UV-B radiation affects chlorophyll and activation of rubisco by rubisco activase in<Emphasis Type="Italic">Canavalia ensiformis</Emphasis> L. leaves

We studied the influence of UV-B radiation on chlorophyll and rubisco activation by rubisco activase in the leaves of jackbean (Canavalia ensiformis). Chlorophyll content was decreased, indicating that the synthesis of those molecules may have been degraded or repressed after exposure. Rubisco content was significantly lower in radiated tissue compared with the untreated control; rubisco activity showed a similar pattern of change. Based on these data, we suggest that rubisco activity is associated with the level of rubisco protein, and that UV-B inhibits its activation and induction, as well as that of rubisco activase. Therefore, we propose that the inhibitory effect of rubisco by UV-B may be caused by rubisco activase.     

Influence of cadmium on rubisco activation in<Emphasis Type="Italic">Canavalia ensiformis</Emphasis> L. leaves

We studied the effect of cadmium on chlorophylls and rubisco activation inCanavalia ensiformis L. leaves. Chlorophyll levels were reduced by 5.0 μM Cd. Rubisco activity at 5.0 μM Cd was significantly smaller than that at no treatment. Rubisco content showed patterns of change similar to rubisco activity. These data suggest that rubisco activity was associated with an amount of rubisco protein, and that the activation and induction of rubisco is inhibited by Cd. The degree of intensity of 50 and 14.5 kD polypeptides identified as the large and small subunit of rubisco by SDS-PAGE analysis at 5.0 μM Cd was significantly lower than that at control, indicating Cd had a effect on both subunits. Under the assumption that effects of Cd on rubisco may be related to rubisco activase, in addition to, its activity and content were determined. The rubisco activase activity at 5.0 μM Cd was more decreased than, the control. A similar change pattern was also observed in content of rubisco activase. Remarkable differences in the intensitiy of both the 45 kD and 41 kD band were found between at control and Cd-treatment. These results suggest that the change in the levels of rubisco activase leads to a subsequent alteration of rubisco levels.     

Influence of benomyl on photosynthetic capacity in soybean leaves

This investigation was performed to study the influence of benomyl on photosynthetic pigments and enzymes in soybean leaves. Chlorophyll and pheophytin levels were reduced by benomyl 45 days after greening. These results indicate that chlorophylla andb, and pheophytin must be controlled by benomyl. SDS-PAGE analysis showed that 50 and 14.5 kD polypeptides represented as the large and small subunits of rubisco. In the both of these subunits, the band intensity of the control was significantly higher than that after benomyl treatment, indicating that these two subunits are affected by benomyl. Benomyl strongly inhibited both the activity and content of rubisco as its concentration was gradually increased. However, it remains unclear whether this reduction of rubisco level was due to a reduced level of rubisco activase. Two major polypeptides of 46 and 42 kD were identified as rubisco activase subunits by SDS-PAGE. The intensity of these two bands was shown to be higher in the control than after benomyl treatment. These results indicate that the rubisco decrease resulting from increased benomyl concentrations was caused by rubisco activase. A significant decrease in both the activity and content of rubisco activase by benomyl was also observed. These results suggest that the decrease in rubisco level caused by benomyl is accompanied by a decrease in both the activity and content of rubisco activase.     

Glyoxylate inhibition of ribulosebisphosphate carboxylase/oxygenase activation in intact,lysed, and reconstituted chloroplasts

At bicarbonate concentrations equivalent to air levels of CO₂, activation of ribulosebisphosphate carboxylase/oxygenase (rubisco) was inhibited by micromolar concentrations of glyoxylate in intact, lysed, and reconstituted chloroplasts and in stromal extracts. The concentration of glyoxylate required for 50% inhibition of light activation in intact chloroplasts was estimated to be 35 micromolar. No direct inhibition by glyoxylate was observed with purified rubisco or rubisco activase at micromolar concentrations. Levels of ribulose 1,5-bisphosphate and ATP increased in intact chloroplasts following glyoxylate treatment. Results from experiments with well-buffered lysed and reconstituted chloroplast systems ruled out lowering of pH as the cause of inhibition. With intact chloroplasts, micromolar glyoxylate did not prevent activation of rubisco at high (10 mM) concentrations of bicarbonate, indicating that rubisco could be spontaneously activated in the presence of glyoxylate. These results suggest the existence of a component of the in vivo rubisco activation system that is not yet identified and which is inhibited by glyoxylate.Abbreviations PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - rubisco ribulosebisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate     

Sucrose regulates growth and activation of rubisco in tobacco leaves<Emphasis Type="Italic">in vitro</Emphasis>

The influence of sucrose onin vitro growth, chlorophyll content, and rubisco/rubisco activase were studied in tobacco leaves. The most pronounced effect onin vitro growth and the chlorophyll content was found at 4% sucrose. The rubisco content increased with increasing concentrations of sucrose, but a point was reached beyond which the increasing concentrations of sucrose caused an inhibition of this enzyme. The rubisco activity showed patterns of change similar to the rubisco content. These data suggest that sucrose may have an affect on the activation and induction of rubisco and that sucrose can be both a positive effector and negative effector depend on its concentration. The degree of intensity of 55 and 15 kD polypeptides, which were identified as the large and small subunit of rubisco, respectively, by SDS-PAGE analysis at 4% sucrose was significantly higher than that of other treatments, indicating that sucrose had an effect on both subunits. We subsequently examined whether the rubisco content and activity of being induced by sucrose is associated with rubisco activase. The rubisco activase content at 4% sucrose was higher than that of the other treatments. A similar change pattern was also observed in the activity of rubisco activase. The intensity of two 52 and 51 kD polypeptide bands at 4% sucrose was higher than that of corresponding bands of other treatments. The stimulatory and inhibitory effects of rubisco by sucrose seemed to be caused by rubisco activase.     

The CbbQO-type rubisco activases encoded in carboxysome gene clusters can activate carboxysomal form IA rubiscos

The CO₂-fixing enzyme rubisco is responsible for almost all carbon fixation. This process frequently requires rubisco activase (Rca) machinery, which couples ATP hydrolysis to the removal of inhibitory sugar phosphates, including the rubisco substrate ribulose 1,5-bisphosphate (RuBP). Rubisco is sometimes compartmentalized in carboxysomes, bacterial microcompartments that enable a carbon dioxide concentrating mechanism (CCM). Characterized carboxysomal rubiscos, however, are not prone to inhibition, and often no activase machinery is associated with these enzymes. Here, we characterize two carboxysomal rubiscos of the form IA^C clade that are associated with CbbQO-type Rcas. These enzymes release RuBP at a much lower rate than the canonical carboxysomal rubisco from Synechococcus PCC6301. We found that CbbQO-type Rcas encoded in carboxysome gene clusters can remove RuBP and the tight-binding transition state analog carboxy-arabinitol 1,5-bisphosphate from cognate rubiscos. The Acidithiobacillus ferrooxidans genome encodes two form IA rubiscos associated with two sets of cbbQ and cbbO genes. We show that the two CbbQO activase systems display specificity for the rubisco enzyme encoded in the same gene cluster, and this property can be switched by substituting the C-terminal three residues of the large subunit. Our findings indicate that the kinetic and inhibitory properties of proteobacterial form IA rubiscos are diverse and predict that Rcas may be necessary for some α-carboxysomal CCMs. These findings will have implications for efforts aiming to introduce biophysical CCMs into plants and other hosts for improvement of carbon fixation of crops.     

The identification and metabolism of GA9 and its metabolites in seed coats and shoots of pea,line G2

[³H]gibberellin A₉ was applied to shoots or seed parts of G2 pea to produce radiolabeled metabolites. These were used as markers during purification for the recovery of endogenous GA₉ and its naturally occurring metabolites. GA₉ and its metabolites were purified by HPLC, derivatized and examined by GC-MS. Endogenous GA₉, GA₂₀, GA₂₉ and GA₅₁ were identified in pea shoots and seed coats. GA₅₁-catabolite and GA₂₉-catabolite were also detected in seed coats. GA₇₀ was detected in seed coats following the application of 1 g of GA₉. Applied [³H]GA₉ was metabolized through both the 13-hydroxylation and 2-hydroxylation pathways. Labeled metabolites were tentatively identified on the basis of co-chromatography on HPLC with endogenous compounds identified by GC-MS. In shoots [³H]GA₅₁ and [³H]GA₅₁-catabolite were the predominant metabolites after 6 hrs, but by 24 hrs there was little of these metabolites remaining, while [³H]GA₂₉-catabolite and an unidentified metabolite predominated. In seed coats [³H]GA₅₁ was the initial product, later followed by [³H]GA₅₁-catabolite and an unidentified metabolite (different from that in shoots), with lesser amounts of [³H]GA₂₀, [³H]GA₂₉ and [³H]GA₂₉-catabolite. [³H]GA₇₀ was a very minor product in both cases. [³H]GA₉ was not metabolized by pea cotyledons.Edited by T.J. Gianfagna.Author for correspondence     

A soluble chloroplast protein catalyzes ribulosebisphosphate carboxylase/oxygenase activation in vivo

Ribulosebisphosphate carboxylase/oxygenase (EC 4.1.1.39) (rubisco) must be fully activated in order to catalyze the maximum rates of photosynthesis observed in plants. Activation of the isolated enzyme occurs spontaneously, but conditions required to observe full activation are inconsistent with those known to occur in illuminated chloroplasts. Genetic studies with a nutant of Arabidopsis thaliana incapable of activating rubisco linked two chloroplast polypeptides to the activation process in vivo. Using a reconstituted light activation system, it was possible to demonstrate the participation of a chloroplast protein in rubisco activation. These results indicate that a specific chloroplast enzyme, rubisco activase, catalyzes the activation of rubisco in vivo.     

GA3 induced flowering in Podophyllum hexandrum Royle: A rare alpine medicinal herb

Podophyllum hexandrum Royle, an important alpine herb, is the source of highly valued podophyllotoxin. The effect of some plant growth substances (GA₃, BAP & ABA), uniconazole (an inhibitor of GA biosynthesis), and a combination of GA₃ and uniconazole were examined in respect to influence on sprouting in rhizomes of P. hexandrum and on induction of flowering at a lower altitude. Amongst the various chemicals tested, GA₃ had a marked effect resulting in uniform sprouting and also induced flowering in about half of the treated rhizomes. While BAP also promoted early sprouting, delayed sprouting was seen in rhizomes treated with ABA. Uniconazole treatment, either alone or with GA₃ was found to inhibit flowering and also resulted in reduced plant height. GA₃ treatment of rhizomes from plants that was maintained for up to 30 months at a lower altitude also induced flowering thus replacing the normal chilling requirement of plants. These results suggest that treatment of GA₃ could be effectively used for inducing uniform sprouting and flowering in rhizomes of P. hexandrum grown at lower altitudes.     

Effects of GA3 on Plant Physiological Properties,Extraction, Subcellular Distribution and Chemical Forms of Pb in Lolium perenne

The effects of growth-promoting hormone gibberellic acid 3 (GA₃) on physiology, Pb phytoextraction, and metal detoxification mechanisms in Lolium perenne were studied. Results showed that addition of GA₃ alone at lower doses (1 or 10 μM) facilitated antioxidant defense of L. perenne under Pb stress, decreased the toxicity of Pb in plant shoot by increasing the proportion of Pb in cell wall, hence significantly enhanced photosynthesis and plant growth, as well as Pb uptake and accumulation in L. perenne (P < 0.05). However, these indicators showed the opposite changes when treated with GA₃ at a higher dose (100 μM). Of the total Pb in plant shoot, 36–51% was associated with cell wall, and 31–40% was soluble fraction, while 41.4–49.7% was NaCl extractable, 24.6–35.4% HAc extractable followed by other fractions. These findings suggest that Pb fixation by pectates and proteins in cell wall and sequestration in vacuole are responsible for Pb detoxification in plant, and the GA₃ at 1 μM appears to be optimal for enhancing Pb phytoextraction by L. perenne from Pb polluted soils.     

Immunoblot analysis of the expression of genes for barley rubisco activase inE. coli

Rubisco activity during photosynthesis is regulated by the rubisco activase, which facilitates the dissociation of RuBP and other inhibitory sugar phosphates from the active site of rubisco in an ATP-dependent reaction. In this paper, barleyRca genes (RcaA1,RcaA2 andRcaB) were expressed inE. coli and the activity of rubisco activase expressed was assayed biochemically by chromatography. Then the protein was identified electrophoretically by SDS-PAGE and detected immunologically by Western blot analysis using polyclonal antibodies raised against the kidney bean rubisco activase as probe. The band pattern of purified proteins on the polyacrylamide gel showed two polypeptides of 46 kD and 42 kD. Anti-rubisco activase antibodies reacted specifically with both polypeptides of 46 kD and 42 kD present in the crude extracts ofE. coli transformants. Therefore, it was found that the genes of barley rubisco activase was successfully expressed inE. coli as active forms of 46 kD and 42 kD.     

Effect of kinetin and GA3 on in vitro ovule embryo culture of Cucumis melo L.

The ability of Cucumis melo embryos of different ages to form plants in vitro was studied in order to rescue hybrid embryos between C. melo and Cucumis metuliferus. Plants were grown in a glasshouse at temperatures ranging from 15°CN-28°CD. Best results were obtained with ovule embryos excised 17 days after pollination. At this age, kinetin of 0.5 mg l^–1 was found optimal for culturing embryo development. Similar results were obtained with ovule embryos excised 14 days after pollination which cultured on 0.5 mg l^–1 kinetin with 0.5 mg l^–1 GA₃.     

Effects of root applications of gibberellic acid on photosynthesis and growth in C3 and C4 plants

The effects of root applications of gibberellic acid (GA₃) on growth and photosynthesis of 12 species of plants including C₃ monocots (Triticum aestivum L., wheat, Hordeum vulgare L., barley and Avena sativa L., oat), C₃ dicots (Vigna radiata L., mung bean, Cucurbita moschata L., squash and Capsicum annuum L., pepper), C₄ monocots (Zea mays L., corn, Sorghum vulgare L., sorghum and Panicum ramosum L., millet) and C₄ dicots (Amaranthus retroflexus L., pigweed, Kochia scoparis L., kochia and Gomphrena celosoides L., gomphrena) were evaluated. Relative growth rates (RGR) of barley, oat, squash, pepper, corn, sorghum, millet, pigweed and kochia were increased above the control by 12.7%, 9.9%, 11.3%, 10.7%, 19.2% 10.1%, 11.5%, 16.4% and 32.7% respectively, four days following optimum GA₃ treatments. There was no effect of GA₃ on RGR in wheat, mung bean, and gomphrena. Gibberellic acid decreased the chlorophyll content expressed on an area basis by 20.0%, 13.9%, 20.9%, 17.1%, 11.9% and 28.0% in barley, squash, pepper, sorghum, pigweed and kochia, respectively, while that of oat, wheat, mung bean, corn, millet and gomphrena remained unchanged. When photosynthetic rates were expressed per mg of chlorophyll, it showed that GA₃ could stimulate photosynthesis in barley, squash, pepper, sorghum, millet, pigweed and kochia by 20.4%, 20.6%, 16.5%, 17.4%, 10.4%, 24.2%, and 29.4%; while there was no effect in oat, wheat, mung bean, corn and gomphrena. An increase in leaf blade area and/or length of sheath were observed in GA₃ treated plants of oat, barley, mung bean, squash, pepper, corn, sorghum, millet and kochia. The transpiration rate remained unchanged following GA₃ treatment in all 12 species.This work was supported in part by the Fair Funds administered by the Pennsylvania Department of Agriculture. Contribution No. 39, Department of Horticulture, The Pennsylvania State University. Authorized for publications as paper no. 6886 in the journal series of the Pennsylvania Agricultual Experiment Station.Research assistant and assistant professor respectively.     

The effect of light quality and gibberellic acid (GA3) on photosynthesis and respiration rates of pea seedlings

CO₂ exchange were measured on pea seedlings (Pisum sativum L. var. Bördi) cultivated from seeds imbibed either in water (C-plants) or in gibberellic acid (GA₃) at the concentration of 25 g/1 (GA-plants), and then grown under 17 W/m² blue light (B-plants) or 11 W/m² red light (R-plants).When measured under the same light conditions as during growth the net photosynthesis (APS) rate in B-plants was about twice higher than that in R-plants. Dark respiration (DR) rate was 70% higher in B- than in R-plants. Red light retarded the development of photosynthetic activity, but GA₃ suppressed this effect. The hormone enhanced net photosynthesis and dark respiration to the same extent.When measured under saturating white light net photosynthesis rate of C-plants was also two times higher in B-plants than in R-plants. Growth conditions had only a slight effect on the APS of GA-plants under white light. APS rates of GA-plants grown under red light were higher under white light than those of C-plants, but lower than those of plants grown under blue light.We assume that blue light induced formation of plants that were adapted to higher light intensity: red light had an opposite effect, whereas gibberellic acid induced formation of plants that were adapted to medium light intensity.     

外源赤霉素对紫斑牡丹种子萌发的影响

以紫斑牡丹种子为试验材料,研究不同浓度的赤霉素(GA_3)处理对种子生根以及生根过程中营养物质、酶活性和内源激素水平变化的影响,为探讨紫斑牡丹种子萌发机制提供依据。结果表明:(1)GA_3处理能够促进种子生根,并以300 mg/L GA_3处理对种子生根效果最好,与对照相比可提前14.67 d生根,生根率可达71.00%。(2)与对照相比,GA_3处理可以在0～15 d时促进种子淀粉水解和可溶性糖的积累,并加速可溶性蛋白的消耗,在0～30 d促进过氧化物酶(POD)活性的提高,从而促进种子萌发生根。(3)在种子沙藏生根过程中,种子脱落酸(ABA)含量呈下降趋势,赤霉素(GA)、玉米素核苷(ZR)和吲哚乙酸(IAA)含量均表现出先上升后下降的趋势,与对照相比,GA_3处理可使种子GA、ZR和IAA的含量在沙藏前期明显上升,以解除种子休眠。研究发现,外源GA_3处理可以调控紫斑牡丹种子内源激素含量和POD活性的变化,促进营养物质转化,从而提前解除种子休眠使其萌发。     

外源GA3对紫斑牡丹幼苗叶片解剖结构与光合特性及内源激素含量的影响

以1年生紫斑牡丹幼苗为试验材料,采用不同浓度(0、100、300、500 mg/L)赤霉素(GA_3)喷施叶片处理,通过透射电镜、扫描电镜、光学显微镜观察幼苗叶片解剖结构,光合仪测定幼苗光合参数并以酶联免疫吸附法测叶片内源激素含量,探究外源GA_3对紫斑牡丹幼苗叶片解剖结构、光合特性和内源激素水平的影响。结果表明:(1)低浓度GA_3处理的紫斑牡丹叶肉细胞增大,栅栏组织外层细胞中叶绿体数量增加,高浓度GA_3处理则与之相反;GA_3处理叶片的栅栏组织/海绵组织比值(P/S)、组织结构紧密度(CTR)均下降,而其组织结构疏松度(SR)增加;GA_3处理的幼苗叶片的叶肉细胞内各叶绿体大小显著大于对照,随着GA_3处理浓度增加,紫斑牡丹叶肉细胞内叶绿体的体积趋于增大,类囊体垛叠凝聚逐渐松散,叶绿体上淀粉颗粒在300 mg/L GA_3处理中较明显;叶片气孔长度、宽度、气孔器大小、气孔开度和气孔密度随着GA_3浓度升高先升高后下降,同时叶片上表皮角质层厚度随GA_3浓度的升高而增加。(2)紫斑牡丹叶片净光合速率(P_n)、气孔导度(Cond)、蒸腾速率(T_r)、水分利用率(WUE)在100和300 mg/L GA_3处理下大都显著高于对照,且300 mg/L GA_3处理显著高于其余处理,而其在500 mg/L GA_3处理下显著低于对照。(3)紫斑牡丹叶片脱落酸(ABA)和吲哚乙酸(IAA)含量均在500 mg/L GA_3下显著高于对照,而在其余浓度处理下不同程度低于对照,叶片内源玉米素核苷(ZR)和GA_3含量均在300 mg/L GA_3处理下显著高于其余处理和对照,而其余处理相比对照均无显著变化;叶片的ZR/ABA、ZR/IAA、ZR/GA_3和(IAA+GA_3+ZR)/ABA比值都在300 mg/L GA_3处理下显著高于其他处理,叶片的IAA/ABA和ABA/GA_3比值均在500 mg/L GA_3处理下显著高于其他处理。研究发现,适宜浓度外源GA_3处理,能显著提高紫斑牡丹幼苗叶片光合速率、水分利用效率及蒸腾速率,调节植物体内源激素的含量及平衡,从而使叶片能合成较多有机物,促进幼苗生长。     

Development of a one-step enzyme immunoassay for the quantitation of gibberellin A3 in barley seeds

The development of a sensitive and specific enzyme immunoassay for GA₃ is reported. This method was based on the use of peroxidase labelled GA₃ and immobilized antibodies. In order to obtain a rapid immunoassay, several steps of purification were analyzed to show their necessity. Barley seed extracts were assayed at different steps of purification to exhibit the effect of extract components on the assay. It was demonstrated that HPLC had to be performed when a selective quantitation of GA₃ was required. This assay allowed GA₃ to be measured with reproducibility as its unmethylated form and the quantitation of GA₃ in barley seeds with this enzyme immunoassay was correlated to a GC-MS method.Abbreviations GA₃ gibberellin A₃ - EIA enzyme immunoassay - DMF dimethylformamide - TEA tri(n)ethylamine - BSA bovine serum albumin - OVA ovalbumine - ECF ethylchloroformate - PB phosphate buffer     

Exogenous GA<Subscript>3</Subscript> overcomes bud deterioration in tulip (<Emphasis Type="Italic">Tulipa gesneriana</Emphasis> L.) bulbs during dry storage by promoting endogenous IAA activity in the internodes

Endogenous free IAA was examined with an immunohistochemical method for its involvement in the reduction of bud deterioration after GA₃ was injected into the bulbs. We found that tulip bulbs stored at 20°C constantly developed severe bud deterioration, whereas the symptoms of deterioration was lighter in the bulbs with GA₃ injection and not observed in the bulbs with 4°C treatment. 73% success in overcoming bud deterioration was achieved in 20°C with GA₃ treatment after 8 weeks of bulb storage, and the success rate was 7% after 12 weeks of storage. IAA was detected in the parenchyma cells in the internodes of the shoot after the bulbs were stored at 4°C or at 20°C with GA₃ injection for 4 weeks, but little was detected in the bulbs stored at 20°C constantly. Moreover, a weak IAA signal was present in between the cells of the internodes irrespective of bulb treatment. After planting, the bulbs that had been treated differently exhibited different flowering ability. The bulbs stored at 4°C for 4, 8 and 12 weeks attained high flowering percentage, which was lower in the 20°C with GA₃ treatment and lowest in the 20°C treatment. It may be concluded that GA₃ injection decreases bud deterioration of tulip bulbs during dry storage at 20°C by promoting the endogenous IAA in the internodes.