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Flowering of Pharbitis nil cv. Violet under different treatments. Seven-day-old seedlings were subjected to various treatments. (A) Control (left) and treatment with theobroxide (right), under LD conditions; (B) exposure with night break (NB, left) and NB+theobroxide (right); (C and D) control (left), cotyledon removal (CR, middle) and CR+theobroxide (right), under SD conditions. Pictures (A, B, and C) were taken at day 26 after beginning treatment, and (D) at day 34.
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Theobroxide, a novel active compound isolated from a fungus, has been reported previously to induce potato tuberization and flower bud formation in Pharbitis nil under non-inductive long-day conditions. Up to date, the action mechanism of theobroxide on flower-bud induction of P. nil, however, is still unknown. In the present study, we observed a r...
Contexts in source publication
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... to the plants with entire cotyledons, the flowering time for the seedlings with cotyledon removal was delayed from days 7 to 9 (Figs. 1C and 2A). This delay was reversed by application of theobroxide on the plants without cotyledons, with 100% of flowering plants at day 11 ( Fig. 2A). Theobroxide also increased the number of flower buds in the seedlings without cotyledons, with an average 11.5 flower buds per seedlings at day 25 whereas those without cotyledons had an average ...
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... elongation was markedly suppressed when both cotyledons were removed under LD conditions or after giving a single 16-h-dark treatment, and this suppression could be nullified by application of GAs. In the present study, we found that the seedlings without cotyledons had shortened stems compared with those with both cotyledons, under SD conditions (Fig. 1C). Moreover, it was also obvious that the stems of seedling grown under SD conditions was much shorter than that under LD conditions, and seedlings exposed to NB treatment displayed stems length intermediate between LD and SD (Figs. 1B and 3). Interestingly, theobroxide spraying signifi- cantly suppressed the stem elongation in the ...
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... it was also obvious that the stems of seedling grown under SD conditions was much shorter than that under LD conditions, and seedlings exposed to NB treatment displayed stems length intermediate between LD and SD (Figs. 1B and 3). Interestingly, theobroxide spraying signifi- cantly suppressed the stem elongation in the seedlings grown under all designed photoperiodic conditions ( Figs. 1 and 3). ...
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... in Fig. 4, GA 1/3 contents were much lower in plants grown under SD conditions than in LD-grown plants at all stages. In theobroxide- treated plants grown under LD conditions, GA 1/3 contents were significantly reduced to almost the same levels as in SD-grown plants. Combined with the results that theobroxide stimulated flower bud formation ( Fig. 1) but reduced stem elongation (Fig. 3) under LD conditions, it appears that theobroxide might mimic the role of SD in flower- bud induction and stem length reduction in P. ...
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... contents of GAs (GA 1/3 ) were quan- tified by the method of direct enzyme-linked immuno-sorbent assay (ELISA) as described by Li et al. (2003). Briefly, the leaves harvested from the plants grown under different conditions were immediately frozen in liquid nitrogen, lyophi- lized, and stored at À 80 1 C until use. Leaves (around 1 g) were extracted with cold 80% (v/v) aqueous methanol (containing 10 mg/L butylated hydroxytoluene to prevent oxidation). The supernatant, after centrifugation at 10,000 g (4 1 C for 20 min), was purified by a Sep-Pak C18 cartridge (Waters, Milford, MA). The eluate was collected and dried under N 2 . The residue was then dissolved in 400 m L of PBS (0.01 mol/L, pH 7.4) and submitted to GA 1/3 ELISA based on monoclonal antibodies that showed highly specific immuno-reactivity with GA 1/3 . The GA 1/3 ELISA kit was kindly provided by the Phytohormone Labora- tory at Nanjing Agricultural University (NAU), PR China. The leaves of five plants were collected and mixed for one sample at indicated time. Each extract was analyzed three times for GA 1/3 contents, and all samples were assayed in triplicate. The values were expressed as the means of three replicates. Consistent with our previous report (Yoshihara et al., 2000), theobroxide showed a stimulating effect on flower-bud formation (Figs. 1A and 2) in P. nil grown under non-inductive LD conditions. The seedlings grown under SD formed flower buds at day 7 after the inductive photoperiod started, and 100% of seedlings produced flower buds at day 9 (Fig. 2A). Flower-bud formation was significantly delayed in the seedlings exposed to a 1-h NB, from days 7 to 19, with an average number of 4.3 of flower buds per seedling at day 25 (Figs. 1B and 2). However, the inhibitory effect of NB on flower-bud formation was abolished completely by spraying with theobroxide (Figs. 1B and 2A), with about 14 flower buds per seedling, about 3.5-fold of plants exposed to a NB, at day 25 after spraying started (Fig. 2B). No flower buds were observed in LD-grown plants, even up to day 25. Compared to the plants with entire cotyledons, the flowering time for the seedlings with cotyledon removal was delayed from days 7 to 9 (Figs. 1C and 2A). This delay was reversed by application of theobroxide on the plants without cotyledons, with 100% of flowering plants at day 11 (Fig. 2A). Theobroxide also increased the number of flower buds in the seedlings without cotyledons, with an average 11.5 flower buds per seedlings at day 25 whereas those without cotyledons had an average 6.9 flower buds (Fig. 2B). P. nil cv. Violet is a taller strain compared with other dwarf strains, such as Sunsmile and Kitachi. Takeno et al. (1996) reported that stem elongation was markedly suppressed when both cotyledons were removed under LD conditions or after giving a single 16-h-dark treatment, and this suppression could be nullified by application of GAs. In the present study, we found that the seedlings without cotyledons had shortened stems compared with those with both cotyledons, under SD conditions (Fig. 1C). Moreover, it was also obvious that the stems of seedling grown under SD conditions was much shorter than that under LD conditions, and seedlings exposed to NB treatment displayed stems length intermediate between LD and SD (Figs. 1B and 3). Interestingly, theobroxide spraying significantly suppressed the stem elongation in the seedlings grown under all designed photoperiodic conditions (Figs. 1 and 3). As shown in Fig. 4, GA 1/3 contents were much lower in plants grown under SD conditions than in LD-grown plants at all stages. In theobroxide- treated plants grown under LD conditions, GA 1/3 contents were significantly reduced to almost the same levels as in SD-grown plants. Combined with the results that theobroxide stimulated flower bud formation (Fig. 1) but reduced stem elongation (Fig. 3) under LD conditions, it appears that theobroxide might mimic the role of SD in flower- bud induction and stem length reduction in P. nil . When the plants were subjected to NB conditions, GA 1/3 contents were decreased markedly compared to those in LD-grown plants (Fig. ...
Citations
... Theobroxide (1, Figure 1) was initially isolated from Lasiodiplodia theobromae as a tuber-inducing compound [1] and was later found to exhibit other unique activities, such as stimulating morning glory flowering [2][3][4] and triggering of ABA accumulation [5]. Because the amount of theobroxide isolated from the culture filtrate of L. theobromae is relatively high and the compound has acceptable solubility in H 2 O, we thought that it might be utilized as a plant chemical activator. ...
... In this study, we examined the metabolite of theobroxide in cowpea (Vigna unguiculata) using [ 2 H 3 -7]theobroxide (2, Figure 1) as a substrate. We found that theobroxide (1) applied exogenously to the roots of cowpea was converted to 3-O--D-glucopyranosyltheobroxide (3). ...
Theobroxide has been isolated from culture filtrates of Lasiodiplodia theobromae as a potato tuber-inducing compound. In this study, the metabolism of theobroxide was investigated using cowpea as an experimental model and [2H3-7]theobroxide as a substrate for analyzing a metabolite, which revealed that theobroxide applied exogenously to the roots was converted into 3-O-β-D-glucopyranosyltheobroxide.
... These highlighted the importance of ABA in the plant's response to drought stress and great attention has been focused on the role of ABA in the stress response. Some analytical methods have been reported to determine this endogenous phytohormone, such as bioassays (Boerjan et al., 1992), gas chromatography (GC) (Du et al., 2000), enzyme linked immunosorbent assay (ELISA) (Gao et al., 2006;Ferreira et al., 2006), spectrometry, electrochemical technique (Carretero and Cruces-Blanco, 2004;Li et al., 2005) and high-performance liquid chromatography (HPLC) (Dobrev et al., 2005), in which HPLC has the highest sensitivity, the most specificity and the best repeatability in comparison with other methods mentioned, thus it is used extensively to determine ABA content in some plants such as soybean, Arabidopsis thaliana and Glycyrrhiza uralensis etc. (Ciha et al., 1977;Ross et al., 2004;Xu et al., 2010). In the present study, an effectual approach based on HPLC method was established to separate and determine the endogenous abscisic acid in cowpea and further relationships were explored between ABA content and drought stress. ...
Cowpea (Vigna unguiculata L.) is one of the most important grain legume supplying necessarily dietary proteins. Although its can tolerate drought environment, still the severe condition can limit the growth and development of this plant. Abscisic acid (ABA) has a crucial function in stress physiology, but little information is available for methods describing ABA determination in cowpea and its relationship to drought stress based on cowpea varieties with different capability of drought resistance. Here, an effectual approach based on high-performance liquid chromatography (HPLC) method was established to separate and determine the endogenous abscisic acid in cowpea and further the cowpea varieties with different capability of drought resistance were used to explore relationships between ABA content and drought stress. The results potentially provided useful reference for the cultivation of cowpea crop. Results-This method showed good liner regression (R2 = 0.9998), coupled with high stability, precision and repeatability. The endogenous ABA decreased under drought treatment and the drought sensitive line had higher ABA content than drought resistant one, suggesting ABA performs a vital function in cowpea’s response and resistance to drought stress. The studies potentially provided useful reference for the cultivation and breeding of cowpea crop. © 2016 Wei Lei, Shian Huang, Shaohu Tang, Xiaorong Shui and Can Chen.
... We subsequently encountered co-metabolite 2 {(1S, 4R, 5S, 6R)-7,9-dioxa-3-methyl-8-oxobicyclo [4.3.0]-2-nonene-4,5-diol} while continuing a survey on potato-tuber inducing substances from a culture of L. theobromae (Matsuura et al., 1998). Compound 1 was proven to inhibit stem elongation in morning glory and spinach through the suppression of gibberellin biosynthesis and stimulation of jasmonic acid biosynthesis (Kong et al., 2005a;Kong et al., 2006;Gao et al., 2006). Immunoblot analysis of key enzymes in JA biosynthesis showed that lipoxygenase, allene oxide synthase and allene oxide cyclase activity, and endogenous levels of JA in Pharbitis nil were induced by 1 (Kong et al., 2005b). ...
Administration of (13)C labeled acetates ([1-(13)C], [2-(13)C] and [1,2-(13)C(2)] to Lasiodiplodia theobromae showed the tetraketide origins of both theobroxide, a potato-tuber inducing substance [1, (1S, 2R, 5S, 6R)-3-methyl-7-oxa-bicyclo[4.1.0]hept-3-en-2,5-diol]) and its carbonyldioxy derivative [2, (1S, 4R, 5S, 6R)-7,9-dioxa-3-methyl-8-oxobicyclo [4.3.0]-2-nonene-4,5-diol]. The incorporation of acetate-derived hydrogen into 1 and 2 was studied using [2-(2)H(3), 2-(13)C]acetate. Three and one deuterium atoms were incorporated at one methyl and epoxy carbons, respectively. The observed loss of deuterium atoms from the methyl group suggests a considerable amount of exchange from the methyl group of [2-(2)H(3), 2-(13)C]acetate during biosynthesis of 1 and 2. Incorporation of [1-(13)C]- and [1,2-(13)C(2)]acetates indicates the carbonyl carbon of the carbonyldioxy derivative is derived from the carboxy carbon of the precursor.
... Interestingly, spraying theobroxide on the leaflets of potatoes (Solanum tuberosum L.) and the leaves of morning glory (Pharbitis nil) induced potato tuber and flower bud formation, respectively, under non-inductive conditions [2]. Furthermore, theobroxide was shown to inhibit stem elongation in spinach (Spinacia oleracea L.) [3] and morning glory [4,5]. In morning glory, theobroxide was proven to inhibit stem elongation through suppressing gibberellin biosynthesis and stimulating jasmonic acid biosynthesis [4]. ...
Theobroxide, a natural product, strongly stimulates the biosynthesis of jasmonic acid (JA) in Pharbitis nil. In this study, we investigated the accumulation of protein by the immunoblot analysis of lipoxygenase (LOX), allene oxide synthase (AOS), and allene oxide cyclase (AOC), key enzymes in JA biosynthesis, and how the endogenous levels of JA in P. nil are affected by theobroxide. The effect of JA on the accumulations of these proteins was monitored simultaneously. The results show that theobroxide treatment led to a high level accumulation of JA, which is due to high accumulations of LOX, AOS, and AOC proteins induced by theobroxide treatment both under short day (SD) and long day (LD) conditions. However, under SD conditions AOS and AOC proteins are not enhanced by JA treatment. Kinetic analysis of protein levels shows that a biphasic activation of AOC protein by theobroxide is displayed and the first activation of AOC protein together with elevated JA levels is observed within 30min after treatment. Meanwhile, AOS and LOX proteins are activated by theobroxide later than AOC protein, suggesting that AOC plays an essential role in the initial JA formation induced by theobroxide. Since theobroxide-increased JA levels also show a biphasic manner similar to AOC activation and AOS, LOX proteins are activated later than AOC, and thus we propose a positive JA feedback regulation. Interestingly, AOS protein, which is also the enzyme for the biosynthesis of 9,10-ketol-octadecadienoic acid (KODA, a flowering inducing factor), accumulates markedly due to the simultaneous involvement of theobroxide and SD conditions, suggesting that AOS probably plays a role in flower bud formation in P. nil.
‘Kidachi’ and ‘Sun Smile’, dwarf cultivars and ‘Violet’, tall cultivar of Pharbitis nil were used for plant materials grown under a long day, non-inducing condition of flower bud. Flowering response was examined according to the concentration, frequency, and treatment time of theobroxide. Theobroxide treatment resulted in promoted flowering and increased flower number even though the flowering response was different depending on cultivars. The most effective condition of theobroxide spray for flowering promotion was 5-10 times of 1 mM for ‘Kidachi’ with 2-3 leaves, 5-10 times of 1 mM for ‘Sun Smile’ with 5-6 leaves, and 5 times of 1-2 mM for ‘Violet’ with 5-6 leaves.
The quenching mechanism of the fluorescence of quantum dots by abscisic acid has been systematically investigated. The quenching
constant K
SV = 5.1 × 1011 / M was obtained under the optimized condition. On the basis of that, a very sensitive method for the determination of abscisic
acid has been developed. The linear equation was F
0/F = 0.9309 + 0.5072 C (pmol/L) and its linear range was 0.2–3.0 pmol/L with a correlation coefficient of 0.9939. The limit of detection was 0.09
pmol/L.
Induction of some plant organs including tubers and flower buds begins with sensing environmental cues, such as photoperiod
and temperature in the leaves. Theobroxide has been shown to induce potato tuberization and flower-bud formation in morning
glory under non-inductive photoperiodic conditions, stimulating the activity of lipoxygenase (LOX) and the synthesis of jasmonic
acid (JA). In the present study, the ability of theobroxide to overcome the inhibitory effect of unfavorable high temperature
on the induction of tubers in potato and flower buds in morning glory was examined. Both tuber induction and flower-bud formation
under non-inductive high temperatures were promoted by the application of theobroxide at a high concentration. However, although
theobroxide treatment resulted in an increase in fresh weight during potato tuber growth at 30°C, morning glory plants treated
with theobroxide at 35°C failed to bloom, implying that theobroxide may assist only in flower-bud formation.
KeywordsJasmonic acid–9,10-ketol-octadecadienoic acid–Lipoxygenase–Temperature–Theobroxide–Tuberonic acid glucoside
A new cyclohexenone compound was isolated as a mixture of enantiomers from a culture filtrate of Lasiodiplodia theobromae. The relative structure was determined to be 4,5-dihydroxy-3-methyl-cyclohex-2-enone on the basis of MS, (1)H-NMR, and (13)C-NMR spectroscopic analyses, including 2D-NMR experiments. Resolution of the enantiomers was conducted by a coupling reaction with (S)-MTPA-Cl followed by HPLC separation.
