Exophiala sp. LHL08 reprograms Cucumis sativus to higher growth under abiotic stresses

Endophytic fungi are potential sources of secondary metabolites; however, they are little known for phytohormones secretion and amelioration of plant growth under abiotic stresses. We isolated a novel endophyte from the roots of Cucumis sativus and identified it as a strain of Exophiala sp. by sequencing internal transcribed spacer/large subunit rDNA and phylogenetic analysis. Prior to identification, culture filtrate (CF) of Exophiala sp. has shown significant growth promotion of Waito‐C [a gibberellins (GAs)‐deficient mutant cultivar] and Dongjin‐byeo (normal GAs biosynthesis cultivar) rice seedlings. CF analysis of Exophiala sp. showed the presence of physiologically active GAs (GA₁, GA₃, GA₄ and GA₇) and inactive GAs (GA₅, GA₈, GA₉, GA₁₂ and GA₂₀). Exophiala sp. had higher GAs in its CF than wild‐type strain of Gibberella fujikuroi except GA₃. Influence of Exophiala sp. was assessed on cucumber plant's growth and endogenous abscisic acid (ABA), salicylic acid (SA) and bioactive GAs under salinity and drought stresses. Exophiala sp.‐treated plants have shown significantly higher growth and rescued the host plants from stress promulgated water deficit, osmotic and cellular damage. The altered levels of stress‐responsive ABA showed low level of stress confined to endophyte‐applied plants than control. Elevated levels of SA and bioactive GAs (GA₃ and GA₄) in endophyte‐associated plants suggest stress‐modulating response toward salinity and drought. In conclusion, symbiotic relations between Exophiala and cucumber have reprogrammed the host plant growth under abiotic stresses, thus indicating a possible threshold role of endophytic fungi in stress alleviation. This study could be extended for improving agricultural productivity under extreme environmental conditions.     

Endophytic Cephalotheca sulfurea AGH07 reprograms soybean to higher growth

Abstract

Gibberellins (GAs) are well known for plant growth promotion. GAs production by fungi has received little attention, although substantial work has been carried out on other aspects of plant growth-promoting fungi (PGPF). We investigated GAs production and plant growth-promoting capacity of an endophytic fungus isolated from the roots of soil grown soybean plants. The endophytic fungus is reported as GAs producer and as PGPF for the first time in this study. Nine endophytic isolates were collected from the roots of soybean, and culture filtrates (CFs) obtained from their pure cultures were screened on Waito-C, a dwarf rice cultivar, for the presence of GAs. Of these, seven fungal isolates promoted shoot length as compared to control (distilled water), while one inhibited it. Three fungal isolates were selected on the basis of higher shoot elongation as compared to wild type Gibberella fujikuroi, which was used as positive control. The growth-prompting capacity of selected fungal isolates SB5-1, SB3-2, and SB3-3 was bio-assayed on soybean cv. Hwangkeumkong. Fungal isolate SB5-1 provided maximum plant height (31.6 cm), shoot length (21.1 cm), whole plant fresh biomass (2.41 g), shoot fresh biomass (1.99 g), and leaf area (24.37 cm²). The CF of isolate SB5-1 was analyzed for the presence of GAs, and it was found that all physiologically active GAs were present (GA₁, 0.15 ng/ml, GA₃, 1.2 ng/ml, GA₄, 7.37 ng/ml, and GA₇, 3.18 ng/ml) in conjunction with physiologically inactive GA₅, GA₉, GA₁₅, GA₁₉, GA₂₀, and GA₂₄. The fungal isolate SB5-1 was identified as a new strain of Cephalotheca sulfurea through molecular and phylogenetic approaches.     

<Emphasis Type="Italic">Chrysosporium pseudomerdarium</Emphasis> produces gibberellins and promotes plant growth

We isolated 10 endophytic fungi from the roots of drought stressed soybean cultivar Hwangkeumkong and bioassyed on waito-c rice and soybean seedlings, in order to identify plant growth-promoting fungi. The fungal isolate D-2-1 provided the best result for plant height and biomass promotion as compared to wild type Gibberella fujikuroi. The D-2-1 culture filtrate (CF) was analyzed for the presence of gibberellins (GAs) and it was observed that all physiologically active GAs, especially gibberellic acid, were present in higher amounts (GA₁, 0.24 ng/ml; GA₃, 8.99 ng/ml; GA₄, 2.58 ng/ml and GA₇, 1.39 ng/ml) in conjunction with physiologically inactive GA₅, GA₉, GA₁₅, GA₁₉, and GA₂₄. The fungal isolate D-2-1 was identified as a new strain of Chrysosporium pseudomerdarium through phylogenetic analysis of 18S rDNA sequence. Plant growth promotion and GAs production capacity of genus Chrysosporium have been reported for the first time in this study.     

Gibberellin production by pure cultures of a new strain of <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis>

Fungal endophytes produce a variety of favorable metabolites for plant growth and survival, but there is limited information on their gibberellin (GA) production capacity. In the current study, we isolated eight endophytic fungi from the roots of a drought stressed soybean cultivar Hwangkeumkong, and screened them on waito-c rice for plant growth promotion. Seven fungal isolates promoted plant growth, while one inhibited it. The culture filtrate (CF) of fungal isolate HK-5-2 provided the best results for growth promotion and was thus bioassayed on soybean. HK-5-2 CF enhanced plant length, plant fresh and dry weight and endogenous bioactive GA₁ and GA₄ contents of soybean as compared to control. The GA analysis of HK-5-2 CF showed the presence of bioactive GA₃ (8.38 ng/ml), GA₄ (2.16 ng/ml) and GA₇ (1.56 ng/ml) in conjunction with physiologically inactive GA₅, GA₁₉ and GA₂₄. Gibberella fujikuroi was used as positive control during this experiment. The fungal isolate HK-5-2 was identified as a new strain of Aspergillus fumigatus through molecular and phylogenetic analysis of 18S and 28S rDNA sequences.     

Endogenous Gibberellins in the Developing Liquid Endosperm of Tea

Changes in the kind and level of endogenous gibberellins (GAs) in the developing liquid endosperm of tea (Camellia sinensis L.) were investigated. Gibberellin A₁ (GA₁), GA₈, GA₁₉, GA₂₀, and GA₄₄ were identified by GC-MS or GC-SIM. Besides these early C-13 hydroxylated GAs, GA₃, iso-GA₃, and GA₃₈ were also identified. Of these GAs, GA₁ and GA₃ were the major gibberellins. The levels of these GAs were at a maximum in the globular embryo stage and then decreased rapidly during embryo maturation.     

Accumulation of C19-gibberellins in the gibberellin-insensitive dwarf mutantgai ofArabidopsis thaliana (L.) Heynh

The endogenous gibberellins (GAs) from shoots of the GA-insensitive mutant,gai, ofArabidopsis thaliana were analyzed and compared with the GAs from the Landsberg erecta (Ler) line. Twenty GAs were identified in Ler plants by full-scan gas chromatography-mass spectrometry (GC-MS) and Kovats retention indices (KRI's). These GAs are members of the early-13-hydroxylation pathway (GA₅₃, GA₄₄, GA₁₉, GA₁₇, GA₂₀, GA₁, GA₂₉, and GA₈), the non-3,13-hydroxylation pathway (GA₁₂, GA₁₅, GA₂₄, GA₂₅, GA₉, and GA₅₁), and the early-3-hydroxylation pathway (GA₃₇, GA₂₇, GA₃₆, GA₁₃, GA₄, and GA₃₄). The same GAs, except GA₅₃, GA₄₄, GA₃₇, and GA₂₉ were detected in thegai mutant by the same methods. In addition, extracts fromgai plants contained GA₄₁ and GA₇₁. Both lines also contained several unknown GAs. In Ler plants these were mainly hydroxy-GA₁₂ derivatives, whereas in thegai mutant hydroxy-GA₂₄, hydroxy-GA₂₅, and hydroxy-GA₉ compounds were detected. Quantification of seven GAs by GC-selected ion monitoring (SIM), using internal standards, and comparisons of the ion intensities in the SIM chromatograms of the other thirteen GAs, demonstrated that thegai mutant had reduced levels of all C₂₀-dicarboxylic acids (GA₅₃, GA₄₄, GA₁₉, GA₁₂, GA₁₅, GA₂₄, GA₃₇, GA₂₇, and GA₃₆). In contrast,gai plants had increased levels of C₂₀-tricarboxylic acid GAs (GA₁₇, GA₂₅, and GA₄₁) and of all C₁₉-GAs (GA₂₀, GA₁, GA₈, GA₉, GA₅₁, GA₄, GA₃₄, and GA₇₁) except GA₂₉. The 3β-hydroxylated GAs, GA₁ and GA₄, and their respective 2β-hydroxylated derivatives, GA₈ and GA₃₄, were the most abundant GAs found in shoots of thegai mutant. Thus, thegai mutation inArabidopsis results in a phenotype that resembles GA-deficient mutants, is insensitive to both applied and endogenous GAs, and contains low levels of C₂₀-dicarboxylic acid GAs and high levels of C₁₉-GAs. This indicates that theGAI gene controls a step beyond the synthesis of an active GA. Thegai mutant is presumably a GA-receptor mutant or a mutant with a block in the transduction pathway between the receptor and stem elongation. We thank Dr. L.N. Mander, Australian National University, Canberra, for providing [²H]gibberellins, Dr. B.O. Phinney, University of California, Los Angeles, USA for [¹³C]GA₈, and Dr. D.A. Gage, MSU-NIH Mass Spectrometry Facility (grant No. DRR00480), for advice with mass spectrometry. This work was supported by a fellowship from the Spanish Ministry of Agriculture (I.N.I.A.) to M.T., by the U.S. Department of Energy under Contract DE-ACO2-76ERO-1338, and by U.S. Department of Agriculture grant No. 88-37261-3434 to J.A.D.Z.     

Gibberellin Metabolism and Transport During Germination and Young Seedling Growth of Pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

The role of gibberellins (GAs) during germination and early seedling growth is examined by following the metabolism and transport of radiolabeled GAs in cotyledon, shoot, and root tissues of pea (Pisum sativum L.) using an aseptic culture system. Mature pea seeds have significant endogenous GA₂₀ levels that fall during germination and early seedling growth, a period when the seedling develops the capacity to transport GA₂₀ from the cotyledon to the shoot and root of the seedling. Even though cotyledons at 0–2 days after imbibition have appreciable amounts of GA₂₀, the cotyledons retain the ability to metabolize labeled GA₁₉ to GA₂₀ and express significant levels of PsGA20ox2 message (which encodes a GA biosynthesis enzyme, GA 20-oxidase). The large pool of cotyledonary GA₂₀ likely provides substrate for GA₁ synthesis in the cotyledons during germination, as well as for shoots and roots during early seedling growth. The shoots and roots express GA metabolism genes (PsGA3ox genes which encode GA 3-oxidases for synthesis of bioactive GA₁, and PsGA2ox genes which encode GA 2-oxidases for deactivation of GAs to GA₂₉ and GA₈), and they develop the capacity to metabolize GAs as necessary for seedling establishment. Auxins also show an interesting pattern during early seedling growth, with higher levels of 4-chloro-indole-3-acetic acid (4-Cl-IAA) in mature seeds and higher levels of indole-3-acetic acid (IAA) in young root and shoot tissues. This suggests a changing role for auxins during early seedling development.     

Bioactive chemical constituents produced by endophytes and effects on rice plant growth

We investigated the bioactive potential of fungal endophytes isolated from the roots of Glycine max (L.) Merr. and Cucumis sativus. Initial screening results showed that endophytes, (Chrysosporium pseudomerdarium, Aspergillus fumigates and Paecilomyces sp., Penicillium sp., Phoma glomerata, and Paecilomyces formosus) were promoting the growth attributes (shoot length, chlorophyll contents, and biomass) of mutant and wild-type rice. To know whether, these endophytes were producing bioactive chemical constituents; the endophytes were grown in potato dextrose, czapek broth, and ICI mediums that resulted in varying mycelia biomass. The advance chromatographic analysis of endophytic fungal culture filtrate showed gibberellins (GA) production namely GA₁ (0.05–10.55 ngml^?1), GA₃ (0.48–9.0 ngml^?1), GA₄ (0.2–8.0 ngml^?1), GA₇ (1.4–6.5 ngml^?1), and GA₉ (0.03–1.05 ngml^?1) while among endophytes, P. glomerata and P. formosus were highly potent in GAs production. Additionally, the endophyte's culture also contained indole-3-acetic acid (0.23–71.51 µgml^?1). Furthermore, oxalic (0.009–0.3 mgl^?1), quinic (0.019–0.22 mgl^?1), malic (1.94–17.2 mgl^?1), and citric (0.012–0.95 mgl^?1) acids were also present in the endophytic cultures. The biochemical potential of endophytes greatly varied depending on nutrient source and pH, however, czapek broth rich in carbon revealed higher potential for bioactive chemical constituent's production. In conclusion, the current findings suggest that endophyte can play a vital role in essential crop plant growth by synthesizing a wide array of bioactive metabolites. Furthermore, an increased production of chemical constituents can be achieved by changing the in vitro growth conditions of endophytes     

Gibberellin-producing Promicromonospora sp. SE188 improves Solanum lycopersicum plant growth and influences endogenous plant hormones

Plant growth-promoting rhizobacteria (PGPR) producing gibberellins (GAs) can be beneficial to plant growth and development. In the present study, we isolated and screened a new strain of Promicromonospora sp., SE188, isolated from soil. Promicromonospora sp. SE188 secreted GAs into its growth medium and exhibited phosphate solubilization potential. The PGPR produced physiologically active (GA₁ and GA₄) and inactive (GA₉, GA₁₂, GA₁₉, GA₂₀, GA₂₄, GA₃₄, and GA₅₃) GAs in various quantities detected by GC/MS-SIM. Solanum lycopersicum (tomato) plants inoculated with Promicromonospora sp. SE188 showed a significantly higher shoot length and biomass as compared to controls where PGPR-free nutrient broth (NB) and distilled water (DW) were applied to plants. The presence of Promicromonospora sp. SE188 significantly up-regulated the non C-13 hydroxylation GA biosynthesis pathway (GA₁₂→GA₂₄→GA₉→GA₄→ GA₃₄) in the tomato plants as compared to the NB and DW control plants. Abscisic acid, a plant stress hormone, was significantly down-regulated in the presence of Promicromonospora sp. SE188. Contrarily, salicylic acid was significantly higher in the tomato plant after Promicromonospora sp. SE188 inoculation as compared to the controls. Promicromonospora sp. SE188 showed promising stimulation of tomato plant growth. From the results it appears that Promicromonospora sp. SE188 has potential as a bio-fertilizer and should be more broadly tested in field trials for higher crop production in eco-friendly farming systems.     

Endogenous gibberellins in flushing buds of three deciduous trees: Alder,aspen, and birch

Endogenous gibberellins (GAs) were extracted from flushing (expanding) vegetative buds of river alder (Alnus tenuifolia), European white birch (Betula pendula), and aspen (Populus tremuloides) and identified by gas chromatography-mass spectrometry with full scans and/or selected ion monitoring. Five 13-hydroxylated GAs were detected from the three trees: GA_{1, 8}, and ₂₀ from alder, GA_{1, 8, 19} and ₂₀ from aspen and GA_{1, 8, 19, 20}, and ₂₉ from birch. Thirteen other GAs previously detected in Salix or common in other plants were specifically investigated but not detected. The presence of GA₁, its probable precursors GA₁₉ and GA₂₀, and its probable metabolite, GA₈, suggests that the early 13-hydroxylated GA biosynthetic pathway is dominant in vegetative buds of these trees. Abundant endogenous GAs of these trees are similar to the principal GAs of willows (various Salix spp.) and poplars (various Populus spp.). This suggests similarities in the GA physiology and is consistent with a common role of GA₁ as a regulator of shoot growth in woody angiosperms.     

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.     

Endogenous gibberellins in the shoots of normal- and bush-typeCucumis sativus L.

Endogenous gibberellins (GAs) in the shoots of normal- (cv. Yomaki, YO) and bush-type (cv. Spacemaster, SP) cultivars of cucumber (Cucumis sativus L.) grown under natural conditions were analyzed. From both YO and SP grown for 40 days, after sowing, a series of C-13-H GAs including GA₄, GA₉, GA₁₅, GA₂₄, GA₂₅, GA₃₄, and GA₅₁ were identified by gas chromatography-mass spectrometry (GC-MS; full scan). In addition to the above GAs, GA₁₂ and GA₇₀ were similarly identified from both YO and SP grown for 61 days after sowing. The endogenous levels of GA₄ and GA₉, which are highly active in promoting cucumber hypocotyl elongation, were quantified by GC-selected ion monitoring (GC-SIM) using [²H₂]GA₄ and [²H₄]GA₉ as internal standards. No remarkable difference in terms of endogenous levels of GA_4/9 was observed between YO and SP in both growth stages (40 and 61 days after sowing).     

Distribution of endogenous gibberellins in vegetative and reproductive organs of Brassica

Recognizing the physiological diversity of different plant organs, studies were conducted to investigate the distribution of endogenous gibberellins (GAs) in Brassica (canola or oilseed rape). GA₁ and its biosynthetic precursors, GA₂₀ and GA₁₉, were extracted, chromatographically purified, and quantified by gas-chromatography-selected ion monitoring (GC-SIM), using [²H₂]GAs as internal standards. In young (vegetative) B. napus cv. Westar plants, GA concentrations were lowest in the roots, increased acropetally along the shoot axis, and were highest in the shoot tips. GA concentrations were high but variable in leaves. GA₁ concentrations also increased acropetally along the plant axis in reproductive plants. During early silique filling, GA₁ concentrations were highest in siliques and progressively lower in flowers, inflorescence stalks (peduncles plus pedicels), stem, leaves, and roots. Concentrations of GA₁₉ and GA₂₀ showed similar patterns of distribution except in leaves, in which concentrations were higher, but variable. Immature siliques were qualitatively rich in endogenous GAs and GA₁, GA₃, GA₄, GA₈, GA₉, GA₁₇, GA₁₉, GA₂₀, GA₂₄, GA₂₉, GA₃₄, GA₅₁, and GA₅₃ were identified by GC-SIM. In whole siliques, GA₁₉, GA₂₀, GA₁, and GA₈ concentrations declined during maturation due to declining levels in the maturing seeds; their concentrations in the silique coats remained relatively constant and low. These studies demonstrate that GAs are differentially distributed in _Brassica with a general pattern of acropetally increasing concentration in shoots and high concentration in actively growing and developing organs.     

Promotive and inhibitory effects of uniconazole and prohexadione on the sprouting of bulbils of Chinese yam,Dioscorea opposita

Gibberellin A₁ (GA₁), GA₃ and GA₄ inhibited the sprouting of nondormant bulbils of Chinese yam, Dioscorea opposita, where the effectiveness of the GAs was as follows: GA₄>GA₁+GA₃. Uniconazole and prohexadione, plant growth retardants, promoted the sprouting of half-dormant bulbils. By contrast, these retardants inhibited the sprouting of nondormant bulbils. Gibberellin A₃ (GA₃) and A₄ (GA₄) which were applied to the stems of the sprouted bulbils, promoted stem elongation, but GAs applied to the bulbous parts inhibited this process. The effectiveness of the GAs on stem elongation was as follows: GA₃+GA₄ for the promotion and GA₄ > GA₃ for the inhibition. Uniconazole applied to the stem inhibited the stem elongation of the sprouted bulbils. These results suggest the possible involvement of endogenous GAs in the induction and maintenance of bulbil dormancy of D. opposita, as well as in the bulbil sprouting and subsequent stem elongation.     

Over-expression of a gibberellin 2-oxidase gene from Phaseolus coccineus L. enhances gibberellin inactivation and induces dwarfism in Solanum species

Gibberellins (GAs) are endogenous hormones that play a predominant role in regulating plant stature by increasing cell division and elongation in stem internodes. The product of the GA 2-oxidase gene from Phaseolus coccineus (PcGA2ox1) inactivates C₁₉-GAs, including the bioactive GAs GA₁ and GA₄, by 2β-hydroxylation, reducing the availability of these GAs in plants. The PcGA2ox1 gene was introduced into Solanum melanocerasum and S. nigrum (Solanaceae) by Agrobacterium-mediated transformation with the aim of decreasing the amounts of bioactive GA in these plants and thereby reducing their stature. The transgenic plants exhibited a range of dwarf phenotypes associated with a severe reduction in the concentrations of the biologically active GA₁ and GA₄. Flowering and fruit development were unaffected. The transgenic plants contained greater concentrations of chlorophyll b (by 88%) and total chlorophyll (11%), although chlorophyll a and carotenoid contents were reduced by 8 and 50%, respectively. This approach may provide an alternative to the application of chemical growth retardants for reducing the stature of plants, particularly ornamentals, in view of concerns over the potential environmental and health hazards of such compounds. C. Dijkstra, E. Adams, A. Bhattacharya and A. F. Page contributed equally to this paper.     

Endophytes Aspergillus caespitosus LK12 and Phoma sp. LK13 of Moringa peregrina produce gibberellins and improve rice plant growth

Two new strains of endophytic fungi were isolated from the bark of Moringa peregrina and identified as Aspergillus caespitosus LK12 and Phoma sp. LK13. These endophytes were identified through amplifying polymerase chain reaction (PCR) and sequencing the 18S internal transcribed spacer of DNA extracted from both endophytes. Pure cultures of endophytic fungi were subjected to extract and isolate gibberellins (GAs). Deuterated standards of [17,17-²H₂]-GA₁, [17,17-²H₂]-GA₃, [17, 17-²H₂]-GA₄ and [17, 17-²H₂]-GA₇ were used to quantify the endophytic fungal GAs. The analysis revealed that both the endophytes are producing bioactive GAs in various quantities (ng mL^?1). A. caespitosus LK12 was producing GA₁ (54.51 ± 1.23), GA₄ (26.5 ± 0.65), and GA₇ (2.87 ± 1.23) while Phoma sp. LK13 was secreting GA₁ (4.8 ± 0.12), GA₃ (8.65 ± 0.21), GA₄ (23.7 ± 0.98), and GA₇ (22.7 ± 0.73). The culture filtrate (CF) of A. caespitosus and Phoma sp. significantly increased the shoot length of GAs-deficient mutant waito-c and normal Dongjin-beyo rice seedlings as compared to control. Application of such growth-promoting and GAs-producing endophytes can ameliorate poorly growing crop plants.     

Characterization of novel mutants with an altered gibberellin spectrum in comparison to different wild-type strains of Fusarium fujikuroi

The rice pathogen Fusarium fujikuroi is known for producing a wide range of secondary metabolites such as pigments, mycotoxins, and a group of phytohormones, the gibberellic acids (GAs). Bioactive forms of these diterpenes are responsible for hyperelongation of rice stems, yellowish chlorotic leaves, and reduced grain formation during the bakanae disease leading to severely decreased crop yields. GAs are also successfully applied in agriculture and horticulture as plant growth regulators to enhance crop yields, fruit size, and to induce earlier flowering. In this study, six F. fujikuroi wild-type and mutant strains differing in GA yields and the spectrum of produced GAs were cultivated in high-quality lab fermenters for optimal temperature and pH control and compared regarding their growth, GA production, and GA gene expression levels. Comparative analysis of the six strains revealed that strain 6314/ΔDES/ΔPPT1, holding mutations in two GA biosynthetic genes and an additional deletion of the 4'-phosphopantetheinyl transferase gene PPT1, exhibits the highest total GA amount. Expression studies of two GA biosynthesis genes, CPS/KS and DES, showed a constantly high expression level for both genes under production conditions (nitrogen limitation) in all strains. By cultivating these genetically engineered mutant strains, we were able to produce not only mixtures of different bioactive GAs (GA₃, GA_4, and GA₇) but also pure GA₄ or GA₇. In addition, we show that the GA yields are not only determined by different production rates, but also by different decomposition rates of the end products GA₃, GA₄, and GA₇ explaining the varying GA levels of genetically almost identical mutant strains.     

Changes in levels of gibberellins,their putative conjugates,and ABA in zygotic embryos during late maturation and dry seed stages of <Emphasis Type="Italic">Brassica napus</Emphasis> cv Topaz

Two late stages [days 35 and 40 after pollination (DAP)] in zygotic embryo (ZE) development of Brassica napus were utilized to quantify, by the stable isotope-labeled dilution method, levels of “free” and “aglycone” gibberellins (GAs), as well as abscisic acid (ABA), during the programmed dehydration of the seed. GAs from both the early 13 hydroxylation and early non-hydroxylation pathways were present in these ZEs of B napus. Between 35 and 40 DAP endogenous ABA dropped precipitously (almost 30-fold) and this drop in ABA was accompanied by a significant reduction in levels of GA₁ and even in levels of the inactive GA catabolites, GA₈ and GA₂₉. Levels of GA₄ and putative GA₈₅ also dropped appreciably, though not significantly. In contrast, the levels of GA₂₀ and GA₉ (the immediate precursors of GA₁ and GA₄, respectively) did not change in the ZEs during this transition. A fungal-derived cellulase was used to hydrolyze the highly water-soluble fraction, which will contain GA conjugates. Relatively high levels of several GAs (GA₉, GA₂₀) were thus quantified after hydrolysis as the aglycones, e.g., 56 and 25 ng/g DW of GA₂₀ and 23 and 5 ng/g DW, of GA₉, respectively at DAP 35 and DAP 40. Other GAs found after hydrolysis of the highly water-soluble fraction remained relatively constant between 35 and 40 DAP. An exception was the putative GA₈₅ aglycone, which increased sixfold (free GA₈₅ decreased by ca. half). The transition to the dry seed stage for ZEs of B. napus is thus accompanied not only by the expected reduction in ABA, but also by reduced levels of many “free” GAs, especially the bioactive, 3β-hydroxylated GAs. In contrast, levels of 3-deoxy GAs remain relatively high, implying a partial block in the 3β-hydroxylation “activation” step of GA biosynthesis.     

Gibberellins in Leaves of Alstroemeria hybrida: Identification and Quantification in Relation to Leaf Age

In alstroemeria (Alstroemeria hybrida), leaf senescence is retarded effectively by the application of gibberellins (GAs). To study the role of endogenous GAs in leaf senescence, the GA content was analyzed by combined gas chromatography and mass spectrometry. Five 13-hydroxy GAs (GA₁₉, GA₂₀, GA₁, GA₈, and GA₂₉) and three non-13-hydroxy GAs (GA₉ and GA₄) were identified in leaf extracts by comparing Kováts retention indices (KRIs) and full scan mass sprectra with those of reference GAs. In addition, GA₁₅, GA₄₄, GA₂₄, and GA₃₄ were tentatively identified by comparing selected ion monitoring results and KRIs with those of reference GAs. A number of GAs were detected in conjugated form as well. Concentrations of GAs in alstroemeria changed with the development of leaves. The proportion of biologically active GA₁ and GA₄ decreased with progressive senescence and the fraction of conjugated GAs increased. Received May 26, 1997; accepted August 12, 1997