The apparent temperature response of leaf respiration depends on the timescale of measurements: a study of two cold climate species.

Productivity and climate models often use a constant Q10 for plant respiration, assuming tight control of respiration by temperature. We studied the temperature response of leaf respiration of two cold climate species (the Australian tree Eucalyptus pauciflora and the subantarctic megaherb Pringlea antiscorbutica, both measured in a field setting) on a short timescale (minutes) during different times within a diel course, and on a longer timescale, using diel variations in ambient temperature. There were great variations in Q10 depending on measuring day, measuring time and measuring method. When Q10 was calculated from short-term (15 min) manipulations of leaf temperature, the resulting values were usually markedly smaller than when Q10 was calculated from measurements at ambient leaf temperatures spread over a day. While for E. pauciflora, Q10 estimates decreased with rising temperature (corroborating the concept of a temperature-dependent Q10), the opposite was the case for P. antiscorbutica. Clearly, factors other than temperature co-regulate both leaf respiration rates and temperature sensitivity and contribute to diel and seasonal variation of respiration.

Negative-ion mass spectrometry of substituted adenine bases and adenosine nucleosides.

The negative-ion chemical ionization (ammonia, 5 Pa source pressure) mass spectra of a series of substituted adenine bases, adenosine nucleosides, and the trimethylsilyl derivatives of the nucleosides are described. Selected ions from these spectra were subject to collisionally activated dissociation with mass-analysed ion kinetic energy (CAD/MIKE) analysis of the products and the spectra assessed for information content. In addition to observing strong peaks due to quasimolecular ions and heterocyclic-base ions, it proved possible to differentiate between 2'-, 3'- and 5'-deoxy and between 2'- and 3'-O-methyl isomers. The negative-ion chemical ionization spectra of four methyladenines are essentially identical, but could be clearly distinguished from each other by CAD/MIKE analysis.

Cellulose synthesis: mutational analysis and genomic perspectives using Arabidopsis thaliana.

Cellulose microfibrils containing crystalline beta-1,4-glucan provide the major structural framework in higher-plant cell walls. Genetic analyses of Arabidopsis thaliana now link specific genes to plant cellulose production just as was achieved some years earlier with bacteria. Cellulose-deficient mutants have defects in several members of one family within a complex glycosyltransferase superfamily and in one member of a small family of membrane-bound endo-1,4-beta-glucanases. The mutants also accumulate a readily extractable beta-1,4-glucan that has short chains which, in at least one case, are lipid linked. Cellulose could be made by direct extension of the glucan chain by the glycosyltransferase or, as the mutant suggests, by an indirect route which makes lipid-linked oligosaccharides. Models discussed incorporate the known enzymes and lipo-glucan and raise the possibility that different CesA glycosyltransferases may catalyse different steps.

Chemical archaeology of kava, a potent brew.

Kava lactones, which are present in the intoxicating Pacific Island drink, kava, have now been detected in a number of archaeological artefacts using selected-ion monitoring techniques in conjunction with gas chromatography/electron impact-mass spectrometry. Thus it is now possible to link unequivocally kava drinking, a major aspect of the ceremonial culture of many Pacific societies, to the archaeological record. In addition, a new variation of the kava lactone skeleton was tentatively identified in the form of 7,8-dihydro-5,6-dehydrokawain and 7,8-dihydro-5,6-dehydromethysticin.

Fractionation of carbohydrates in Arabidopsis root cell walls shows that three radial swelling loci are specifically involved in cellulose production.

Three non-allelic radial swelling mutants (rsw1, rsw2 and rsw3) of Arabidopsis thaliana L. Heynh. were shown to be specifically impaired in cellulose production. Fractionation methods that identify, characterise and quantify some of the major cell wall polysaccharides in small quantities of seedlings demonstrated that changes in the production of cellulose are much more pronounced than changes in the production of non-cellulosic polysaccharides. A crude cell wall pellet was sequentially extracted with chloroform methanol (to recover lipids), dimethyl sulphoxide (starch), ammonium oxalate (pectins) and alkali (hemicelluloses). Crystalline cellulose remained insoluble through subsequent treatments with an acetic/nitric acid mixture and with trifluoroacetic acid. Cetyltrimethylammonium bromide precipitation resolved neutral and acidic polymers in the fractions, and precipitation behaviour, monosaccharide composition and glycosidic linkage patterns identified the major polysaccharides. The deduced composition of the walls of wild-type seedlings and the structure and solubility properties of the major polymers were broadly typical of other dicots. The three temperature-sensitive, radial swelling mutants produced less cellulose in their roots than the wild type when grown at their restrictive temperature (31 degrees C). There were no significant differences at 21 degrees C where no radial swelling occurs. The limited changes seen in the monosaccharide compositions, glycosidic linkage patterns and quantities of non-cellulosic polysaccharides support the view that the RSW1, RSW2 and RSW3 genes are specifically involved in cellulose synthesis. Reduced deposition of cellulose was accompanied by increased accumulation of starch.

Hawkinsinuria--identification of quinolacetic acid and pyroglutamic acid during an acidotic phase.

A second Australian family with the genetic disease Hawkinsinuria has been identified. Affected members excrete hawkinsin and cis- and trans-4-hydroxycyclohexylacetic acid. An infant in this family presented with metabolic acidosis and excreted quinolacetic acid and pyroglutamic acid in the urine together with the tyrosine derived phenolic acids reported in the original index case. It is thought that quinolacetic acid is accumulated as a by-product of the partially defective enzyme, 4-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27) and that pyroglutamic acid indicated lowered glutathione levels.

Do rhizobia produce cytokinins?

Two Rhizobium strains were cultured on a defined medium; one was a normal strain of the cowpea group (ANU240) while the other (IC3342) was an unusual but related strain of the same group which induced abnormal shoot development, including proliferation of lateral buds, in nodulated plants. Culture supernatants were examined for the presence of cytokinins by mass spectrometry using deuterium-labelled internal standards and by radioimmunoassay. In culture supernatants of both strains a range of cytokinins was detected and quantified, but N6-(2-isopentenyl)adenine (iP) and zeatin (Z) were the dominant cytokinins. The levels of Z and iP in supernatants of strain IC3342 were 26 and 8 times, respectively, those in supernatants of the strain ANU240. These results appear to provide the first unambiguous identifications of cytokinins in Rhizobium culture media. The cytokinin level in xylem sap of pigeonpea plants inoculated with strain IC3342 was markedly greater than that in plants inoculated with a normal nodulating strain. The abnormal proliferation of lateral buds in the former plants is probably linked to the elevation of cytokinin level in xylem sap caused by strain IC3342.

Effects of elevated [CO(2)] and nitrogen nutrition on cytokinins in the xylem sap and leaves of cotton.

We measured the level of xylem-derived cytokinins (CKs) entering a cotton leaf, and the CK levels in the same leaf, thus enabling xylem sap and foliar CKs to be compared concurrently. Although zeatin was the dominant CK in xylem sap, zeatin, dihydrozeatin, and N(6)-(2-isopentenyl) adenine were present in approximately equimolar levels in leaves. Elevated [CO(2)] (EC) has an effect on the levels of cytokinins in sap and leaf tissues. This effect was modulated by the two levels of root nitrogen nutrition (2 and 12 mM nitrate). Growth enhancement (70%) in EC over plants in ambient [CO(2)] (AC) was observed for both nitrogen nutrition treatments. Low-nitrogen leaves growing in EC exhibited photosynthetic acclimation, whereas there was no sign of photosynthetic acclimation in high-nitrogen grown leaves. Under these prevailing conditions, xylem sap and leaf tissues were obtained for CK analysis. Higher nitrogen nutrition increased the delivery per unit leaf area of CKs to the leaf at AC. EC caused a greater increase in CK delivery to the leaf at low nitrogen conditions (106%) than at high nitrogen conditions (17%). EC induced a significant increase in CK content in low-nitrogen leaves, whereas CK content in leaf tissues was similar for high-nitrogen leaves growing in AC and EC.

Mass spectrometry and chromatography of t-butyldimethylsilyl derivatives of cytokinin bases.

Di-(t-butyldimethylsilyl) derivatives of the cytokinin bases zeatin, cis-zeatin, and dihydrozeatin may be prepared quantitatively in the presence of dimethylaminopyridine. These derivatives have good gas chromatographic properties and are very suitable for gas chromatography-mass spectrometry analysis of cytokinin bases. The t-butyldimethylsilyl (tBuDMS) group at N-9 may be selectively hydrolyzed and the resulting mono-O-silyl derivatives are sufficiently stable to be subjected to thin-layer chromatography and high-performance liquid chromatography. The mass spectral fragmentation of the mono- and di-tBuDMS derivatives of adenine, zeatin, cis-zeatin, and dihydrozeatin and also of the mono-tBuDMS derivatives of N6-isopentenyladenine and 6-benzylaminopurine have been rationalized. The 9-tBuDMS moiety was characterized by an elimination of isobutene (M-56) and of isobutene plus a methyl radical (M-56-15).

Posttranscriptional modification of tRNA in thermophilic archaea (Archaebacteria).

Nucleoside modification has been studied in unfractionated tRNA from 11 thermophilic archaea (archaebacteria), including phylogenetically diverse representatives of thermophilic methanogens and sulfur-metabolizing hyperthermophiles which grow optimally in the temperature range of 56 (Thermoplasma acidophilum) to 105 degrees C (Pyrodictium occultum), and for comparison from the most thermophilic bacterium (eubacterium) known, Thermotoga maritima (80 degrees C). Nine nucleosides are found to be unique to the archaea, six of which are structurally novel in being modified both in the base and by methylation in ribose and occur primarily in tRNA from the extreme thermophiles in the Crenarchaeota of the archaeal phylogenetic tree. 2-Thiothymine occurs in tRNA from Thermococcus sp., and constitutes the only known occurrence of the thymine moiety in archaeal RNA, in contrast to its near-ubiquitous presence in tRNA from bacteria and eukarya. A total of 33 modified nucleosides are rigorously characterized in archaeal tRNA in the present study, demonstrating that the structural range of posttranscriptional modifications in archaeal tRNA is more extensive than previously known. From a phylogenetic standpoint, certain tRNA modifications occur in the archaea which are otherwise unique to either the bacterial or eukaryal domain, although the overall patterns of modification are more typical of eukaryotes than bacteria.

Temperature-sensitive alleles of RSW2 link the KORRIGAN endo-1,4-beta-glucanase to cellulose synthesis and cytokinesis in Arabidopsis.

An 8.5-kb cosmid containing the KORRIGAN gene complements the cellulose-deficient rsw2-1 mutant of Arabidopsis. Three temperature-sensitive alleles of rsw2 show single amino acid mutations in the putative endo-1,4-beta-glucanase encoded by KOR. The F1 from crosses between kor-1 and rsw2 alleles shows a weak, temperature-sensitive root phenotype. The shoots of rsw2-1 seedlings produce less cellulose and accumulate a short chain, readily extractable glucan resembling that reported for rsw1 (which is defective in a putative glycosyltransferase required for cellulose synthesis). The double mutant (rsw2-1 rsw1) shows further reductions in cellulose production relative to both single mutants, constitutively slow root growth, and enhanced temperature-sensitive responses that are typically more severe than in either single mutant. Abnormal cytokinesis and severely reduced birefringent retardation in elongating root cell walls of rsw2 link the enzyme to cellulose production for primary cell walls and probably cell plates. The Rsw2(-) phenotype generally resembles the Kor(-) and cellulose-deficient Rsw1(-) phenotypes, but anther dehiscence is impaired in Rsw2-1(-). The findings link a second putative enzyme activity to cellulose synthesis in primary cell walls of Arabidopsis and further increases the parallels to cellulose synthesis in Agrobacterium tumefaciens where the celA and celC genes are required and encode a putative glycosyltransferase and an endo-1,4-beta-glucanase related to RSW1 and KOR, respectively.