Willy Hilgenberg

Glucosinolate content in susceptible and resistant Chinese cabbage varieties during development of clubroot disease.

Abstract The glucosinolate content in Chinese cabbage (Brassica campestris ssp. pekinensis) during the development of clubroot disease caused by the obligate biotroph Plasmodiophora brassicae was investigated. Two Plasmodiophora-resistant and two susceptible varieties of Chinese cabbage were used and three classes of glucosinolates, aliphatic (=alkenyl), aromatic and indolic were analysed. Between the susceptible varieties ‘Granat’ and ‘Osiris’ and the resistant varieties ‘Parkin’ and ‘Yuki’ there were significant differences in glucosinolate pattern. The total glucosinolate content in roots of the two susceptible varieties was higher throughout the experimental period than in roots of the two resistant varieties. ‘Osiris’ showed the highest glucosinolate content of all the varieties investigated (ca three-fold higher than ‘Granat’ and ca five-fold higher than ‘Parkin’ and ‘Yuki’). After infection with P. brassicae the indole glucosinolates increased after 14 and 20 days in roots of ‘Granat’ and ‘Osiris’, respectively, whereas ther was no difference between infected and control roots in ‘Parkin’ and ‘Yuki’. The aliphatic glucosinolates were also enhanced in infected roots of ‘Granat’, whereas ‘Osiris’ showed a very high content of aliphatic glucosinolates during the whole experimental period. Roots of ‘Parkin’ and ‘Yuki’ grown in the presence of Plasmodiophora spores showed an elevated concentration of aromatic glucosinolates after 14 and 30 days, respectively, which was not found in ‘Granat’ and ‘Osiris’. Total seed glucosinolate content appeared to be correlated with the susceptibility of the Chinese cabbage varieties tested. Eight different susceptible varieties showed higher total glucosinolate contents than the two resistant varieties. Treatment of plants of the varieties ‘Parkin’ and ‘Granat’ with salicylic acid and jasmonic acid resulted in increased amounts of glucosinolates, although differences in the response were observed between the two treatment. Jasmonic acid induced mainly indole glucosinolates in the leaves, whereas salicylic acid induced indole glucosinolates also in the roots of both varieties. In the variety ‘Parkin’, we also observed induction of aliphatic and aromatic glucosinolates after jasmonate treatment. Although the variety ‘Parkin’ showed no clubroot symptoms, we were able to detect fungal structures within the roots using scanning electron microscopy. We would, therefore, rather describe this variety as tolerant not resistant to clubroot disease. The potential role of different glucosinolates in plant-pathogen interactions is discussed.

A soluble protein factor from chinese cabbage converts indole-3-acetaldoxime to iaa

Abstract A soluble enzyme preparation from Chinese cabbage seedlings ( Brassica campestris ssp. pekinensis ) which catalyses the conversion of indole-3-acetaldoxime (IAOX) to IAA was partially purified by ion exchange chromatography. After purification enzyme activity was stable for more than 6 hr. Substrate kinetics showed a K m value of 50 μM; the pH optimum was 7. The conversion of IAOX to IAA was increased by NAD, NADP or FAD, but none of them seemed to be a preferential co-substrate. Besides IAA some labelled indole-3-acetaldehyde (IAALD) could be extracted from the reaction mixture. Addition of unlabelled IAALD at 100 nmol/ml led to a significant inhibition of IAA formation while some label accumulated in the aldehyde, Indole-3-acetonitrile was never detected as a reaction product. The results are compared with those from earlier in vivo experiments and are discussed in view of their significance for IAA biosynthesis in the Brassicaceae.

Developmental regulation of aldoxime formation in seedlings and mature plants of Chinese cabbage (Brassica campestris ssp. pekinensis) and oilseed rape (Brassica napus): glucosinolate and IAA biosynthetic enzymes

The first steps in the biosynthesis of glucosinolates and indole-3-acetic acid (IAA) in oilseed rape (Brassica napus L.) and Chinese cabbage (Brassica campestris ssp. pekinensis) involve the formation of aldoximes. In rape the formation of aldoximes from chain-extended amino acids, for aromatic and aliphatic glucosinolate biosynthesis, is catalysed by microsomal flavin-containing monooxygenases. The formation of indole-3-aldoxime from l-tryptophan, the potential precursor of both indole-3-acetic acid and indolyl-glucosinolates, is catalysed by several microsomal peroxidases. The biosynthesis of glucosinolates and indole-3-acetic acid was shown to be under developmental control in oilseed rape and Chinese cabbage. No monooxygenase activities were detected in cotyledons or old leaves of either species. The highest monooxygenase activities were found in young expanding leaves; as the leaves reached full expansion and matured the activities decreased rapidly. The indole-aldoxime-forming activity was found in all of the tissues analysed, but there was also a clear decrease in foliar activity with maturity in leaves of rape and Chinese cabbage. Partial characterisation of the Chinese cabbage monooxygenases showed that they have essentially identical properties to the previously characterised rape enzymes; they are not cytochrome P450-type enzymes, but resemble flavin-containing monooxygenases. No monooxygenase inhibitors were detected in microsomes prepared from either cotyledons or old leaves.

Plasma membrane bound high pI peroxidase isoenzymes convert tryptophan to indole-3-acetaldoxime

Abstract -Several peroxidase isoenzymes (pI 8–9) from phase-partitioned Chinese cabbage plasma membrane oxidized l -tryptophan to indole-3-acetaldoxime as shown by (i) correlation of catalytic activity with benzidineguaiacol staining on isoelectric focussing (IEF) gels, (ii) inhibition of enzyme activity by polyclonal antisera directed against tobacco peroxidase isoenzymes (which cross-react with Chinese cabbage peroxidases) and (iii) inhibition of tetraguaiacol formation by l -tryptophan. Treatment of the outside-out plasma membrane vesicles with 1 M sodium chloride and sonification released only part of the membrane-bound tryptophan oxidizing enzyme activity, suggesting little non-specific binding of peroxidases to (or trapping in) the membrane vesicles.

Metabolism of 14C-indole-3-acetaldoxime by hypocotyls of Chinese cabbage

Abstract The metabolism of 14C-indole-3-acetaldoxime by Chinese cabbage hypocotyls was investigated using labelled tracer and incubation times less than 1 hr. Indole-3-acetonitrile, indole-3-methylglucosinolate and desulpho-indole-3-methylglucosinolate were the major metabolites, while IAA or other IAA precursors were not detected. The kinetics of the conversion of the aldoxime to the three metabolites was different under continuous feeding and pulse feeding conditions. The apparent Km for the conversion of the aldoxime to the nitrile and the glucosinolate were 3.3 and 5.0 μM, respectively. Tissues of Isatis tinctoria, Helianthus annuus and Zea mays also formed significant amounts of the nitrile and Zea mays formed small amounts of indole-3-acetaldehyde.

Infection with the Obligate Biotroph Plasmodiophora braßicae, the Causal Agent of the Club Root Disease, does not Affect Expreßion of NIT-1/2-related Nitrilases in Roots of Chinese Cabbage

Summary The infection of Chinese cabbage ( Brasica rapa sp. pekinensis cv. Granaat) with the causal agent of the club root disease, Plasmodiophora brasicae Wor., leads to tumorous swellings of the root as a result of increased cell division and hypertrophy. An increased auxin formation in infected tisue by enzymatic hydrolysis of the precursor indole-3-acetonitrile has previously been postulated. Based on a recently published nitrilase cDNA sequence of Arabidopsis thaliana , we have amplified a partial nitrilase cDNA fragment from Chinese cabbage Lstrand cDNA, synthesized from hypocotyl total RNA, by polymerase chain reaction. Also, genomic nitrilase fragments from Chinese cabbage and Brasica oleracea, another host for P. brasicae , were amplified with the same set of primers. Sequence comparisons indicate high homology in exon and intron regions. The sequenced isoform of Chinese cabbage is most closely related to the NIT isoforms 1 and 2 of A. thaliana . Using the homologous nitrilase cDNA fragment (504 by of coding region) as a probe, nitrilase mRNA was determined in total RNA from P. brasicae -infected and healthy root tisue of Chinese cabbage. In both tisues a mRNA of approx. 1.3 kB was detected with similar amounts in infected and healthy tisues. Our results suggest that the biotrophic pathogen P. brasicae does not affect expresion of NIT 1/2-related nitrilase(s) at the transcriptional level.

The in vitro biosynthesis of indole-3-butyric acid in maize

Abstract The in vitro biosynthesis of indole-3-butyric acid (IBA) was investigated in maize ( Zea mays L.). Incubation of microsomal membranes from shoots and roots of dark-grown maize with labelled indole-3-acetic acid (IAA), acetyl coenzyme A (acetyl CoA) and ATP resulted in the formation of an unknown labelled reaction product, product X. Other coenzymes had very little effect on the formation of this product. No IBA was detected in this system. The labelled product was purified and fed to other cell fractions of dark-grown maize. Incubation with the organelle fraction using NADPH as a cofactor resulted in the conversion of product X to a new compound which had the retention time of IBA in HPLC. Attempts to identify product X led to the hypothesis that it is a conjugate of IAA with ADP. Microsomal membranes from light-grown shoots and roots converted IAA to IBA, as demonstrated by HPLC analysis. The identity of the IBA peak was confirmed by GC-mass spectrometry. Microsomal membranes from light-grown roots formed both IBA and little of product X. For the direct in vitro biosynthesis of IBA acetyl CoA was needed as a cofactor, and the reaction as enhanced by the addition of ATP or NADPH. The optimum pH of the in vitro formation of IBA was 6–7 in the roots and 5 in the shoots. Product X was formed only at neutral pH (7–8). The K m value for IAA in the synthesis of IBA was 16 μ m .

Uptake and Transport of Indole-3-Butyric Acid in Arabidopsis thaliana: Comparison with Other Natural and Synthetic Auxins

Summary The uptake of3 H-indole-3-butyric acid (IBA) in Arabidopsis thalianaseedlings grown in liquid culture was studied and compared with other natural and synthetic auxins. It was shown that the uptake of IBA and also of indole-3-acetic acid (IAA) was linear during 1 h, but it was saturable after 5 and 12 h, respectively. The Km value for the saturable in vivo transport of IBA was 0.18 μmol/L, compared with 0.19 μmol/L for IAA, 3.1 μmol/L for naphthylacetic acid (NAA) and 1.2 μmol/L for phenylacetic acid (PAA). The uptake of 3 H-IBA was strongly inhibited by unlabelled IAA. The K; value for IAA was determined as 1.7 μmol/L. Long distance transport of IAA and IBA (acropetal and basipetal) has been investigated using 10 cm stem segments of Arabidopsis. In general, both hormones were transported better acropetally than basipetally. IBA was taken up better than IAA and was also transported more rapidly, although for both hormones a transport further than 6 cm after 4.5 h in the stem could not be detected. Our results indicate no prominent differences between IAA and IBA transport in Arabidopsis thaliana.

Partial purification of nitrilase from Chinese cabbage.

Abstract Nitrilase was purified ca 28-fold from Chinese cabbage seedlings. Km values of 5.2 × 10−4 and 2.6 × 10−3 M were obtained for indoleacetonitrile (IAN) and 3-cyanopyridine (3-CP) as substrates. For hydrolysis of 3-CP, the maximal velocity was 44 times higher than for the natural substrate IAN. The pH optimum is at 7.5. IAA concentrations from 10−6 to 10−3 M did not inhibit the partially purified enzyme. Nitrilase activity was investigated during development of seedlings grown under continuous light. Roots with hypocotyls exhibited only slightly lower activity than cotyledons based on fresh weight, although their specific activity was ca 5 times higher. Etiolated seedlings showed a very similar distribution of nitrilase activity. The significance of the results for IAA biosynthesis is discussed.

High-performance liquid chromatographic separation and some properties of (e)- and (z)-3-idoleacetaldoxime

Abstract An isocratic reversed-phase high-performance liquid chromatographic (HPLC) system for the separation of ( E )- and ( Z )-3-idoleacetaldoxime was developed. The indentities of the isomers were confirmed by NMR spectrometry. Both isomers could be converted into 3-indoleacetonitrile with acetic anhydride. UV irradiation of both isomers in ethanolic solution led to rapid photolysis. For the identification of the degradation products an isocratic silica gel HPLC system was developed, which showed that 3-methylindole was one major product whereas neither 3-indoleacetonitrile nor 3-indoleacetamide was formed in significant amounts. Conversely, in acetic acid both isomers were relatively stable to UV light. The implications of the results for studies of the role of 3-indoleacetaldoxime as a natural compound in higher plans are discussed.