Endogenous Diterpenes Derived from ent-Kaurene,a Common Gibberellin Precursor,Regulate Protonema Differentiation of the Moss Physcomitrella patens

Gibberellins (GAs) are a group of diterpene-type plant hormones biosynthesized from ent-kaurene via ent-kaurenoic acid. GAs are ubiquitously present in seed plants. The GA signal is perceived and transduced by the GID1 GA receptor/DELLA repressor pathway. The lycopod Selaginella moellendorffii biosynthesizes GA and has functional GID1-DELLA signaling components. In contrast, no GAs or functionally orthologous GID1-DELLA components have been found in the moss Physcomitrella patens. However, P. patens produces ent-kaurene, a common precursor for GAs, and possesses a functional ent-kaurene synthase, PpCPS/KS. To assess the biological role of ent-kaurene in P. patens, we generated a PpCPS/KS disruption mutant that does not accumulate ent-kaurene. Phenotypic analysis demonstrates that the mutant has a defect in the protonemal differentiation of the chloronemata to caulonemata. Gas chromatography-mass spectrometry analysis shows that P. patens produces ent-kaurenoic acid, an ent-kaurene metabolite in the GA biosynthesis pathway. The phenotypic defect of the disruptant was recovered by the application of ent-kaurene or ent-kaurenoic acid, suggesting that ent-kaurenoic acid, or a downstream metabolite, is involved in protonemal differentiation. Treatment with uniconazole, an inhibitor of ent-kaurene oxidase in GA biosynthesis, mimics the protonemal phenotypes of the PpCPS/KS mutant, which were also restored by ent-kaurenoic acid treatment. Interestingly, the GA₉ methyl ester, a fern antheridiogen, rescued the protonemal defect of the disruption mutant, while GA₃ and GA₄, both of which are active GAs in angiosperms, did not. Our results suggest that the moss P. patens utilizes a diterpene metabolite from ent-kaurene as an endogenous developmental regulator and provide insights into the evolution of GA functions in land plants.GAs are a large family of tetracyclic diterpenoids, and bioactive GAs play crucial roles in aspects of plant growth and development, including seed germination, stem elongation, leaf expansion, trichome development, and flower and fruit development (Olszewski et al., 2002). GAs are biosynthesized from ent-kaurene, the key intermediate of the GA biosynthetic pathway (Olszewski et al., 2002; Yamaguchi, 2008; Fig. 1). ent-Kaurene is synthesized via sequential cyclization steps of geranylgeranyl diphosphate (GGDP) by ent-copalyl diphosphate synthase (CPS; Sun and Kamiya, 1994) and ent-kaurene synthase (KS; Yamaguchi et al., 1996, 1998). The bioactive GAs (GA₁ and GA₄) are synthesized through a series of oxidation reactions of ent-kaurene by two types of oxidases. Both ent-kaurene oxidase and ent-kaurenoic acid oxidase are cytochrome P450 monooxygenases that successively convert ent-kaurene to GA₁₂. GA₁₂ is further converted to bioactive GAs by two 2-oxoglutarate-dependent dioxygenases, GA 20-oxidase and GA 3-oxidase (Phillips et al., 1995; Olszewski et al., 2002; Yamaguchi, 2008; Fig. 1). GA 2-oxidase is another member of the 2-oxoglutarate-dependent dioxygenase family and is responsible for GA inactivation (Fig. 1). The active GAs can bind to the soluble GA receptor, GID1, and promote the interaction of GID1 with DELLA repressors, which are negative regulators of GA signaling (Ueguchi-Tanaka et al., 2005; Nakajima et al., 2006). This GA-promoted GID1-DELLA interaction triggers the degradation of DELLA repressors via the SCF^GID2/SLY1 proteasome pathway and consequently activates GA signaling (Ueguchi-Tanaka et al., 2007).Open in a separate windowFigure 1.The biosynthetic pathway of GA. The enzyme names are shown in boldface below or to the right of each arrow. AMO-1618 is an angiosperm inhibitor of CPS. Uniconazole, a GA biosynthesis inhibitor, blocks ent-kaurene oxidase activity. GA₁ and GA₄ are the bioactive GAs, and GA₈ and GA₃₄ are their inactive catabolites, respectively. KAO, ent-Kaurenoic acid oxidase.In nonseed land vascular plants, auxin, cytokinin, and abscisic acid function as regulators of plant growth and development (Chopra and Kumra, 1988; Raghavan, 1989). Various physiological responses to these phytohormones are investigated in nonseed land plants, especially in the model moss Physcomitrella patens (Cove et al., 2006). Auxin and cytokinin function in developmental phase changes of chloronemata, caulonemata, and gametophores as well as in cellular growth regulation in P. patens (Imaizumi et al., 2002; Sakakibara et al., 2003; Decker et al., 2006). Abscisic acid mediates the establishment of tolerance to dehydration, cold temperature, and osmotic stresses in P. patens as in angiosperms (Decker et al., 2006; Cho et al., 2009; Khandelwal et al., 2010). In contrast to these hormones, there are only a few studies on the physiological activity of GA in mosses (Von Maltzahn and Macquarrie, 1958; Chopra and Mehta, 1987; Vandenbussche et al., 2007), and the GA function and signaling pathways are still unclear.Recent progress in plant molecular biology and chemical analysis of GA revealed the biosynthesis, perception, and signaling of GA in P. patens and the lycopod Selaginella moellendorffii (Hirano et al., 2007; Vandenbussche et al., 2007; Yasumura et al., 2007). Genome sequence for these organisms has enabled the identification of genes orthologous to flowering plant genes encoding GA biosynthetic enzymes and GA signaling components involved in the GID1-DELLA pathway (Hirano et al., 2007; Vandenbussche et al., 2007). Recently, two reports demonstrated that GID1-DELLA-mediated signaling is functionally conserved in the fern Selaginella and in angiosperms (Hirano et al., 2007; Yasumura et al., 2007). GA-dependent protein-protein interactions were observed between SmGID1 and SmDELLA proteins, the S. moellendorffii proteins orthologous to the rice (Oryza sativa) GID1 and DELLA proteins, respectively. The introduction of either the SmGID1a or SmGID1b gene rescued the rice Osgid1-3 mutant, and the overproduction of SmDELLA1 suppressed GA action in the wild-type background. These reports indicate that the GID1 and DELLA proteins function similarly in S. moellendorffii and in angiosperms. Additionally, S. moellendorffii has functional GA biosynthetic enzymes similar to the angiosperm GA 20- and GA 3-oxidases and endogenous active GA₄ detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. However, endogenous GAs were not detected in P. patens by LC-MS/MS analysis, and the putative P. patens GA oxidases did not show any enzymatic activity on the known substrate for the orthologous angiosperm GA oxidases (Hirano et al., 2007). Furthermore, the PpGID1-like and PpDELLA-like proteins did not interact in the presence of active GA in yeast cells, and the PpDELLA-like protein did not complement the rice DELLA function. These findings suggest that GID1-DELLA-mediated GA signaling evolved in the vascular plant lineage after bryophyte divergence (Hirano et al., 2008).GA₁ and GA₄ are recognized as major biologically active GAs in angiosperms. S. moellendorffii biosynthesizes GA₄ as an active GA. Additionally, the Schizaeaceae family of ferns utilize GA methyl esters (methyl esters of GA₉, GA₂₀, and GA₇₃) as regulators of antheridium development, whereas these GA methyl esters are inactive in angiosperms (Yamauchi et al., 1996, 1997; Kurumatani et al., 2001). The biologically active GAs present in angiosperms were not detected in P. patens (Hirano et al., 2007). Although diverse GA metabolites have been found in plants and fungi, all the GA metabolites are thought to be derived from ent-kaurene, a common intermediate in early GA biosynthetic steps in both land plants and fungi (Kawaide, 2006). In angiosperms, two separate enzymes (CPS and KS) are involved in ent-kaurene synthesis from GGDP via ent-copalyl diphosphate as a reaction intermediate (Fig. 1). We have reported that PpCPS/KS, catalyzing the direct cyclization of GGDP to ent-kaurene, was a bifunctional diterpene cyclase with both CPS and KS activities in a single polypeptide (Hayashi et al., 2006). This type of bifunctional ent-kaurene synthase was also found in GA-producing fungi but was not identified in angiosperms (Kawaide et al., 1997; Toyomasu et al., 2000). The P. patens genome contains a single CPS/KS homolog, and no diterpene cyclase gene was found on the basis of sequence similarity in this organism. Anterola et al. (2009) reported that AMO-1618, an inhibitor of CPS, suppressed spore germination in P. patens; the suppression was recovered by exogenous ent-kaurene application. These results led the authors to hypothesize a role for ent-kaurene in regulating spore germination (Anterola et al., 2009). However, the hypothesis should be examined because the AMO-1618 inhibitory effect was not fully recovered by ent-kaurene application, probably because of the unspecific inhibitory effect of AMO-1618 on spore germination (Anterola et al., 2009).To assess the biological role of ent-kaurene and its metabolites in P. patens, we performed an insertional knockout of the ent-kaurene synthase gene, CPS/KS, in P. patens; the loss of ent-kaurene production was confirmed by gas chromatography-mass spectrometry (GC-MS) analysis. We also determined the abundance of all possible GAs and their precursors in P. patens by LC-MS/MS analysis. The PpCPS/KS disruption mutant (Ppcps/ks KO) lines have a defect in protonemal development. The differentiation of chloronemata to caulonemata was suppressed in the Ppcps/ks KO mutants, and the defect was recovered by the exogenous application of ent-kaurene or ent-kaurenoic acid. Furthermore, the GA₉ methyl ester, an antheridiogen of schizaeaceous ferns, rescued the protonemal defect of the mutants, but GA₃ and GA₄, the representative active GAs for angiosperm, did not. Our results demonstrate that P. patens utilizes GA-type diterpenes synthesized from ent-kaurene as an endogenous growth regulator in protonemal development.     

Gibberellin biosynthetic pathway and the physiologically active gibberellin in the shoot ofCucumis sativus L.

[²H₂]Gibberellin A₂₄ (GA₂₄) and [²H₄]-GA₉ were applied to the apices of normal-type cucumber (Cucumis sativus L. cv. Yomaki) seedlings treated with uniconazole, an inhibitor of GA biosynthesis. The metabolites from these feeds were identified by full-scan gas chromatography-mass spectrometry (GC-MS) to confirm the conversions of [²H₂]GA₂₄ to [²H₂]GA₉ and of [²H₄]GA₉ to [²H₄]GA₄. The results show that GA₄ is biosynthesized from GA₂₄ via GA₉. In a cucumber hypocotyl elongation bioassay using cv. Yomaki, prohexadione (DOCHC), an inhibitor of 2-oxoglutaratedependent dioxygenase, inhibited the hypocotyl elongation caused by application of GA₉, while DOCHC enhanced the elongation caused by application of GA₄. These results indicate that GA₄ is a physiologically active GA and that the activity of GA₉ is due to its conversion to GA₄ in cucumber shoots.     

Gibberellins in shoots and developing capsules of Populus species

Extracts of stems of growing shoots of Populus deltoides and P. trichocarpa, and developing capsules of P. deltoides were analysed for gibberellins (GAs) by gas chromatography-mass spectrometry. The following known GAs were identified by comparison of their Kovats retention indices (KRIs) and mass spectra with those of standards: GA1, GA8, GA9, GA19, GA20, 16 beta,17-dihydro-17-hydroxy GA20, GA23, GA28, GA29, GA34, GA44, and GA97. Several of these have not been previously reported from Populus. In addition, two new GAs were identified as 12 beta-hydroxy GA53 (GA127) and 16 beta,17-dihydro-17-hydroxy GA53 and their structures were confirmed by partial synthesis. Evidence was found of 16,17-dihydro-16,17-dihydroxy GA9, 16,17-dihydro-16,17-dihydroxy GA12, 12-hydroxy GA14, and GA34-catabolite by comparison of mass spectra and KRIs with published data. Several putative GAs (hydroxy- and dihydroxy-GA12-like) were also found. The catabolites of active GAs or of key precursors, hydroxylated at C-2 in stems and either C-2, C-12, C-17, or C-16,17 in capsules, were the major proportion of the GAs.     

The role of gibberellin biosynthesis in the control of growth and flowering in Matthiola incana

Recently, it was found that stem elongation and flowering of stock Matthiola incana (L.) R. Br. are promoted by exogenous gibberellins (GAs), including GA₄, and also by acylcyclohexanedione inhibitors of GA biosynthesis, such as prohexadione‐calcium (PCa) and trinexapac‐ethyl (TNE). Here, because it was unclear how GA biosynthetic inhibitors could promote stem elongation and flowering, their effect on GA biosynthesis has been examined by quantifying endogenous GA levels; also, the sensitivity of stem elongation and flowering to various GAs in combination with the inhibitors was examined. Stem elongation and flowering were most effectively promoted by GA₄ when combined with PCa and, next in order, by 2,2‐dimethyl‐GA₄, PCa, GA₄+TNE, TNE, GA₉+PCa and by GA₄. There was little or no promotion by GA₁, GA₃, GA₉, GA₁₃, GA₂₀ and 3‐epi‐2,2‐dimethyl‐GA₄. Both the promotive effects of the acylcyclohexanediones on stem elongation and flowering, particularly when applied with GA₄, and the fact that TNE caused a build‐up of endogenous GA₄ imply that one effect of TNE at the lower dose involved an inhibition of 2β‐hydroxylation of GA₄ rather than an inhibition of 20‐oxidation and 3β‐hydroxylation of GAs which were precursors of GA₄. Overall, these results indicate that: (1) GAs with 3β‐OH and without 13‐OH groups (e.g. GA₄) are the most important for stem elongation and flowering in M. incana; (2) growth promotion rather than inhibition can result if an acylcyclohexanedione acts predominantly to slow 2β‐hydroxylation and so slows inactivation of active gibbberellins, including GA₄. It follows that a low dose of an acylcyclohexanedione can be a ‘growth enhancer’ for any applied GA that is liable to inactivation by 2β‐hydroxylation.     

Ferrous Sulfate Supplementation Causes Significant Gastrointestinal Side-Effects in Adults: A Systematic Review and Meta-Analysis

BackgroundThe tolerability of oral iron supplementation for the treatment of iron deficiency anemia is disputed.ObjectiveOur aim was to quantify the odds of GI side-effects in adults related to current gold standard oral iron therapy, namely ferrous sulfate.MethodsSystematic review and meta-analysis of randomized controlled trials (RCTs) evaluating GI side-effects that included ferrous sulfate and a comparator that was either placebo or intravenous (IV) iron. Random effects meta-analysis modelling was undertaken and study heterogeneity was summarised using I² statistics.ResultsForty three trials comprising 6831 adult participants were included. Twenty trials (n = 3168) had a placebo arm and twenty three trials (n = 3663) had an active comparator arm of IV iron. Ferrous sulfate supplementation significantly increased risk of GI side-effects versus placebo with an odds ratio (OR) of 2.32 [95% CI 1.74–3.08, p<0.0001, I² = 53.6%] and versus IV iron with an OR of 3.05 [95% CI 2.07-4.48, p<0.0001, I² = 41.6%]. Subgroup analysis in IBD patients showed a similar effect versus IV iron (OR = 3.14, 95% CI 1.34-7.36, p = 0.008, I² = 0%). Likewise, subgroup analysis of pooled data from 7 RCTs in pregnant women (n = 1028) showed a statistically significant increased risk of GI side-effects for ferrous sulfate although there was marked heterogeneity in the data (OR = 3.33, 95% CI 1.19-9.28, p = 0.02, I² = 66.1%). Meta-regression did not provide significant evidence of an association between the study OR and the iron dose.ConclusionsOur meta-analysis confirms that ferrous sulfate is associated with a significant increase in gastrointestinal-specific side-effects but does not find a relationship with dose.