If older plant material was used, the plant organs were harvested from plants grown in a greenhouse on a mixture of soil and sand for 4—6 weeks in short days 8 h photoperiod. Thereafter, plants were transferred to long day conditions 16 h photoperiod. Three biological replicates were harvested and frozen into liquid N2 prior to RNA extraction. Variation between replicates was accounted for by using the LIMMA model, which allows correlation of expression levels within each biological replicate and where information is drawn across genes by fixing the within-biological clone correlation Smyth, Differentially expressed genes were identified by the modified t-test implemented in the LIMMA package.
A p-value of 0. A more classical adjusted p-value of 0. Of the differentially regulated genes a total number of genes could be assigned to GO ontologies and used for BiNGO analysis Supplemental Data Sheet 2 using a hypergeometric test with a traditional Benjamini-Hochberg FDR correction and a post-correction selection significance level of 0.
After identifying a clone harboring the NIT1:c-myc fusion in the correct orientation pEXP1-nit1 , this clone served as a preliminary control to check the enzymatic activity of the NIT1:c-myc fusion protein prior to plant transformation. In case of the construct to induce RNAi in A. Heterologous Expression of Recombinant NIT1:c-myc To investigate the general feasibility of NIT1:c-myc fusion constructs, the fusion protein was at first expressed in Escherichia coli, strain XL1-blue, and, thereafter, the nitrilase activity of the crude extracts was compared.
Afterwards, adding 0. Hereafter, complete cell disruption was achieved by six burst of ultrasound 20 s. Incorporation of the binary vectors was verified by restriction analysis and polymerase chain reaction, using primers specific for the recombinant construct. Transgenic plants were generated following the protocol of Clough and Bent Seeds of transformed T0-plants were harvested in bulk, sown on soil, and selected by spraying with 0.
Of those 20 showed no significant transgene expression, 41 showed weak NIT1:c-myc expression and 8 showed substantial expression of the NIT1:c-myc construct.
Western blots were utilized to compare the relative NIT1:c-myc content in the independent lines. From the latter group, three lines showing both stable and high level expression of recombinant NIT1:c-myc were selected for all further experiments. In brief, the samples were pre-cleaned by micro-scale solid phase extraction on custom-made cartridges containing a silica-based aminopropyl matrix. Subsequently samples were transferred to autosampler vials and excessive diazomethane and solvent was removed in a gentle stream of nitrogen.
About 20 seedlings were used per replicate and three replicates were made for each treatment. On the one hand, the proteins were utilized in immunoblot analysis of transgenic lines.
On the other hand, the extracts were employed to estimate the nitrilase activity in NIT1ox lines compared to appropriate controls, i. For the extraction, 30— mg of plant tissue was frozen in micro-reaction tubes in liquid nitrogen before the tissue was disrupted using micro pestles Sigma, Munich, Germany.
Protein concentrations were determined according to Bradford using serum albumin as a protein standard. Either a rabbit antiserum raised against native NIT1 Schmidt et al. E10 Evan et al. Although it has previously described that IAN is not the preferred substrate of nitrilases in vitro Vorwerk et al. Statistical Analysis The data were analyzed using either Student's t-test when two means were compared or one-way ANOVA followed by Tukey's B post-hoc test to allow for comparisons among all means.
Results Analysis of Transcriptional Perturbations of Auxin Biosynthesis-Related Genes through a Blockade of the IndoleAcetaldoxime Pathway Previous work suggested the operation of nitrilases downstream of glucobrassicin Barlier et al. Herein, we were particularly interested in transcriptional changes of genes known to be involved in auxin biosynthesis, transport, and signaling.
From these genes appeared to be up regulated, while were significantly down regulated. Expectedly, both CYP79B2 and CYP79B3 were identified among the top-ranked down-regulated genes, due to the lack of these transcripts in the double mutant. To resolve which biological processes are mostly affected in the mutant, the two groups of up- and down-regulated genes were sorted with respect to their putative involvement in different biological processes using the gene ontology program BiNGO Maere et al.
The tested up-regulated genes were found to fall into three major groups, including stress responses, such as the defense responses to fungi and responses to abiotic stresses like light, salt and cold, responses to chemical stimuli, like, e. When examining the under-represented groups, expectedly, metabolic processes related with secondary metabolites, including the biosynthesis of glycosinolates, glucosinolates and phenylpropanoids became evident.
Along with these processes, cell wall modification related processes could be detected. However, neither among the over- nor under-represented genes there was an enrichment of directly auxin-related gene groups detectable. Surprisingly, nearly no significant changes in the IPyA pathway were determined Table 1. In line with the results presented by Morant et al. Here we show that the reason for this is most likely not the compensation of the loss an auxin biosynthetic pathway by other metabolic routes that lead to IAA, but rather that the IAOx pathway does not contribute to general auxin biosynthesis under normal growth conditions.
However, the experiments provided evidence for an involvement of the IAOx pathway in numerous stress responses, which gives reason to assume that this pathway is activated under particular circumstances, providing extra auxin if required for adequate responses to environmental stress factors. Intriguingly, we detected the induction of a small number of genes that contribute to Trp biosynthesis, indicating an enhanced metabolic activity of this route. In addition, we found the induction of two Gretchen Hagen 3 genes that are assumed to be involved in maintaining auxin homeostasis by conjugating excess auxin to amino acids.
Together, these findings support the notion of enhanced auxin production. Generation of Gain- and Loss-of-Function Lines The conducted microarray analysis did not provide considerable evidence supporting a general role of the IAOx pathway in auxin formation. However, based on publicly available expression data, NIT1 is not only expressed under stress conditions, but rather shows a broad expression pattern throughout plant development Winter et al.
These phenomena made it tempting to speculate that NIT1 may possess a more general role in plant growth and development, rather than being restricted to stress responses.
In order to investigate the role of NIT1 in more detail, we decided to take a genetic approach, testing the impact of both the constitutive over-expression of the gene and the RNA interference RNAi -mediated knockdown of the entire NIT1-subfamily. To this end, we engineered a construct to produce c-myc epitope-tagged NIT1 under the control of a 35S promoter that facilitates the specific immunological detection of the recombinant protein in corresponding mutant plants.
Prior to the generation of stable transformants, the enzymatic properties of the chimeric NIT1:c-myc protein were analyzed. As preliminary experiments showed that N-terminally Strep-tagged NIT1 considerably lost activity compared to both wild type and C-terminally Strep-tagged variants of the protein data not shown , we fused the c-myc epitope to the C-terminus of NIT1.
To prove the functionality of the fusion protein, the NIT1:c-myc fragment was cloned into an appropriate bacterial over-expression system Raymond et al.
After this initial proof of general applicability, the above mentioned NIT1:c-myc fragment was introduced into a binary vector downstream of a cauliflower mosaic virus 35S promoter that was subsequently used to transform Arabidopsis plants. BASTA selection was used to select for transformed individuals.
It was possible to obtain 69 BASTA resistant lines of which 61 lines showed either no or only a moderate expression of the transgene. From the remaining 8 individual lines that showed a clear over-expression of the NIT1 construct three lines have been selected for further analyses, i. A Three individual plants of A.
The bottom panel shows a Coomassie blue-stained gel, indicating even loading loading control, LC. Apart of the analysis of the consequences of the over-expression of NIT1, we aimed at the examination of plants that lack the activity of nitrilases of the NIT1-subgroup. The available nit mutant shows, except of the increased tolerance against exogenously applied IAN, no significant changes compared to wild-type plants Normanly et al.
However, the NIT1-clade isogenes share a very high sequence similarity. Hence, a transitive RNAi approach was applied to suppress the expression of the complete isogene family Sidahmed and Wilkie, For this, an appropriate construct, comprising a highly conserved cDNA fragment, was cloned and introduced into A. Thus, lines 8 and 16 were used in all following experiments. In contrast, the root phenotype of young NIT1ox seedlings differed remarkably from that of wild-type plants, nit knockout mutants, and the NIT2RNAi lines, respectively.
A closer inspection of the root morphology revealed that in NIT1ox the number of lateral roots slightly increased, while the lateral roots are apparently shorter Figure 4A. Although different plant species may have unique strategies and modifications to optimize their metabolic pathways, it would seem reasonable that plants would share evolutionarily conserved core mechanisms for auxin biosynthesis because IAA is a fundamental substance in the plant life cycle.
Green arrows indicate the tryptophan synthetic pathway in the chloroplast. A thin dashed black arrow denotes the tryptophan-independent IAA biosynthetic pathway. Blue arrows indicate steps for which the gene and enzymatic function are known in the tryptophan-dependent IAA biosynthetic pathway.
Red arrows indicate the indole alkaloid and serotonin biosynthetic pathway. Mustard-coloured arrows indicate the Brassicaceae species-specific pathway. Black arrows indicate steps for which the gene s and enzymatic function s are unknown. Dashed mustard-coloured arrows indicate steps for which the gene and enzymatic function s remain poorly understood.
Letters in italics show genes involved in the conversion process. Lower case letters in italics indicate bacterial genes. In this review, the focus is on assessing the consensus pathway for auxin biosynthesis, and the genes and metabolites involved in auxin biosynthesis in the plant kingdom are summarized.
Multiple pathways postulated for auxin biosynthesis in plants Two major pathways for IAA biosynthesis have been proposed in plants: the tryptophan Trp -independent and Trp-dependent pathways Woodward and Bartel, ; Chandler, ; Normanly, In Trp-independent IAA biosynthesis, indoleglycerol phosphate or indole is the likely precursor, but little is known about the biochemical pathway to IAA Ouyang et al.
Genes that have been suggested to participate in IAA biosynthesis in plants and their respective functions are summarized in Table 1.Previous studies have demonstrated that yucasin suppressed high-auxin phenotypes in YUC overexpression plants, but wild-type plants treated with yucasin showed faint root growth inhibition Supplemental Fig. Nitrilase activities have early been described from a broad variety of plant families including Cruciferae, Gramineae, and Musaceae Mahadevan and Thimann, ; Thimann and Mahadevan, Although IAA was the first plant hormone identified, the biosynthetic pathway at the genetic level has remained unclear.
The bifunctional photoaffinity probe has aryl azido and alkyl azido groups at the m-position of the phenyl ring of yucasin Fig. A p-value of 0. These data suggest that the introduction of a fluorine group improves the chemical stability of yucasin-type inhibitors.
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. However, the analysis of auxin contents in sterile grown seedlings, which show phenotypic differences, revealed very intriguing aspects. The loss-of-functional cyp79b2 79b3 double mutant displayed more severe defects in the presence of YDF, L-Kyn and the biosynthesis inhibitor cocktail Fig. YUC enzymes encode flavin monooxygenase and a part of a large gene family consisting of 11 members in Arabidopsis 11 ,
De novo auxin biosynthesis plays an essential role in many developmental processes. L-Kyn was reported to be a competitive TAA inhibitor Obviously, this initial assumption did not stand the proof of time, since we know today that other enzymes, such as the members of the YUCCA-family, are the main players in auxin biosynthesis Stepanova et al. Thus, lines 8 and 16 were used in all following experiments.
In a previous study, we demonstrated that yucasin 5-[4-chlorophenyl]-2,4-dihydro-[1,2,4]-triazolethione was identified as a YUC inhibitor from synthetic chemical library. From the remaining 8 individual lines that showed a clear over-expression of the NIT1 construct three lines have been selected for further analyses, i. In addition, the amount of root hairs in the NIT1ox line is substantially higher relative to the wild type Figure 4B. Expression of camalexin biosynthesis-associated genes is strongly induced by pathogens at the infection site but remains low under normal conditions Glawischnig,
This seems to be affected by the alteration of the NIT1 content in planta in an either direct or indirect fashion. Lower case letters in italics indicate bacterial genes.
Off-target effects should especially be carefully considered when the pathway is completely blocked with high concentrations of inhibitors. IAOx is considered to be an important metabolic branching point from which a number of different pathways emerge that can eventually lead to the production of either IAA or defense-related secondary metabolites, such as camalexin or indole glucosionlates IGs Zhao et al. The structure-activity study for yucasin revealed that m-substituted analogs 7, 12, and 15 retain YUC inhibitory activity Fig. Seeds of transformed T0-plants were harvested in bulk, sown on soil, and selected by spraying with 0. Moreover, auxins also contribute to photo- and gravitropic responses, fruit ripening and senescence Thimann, ; Davies,
S6 , but wavy and slight agravitropic root phenotypes were observed.
The fluorescent images of root tips were immediately obtained using fluorescence microscopy. The function of target proteins can be spatiotemporally modulated by small molecules in any tissue or cell. Multiple pathways postulated for auxin biosynthesis in plants Two major pathways for IAA biosynthesis have been proposed in plants: the tryptophan Trp -independent and Trp-dependent pathways Woodward and Bartel, ; Chandler, ; Normanly, The fluorescent signal disappeared when the probe was photoactivated in the presence of the substrates Fig. Recently, several auxin biosynthesis inhibitors targeting the IPA pathway have been discovered 16 , 17 ,
In this study, we synthesized various analogs of yucasin and investigated their structure—activity relationships SARs. In brief, the samples were pre-cleaned by micro-scale solid phase extraction on custom-made cartridges containing a silica-based aminopropyl matrix.
It can be concluded that under these conditions the levels of IAN are likely too low for an efficient conversion to IAA by the recombinant nitrilase. The concentration of the IAN content in the plates is indicated. Finally, we simultaneously blocked the IPA and CYP79B auxin biosynthesis pathways using a combination of the inhibitors and mutants for auxin biosynthesis and signaling, and the results suggested auxin null phenotype of Arabidopsis plants. Plant genes thought to be involved in IAA biosynthesis Gene.
Determining the molecular mechanisms of auxin biosynthesis may provide new tools for solving difficult plant development questions, defining the roles of auxin in plant development, understanding auxin transport, and studying the mechanisms of auxin in regulating plant development. In Trp-independent IAA biosynthesis, indoleglycerol phosphate or indole is the likely precursor, but little is known about the biochemical pathway to IAA Ouyang et al. Despite looking quite like wild type under normal growth conditions, the knockdown line showed the expected increased tolerance toward IAN in the solidified growth medium, while showing wild type reactions when grown on medium containing IAA. The concentration of the IAN content in the plates is indicated.