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Preparation of monoclonal antibodies specific for methyl esters of gibberellins A7 and A4.

According to the heterogeneity of the immunogen of GA3-3-O-HSA, which contained other components such as GA3-7-CONH-HSA, two kinds of monoclonal antibodies (MAbs) against different antigen determinants of GA3 were prepared in a single process using a two-step screening assay for hybridomas. The results from a cross-reactivity experiment showed that MAb BG2 was specific for GA7/4 methyl esters (GA7/4me) with high affinity. Its affinity for GA7me was over 100 and 200 times higher than for GA3me and GA1me, respectively. Methylation of the 7-oic acid significantly increased the binding of MAb BG2 with GAs. On the contrary, the absence of a double bond or 3beta-OH in ring A and the breakdown of 19,10-gamma-lactone as well as the presence of 13-OH in ring D greatly reduced the binding of MAb BG2 with GAs. This antibody with high specificity can be used effectively to quantify and localize the main active GA7 and GA4 from the early non-hydroxylation pathway of GAs metabolism. Using this antibody, enzyme immunoassays with high sensitivity were developed which displayed linear ranges from 1.0 x 10(-14) to 1.0 x 10(-12) mol for GA4me and from 2.0 x 10(-15) to 2.0 x 10(-13) mol for GA7me.

Antibodies, Monoclonal↗

Gibberellin-regulated expression in oat aleurone cells of two kinases that show homology to MAP kinase and a ribosomal protein kinase.

cDNA fragments from ten different protein kinases expressed in Avena sativa aleurone cells were amplified from mRNA by RT-PCR with degenerate primers. These could be classified into five groups: Aspk1-3 showed homology to the Snf1-related protein kinases, Aspk4-5 to a wheat ABA up-regulated protein kinase, Aspk6-8 to the Ca-dependent, calmodulin-independent protein kinase family, Aspk9 encoded a MAP kinase and Aspk10 was closely related to a novel Arabidopsis ribosomal protein kinase. GA caused a rapid increase in transcripts hybridising to Aspk10, while inhibiting the dramatic accumulation of transcripts hybridising to Aspk9 that occurred in the absence of GA.

Amino Acid Sequence↗

Gibberellins

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Journal Article↗

Effects of gibberellin A(4 + 7) and bark-ringing on cone initiation in mature Picea sitchensis grafts.

Large numbers of cones (strobili) were induced in a 10-year-old plot of mature grafts of Picea sitchensis (Bong.) Carr. All trees injected with 20 mg GA(4 + 7) in June initiated female and male cones in the same year. This treatment increased the number of female cones per plant 12-fold above the controls, more than doubling the percentage of cones that were female. Complete bark-ringing (done in May of the previous year) showed an additive effect with GA on the number of female cones formed, but a negative interaction on the number of male cones induced. Ringing promoted male cone production most when used alone. Treatments, singly or combined, also increased the proportion of cones that were lateral, compared with the preponderance of terminal male cones in the controls. The effects were apparently not directly associated with alterations in vegetative vigor, although these occurred causing a reduction in the proportion of buds containing vegetative shoots the following year. The clones differed in most characteristics, but both sparse and prolific clones were induced to reproductive activity. Viable seed yields per tree, and notional production from seed-orchards were enhanced almost 10-fold by GA injection, and about 4-fold by bark-ringing and GA + bark-ringing.

Journal Article↗

Aleurones from a Barley with Low [alpha]-Amylase Activity Become Highly Responsive to Gibberellin When Detached from the Starchy Endosperm.

The physiological and molecular bases for contrasting [alpha]-amylase phenotypes were examined in germinating seeds of two barley (Hordeum vulgare L.) cultivars, Morex and Steptoe. Morex is a high-quality malting barley that develops high [alpha]-amylase activity soon after germination. Steptoe is a feed barley that develops only low [alpha]-amylase activity levels during this period. The expression of all high- and low-isoelectric point (pl) [alpha]-amylase isozymes is reduced in Steptoe. The amount of [alpha]-amylase mRNA per gram of seedling tissue is correspondingly lower in Steptoe. Southern blot analysis revealed that the cultivars have the same copy number and organization for most high- and low-pl genes. Steptoe seedlings or embryoless half-seeds produce little [alpha]-amylase in response to exogenous applications of gibberellic acid (GA3) compared with Morex. However, when isolated aleurones of both cultivars are treated with GA3, they produce similar amounts of high- and low-pl [alpha]-amylase RNAs. This suggests that a factor in the starchy endosperm is responsible for lowered [alpha]-amylase response in Steptoe. The factor is probably not abscisic acid (ABA), since the two cultivars have similar concentrations of ABA during germination.

Journal Article↗

Perception of Gibberellin and Abscisic Acid at the External Face of the Plasma Membrane of Barley (Hordeum vulgare L.) Aleurone Protoplasts.

The response of protoplasts isolated from aleurone layers of barley (Hordeum vulgare L. cv Himalaya) to internally and externally applied hormone was analyzed to localize the site of perception of the hormonal signal. Protoplasts responded to externally applied gibberellic acid (GA3) with increased synthesis and secretion of [alpha]-amylase, transient expression of the glucuronidase reporter gene fused to the hormone-responsive elements of the [alpha]-amylase promoter, and the vacuolation typical of GA3-treated aleurone cells. When up to 250 [mu]M GA3 was microinjected into the protoplast cytoplasm, none of these responses were observed. This did not reflect damage to the protoplasts during the microinjection procedure, since microinjected protoplasts remained responsive to externally applied hormone. Nor did it reflect loss of microinjected GA3 from the protoplast, since 50% of microinjected [3H]GA20 was retained by protoplasts for at least 24 h. Externally applied abscisic acid (ABA) could reverse the stimulation of [alpha]-amylase synthesis and secretion, whereas microinjecting up to 250 [mu]M ABA was ineffective at antagonizing the stimulatory effect of GA3. These results suggest that the site of perception of GA3 and ABA in the barley aleurone protoplast is on the external face of the plasma membrane.

Journal Article↗

Identification of Endogenous Gibberellins in Petunia Flowers (Induction of Anthocyanin Biosynthetic Gene Expression and the Antagonistic Effect of Abscisic Acid).

The elongation and pigmentation of corollas of Petunia hybrida requires the presence of anthers. The ability of exogenous gibberellic acid (GA3) to substitute for the anthers suggests a role for endogenous GAs. Here we report the identification of endogenous GAs in corollas and in anthers and show that both tissues contain detectable levels of GA1, GA4, and GA9, of which GA4 is the most abundant. These GAs stimulate corolla pigmentation, chalcone synthase (chs) mRNA accumulation, and chs transcription in an in vitro flower bud culture system. Methyl ester derivatives of GA3 and GA4 were not active but did not inhibit the bioactive GAs. Even though it is unknown whether abscisic acid (ABA) is involved in corolla maturation, ABA inhibited pigmentation of intact flowers, overruling the effect of the anthers. In detached flower buds it was shown that ABA prevented activation of the chs promoter by GA3. The synthesis of anthocyanin pigments requires the coordinate expression of at least 15 structural genes. Expression of early biosynthetic genes and of late biosynthetic genes are regulated by different transcriptional activators. GA induces both classes of genes with similar kinetics, indicating that GA acts relatively early in the signaling pathway.

Journal Article↗

Effect of Gibberellin and Heat Shock on the Lipid Composition of Endoplasmic Reticulum in Barley Aleurone Layers.

The heat-shock responses of barley (Hordeum vulgare L. cv Hi- malaya) aleurone layers incubated with or without gibberellic acid (GA3) were compared. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that heat shock blocked the synthesis and secretion of secretory proteins from GA3-treated layers but not untreated layers. This suppression of secretory protein synthesis has been correlated with changes in endoplasmic reticulum (ER) membranes (F.C. Belanger, M. R. Brodl, T.-h.D. Ho [1986] Proc Natl Acad Sci USA 83: 1354-1358; L. Sticher, A.K. Biswas, D.S. Bush, R.L. Jones [1990] Plant Physiol 92: 506-513). Our secretion data suggested that the ER membranes of aleurone layers incubated without GA3 may be more heat shock tolerant. To investigate this, the lipid profiles of membrane extracts in aleurone layers labeled with [14C]glycerol were examined. Heat shock markedly increased [14C]glycerol incorporation into phosphatidylcholine (PC), and gas chromatography revealed an increase in the amount of saturated fatty acids associated with thin layer chromatography-purified PC in GA3-treated layers. In contrast, aleurone layers incubated without GA3 at normal temperature contained PC-associated fatty acids with a greater degree of saturation than GA3-treated layers. Heat shock modestly increased the degree of fatty acid saturation in untreated aleurone layers. This same trend was noted in fatty acids isolated from ER membranes purified by continuous sucrose density centrifugation. We propose that increased fatty acid saturation may help sustain ER membrane function in heat-shocked aleurone layers incubated in the absence of GA3.

Journal Article↗