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Guaranteeing successful flowering is very important in economic plant species, especially apple (Malus domestica Borkh.), which is difficult to induce to flower. However, the gene expression and networks involved in flowering have not been totally characterized. Here, we employed mRNA and microRNA (miRNA) sequencing to understand the different responses to gibberellin- and its inhibitor paclobutrazol- (PAC) mediated flower induction. Significant opposite cytological and morphological changes were observed in treated terminal buds, which led to a reduced flowering rate under gibberellin and an increased flowering rate under PAC. We also found that the differentially expressed mRNAs, miRNAs and miRNA target genes participated in different biological networks including hormones, photosynthesis, redox state and other metabolic processes, which provided important clues to understand the complex networks involved in apple flower induction. Additionally, we subsequently focused on one important candidate, MdSPL3, which is one of 31 apple SPL gene family members and whose transcription was inhibited by gibberellin but promoted by PAC. Functional investigation showed that MdSPL3 was located in the nucleus, and ectopic MdSPL3 activated floral meristem identity genes, promoted the formation of floral primordia and led to an earlier flowering phenotype in Arabidopsis. Our research identified critical mRNA and miRNA responsive to gibberellin or PAC, and provided a candidate framework for flower induction. This carefully orchestrated regulatory cross-talk highlighted potential targets for developing regulatory techniques and genetic improvement of flower induction in apple.

Plant hormones are a group of structurally diverse small compounds that orchestrate the cellular processes governing proper plant growth and environmental adaptation. To understand the details of hormonal activity, we must study not only their inherent activities but also the cross-talk among plant hormones. In addition to their use in agriculture, plant chemical activators, such as probenazole and uniconazole, have made great contributions to understand hormonal cross-talk. However, the use of plant chemical activators is limited due to the lack of activators for certain hormones. For example, to the best of our knowledge, there are only a few chemical activators previously known to stimulate the accumulation of ABA in plants, such as absinazoles and proanthocyanidins. In many cases, antagonistic effects have been examined in experiments using exogenously applied ABA, although these studies did not account for biologically relevant concentrations. In this report, it was found that a natural product, theobroxide, had potential as a plant chemical activator for stimulating the accumulation of ABA. Using theobroxide, the antagonistic effect of ABA against GAs was proved without exogenously applying ABA or using mutant plants. Our results suggest that ABA levels could be chemically controlled to elicit ABA-dependent biological phenomena., (© The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background and Aims: Angiosperm trees generally form tension wood on the upper sides of leaning stems. The formation of tension wood is an important response to gravitational stimulus. Gibberellin appears to be involved in the differentiation of secondary xylem, but it remains unclear whether gibberellin plays a key role in the formation of tension wood and plant gravitropism. Therefore, a study was designed to investigate the effects of gibberellin and of inhibitors of the synthesis of gibberellin, namely paclobutrazole and uniconazole-P, on the formation of tension wood and negative stem gravitropism in Acacia mangium seedlings., Methods: Gibberellic acid (GA(3)), paclobutrazole and uniconazole-P were applied to seedlings via the soil in which they were growing. Distilled water was applied similarly as a control. Three days after such treatment, seedlings were tilted at an angle of 45° from the vertical, and samples of stems were collected for analysis 2 weeks, 2 months and 6 months after tilting. The effects of treatments on the stem recovery degree (Rº) were analysed as an index of the negative gravitropism of seedlings, together the width of the region of tension wood in the upper part of inclined stems., Key Results: It was found that GA(3) stimulated the negative gravitropism of tilted seedling stems of A. mangium, while paclobutrazole and uniconazole-P inhibited recovery to vertical growth. Moreover, GA(3) stimulated the formation of tension wood in tilted A. mangium seedlings, while paclobutrazole and uniconazole-P strongly suppressed the formation of tension wood, as assessed 2 weeks after tilting., Conclusions: The results suggest that gibberellin plays an important role at the initial stages of formation of tension wood and in stem gravitropism in A. mangium seedlings in response to a gravitational stimulus.

In 2007-2008, the "annual bluegrass weevil," Listronotus maculicollis Kirby (Coleoptera: Curculionidae), a serious pest of Poa annua L. (Poales: Poaceae) on U.S. golf courses, was shown to be resistant to two pyrethroids, bifenthrin and lambda-cyhalothrin. In 2008, we showed that bifenthrin resistance was principally mediated by oxidase detoxification (cytochrome P450 [P450]). P450s can be inhibited by demethylation inhibitor fungicides and gibberellin inhibitor plant growth regulators, both of which are commonly used on golf courses. We tested these compounds for synergistic activity with bifenthin against a pyrethroid-resistant population of L. maculicollis. The LD50 value for bifenthrin was significantly reduced from 87 ng per insect (without synergists) to 9.6-40 ng per insect after exposure to the fungicides fenarimol, fenpropimorph, prochloraz, propiconazole, and pyrifenox and the plant growth regulators flurprimidol, paclobutrazol, and trinexapac-ethyl. Simulated field exposure with formulated products registered for use on turf revealed enhanced mortality when adult weevils were exposed to bifenthrin (25% mortality, presented alone) combined with field dosages of propiconizole, fenarimol, flurprimidol, or trinexapac-ethyl (range, 49-70% mortality).

Intact seeds from freshly harvested fruits of Myrica rubra (Sieb et Zucc.) were dormant and required 8 weeks of warm stratification followed by 12 weeks of cold stratification for germination. Exogenous application of gibberellic acid (GA(3)) to intact fresh seeds was effective in breaking dormancy, with > 70% of seeds germinating when treated with 5.2 mM GA(3) and incubated at a day/night temperature of 30/20 degrees C for 20 weeks. Removing the hard endocarp or endocarp plus seed coat of fresh seeds promoted germination, and addition of GA(3) to the embryo accelerated germination. The gibberellins GA(1) and GA(4) were more effective than GA(3) in promoting germination of seeds with the endocarp removed. Endogenous contents of GA(1), GA(3), GA(4), GA(7) and GA(20) were quantified by gas chromatography-mass spectrometry-selected ion monitoring in the endocarps, seed coats and embryos of fresh seeds treated with 5.2 mM GA(3). The content of GA(3) decreased in the endocarp during incubation, whereas GA(1) contents increased in the endocarp and seed coat. A high GA(1) content was detected in the endocarps and embryos of newly germinated seeds. We speculate that GA(3) was converted to GA(1) during incubation and that GA(1) is involved in seed germination. Endogenous abscisic acid (ABA) contents were measured in fresh seeds and in warm and cold stratified seeds. The ABA content in fresh seeds was distributed in the order endocarp > seed coat > embryo, with the content in the endocarp being about 132-fold higher than in the seed coat and embryo. Total ABA content of seeds subjected to warm or cold stratification, or both, was 8.7- to 14.0-fold lower than that of fresh seeds. Low contents of endogenous GA(1), GA(3), GA(7) and GA(20), but elevated contents of GA(4), were found in the seed coats and endocarps of warm plus cold stratified seeds and in the seed coats and embryos of newly germinated seeds. These observations, coupled with the finding that GA stimulated germination of dormant Myrica seeds, provide evidence that endogenous ABA inhibited release of dormancy and that endogenous gibberellins, especially GA(4) or GA(1), or both, are involved in germination.

Arabinogalactan proteins (AGPs) are hydroxyproline-rich glycoproteins present at the plasma membrane and in extracellular spaces. A synthetic chemical, beta-glucosyl Yariv reagent (beta-GlcY), binds specifically to AGPs. We previously reported that gibberellin signaling is specifically inhibited by beta-GlcY treatment in barley aleurone protoplasts. In the present study, we found that beta-GlcY also inhibited gibberellin-induced programmed cell death (PCD) in aleurone cells. We examined the universality and specificity of the inhibitory effect of beta-GlcY on gibberellin signaling using microarray analysis and found that beta-GlcY was largely effective in repressing gibberellin-induced gene expression. In addition, >100 genes were up-regulated by beta-GlcY in a gibberellin-independent manner, and many of these were categorized as defense-related genes. Defense signaling triggered by several defense system inducers such as jasmonic acid and a chitin elicitor could inhibit gibberellin-inducible events such as alpha-amylase secretion, PCD and expression of some gibberellin-inducible genes in aleurone cells. Furthermore, beta-GlcY repressed the gibberellin-inducible Ca2+-ATPase gene which is important for gibberellin-dependent gene expression, and induced known repressors of gibberellin signaling, two WRKY genes and a NAK kinase gene. These effects of beta-GlcY were also phenocopied by the chitin elicitor and/or jasmonic acid. These results indicate that gibberellin signaling is under the regulation of defense-related signaling in aleurone cells. It is also probable that AGPs are involved in the perception of stimuli causing defense responses.

The co-ordination of cell wall synthesis with plant cell expansion is an important topic of contemporary plant biology research. In studies of cell wall synthesis pathways, cellulose synthesis inhibitors are broadly used. It is demonstrated here that ancymidol, known as a plant growth retardant primarily affecting gibberellin biosynthesis, is also capable of inhibiting cellulose synthesis. Its ability to inhibit cellulose synthesis is not related to its anti-gibberellin action and possesses some unique features never previously observed when conventional cellulose synthesis inhibitors were used. It is suggested that ancymidol targets the cell wall synthesis pathway at a regulatory step where cell wall synthesis and cell expansion are coupled. The elucidation of the ancymidol target in plant cells could potentially contribute to our understanding of cell wall synthesis and cell expansion control.

Growing potato tubers or freshly harvested mature tubers have a dormant apical bud. Normally, this dormancy is spontaneously broken after a period of maturation of the tuber, resulting in the growth of a new sprout. Here it is shown that in in vitro-cultured growing and maturing tubers, ethanol can rapidly break this dormancy and re-induce growth of the apical bud. The in vivo promoter activity of selected genes during this secondary growth of the apical bud was monitored, using luciferase as a reporter. In response to ethanol, the expression of carbohydrate-storage, protein-storage, and cell division-related genes are rapidly down-regulated in tuber tissue. It was shown that dormancy was broken by primary but not by secondary alcohols, and the effect of ethanol on sprouting and gene expression in tuber tissue was blocked by an inhibitor of alcohol dehydrogenase. By contrast, products derived from alcohol dehydrogenase activity (acetaldehyde and acetic acid) did not induce sprouting, nor did they affect luciferase reporter gene activity in the tuber tissue. Application of an inhibitor of gibberellin biosynthesis had no effect on ethanol-induced sprouting. It is suggested that ethanol-induced sprouting may be related to an alcohol dehydrogenase-mediated increase in the catabolic redox charge [NADH/(NADH+NAD+)].

In Arabidopsis thaliana (L.) Heynh ecotype Landsberg, levels of soluble acid invertase activity are closely related to the progress of seed germination. To study the mechanism(s) of the development of these enzymes, two cDNA clones that encode putative vacuolar acid invertases were isolated from germinating seeds and very young seedlings using reverse-transcription polymerase chain reactions with degenerate primers. These fragments corresponded to the genes At beta fruct3 and At beta fruct4 from the Columbia ecotype. An apoplasmic invertase gene corresponding to At beta fruct1/ATCWINV1 was also isolated from these samples. Northern blot analyses showed that At beta fruct3 and At beta fruc4 are expressed concomitantly with germination and the subsequent seedling growth. In contrast, the At beta fruct1/AtcwINV1 mRNA is translated before germination. These expression patterns are regulated by phytochrome, which perceives red light and in turn triggers de novo synthesis of gibberellin, initiating Arabidopsis seed germination. To test the effects of gibberellin on the expression of these genes, seed were treated with a gibberellin biosynthesis inhibitor, uniconazole or prohexadione. These chemicals inhibited both seed germination and expression of the above genes, but subsequently applied GA(4), an active gibberellin, reversed the inhibition. These results suggest that the transcription of genes encoding the vacuolar invertases, At beta fruct3 and At beta fruct4 and a gene encoding the apoplasmic invertase, At beta fruct1/AtcwINV1, are induced by gibberellin synthesized de novo following irradiation with red light.

The role of gibberellins (GAs) during early embryo development was examined using microspore-derived embryos (MDEs) of Brassica napus. At the globular stage of development, 10 d after initial culture (DAC) when endogenous GA(1) levels are increasing rapidly, a triazole, uniconazole, was used at 1, 33 and 100 microM to inhibit GA biosynthesis. Within this dose range there was no apparent effect of the inhibitor on embryo growth through to the early torpedo stage. However, by 25 DAC uniconazole-treated MDEs showed significantly reduced (50%) axis elongation. Addition of GA(1) at 33 microM on 14 DAC to embryos pretreated with 1 microM uniconazole on 10 DAC prevented this reduction in axis length, giving axis elongation equivalent to untreated MDEs. Application of GA(1) alone, however, did not significantly increase axis elongation. The reduced axis growth seen with uniconazole treatment was due to reduced cell elongation, but not cell number, and the co-applied GA(1) thus prevented the uniconazole-induced reduction in cell length. The elongating axis of MDEs may thus be a useful tool for examining the role of GAs in cell elongation.

Bulbing was studied in shallot plants cultured in vitro. Bulbing occurred under a 16 h photoperiod with fluorescent + incandescent light and 30-50 g 1(-1) sucrose in the culture medium. Exogenous gibberellin (10 microM GA3) inhibited leaf and root growth and bulbing. When added to the medium at a concentration of 10 microM, three inhibitors of gibberellin biosynthesis (ancymidol, flurprimidol and paclobutrazol) promoted bulb formation and the percentage of bulbing. When ancymidol was used in combination with GA3, it did not reverse the effect of GA3 applied alone. Under treatments with 30-70 g l(-1) sucrose, bulbing ratios greater than those found in control plants were achieved by addition of ancymidol, and bulb fresh weight was increased in the same way. Ancymidol caused a 66% decrease in sucrose content in leaf bases but greatly increased the glucose, fructose and fructan contents. The increase in fructan content by ancymidol could result from the three-fold rise in total [14C]sucrose uptake per plant from the culture medium associated with a marked increase in leaf base labelling at the expense of root labelling. The possible role of ancymidol is discussed and evidence supports a major regulatory role for gibberellins in bulbing.

The antagonism between gibberellins (GA) and abscisic acid (ABA) is an important factor regulating the developmental transition from embryogenesis to seed germination. In barley aleurone layers, the expression of genes encoding alpha-amylases and proteases is induced by GA but suppressed by ABA. It has been shown that an ABA-induced protein kinase, PKABA1, mediates the ABA suppression of alpha-amylase expression. Using a barley aleurone transient expression system, we have now localized the site of action of PKABA1 relative to other signal transduction components governing the expression of alpha-amylase. The expression of alpha-amylase can be transactivated by the transcription factor GAMyb, which is itself induced by GA. A truncated GAMyb containing the DNA binding domain but lacking the transactivation domain prevents the GA induction of alpha-amylase, further supporting the notion that GAMyb mediates the GA induction of alpha-amylase expression. Although ABA and PKABA1 strongly inhibit the GA induction of alpha-amylase, they have no effect on GAMyb-transactivated alpha-amylase expression. Using a GAMyb promoter--beta-glucuronidase construct, we also show that both ABA and PKABA1 repress the GA induction of GAMyb. In the slender mutant, GAMyb and alpha-amylase are highly expressed, even in the absence of GA. However, this constitutive expression can still be inhibited by ABA, PKABA1, or an inhibitor of cGMP synthesis. On the basis of these observations, we suggest that PKABA1 acts upstream from the formation of functional GAMyb but downstream from the site of action of the Slender gene product. Because PKABA1 inhibits the GA induction of the GAMyb promoter--beta-glucuronidase construct, it appears that at least part of the action of PKABA1 is to downregulate GAMyb at the transcriptional level.

Hormonal control of root growth was studied in Lemna minor. Although addition of gibberellic acid (GA3) to the culture medium did not promote the root growth, a gibberellin biosynthesis inhibitor, uniconazole P (Un-P), significantly inhibited root growth. Both length and diameter of roots in Un-P-treated plants were significantly smaller than those in control plants, mainly caused by inhibition of cell division. In epidermal cells, the length was slightly decreased and the width increased by Un-P treatment, indicating inhibition of elongation growth. GA3 completely nullified the inhibition caused by Un-P. Transverse cortical microtubules (CMTs) of epidermal cells in the elongation zone were significantly fragmented by treatment with Un-P, but not by that in the presence of GA3. The cellulose microfibril array in the Un-P-treated cells was more random and more oblique than that in the control cells. These results suggested that root growth in L. minor is regulated by endogenous gibberellin.

The gibberellin (GA) biosynthetic pathway includes four apparent cytochrome P450-mediated steps that convert kaurene to 7 alpha-hydroxykaurenoic acid. One of these reactions, the hydroxylation of kaurenoic acid to 7 alpha-hydroxykaurenoic acid, is mediated by kaurenoic acid hydroxylase. This reaction can be catalyzed in vitro by microsomal preparations from the fungus Gibberella fujikuroi (Saw.) Wr. and monitored by HPLC. Cultures grown in the presence of 84 microM AMO-1618 (an inhibitor of kaurene synthesis) had reduced levels of GA3 in fungal filtrates and decreased cell-free kaurenoic acid hydroxylase activity. However, the level of hydroxylase activity from AMO-1618-treated cultures could be induced several-fold by growing cultures in the presence of 350 microM kaurene. Since transcripts related to GA biosynthesis might be decreased in AMO-1618-treated cultures, a subtractive hybridization procedure was used to enrich cDNA fragments corresponding to messages that are more abundant in untreated than treated cultures. A fungal cDNA library was screened with the subtraction products and a clone was isolated that corresponds to two down-regulated transcripts in AMO-1618-treated cultures. This cDNA does not encode a cytochrome P450 but may be associated with GA biosynthesis.

The biological activity of cell wall-derived feruloyl oligosaccharides was investigated using a modified micro-drop bioassay. A feruloyl arabinoxylan trisaccharide (FAXX) and a feruloyl arabinoxylan tetrasaccharide (FAXXX) were found to inhibit the gibberellin-induced elongation of dwarf rice (Oryza sativa L., cv, Tan-ginbozu) that had been treated with uniconazole (S-3307), an inhibitor of the biosynthesis of gibberellins. An arabinoxylan trisaccharide (AXX) was ineffective. The growth-inhibitory effect of feruloyl oligosaccharides depended on their feruloyl and glycosyl moieties. The amount of esterified diferulic acid residues in the cell walls of the second leaf sheath of rice seedlings that had been treated with FAXX was almost same as that of controls. Feruloyl oligosaccharides did not inhibit the incorporation of [14C]leucine into acid-precipitable proteins by suspension-cultured maize cells, whereas cinnamic acid and its derivatives strongly inhibited such incorporation.

Experiments were carried out to explore the involvement of gibberellins (GAs) in the light-induced germination of Arabidopsis thaliana (L.) Heynh, using wild type (WT) and phytochrome-deficient mutants (phyA, phyB and phyAphyB deficient in phytochrome A, B and A plus B, respectively). Seed germination of WT and phytochrome-deficient mutants was inhibited by uniconazole (an inhibitor of an early step in biosynthesis of GA, the oxidation of ent-kaurene) and prohexadione (an inhibitor of late steps, namely, 2 beta- and 3 beta-hydroxylation). This inhibition was overcome by simultaneous application of 10(-5) M GA4. The relative activity of GAs for promoting germination of uniconazole-treated seeds was GA4 > GA1 = GA9 > GA20. The wild type and the phyA and phyB mutants had an increased response to a red light pulse in the presence of GA1, GA4, GA9, GA20 and GA24 but there were no significant differences in activity of each GA between the mutants. Therefore, neither phytochrome A nor hytochrome B appears to regulate GA biosynthesis from GA12 to GA4 during seed germination, since the conversion of GA12 to GA9 is regulated by one enzyme (GA 20-oxidase). However, GA responsiveness appears to be regulated by phytochromes other than phytochromes A and B, since the phyAphyB double mutant retains the photoreversible increased response to GAs after a red light pulse.

The objective of this research was to determine if gibberellic acid (GA) and/or abscisic acid (ABA) are necessary for graviresponsiveness by primary roots of Zea mays. To accomplish this objective we measured the growth and graviresponsiveness of primary roots of seedlings in which the synthesis of ABA and GA was inhibited collectively and individually by genetic and chemical means. Roots of seedlings treated with Fluridone (an inhibitor of ABA biosynthesis) and Ancymidol (an inhibitor of GA biosynthesis) were characterized by slower growth rates but not significantly different gravicultures as compared to untreated controls. Gravicurvatures of primary roots of d-5 mutants (having undetectable levels of GA) and vp-9 mutants (having undectable levels of ABA) were not significantly different from those of wild-type seedlings. Roots of seedlings in which the biosynthesis of ABA and GA was collectively inhibited were characterized by gravicurvatures not significantly different for those of controls. These results (1) indicate that drastic reductions in the amount of ABA and GA in Z. mays seedlings do not significantly alter root graviresponsiveness, (2) suggest that neither ABA nor GA is necessary for root gravicurvature, and (3) indicate that root gravicurvature is not necessarily proportional to root elongation.