Are xylem radial development and hydraulic conductivity in downwardly-growing grapevine shoots influenced by perturbed auxin metabolism?

Downwardly-growing grapevine shoots have smaller and more frequent vessels than upwardly-growing ones and, as a consequence, a lower hydraulic conductivity. Here, grapevine (Vitis vinifera L.) shoot growth orientation was manipulated to test whether downward shoot orientation negatively affects vessel growth in the apex via a shortage of water and nutrients. The orientation of the central vine shoot portion was inverted by two consecutive 135 degrees bends, resulting in double-bent N-shaped vines; the central downward shoot portion was of different lengths in the experimental treatments to induce increasing reductions of shoot conductivity. These treatments reduced shoot conductivity and water flow, but had no effects on vessel development and frequency in the apex. In a second experiment, auxin concentration was assessed in shoots of upwardly- and downwardly-growing plants. IAA concentration at the apical internodes was higher in downwardly-oriented shoots than in shoots growing upwards. In addition, a higher density and a lower vessel diameter were observed in the lower, than the upper side, of the downwardly-oriented shoot, suggesting increased accumulation of auxin in the lower side. These results suggest that the downward orientation induces accumulation of auxin in the apex, which in turn affects the density and the size of the xylem vessels, causing reduction of hydraulic conductivity.     

Adventitious rooting: examining the role of auxin in an easy- and a difficult-to-root plant

Therooting responses of cuttings of difficult-to-root lilac (Syringavulgaris) and easy-to-root forsythia(Forsythia×intermedia)were compared. The rooting ability of lilac cuttings declined over the growingseason (May–June). There was also a decline in the initial concentrationof free IAA at the base of the cuttings, but there was not a tight relationshipbetween basal IAA concentration and rooting ability. Polar auxin transportability was measured in lilac and forsythia during the period of maximum growthby [³H]IAA application to stem internodal tissue. Transport abilitydeclined in lilac over this time period, particularly in terms of transportintensity and percentage of [³H]IAA transported. In contrast thechanges in polar auxin transport ability in forsythia were less marked. Thisdifference between species was maintained in winter hardwood cuttings, withforsythia tissue showing greater polar auxin transport ability than lilac. Theimportance of polar auxin transport for adventitious rooting was demonstratedinboth lilac and forsythia softwood cuttings by use of the polar transportinhibitor 2,3,5-triiodobenzoic acid (TIBA). Overall the results indicate thatdifferences in polar auxin transport ability between lilac and forsythiacontribute to differences in rooting ability.     

Influence of amino acid numbers between two ligand cysteines of zinc finger proteins on affinity and specificity of DNA binding

Hypaphorine, an indolic alkaloid from an ectomycorrhizal fungus is a putative antagonist of indole-3-acetic acid (IAA) known to inhibit the effect of IAA in growing roots of Eucalyptus seedling. Previously we have used horseradish peroxidase-C (HRP) as a sensitive reporter of IAA-binding to the IAA-binding domain, and reported that hypaphorine specifically inhibits the HRP-catalyzed superoxide generation coupled to oxidation of IAA [Kawano et al., Biochem. Biophys. Res. Commun. 288]. Since binding of IAA to the auxin-binding domain is the key step required for IAA oxidation by HRP, it was assumed that the inhibitory effect of hypaphorine is due to its competitive binding to the auxin-binding domain in HRP. Here, we obtained further evidence in support of our assumption that hypaphorine specifically inhibits binding of IAA to HRP. In this study, HRP arrested at the temporal inactive form known as Compound III was used as a sensitive indicator for binding of IAA to HRP. Addition of IAA to the preformed Compound III resulted in rapid decreases in absorption maxima at 415, 545, and 578 nm characteristic to Compound III, and in turn a rapid increase in absorption maximum at 670 nm representing the formation of P-670, the irreversibly inactivated form of hemoproteins, was induced. In contrast, the IAA-dependent irreversible inactivation of HRP was inhibited in the presence of hypaphorine. In addition, the mode of interaction between IAA and hypaphorine was determined to be competitive inhibition, further confirming that hypaphorine is an IAA antagonist which specifically compete with IAA in binding to the IAA-binding site in plant peroxidases.     

Studies on cell wall loosening enzymes in hormone treated internodes of <Emphasis Type="Italic">Merremia emarginata</Emphasis>

The cell wall loosening enzymes viz. glycosidases, polygalacturonase and xylanase were analyzed in cytoplasmic and wall bound fractions extracted from control and hormone (GA₃ NAA, PAA) treated internodes, as they are known to play a key role in cell wall metabolism. Among the glycosidases, wall bound β-glucosidase and α-galactosidase activities were significantly correlated with age of control internodes. Cytoplasmic α-galactosidase showed significant correlation in hormone treated internodes. Maximum correlation was observed in GA₃, followed by PAA and NAA. Wall bound xylanase activity was well correlated with length only in NAA treated internodes and less after GA₃ treatment while cytoplasmic xylanase showed correlation with intrnode length only in control and after NAA treatment. Cytoplasmic polygalacturonase showed correlation with internode length only after GA₃ treatment while wall bound polygalacturonase showed correlation with internode length after NAA treatment. The possible role of these enzymes in internode development is discussed.     

Aromatic amino acid aminotransferase activity and indole-3-acetic acid production by associative nitrogen-fixing bacteria

In this work, we report the detection of aromatic amino acid aminotransferase (AAT) activity from cell-free crude extracts of nine strains of N(2)-fixing bacteria from three genera. Using tyrosine as substrate, AAT activity ranged in specific activity from 0.084 to 0.404 micromol min(-1)mg(-1). When analyzed under non-denaturating PAGE conditions; and using tryptophan, phenylalanine, tyrosine, and histidine as substrates Pseudomonas stutzeri A15 showed three isoforms with molecular mass of 46, 68 and 86 kDa, respectively; Azospirillum strains displayed two isoforms which molecular mass ranged from 44 to 66 kDa and Gluconacetobacter strains revealed one enzyme, which molecular mass was estimated to be much more higher than those of Azospirillum and P. stutzeri strains. After SDS-PAGE, some AAT activity was lost, indicating a differential stability of proteins. All the strains tested produced IAA, especially with tryptophan as precursor. Azospirillum strains produced the highest concentrations of IAA (16.5-38 microg IAA/mg protein), whereas Gluconacetobacter and P. stutzeri strains produced lower concentrations of IAA ranging from 1 to 2.9 microg/mg protein in culture medium supplemented with tryptophan. The IAA production may enable bacteria promote a growth-promoting effect in plants, in addition to their nitrogen fixing ability.     

The effect of light on IAA metabolism in different parts of maize seedlings in correlation with their growth

The inhibitory effect of light on the growth of plantscorrelates with a decrease of free IAA content in their tissues andmight be mediated through changes of IAA metabolism. In different partsof Zea mays L. seedlings (roots, mesocotyls and coleoptiles)that respond to light with a different growth rate, the effect of lighton the formation of IAA metabolites was examined in feeding experimentswith ¹⁴C-IAA. In all tissues, IAA was taken up andmetabolised mainly into six compounds, four of them were tentativelyidentified as IAA-1-O-glucose (IAGlc), IAA-myo-inositol, indoleacetamide and IAA-aspartate (IAAsp). IAA was metabolised most slowly inthe roots. In coleoptiles and mesocotyls, IAGlc was the most abundantmetabolite, except for mesocotyls in the light. In roots, a relativelylarge amount of IAA was also metabolised into IAAsp. Light stimulatedthe rate of IAA metabolism in all tissues, but its effect on theconversion of IAA was exceptionally high in mesocotyls. In mesocotyltissue the conversion into IAAsp was greatly stimulated by light.Conversely, the content of IAGlc in mesocotyls was decreased by light.Since light inhibited mesocotyl growth significantly and specifically,it is possible that the high capacity of mesocotyls to synthesise IAAspin the light may have caused a depletion of free IAA, which then led toan inhibition of growth. In mesocotyls from the light-grown plants IAAconjugated into IAGlc was probably used for IAAspbiosynthesis.     

Transgenic tobacco plants co-expressing Agrobacterium iaa and ipt genes have wild-type hormone levels but display both auxin- and cytokinin-overproducing phenotypes

Transgenic tobacco lines simultaneously expressing the Agrobacterium iaaM, iaaH and ipt genes, obtained by crossing lines expressing ipt with lines expressing iaaM and iaaH, were used to study in planta interactions between auxin and cytokinins. All phenotypic traits of the respective parental lines characteristic of cytokinin and auxin overproduction were present in the cross. Indole-3-acetic acid (IAA) and combined zeatin riboside (ZR) and zeatin riboside-5'-monophosphate (ZRMP) contents were analysed by mass spectrometry in young, developing leaves from the cross, the parental lines and the wild type. Unexpectedly, hormone levels in the cross were very similar to wild-type levels. Thus IAA levels in the cross were much lower throughout vegetative development than in the parental IAA overproducing line, although expression of the bacterial IAA biosynthesis genes was not reduced. The results suggest that effects on apical dominance, adventitious root formation, leaf morphology and other traits commonly +/- associated with IAA and cytokinin overproduction, and observed in the iaa E ipt cross, cannot be explained solely by analysis of auxin and cytokinin contents in individual organs. As traits associated with both hormones are expressed in close spatial and temporal proximity, it is likely that cellular resolution of hormone contents is essential to explain physiological responses to auxins and cytokinins.