Biosynthesis of Camalexin from Tryptophan Pathway Intermediates in Cell-Suspension Cultures of Arabidopsis1

Camalexin (3-thiazol-2���-yl-indole) is the principal phytoalexin that accumulates in Arabidopsis after infection by fungi or bacteria. Camalexin accumulation was detectable in Arabidopsis cell-suspension cultures 3 to 5 h after inoculation with Cochliobolus carbonum (Race 1), and then increased rapidly from 7 to 24 h after inoculation. Levels of radioactivity incorporated into camalexin during a 1.5-h pulse labeling with [14C]anthranilate also increased with time after fungal inoculation. The levels of radioactive incorporation into camalexin increased rapidly between 7 and 18 h after inoculation, and then decreased along with camalexin accumulation. Relatively low levels of radioactivity from [14C]anthranilate incorporated into camalexin in the noninoculated controls. Autoradiographic analysis of the accumulation of chloroform-extractable metabolites labeled with [14C]anthranilate revealed a transient increase in the incorporation of radioactivity into indole in fungus-inoculated Arabidopsis cell cultures. The time-course measurement of radioactive incorporation into camalexin during a 1.5-h pulse labeling with [14C]indole was similar to that with [14C]anthranilate. These data suggest that indole destined for camalexin synthesis is produced by a separate enzymatic reaction that does not involve tryptophan synthase.

Cardiolipin is a normal component of human plasma lipoproteins

Anticardiolipin (anti-CL) antibodies, diagnostic for antiphospholipid antibody syndrome, are associated with increased risks of venous and arterial thrombosis. Because CL selectively enhances activated protein C/protein S-dependent anticoagulant activities in purified systems and because CL is not known to be a normal plasma component, we searched for CL in plasma. Plasma lipid extracts [chloroform/methanol (2:1, vol/vol)] were subjected to analyses by using TLC, analytical HPLC, and MS. A plasma lipid component was purified that was indistinguishable from reference CL (M:1448). When CL in 40 fasting plasma lipid extracts (20 males, 20 females) was quantitated by using HPLC, CL (mean �� SD) was 14.9 �� 3.7 ��g/ml (range 9.1 to 24.2) and CL was not correlated with phosphatidylserine (3.8 �� 1.7 ��g/ml), phosphatidylethanolamine (64 �� 20 ��g/ml), or choline-containing phospholipid (1,580 �� 280 ��g/ml). Based on studies of fasting blood donors, CL (���94%) was recovered in very low density, low density, and high density lipoproteins (11 �� 5.3%, 67 �� 11.0%, and 17 �� 10%, respectively), showing that the majority of plasma CL (67%) is in low density lipoprotein. Analysis of relative phospholipid contents of lipoproteins indicated that high density lipoprotein is selectively enriched in CL and phosphatidylethanolamine. These results shows that CL is a normal plasma component and suggest that the epitopes of antiphospholipid antibodies could include CL or oxidized CL in lipoproteins or in complexes with plasma proteins (e.g., ��2-glycoprotein I, prothrombin, protein C, or protein S) or with platelet or endothelial surface proteins.

Callose Deposition Is Responsible for Apoplastic Semipermeability of the Endosperm Envelope of Muskmelon Seeds1

Semipermeable cell walls or apoplastic ���membranes��� have been hypothesized to be present in various plant tissues. Although often associated with suberized or lignified walls, the wall component that confers osmotic semipermeability is not known. In muskmelon (Cucumis melo L.) seeds, a thin, membranous endosperm completely encloses the embryo, creating a semipermeable apoplastic envelope. When dead muskmelon seeds are allowed to imbibe, solutes leaking from the embryo are retained within the envelope, resulting in osmotic water uptake and swelling called osmotic distention (OD). The endosperm envelope of muskmelon seeds stained with aniline blue, which is specific for callose (��-1,3-glucan). Outside of the aniline-blue-stained layer was a Sudan III- and IV-staining (lipid-containing) layer. In young developing seeds 25 d after anthesis (DAA) that did not exhibit OD, the lipid layer was already present but callose had not been deposited. At 35 DAA, callose was detected as distinct vesicles or globules in the endosperm envelope. A thick callose layer was evident at 40 DAA, coinciding with development of the capacity for OD. Removal of the outer lipid layer by brief chloroform treatment resulted in more rapid water uptake by both viable and nonviable (boiled) seeds, but did not affect semipermeability of the endosperm envelope. The aniline-blue-staining layer was digested by ��-1,3-glucanase, and these envelopes lost OD. Thus, apoplastic semipermeability of the muskmelon endosperm envelope is dependent on the deposition of a thick callose-containing layer outside of the endosperm cell walls.

Identification and Partial Characterization of the Pectin Methyltransferase ���Homogalacturonan-Methyltransferase��� from Membranes of Tobacco Cell Suspensions1

A membrane preparation from tobacco (Nicotiana tabacum L.) cells contains at least one enzyme that is capable of transferring the methyl group from S-adenosyl-methionine (SAM) to the C6 carboxyl of homogalacturonan present in the membranes. This enzyme is named homogalacturonan-methyltransferase (HGA-MT) to distinguish it from methyltransferases that catalyze methyletherification of the pectic polysaccharides rhamnogalacturonan I or rhamnogalacturonan II. A trichloroacetic acid precipitation assay was used to measure HGA-MT activity, because published procedures to recover pectic polysaccharides via ethanol or chloroform:methanol precipitation lead to high and variable background radioactivity in the product pellet. Attempts to reduce the incorporation of the 14C-methyl group from SAM into pectin by the addition of the alternative methyl donor 5-methyltetrahydrofolate were unsuccessful, supporting the role of SAM as the authentic methyl donor for HGA-MT. The pH optimum for HGA-MT in membranes was 7.8, the apparent Michaelis constant for SAM was 38 ��m, and the maximum initial velocity was 0.81 pkat mg���1 protein. At least 59% of the radiolabeled product was judged to be methylesterified homogalacturonan, based on the release of radioactivity from the product after a mild base treatment and via enzymatic hydrolysis by a purified pectin methylesterase. The released radioactivity eluted with a retention time identical to that of methanol upon fractionation over an organic acid column. Cleavage of the radiolabeled product by endopolygalacturonase into fragments that migrated as small oligomers of HGA during thin-layer chromatography, and the fact that HGA-MT activity in the membranes is stimulated by uridine 5���-diphosphate galacturonic acid, a substrate for HGA synthesis, confirms that the bulk of the product recovered from tobacco membranes incubated with SAM is methylesterified HGA.