Proteome analysis of rice root proteins regulated by gibberellin

To gain an enhanced understanding of the mechanism by which gibberellins （GAs） regulate the growth and development of plants, it is necessary to identify proteins regulated by GA. Proteome analysis techniques have been applied as a direct, effective, and reliable tool in differential protein expressions. In previous studies, sixteen proteins showed differences in accumulation levels as a result of treatment with GA3, uniconazole, or abscisic acid （ABA）, and/or the differences between the GA-deficient semi-dwarf mutant, Tan-ginbozu, and normal cultivars. Among these proteins, aldolase increased in roots treated with GA3, was present at low levels in Tan-ginbozu roots, and decreased in roots treated with uniconazole or ABA. In a root elongation assay, the growth of aldolase-antisense transgenic rice was half of that of vector control transgenic rice. These results indicate that increases in aldolase activity stimulate the glycolytic in the GA-induced growth of roots. In among GA, aldolase, and root growth. pathway and may play an important role this review, we discuss the relationship among GA, aldolase, and root growth.     

Characterization of fructose-bisphosphate aldolase regulated by gibberellin in roots of rice seedling

Fructose-bisphosphate aldolase is a glycolytic enzyme whose activity increases in rice roots treated with gibberellin (GA). To investigate the relationship between aldolase and root growth, GA-induced root aldolase was characterized. GA₃ promoted an increase in aldolase accumulation when 0.1 M GA₃ was added exogenously to rice roots. Aldolase accumulated abundantly in roots, especially in the apical region. To examine the effect of aldolase function on root growth, transgenic rice plants expressing antisense aldolase were constructed. Root growth of aldolase-antisense transgenic rice was repressed compared with that of the vector control transgenic rice. Although aldolase activity increased by 25% in vector control rice roots treated with 0.1 M GA₃, FBPA activity increased very little by 0.1 M GA₃ treatment in the root of aldolase-antisense transgenic rice. Furthermore, aldolase co-immunoprecipitated with antibodies against vacuolar H⁺-ATPase in rice roots. In the root of OsCDPK13-antisense transgenic rice, aldolase did not accumulate even after treatment with GA₃. These results suggest that the activation of glycolytic pathway function accelerates root growth and that GA₃-induced root aldolase may be modulated through OsCDPK13. Aldolase physically associates with vacuolar H-ATPase in roots and may regulate the vacuolar H-ATPase mediated control of cell elongation that determines root length.     

Seasonal Changes of GA1, GA19 and Abscisic Acid in Three Rice Cultivars

The levels of endogenous gibberellin A₁ and A₁₉ (GA₁ and GA₁₉)and abscisic acid (ABA) in three rice (Oryza sativa L.) cultivars,Nihonbare (normal), Tan-ginbozu (semid-warf) and Tong-il (semi-dwarf),were measured at various stages of internode elongation andear development. GA₁₉ was the main GA in Nihonbare and Tong-ilthroughout the life cycle but was not detected in Tan-ginbozu.The levels of GA₁ in the ears of all three cultivars were lowand reached their maxima after anthesis. Similarly, the earsof all three cultivars contained high ABA levels which peakedafter anthesis. Shoots contained low quantities of ABA throughoutthe life cycle. The roles of GAs and ABA are discussed withrespect to physiological phenomena, such as internode elongation,ear development and dwarfism. (Received May 9, 1981; Accepted July 18, 1981)     

A Step in the Biosynthesis of Gibberellins that Is Controlled by the Mutation in the Semi-Dwarf Rice Cultivar Tan-Ginbozu

Genetic analysis and a comparison of endogenous levels of gibberellinsbetween the semi-dwarf rice cultivar Tan-ginbozu and the correspondingnormal cultivar Ginbozu have confirmed that Tan-ginbozu is agibberellin deficient mutant and that the semi-dwarfism of Tan-ginbozuis controlled by a single recessive gene. A step in the biosynthesisof gibberellins that is blocked by the mutation in Tan-ginbozuhad been considered to be the synthesis of ent-kaurene or anearlier step. However, the rate of production of ent-kaureneby Tan-ginbozu was almost the same as that by Ginbozu. By contrast,accumulation of only a small amount of ent-kaurene was detectedin Tan-ginbozu, and the amount that accumulated was similarto that in Ginbozu that had been treated with 6.9 x 10^-8 M uniconazole-P(an effective inhibitor of three oxidative steps in the pathwayfrom ent-kaurene to ent-kaurenoic acid via entkaurenol and ent-kaurenal).The height of the treated Ginbozu plants was reduced to thesame as that of Tanginbozu plants. Resembling Tan-ginbozu plants,Ginbozu plants that had been treated with uniconazole-P respondedwell to ent-kaurenoic acid and slightly to ent-kaurene and ent-kaurenol.Since the growth-promoting activity of enf-kaurenal in Tan-ginbozuwas similar to that of ent-kaurene, our results suggest thatthe mutation in Tan-ginbozu blocks the three oxidative stepswhereby ent-kaurene is converted to ent-kaurenoic acid. (Received June 9, 1995; Accepted February 15, 1996)     

The involvement of gibberellin signalling in the effect of soil resistance to root penetration on leaf elongation and tiller number in wheat

Background and aims

The concept of root-sourced chemical signals that affect shoot growth in response to drought is widely reported; in particular the role of ABA in regulating stomatal conductance has received much attention. ABA, alone, does not fully explain all the effects of abiotic stresses in the root zone on shoot architecture. An increase in mechanical impedance, which can occur on even relatively modest soil drying, results in reduced root and shoot growth, processes that are potentially regulated by gibberellins (GAs).

Methods

In this study we explored the role of mechanical impedance and exogenous gibberellin (GA₃) on root and shoot architecture in wheat seedlings containing the Rht-B1a (tall), Rht-B1b (semi-dwarf) or Rht-B1c (dwarf) alleles in the April-Bearded or Mercia backgrounds. Our experiments were based on the use of the sand culture system which allows the mechanical impedance of the root growth environment to remain constant and independent of water and nutrient availability. We investigated the effects of the application of exogenous GA₃ to the root system.

Results

We found that impeding soil reduced leaf elongation in the tall and semi-dwarf lines, confirming the stunting effect of mechanical impedance which is widely reported. However, leaf elongation in the dwarf lines was not affected by root impedance. Application of GA₃ to the roots restored leaf elongation in the tall and semi-dwarf lines growing in impeding soil, with some growth response even in the dwarf line, the longest leaves being obtained when GA was applied to impeded roots of a tall line. Both exogenous GA and root impedance reduced the number of tillers, but there was no interaction with the Rht genotype. The genetic background did not affect the results.

Conclusion

We suggest that the GA signalling pathway has an unidentified role in the leaf elongation response to mechanical impedance to root growth.     

Endogenous gibberellin- and cytokinin-like substances in cultured shoot tissues of apple,Malus pumila cv. Jonathan,in relation to adventitious root formation

The frequencies of adventitious root formation in vitro of isolated shoots from bud cultures of apple (Malus pumila cv. Jonathan) after 1, 7 and 31 subcultures (weeks 5, 29 and 109 after the initial culture) were 5, 78 and 95% respectively. Endogenous gibberellin-like substances (GA) were extracted, chromatographed on SiO₂ partition columns, and assayed on dwarf rice (Oryza sativa cv. Tan-ginbozu). The levels of GA in shoots from the 1st, 7th and 31st subcultures were 40, 19 and 14 ng GA₃ eq./g dry weight of tissue, respectively, a trend which suggests an inverse relationship between endogenous GA level and rooting ability. This is consistent with the fact that applied GA₃ inhibits rooting in apple and many other species. The major peak of GA activity eluted coincidentally with GA₁/GA₃/GA₁₉. Endogenous cytokinin-like substances (CK) were chromatographed on paper and assayed with soybean hypocotyl sections. In contrast to the decrease in GA activity, CK activity increased 1.5–2.7 fold in the later subcultures (cytokinin activity per shoot, however, declined).     

Abscisic acid and gibberellin-like substances in roots and root nodules ofGlycine max

Summary The content of endogenous gibberellin (GA)-like substances of roots and root nodules of SOya, and GA production byRhizobium japonicum cultures, were investigated by a combined thin layer chromatographic (TLC)-dwarf pea epicotyl bioassay technique. GAs were more concentrated in root nodules than in the roots, totalling 1.34 and 0.16 nM GA₃ equivalents g⁻¹ dry wt. respectively. GA production byR. japonicum cultures was demonstrated (1.00 nM GA₃ equivalentsl ⁻¹) and comparison of the GA components of plant and bacterial culture medium extracts, suggested that rhizobial GA production may contribute to the nodule GA content. Cis-trans abscisic acid (ABA) was identified in root and nodule extracts by TLC-gas liquid chromatography (GLC), and amounted to 0.18 and 2.21 nM g⁻¹ dry wt. respectively, whereas 0.30 and 4.63 nM ABA equivalents g⁻¹ dry wt. were detected by a TLC-wheat embryo bioassay technique. ABA was not detected in extracts of bacterial cultures.     

Detection and identification of gibberellins in needles of silver fir (Abies alba Mill.) by combined gas chromatography-mass spectrometry

Extracts of mature silver fir (Abies alba Mill.) needles, current-year, and one-year old (A) and seven to nine-year old (B), were purified by reversed and normal phase HPLC. Gibberellin (GA)-like compounds were detected by the Tan-ginbozu dwarf rice micro-drop bioassay and corresponding fractions were analyzed by GC-MS. GA₉ was present in small amounts, while a major component was a cellulase-hydrolysable GA₉ conjugate which was assumed to be GA₉ glucosyl ester. It is proposed that GA₉ glucosyl ester plays a key role in the regulation of endogenous GA levels in silver fir needles.     

Qualitative and Quantitative Analysis of Endogenous Gibberellins in Onion Plants and Their Effects on Bulb Development

Evidence has been reported that bulb development in onion plants (Allium cepa L.) is controlled by endogenous bulbing and anti-bulbing hormones, and that gibberellin (GA) is a candidate for anti-bulbing hormone (ABH). In this study, we identified a series of C-13-H GAs (GA₁₂, GA₁₅, GA₂₄, GA₉, GA₄, GA₃₄, and 3-epi-GA₄) and a series of C-13-OH GAs (GA₄₄, GA₂₀, GA₁ and GA₈) from the leaf sheaths including the lower part of leaf blades of onion plants (cv. Senshu-Chuko). These results suggested that two independent GA biosynthetic pathways, the early-non-hydroxylation pathway to GA₄ (active GA) and early-13-hydroxylation pathway to GA₁ (active GA), exist in onion plants. It was also suggested that GA₄ and GA₁ have almost the same ability to inhibit bulb development in onion plants induced by treatment with an inhibitor of GA biosynthesis, uniconazole-P. The endogenous levels of GA₁ and GA₄, and their direct precursors, GA₂₀ and GA₉, in leaf blades, leaf sheaths, and roots of 4-week-old bulbing and non-bulbing onion plants were measured by gas chromatography/selected ion monitoring with the corresponding [²H]labeled GAs as internal standards. In most cases, the GA levels in long-day (LD)-grown bulbing onion plants were higher than those of short-day (SD)-grown non-bulbing onion plants, but the GA₁ level in leaf blades of SD-grown onion plants was rather higher than that of LD-grown onion plants. Relationship between the endogenous GAs and bulb development in onion plants is discussed.     

Interactions between abscisic acid, ethylene and gibberellin in internodal elongation in floating rice: the promotive effect of abscisic acid at low humidity

The enhancement of internodal elongation in floating or deepwater rice (Oryza sativa L. cv. Habiganj Aman II) by treatment with ethylene or gibberellic acid (GA₃) at high relative humidity (RH) is inhibited by abscisic acid (ABA). Here, we examined the interactive effects of ethylene, gibberellin (GA) and ABA at low RH on internodal elongation of deepwater rice stem segments. Although ethylene alone hardly promoted internodal elongation of stem sections at 30% RH, it enhanced the internodal elongation induced by GA₃. Application of ABA alone to stem segments had no effect on internodal elongation. However, in the presence of ethylene and GA₃ at 30% RH, ABA further promoted internodal elongation. This promotive effect of ABA was not found in the internodes of stem segments treated either with ethylene or with GA₃ at 30% RH or in the internodes of stem segments treated with ethylene and/or GA₃ at 100% RH.     

Restoration of gibberellin biosynthesis by 2,6-diisopropylphenoxyacetic acid in uniconazole-treated rice plants

2,6-Diisopropylphenoxyacetic acid (DIPA), a promoter of growth and flowering of Sagittaria species, was found to improve the shoot growth of rice plants treated with uniconazole, an inhibitor of gibberellin (GA) biosynthesis. In a modified micro-drop bioassay using semi-dwarf rice, Oryza sativa L. cv. Tan-ginbozu, in which uniconazole had been incorporated into the agar medium, a significant recovery from growth inhibition was observed for both the 3rd and the 4th leaf sheaths but not for the 2nd sheath. In greenhouse experiments, uniconazole-treated rice plants partially recovered from growth inhibition when DIPA was applied after uniconazole treatment, whereas DIPA applied with, or before, uniconazole treatment did not improve growth. The levels of GA₁ and GA₂₀ in the rice plants treated with uniconazole plus DIPA were almost equal to those of the untreated controls, indicating that the observed growth recovery is attributable to the restoration of GA biosynthesis by DIPA.     

Quantitative Analysis of Endogenous Gibberellins in Normal and Dwarf Cultivars of Rice

Endogenous levels of gibberellins in shoots and ears of twodwarf rice (Oryza sativa L.) cultivars, Tan-ginbozu (dx mutant)and Waito-C (dy mutant), were analyzed and compared with thoseof normal rice cultivar, Nihonbare. The endogenous levels of13-hydroxylated gibberellins in Tan-ginbozu were much lowerthan those in Nihonbare. In Waito-C, the levels of GA₁₉ andGA₂₀ in the shoots were higher but that of GA₁ was lower thanthe levels of these gibberellins in Nihonbare. These resultssupport the hypothesis that the dy gene controls the 3ß-hydroxylationof GA₂₀ to GA₁ while the dx gene controls a much earlier stepin the gibberellin biosynthesis. Our results indicate that GA₁is the active gibberellin that regulates the vegetative growthof rice. The endogenous levels of GA₄ in the ears of the twodwarf cultivars of rice were higher than the level of GA₄ inthe ears of the normal cultivar, Nihonbare suggesting that thebiosynthesis of gibberellin is not blocked in the anthers ofthe dwarf rice. (Received April 27, 1989; Accepted July 12, 1989)     

The effect of phytohormones on growth and artemisinin production in <Emphasis Type="Italic">Artemisia annua</Emphasis> hairy roots

Summary Few studies have focused on the effect of a broad range of phytohormones on growth and secondary metabolism of a single hairy root species. We measured growth, development, and production of the antimalarial drug, artemisinin, in Artemisia annua hairy roots in response to the five main hormones: auxins, cytokinins, ethylene, gibberellins (GA), and abscisic acid (ABA). Single roots grown in six-well plates in medium B5 with 0.01 mgl⁻¹ (0.029 μM) GA₃ produced the highest values overall in terms of the number of lateral roots, length of the primary root, lateral root tip density, total lateral root length, and total root length. When the total root lengths are compared, the best conditions for stimulating elongation appear to be: GA 0.01 mgl⁻¹ (0.029μM)> ABA 1.0 mgl⁻¹ (3.78μM)=GA 0.02 mgl⁻¹ (0.058μM). Bulk yields of biomass were inversely proportional to the concentration of each hormone tested. All cultures provided with ABA yielded the highest amount of biomass. Both 6-benzylaminopurine and 2-isopentenyladenine inhibited root growth, however, only 2-isopentenyladenine stimulated artemisinin production, more than twice that of the B5 controls, and more than any other hormone studied. These results will prove useful in increasing hairy root growth and artemisinin production.     

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.     

Gibberellins and Abscisic Acid Promote Carbon Allocation in Roots and Berries of Grapevines

Carbon allocation within grapevines may affect berry growth and development. The plant hormones gibberellins (GAs) and abscisic acid (ABA) control various processes across the plant life and both have been involved in assimilate production and transport in different species. Hence, this work examined the distribution of sugars (sucrose, fructose, and glucose) and starch in grapevines at veraison after foliar applications of GA₃, ABA, and an inhibitor of GA biosynthesis, paclobutrazol (PBZ). The results demonstrated that GA₃ increased total grapevine mass, with carbon allocated to the whole grapevine (as structural and soluble carbohydrates). Both GA₃ and ABA increased monosaccharide (glucose and fructose) levels in berries (up to tenfold) and roots (up to threefold). However, GA₃ increased the net carbon fixation whereas ABA did not. PBZ diminished most growth parameters except grapevine mass, and allocated more carbohydrates to roots (up to threefold more sucrose and starch). Such results indicate that GAs promote net carbon fixation and transport, whereas ABA as a stress signal only enhances sugar transport; notwithstanding the two hormones promoted carbon allocation toward roots and berries.     

Profile of Plant Hormones and their Metabolites in Germinated and Ungerminated Canola (Brassica napus) Seeds Imbibed at 8°C in either GA4+7, ABA, or a Saline Solution

Abscisic acid (ABA) and gibberellins (GAs) are two major phytohormones that regulate seed germination in response to internal and external factors. In this study we used HPLC-ESI/MS/MS to investigate hormone profiles in canola (Brassica napus) seeds that were 25, 50, and 75% germinated and their ungerminated counterparts imbibed at 8°C in either water, 25 μM GA₄₊₇, a 80 mM saline solution, or 50 μM ABA, respectively. During germination, ABA levels declined while GA₄ levels increased. Higher ABA levels appeared in ungerminated seeds compared to germinated seeds. GA₄ levels were lower in seeds imbibed in the saline solution compared to seeds imbibed in water. Ungerminated seeds imbibed in ABA had lower GA₄ levels compared to ungerminated seeds imbibed in water; however, the levels of GA₄ were similar for germinated seeds imbibed in either water or ABA. The ABA metabolites PA and DPA increased in seeds imbibed in either water, the saline solution, or ABA, but decreased in GA₄₊₇-imbibed seeds. In addition, ABA inhibited GA₄ accumulation, whereas GA had no effect on ABA accumulation but altered the ABA catabolism pathway. Information from our studies strongly supports the concept that the balance of ABA and GA is a major factor controlling germination.     

Endogenous gibberellin A1 level and α-amylase activity in germinating rice seeds

The role of endogenous gibberellin A₁ (GA₁) in the induction of -amylase activity was investigated during germination of rice (Oryza sativa L.) seeds. The level of endogenous GA₁ and the -amylase activity in the seeds of normal rice, cv. Nipponbare, increased simultaneously from 3 days after the imbibition of water. The -amylase activities in the dwarf rice, cv. Waito-C and Tan-ginbozu, were less than that in the normal rice. The level of endogenous GA₁ and -amylase activity were decreased in proportion to the concentration of a growth retardant, uniconazole. The retardation in -amylase activity caused by the treatment of uniconazole was recovered by the application of exogenous GA₁. These results indicate that the endogenous GA₁ biosynthesized de novo regulates -amylase production in germinating rice seeds.Abbreviations GA(s) gibberellin(s) - ABA abscisic acid - AE fraction acidic ethyl acetate-soluble fraction - HPLC high performance liquid chromatography - R _t retention time - GC-SIM gas chromatography-selected ion monitoring     

Calcium and calmodulin inhibitor effects on the growth of carrot (Daucus carota L.) and radish (Raphanus sativus L.) in nutrient culture

Growth of carrot and radish seedlings in nutrient culture was inhibited by pretreatment with three calmodulin inhibitors. There was little selective effect on specific organs, shoots, tap root and fibrous roots over a range of concentrations. Although pretreatment with CaCl₂ (0.5 mM) did not affect growth of untreated seedlings, it partially reduced the inhibitory effects of trifluoperazine over the concentration range 0.01–0.05 mM. Trifluoperazine reduced the growth of GA₃-treated seedlings but did not overcome the modifying effect of GA₃ in favouring shoot/root ratio; ABA exacerbated its inhibitory effect on overall seedling growth and particularly on tap root development.Abbreviations GA₃ gibberellic acid - ABA abscisic acid - CaCl₂ calcium chloride - GAs gibberellins - Tfp trifluoperazine     

Physiological characteristics and related gene expression of after‐ripening on seed dormancy release in rice

After‐ripening is a common method used for dormancy release in rice. In this study, the rice variety Jiucaiqing (Oryza sativa L. subsp. japonica) was used to determine dormancy release following different after‐ripening times (1, 2 and 3 months). Germination speed, germination percentage and seedling emergence increased with after‐ripening; more than 95% germination and 85% seedling emergence were observed following 1 month of after‐ripening within 10 days of imbibition, compared with <45% germination and 20% seedling emergence in freshly harvested seed. Hence, 3 months of after‐ripening could be considered a suitable treatment period for rice dormancy release. Dormancy release by after‐ripening is mainly correlated with a rapid decline in ABA content and increase in IAA content during imbibition. Subsequently, GA₁/ABA, GA₇/ABA, GA₁₂/ABA, GA₂₀/ABA and IAA/ABA ratios significantly increased, while GA₃/ABA, GA₄/ABA and GAs/IAA ratio significantly decreased in imbibed seeds following 3 months of after‐ripening, thereby altering α‐amylase activity during seed germination. Peak α‐amylase activity occurred at an earlier germination stage in after‐ripened seeds than in freshly harvested seeds. Expression of ABA, GA and IAA metabolism genes and dormancy‐related genes was regulated by after‐ripening time upon imbibition. Expression of OsCYP707A5, OsGA2ox1, OsGA2ox2, OsGA2ox3, OsILR1, OsGH3‐2, qLTG3‐1 and OsVP1 increased, while expression of Sdr4 decreased in imbibed seeds following 3 months of after‐ripening. Dormancy release through after‐ripening might be involved in weakening tissues covering the embryo via qLTG3‐1 and decreased ABA signalling and sensitivity via Sdr4 and OsVP1.