributed in the plant kingdom. Plant nitrilases are mainly involved in the detoxification of ß-cyanoalanine, a side-product of ethylene biosynthesis. In the model plant Arabidopsis thaliana a second group of Brassicaceae-specific nitrilases (NIT1-3) has been found. This so-called NIT1-subfamily has been associated with the conversion of indole-3-acetonitrile (IAN) into the major plant growth hormone, indole-3-acetic acid (IAA). However, apart of reported functions in defense responses to pathogens and in responses to sulfur depletion, conclusive insight into the general physiological function of the NIT-subfamily nitrilases remains elusive. In this report, we test both the contribution of the indole-3-acetaldoxime (IAOx) pathway to general auxin biosynthesis and the influence of altered nitrilase expression on plant development. Apart of a comprehensive transcriptomics approach to explore the role of the IAOx route in auxin formation, we took a genetic approach to disclose the function of NITRILASE 1 (NIT1) of A. thaliana. We show that NIT1 over-expression (NIT1ox) results in seedlings with shorter primary roots, and an increased number of lateral roots. In addition, NIT1ox plants exhibit drastic changes of both free IAA and IAN levels, which are suggested to be the reason for the observed phenotype. On the other hand, NIT2RNAi knockdown lines, capable of suppressing the expression of all members of the NIT1-subfamily, were generated and characterized to substantiate the above-mentioned findings. Our results demonstrate for the first time that Arabidopsis NIT1 has profound effects on root morphogenesis in early seedling development.

Characterization of Four Bifunctional Plant IAM/PAM-Amidohydrolases Capable of Contributing to Auxin Biosynthesis.

Amidases [EC 3.5.1.4] capable of converting indole-3-acetamide (IAM) into the major plant growth hormone indole-3-acetic acid (IAA) are assumed to be involved in auxin de novo biosynthesis. With the emerging amount of genomics data, it was possible to identify over forty proteins with substantial homology to the already characterized amidases from Arabidopsis and tobacco. The observed high conservation of amidase-like proteins throughout the plant kingdom may suggest an important role of theses enzymes in plant development. Here, we report cloning and functional analysis of four, thus far, uncharacterized plant amidases from Oryza sativa, Sorghum bicolor, Medicago truncatula, and Populus trichocarpa. Intriguingly, we were able to demonstrate that the examined amidases are also capable of converting phenyl-2-acetamide (PAM) into phenyl-2-acetic acid (PAA), an auxin endogenous to several plant species including Arabidopsis. Furthermore, we compared the subcellular localization of the enzymes to that of Arabidopsis AMI1, providing further evidence for similar enzymatic functions. Our results point to the presence of a presumably conserved pathway of auxin biosynthesis via IAM, as amidases, both of monocot, and dicot origins, were analyzed.