Immunocytochemical localization of cytokinins in Craigella tomato and a sideshootless mutant

Post-embedding immunocytochemical techniques using peroxidase-antiperoxidase or immunoglobulin G-gold as markers were used for the localization of cytokinins (CKs) in two isogenic lines, Craigella (C) and Craigella lateral suppressor (Cls), of tomato Lycopersicon esculentum Mill. Terminal buds, nodes, hypocotyl segments and root tips were submitted to a periodate-borohydride procedure, to obtain the coupling of isopentenyladeosine and zeatin riboside to cellular proteins, followed by a fixative step with a paraformaldehyde and glutaraldehyde mixture. Enzyme-linked immunosorbent assay tests performed on ovalbumin-coated microtitration plates have shown that this method was effective for CK riboside and base coupling to proteins. Paraffin-wax- or Spurr's-resin-embedded sections were cleared of wax or resin before incubation with anti-zeatin riboside or anti-isopentenyladenosine antibodies. The procedure was thoroughly investigated and many controls were done in order to eliminate artefacts. The immunostaining patterns observed along the plants showed a basipetally decreasing gradient of CKs along the stem and in the roots. Immunolabelling was higher in the actively growing regions of the stem bud and root apices. Terminal buds of Cls appeared to be less immunoreactive than C, whereas no differences were detected in root-tip immunolabelling. The staining patterns are consistent with the idea that root and bud apices have a different CK metabolism. The absence of axillary bud formation in Cls is correlated with low CK levels in the organogensis sites.Abbreviations C Craigella, isogenic line - CK cytokinin - Cls Craigella lateral suppressor - EDC 1-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride - ELISA enzyme-linked immunosorbent assay - 2iP isopentenyladenine - 2iPA isopentenyladenosine - PAP peroxidase-anti-peroxidase - PFAG paraformaldehyde/glutaraldehyde mixture - Z zeatin - ZR zeatin riboside     

Analysis of phytochrome- and ABA-deficient mutants suggests that ABA degradation is controlled by light in Nicotiana plumbaginifolia

The phytochrome chromophore-deficient mutant, pew1, of Nicotiana plumbaginifolia exhibited decreased germination and slower dehydration of detached leaves during water stress as compared with the wild-type. These physiological processes are controlled by abscisic acid (ABA) and we examined, therefore, whether phytochrome plays a specific role in the regulation of ABA metabolism using the pew1 mutant. The ABA contents of mature seeds and young leaves were analysed and in both cases mutant material was found to contain higher amounts of ABA as compared with the wild-type. This indicates that the phytochrome activation can lead to a decrease of the ABA level in the wild-type plant. The role of phytochromes was investigated in greater detail using the ABA-deficient mutant aba1 of N. plumbaginifolia exhibiting an early and synchronous germination. This mutant accumulates at very high levels a metabolite derived from a precursor (ABA-aldehyde) in the ABA biosynthetic pathway. The first biochemical characterization of this molecule, which corresponds to the glucose-conjugated ABA-alcohol (ABA-AG) is described. A pew1-aba1 double mutant exhibiting both an etiolated growth and early germination was also obtained. The comparable accumulation of ABA-AG in the pew1-aba1 double mutant as compared with the aba1 mutant allowed the proposition that, in a wild-type plant, the phytochrome-mediated light signal enhances ABA degradation rather than inhibits its biosynthesis.     

The differential effect of N6-benzyl-adenine and N6-(Δ2-isopentenyl)-adenine on in vitro propagation of Paeonia suffruticosa Andr. is correlated with different hormone contents

Summary N⁶-benzyl-adenine (BA) enhanced phyllogenesis and axillary bud development of Paeonia suffruticosa during in vitro culture allowing good propagation while N⁶-(²isopentenyl)adenine (iP) did not. During the first five days of culture, the mitotic activity of BA-treated explants was higher than in the iP-treated ones. High BA levels were detected in the BA-treated explants, and this was correlated with the absence of or the low indole-3-acetic acid (IAA) content. The low iP levels measured in iP-treated explants were correlated with high endogenous IAA content; the new cytokinin / auxin ratio could explain the lack of axillary buds and the development of only one leaf. Abscisic acid (ABA) was detected neither in the controls nor in the cytokinin-treated explants during the first week. However, intensive restoration of ABA accumulation was observed in controls from the third week onwards. Both BA and iP-treated explants accumulated less ABA than the controls but this hormone appeared later in the BA-treated explants than in the iP-treated ones.Abbreviations ABA abscisic acid - BA N⁶-benzyl-adenine - BHT butyl-hydroxy-toluene - ELISA enzyme linked immunosorbent assay - FM fresh mass - HPLC high performance liquid chromatography - IAA indole-3-acetic acid - iP N⁶-(²-isopentenyl)adenine - MI mitotic index - 9RBA 9-ß-D-ribofuranosyl-BA - 9RiP 9-ß-Dribofuranosyl-iP - 9RZ 9-ß-D-ribofuranosyl-zeatin - Z zeatin     

Abscisic acid, indole-3-acetic acid and cytokinin changes in buds of Pseudotsuga menziesii during bud quiescence release

Bud quiescence release, considered as the ultimate dormancy breaking phase, was achieved in Pseudotsuga menziesii (Mirb.) Franco by a 9-week cold (5°C) treatment, under short daylength (9 h) followed by a transfer to mild temperature (22°C) under long daylength (16 h). Indole-3-acetic acid (IAA), abscisic acid (ABA), zeatin-type (Z) and isopentenyladenine-type (iPA) cytokinin (CK) levels were measured by means of an ELISA technique performed on HPLC-fractionated extracts of terminal and axillary buds. During the cold period, all hormones except IP-type CK levels decreased, whereas the opposite observation was made after transfer to mild temperature and long daylength, when buds started to grow. Some other immunoreactive compounds were also detected and quantified. The ABA-glucosyl ester (ABA-GE) level pattern was similar to that of ABA, but no accumulation occurred at mild temperatures. A putative IAA conjugate, more polar than IAA, was also detected. Its level increased transiently like IAA in terminal buds and, to a lesser extent, in axillary buds during the 10th week of the experiment. In terminal buds, isopentenyladenosine ([9R]-iP) was released by alkaline hydrolysis of a polar immunoreactive compound detected with anti-[9R]iP antibodies. This compound accumulated during the cold period and quickly dropped at 22°C. Relationships between environmental conditions and endogenous hormones are discussed.     

Control of seed dormancy in Nicotiana plumbaginifolia: post-imbibition abscisic acid synthesis imposes dormancy maintenance

The physiological characteristics of seed dormancy in Nicotiana plumbaginifolia Viv. are described. The level of seed dormancy is defined by the delay in seed germination (i.e the time required prior to germination) under favourable environmental conditions. A wild-type line shows a clear primary dormancy, which is suppressed by afterripening, whereas an abscisic acid (ABA)-deficient mutant shows a non-dormant phenotype. We have investigated the role of ABA and gibberellic acid (GA₃) in the control of dormancy maintenance or breakage during imbibition in suitable conditions. It was found that fluridone, a carotenoid biosynthesis inhibitor, is almost as efficient as GA₃ in breaking dormancy. Dry dormant seeds contained more ABA than dry afterripened seeds and, during early imbibition, there was an accumulation of ABA in dormant seeds, but not in afterripened seeds. In addition, fluridone and exogenous GA₃ inhibited the accumulation of ABA in imbibed dormant seeds. This reveals an important role for ABA synthesis in dormancy maintenance in imbibed seeds. Received: 31 December 1998 / Accepted: 9 July 1999     

Evidence for ACD5 ceramide kinase activity involvement in Arabidopsis response to cold stress

Although sphingolipids emerged as important signals for plant response to low temperature, investigations have been limited so far to the function of long‐chain base intermediates. The formation and function of ceramide phosphates (Cer‐Ps) in chilled Arabidopsis were explored. Cer‐Ps were analysed by thin layer chromatography (TLC) following in vivo metabolic radiolabelling. Ceramide kinase activity, gene expression and growth phenotype were determined in unstressed and cold‐stressed wild type (WT) and Arabidopsis ceramide kinase mutant acd5. A rapid and transient formation of Cer‐P occurs in cold‐stressed WT Arabidopsis plantlets and cultured cells, which is strongly impaired in acd5 mutant. Although concomitant, Cer‐P formation is independent of long‐chain base phosphate (LCB‐P) formation. No variation of ceramide kinase activity was measured in vitro in WT plantlets upon cold stress but the activity in acd5 mutant was further reduced by cold stress. At the seedling stage, acd5 response to cold was similar to that of WT. Nevertheless, acd5 seed germination was hypersensitive to cold and abscisic acid (ABA), and ABA‐dependent gene expression was modified in acd5 seeds when germinated at low temperature. Our data involve for the first time Cer‐P and ACD5 in low temperature response and further underline the complexity of sphingolipid signalling operating during cold stress.     

Auxin metabolism and rooting in young and mature clones of Sequoia sempervirens

The capacity of young and mature Sequoia sempervirens clones to produce roots in vitro was studied after wounding and indole-3-butyric acid (IBA) treatments. Rooting was not observed in mature or in young cuttings cultivated for 30 days in medium without IBA. The presence of 25 μ M IBA in the medium resulted in the appearance of roots at the base of the cuttings. More roots appeared and grew faster on cuttings of the young than on the mature clone. This difference in rooting capacity between young and mature cuttings may be related to differences in the hormone levels at the base of the 5 mm long cuttings during the first 4 days of the root inductive period. After HPLC fractionation. IAA. IBA and related compounds, including indole-3-aspartic acid (IAAsp) and IBA-glucose ester (IBA-GE), were determined by MS and MS-MS and their levels measured by ELISA. Another immunoreactive compound was also found and determined to be N,N-dimethyltryptophan (DMT), a compound previously reported to inhibit auxin-enhanced ethylene production. Wounding of the stem without IBA treatment revealed a transient increase in IAA, IAAsp and DMT levels in young cuttings while a dramatic increase in the levels of DMT was observed in mature cuttings. Following IBA treatment. IAA levels increased in both clones, but higher levels were measured in the young than in the mature clone. IBA and IBA-GE were also found but in higher levels in the mature clone. Thus, the difficult-to-root mature clone differs from the young clone in its auxin metabolism.     

Engineering seed dormancy by the modification of zeaxanthin epoxidase gene expression

Abscisic acid (ABA) is a plant hormone synthesized during seed development that is involved in the induction of seed dormancy. Delayed germination due to seed dormancy allows long-term seed survival in soil but is generally undesirable in crop species. Freshly harvested seeds of wild-type Nicotiana plumbaginifolia plants exhibit a clear primary dormancy that results in delayed germination, the degree of primary dormancy being influenced by environmental culture conditions of the mother plant. In contrast, seeds, obtained either from ABA-deficient mutant aba2-s1 plants directly or aba2-s1 plants grafted onto wild-type plant stocks, exhibited rapid germination under all conditions irrespective of the mother plant culture conditions. The ABA biosynthesis gene ABA2 of N. plumbaginifolia, encoding zeaxanthin epoxidase, was placed under the control of the constitutive 35S promoter. Transgenic plants overexpressing ABA2 mRNA exhibited delayed germination and increased ABA levels in mature seeds. Expression of an antisense ABA2 mRNA, however, resulted in rapid seed germination and in a reduction of ABA abundance in transgenic seeds. It appears possible, therefore, that seed dormancy can be controlled in this Nicotiana model species by the manipulation of ABA levels.