Evaluation of several RT-PCR primer pairs for the detection of Apple stem pitting virus.

Detection of Apple stem pitting virus (ASPV) using RT-PCR based methods was studied in infected apple and pear trees. Three virus-specific primers (ASPF1CP, ASPF2CP, ASPR3CP) were designed to target the most conservative regions of the coat protein gene of 10 virus isolates in Poland and 7 other ASPV sequences available in GenBank. The suitability of the primer pairs ASPF1CP-ASPR3CP and ASPF2CP-ASPR3CP for detection of 19 virus isolates was checked. Both new primer pairs initiated amplification of a specific product from all sources tested. From 1 to 11 isolates were not detected with the primer sets published previously. Detection of the virus in the samples collected in March, using ASPF1CP-ASPR3CP primer pair, was possible up to 512 times dilution. For the samples collected in July, virus was detected in the extracts from infected plants diluted eight times. More than 100-fold increase of sensitivity could be obtained by semi-nested PCR with primers ASPF2CP-ASPR3CP following the first round with ASPF1CP-ASPR3CP. Identification of virus isolates with different number of deletions in the coat protein gene was possible using RT-PCR with newly designed reverse primer ASPDEL in combination with the published primer ASPV7956. Besides, the comparative analysis of silicacapture-RT-PCR (SC-RT-PCR) versus immunocapture-RT-PCR (IC-RT-PCR) assays was carried out. Few ASPV isolates escaped detection by IC-RT-PCR, while all isolates tested were detected using the SC-RT-PCR with the new primers.

Regulation of indole-3-acetic Acid biosynthetic pathways in carrot cell cultures.

2,4-Dichlorophenoxyacetic acid (2,4-D) promotes the accumulation of tryptophan-derived indole-3-acetic acid (IAA) in carrot cell cultures during callus proliferation by a biosynthetic pathway that is apparently not active during somatic embryo formation. The effects of 2,4-D were examined by measuring the isotopic enrichment of IAA due to the incorporation of stable isotope-labeled precursors (deuterium oxide, [(15)N]indole, and (2)H(5)-l-tryptophan). Enrichment of IAA from deuterium oxide is similar in both cultured hypocotyls and cell suspension cultures in the presence and absence of 2,4-D, despite the large differences in absolute IAA concentrations. The enrichment of IAA due to the incorporation of [(15)N]indole is also similar in callus proliferating in the presence of 2,4-D and in embryos developing in the absence of 2,4-D. The incorporation of (2)H(5)-l-tryptophan into IAA, however, is at least 7-fold higher in carrot callus cultures proliferating in the presence of 2,4-D than in embryos developing in the absence of 2,4-D. Other experiments demonstrated that this differential incorporation of (2)H(5)-l-tryptophan into IAA does not result from differential tryptophan uptake or its subsequent compartmentation. Thus, it appears that differential pathways for IAA synthesis operate in callus cultures and in developing embryos, which may suggest that a relationship exists between the route of IAA biosynthesis and development.

Auxin Biosynthesis during Seed Germination in Phaseolus vulgaris.

The relative roles of de novo biosynthesis of indoleacetic acid (IAA) and IAA conjugates stored in mature seeds (Phaseolus vulgaris L.) in supplying auxin to germinating bean seedlings were studied. Using (2)H oxide and 2,4,5,6,7-[(2)H]l-tryptophan as tracers of IAA synthesis, we have shown that de novo biosynthesis of IAA, primarily from tryptophan, is an important source of auxin for young bean seedlings. New synthesis of IAA was detected as early as the second day of germination, at which time the seedlings began to accumulate fresh weight intensively and the total content of free IAA began to increase steadily. IAA conjugates that accumulate in large amounts in cotyledons of mature seeds may thus be considered to be only one of the possible sources of IAA required for the growth of bean seedlings.

Indole-3-Acetic Acid Biosynthesis in the Mutant Maize orange pericarp, a Tryptophan Auxotroph.

The maize mutant orange pericarp is a tryptophan auxotroph, which results from mutation of two unlinked loci of tryptophan synthase B. This mutant was used to test the hypothesis that tryptophan is the precursor to the plant hormone indole-3-acetic acid (IAA). Total IAA in aseptically grown mutant seedlings was 50 times greater than in normal seedlings. In mutant seedlings grown on media containing stable isotopelabeled precursors, IAA was more enriched than was tryptophan. No incorporation of label into IAA from tryptophan could be detected. These results establish that IAA can be produced de novo without tryptophan as an intermediate.

Enzymic synthesis of indol-3-ylacetyl-myo-inositol galactoside.

Extracts of immature kernels of Zea mays catalysed the synthesis of indol-3-ylacetyl-myo-inositol galactoside from indol-3-ylacetyl-myo-inositol and UDP-galactose. Addition of 2-mercaptoethanol was required for stability of the catalytic activity during dialysis. The enzyme could be fractionated with (NH4)2SO4, and 55% of the activity was recovered in the 30-60%-saturation fraction. The product of the reaction contained radioactivity from UDP-[U-14C]galactose and was identified as indol-3-ylacetyl-myo-inositol galactoside by gas chromatography-mass spectrometry. Therefore a UDP-galactose:indol-3-ylacetyl-myo-inositol galactosyltransferase (indol-3-ylacetyl-myo-inositol galactoside synthase) is present in developing kernels of Zea mays. The description of this enzyme, together with the enzymes described in the accompanying paper [Michalczuk & Bandurski (1982) Biochem. J. 207, 273-281] for the synthesis of indol-3-ylacetylglucose and indol-3-ylacetyl-myo-inositol, now provides mechanisms for the biosynthesis of one-half of the low-molecular-weight esters of indol-3-ylacetic acid in Zea mays.

Enzymic synthesis of 1-O-indol-3-ylacetyl-beta-D-glucose and indol-3-ylacetyl-myo-inositol.

An enzyme fraction from extracts of immature kernels of Zea mays catalyses the formation of 1-O-indol-3-ylacetyl-beta-D-glucose from indol-3-ylacetic acid and UDP-glucose. A second enzyme fraction catalyses the formation of indol-3-ylacetyl-myo-inositol from 1-O-indol-3-ylacetyl-beta-D-glucose and myo-inositol. To our knowledge, this is the first example of hydroxy-group acylation by a 1-O-acyl sugar. The following reaction sequence is proposed: Indol-3-ylacetic acid + UDP-glucose leads to indol-3-ylacetylglucose + UDP (1) Indol-3-ylacetylglucose + myo-inositol leads to indol-3-ylacetyl-myo-inositol + glucose (2) The enzyme catalysing reaction (1) is called UDP-glucose:indol-3-ylacetate glucosyl-transferase (indol-3-ylacetylglucose synthase), and that catalysing reaction (2) is indol-3-ylacetylglucose:myo-inositol indol-3-ylacetyltransferase (indol-3-ylacetyl-myo-inositol synthase). We further show that indol-3-ylacetylglucose synthase is specific for UDP-glucose and, at the stage of purity tested, the enzyme will use either indol-3-ylacetic acid or naphthalene-1-acetic acid, but not 2.4-dichlorophenoxyacetic acid, as glucose acceptor. The indol-3-ylacetyl-myo-inositol synthase is specific for indol-3-ylacetyl-glucose and will not use naphthalene-1-acetylglucose as substrate, and it is specific for myo-inositol among the alcohol acceptors tested. Thus, of the auxins tested, only indol-3-ylacetic acid forms the myo-inositol ester.