The foliage of fifteen day-old Vigna radiata seedlings was sprayed with water (control) or with a 10–10 M, 10–8Mor 10–6 Maqueous solution of 28-homobrassinolide (HBR). Samples of the treated plant material, collected at 30/50 days after sowing (DAS), were assessed
for the activities of carbonic anhydrase (CA) and nitrate reductase (NR); leaf chlorophyll content (Chl); stomatal conductance (gs); carboxylation efficiency (CE), and net photosynthetic rate (PN). 28-homobrassinolide generated a significant impact on all these
characteristics and on seed yield at harvest. Among the treatments, 10–8Mproved best, in which case the values for the above six physiological parameters in 30 days-old plants increased by, respectively, 31, 29, 27, 28, 29 and 33% over the control. Moreover, the harvest weight of the seeds of these plants was 27% larger than in the control

A. Ahmad

B. Ali

Q. Fariduddin

S. Hayat

The foliage of fifteen day-old Vigna radiata seedlings was sprayed with water (control) or with a 10–10 M, 10–8Mor 10–6 Maqueous solution of 28-homobrassinolide (HBR). Samples of the treated plant material, collected at 30/50 days after sowing (DAS), were assessed

for the activities of carbonic anhydrase (CA) and nitrate reductase (NR); leaf chlorophyll content (Chl); stomatal conductance (gs); carboxylation efficiency (CE), and net photosynthetic rate (PN). 28-homobrassinolide generated a significant impact on all these

characteristics and on seed yield at harvest. Among the treatments, 10–8Mproved best, in which case the values for the above six physiological parameters in 30 days-old plants increased by, respectively, 31, 29, 27, 28, 29 and 33% over the control. Moreover, the harvest weight of the seeds of these plants was 27% larger than in the control

Fariduddin, Q.

Hayat, S.

Ali, B.

Ahmad, A.

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Acta Bot. Croat. 65 (1), 19–23, 2006 CODEN:ABCRA25ISSN 0365–0588Effect of 28-homobrassinolide on the nitratereductase, carbonic anhydrase activities andnet photosynthetic rate in Vigna radiataQAZI FARIDUDDIN, SHAMSULHAYAT, BARKETALI, AQILAHMAD*Plant Physiology Section, Department of Botany, Aligarh Muslim University,Aligarh-202 002, IndiaThe foliage of fifteen day-old Vigna radiata seedlings was sprayed with water (control) orwith a 10–10 M, 10–8 M or 10–6 M aqueous solution of 28-homobrassinolide (HBR). Sam-ples of the treated plant material, collected at 30/50 days after sowing (DAS), were as-sessed for the activities of carbonic anhydrase (CA) and nitrate reductase (NR); leaf chlo-rophyll content (Chl); stomatal conductance (gs); carboxylation efficiency (CE), and netphotosynthetic rate (PN). 28-homobrassinolide generated a significant impact on all thesecharacteristics and on seed yield at harvest. Among the treatments, 10–8 M proved best, inwhich case the values for the above six physiological parameters in 30 days-old plants in-creased by, respectively, 31, 29, 27, 28, 29 and 33% over the control. Moreover, the har-vest weight of the seeds of these plants was 27% larger than in the control.Key words: Brassinosteroids, carbonic anhydrase, nitrogen, nitrate reductase, photosyn-thetic rate, seed, yield, Vigna radiataIntroductionBrassinosteroids (BRs) are steroidal plant hormones, actively involved in various phys-iological processes and are essential for plant growth and development (BISHOP andYOKOTA2001, SASSE 2003). BRs have pleiotropic effects and can induce a broad spectrumof cellular responses including stem elongation, pollen tube growth, leaf bending andepinasty, root inhibition, induction of ethylene biosynthesis, proton pump activation, xy-lem differentiation and the regulation of gene expression (LI and CHORY1999,MUSSIG et al.2002, SASSE 2003). BRs have been identified in 27 families of higher plants and three fami-lies of lower plants. They are distributed in various plant parts including roots (SASSE2003). BRs are also employed in agricultural production. Previous studies have demon-strated that BRs influence seed germination, plant growth, nitrogen fixation, senescence,and leaf abscission and enhance tolerance to cold stress, salt stress, and diseases (CLOUSEand SASSE 1998, KHRIPACH et al. 2000, RAO et al. 2002, HAYAT et al. 2003). In the presentstudy, dilute aqueous solutions, of 28-homobrassinolide were applied to the foliage, thesite of active metabolism, to influence plant growth and productivity.ACTA BOT. CROAT. 65 (1), 2006 19* Corresponding author, e-mail: aqil_ahmad@lycos.comMaterial and methodsThe seeds of Vigna radiata (L.) Wilczeck cv. T 44 were obtained from the NationalSeed Corporation Ltd., New Delhi. Vigna radiata belongs to the family Fabaceae. Healthyseeds were surface sterilized with 0.01% mercuric chloride followed by inoculation withRhizobium sp. The seeds were then sown in earthen pots (25 cm2), filled with a mixture ofsandy loam and farmyard manure in a ratio of 9:1. Fifteen day-old seedlings were sprayedwith water (control) or with a 10–10 M, 10–8 M or 10–6 M aqueous solution of 28-homo-brassinolide (HBR). Each treatment was replicated thrice. The plants were sampled at 30and 50 d after sowing (DAS) to study various characteristics. Shoot dry mass was deter-mined after dehydrating the plants at 80 °C for 24 h. Nitrate reductase (NR) activity was de-termined in fresh leaf samples, following the method of JAWORSKI (1971). Leaf nitrogenwas estimated following the procedure of LINDNER (1944). Chlorophyll content was quan-tified by the procedure of MACKINNEY (1941). The net photosynthetic rate and stomatalconductance were measured using a LI-6200 portable photosynthetic system (LI-COR,NE, USA). The carboxylation efficiency was calculated by employing a formula given byTIWARI et al. (1998). The CA activity in the leaves was measured following the proceduredescribed by DWIVEDI and RANDHAWA (1974). The data were analyzed for variance byone-way ANOVA. Mean values were tested by least significant difference at the 0.05 levelof probability using the statistical package SPSS 7.5.Results and discussionThe leaves of 30 and 50 day-old plants, sprayed with 28-homobrassinolide (HBR) atday 15, photosynthesized at a significantly higher rate than the control (Tab. 1). Moreover,a maximum increase of 33 and 30 %, over the control, at 30 and 50 DAS was recorded inthe plants treated with 10–8 MHBR. Similarly, BRAUNandWILD (1984a) reported enhance-ment in the photosynthetic parameters in BR-treated wheat and mustard plants because ofthe activation of CO2 fixation. However, we are of the opinion that, like other hormones,HBR might be acting at multiple points. One of them, which has been approached from se-lected angles in the present study, may be the facilitated diffusion of CO2 followed by its re-duction. A clear-cut response of the plants to applied HBR is evident from an increase inthe stomatal conductance (Tab. 1), facilitating the diffusion of carbon dioxide into thestomatal cavity. The next important stage is the entry of CO2 across the membrane of thechloroplast, which is catalyzed by the enzyme carbonic anhydrase (CA). Finally, the CO2 isreduced by RuBPCO in the chloroplast stroma. The values of carbonic anhydrase (Tab. 1)and of RuBPCO (BRAUN and WILD 1984b, YU et al. 2004) are elevated by BRs. The re-sponse of the plants, in terms of CA activity, was best at 10–8 M HBR exhibiting, respec-tively, a 31 and 26% increase over the control, at 30 and 50 DAS. The exact nature bywhich HBR exerts its influence on enzyme activity is not understood but KHRIPACH et al.(1999) suggested that BRs affect gene expression and/or membrane functions. The formerwould be corroborated by the observed (Tab. 1) increase in the activity of a further enzyme,nitrate reductase (NR). Comparable observations have also been reported in rice (MAI et al.1989), maize (SHEN et al. 1990) and wheat (SAIRAM 1994), treated with BRs. This specificprotein is responsible for the initiation of nitrogen metabolism and, consequently, for thelevel of protein synthesis. KALINICHet al. (1985) have reported higher protein content in theplants of Phaseolus vulgaris and Phaseolus aureus, treated with BRs and assigned the im-20 ACTA BOT. CROAT. 65 (1), 2006FARIDUDDIN Q., HAYAT S., ALI B., AHMAD A.pact of the hormone on the process of protein synthesis, as a possible reason. Moreover,high nitrogen content (Tab. 1) may be an additional factor involved in increased NR activ-ity, as nitrate is known to induce functional NR, essential for nitrate reduction (SAROOP etal. 1998) by producing ‘nitrate sensing’proteins of an unknown nature which possibly bindwith the regulatory regions at the NR-genes and turn on the expression of NR-mRNA(REDINBAUGH and CAMPBELL1991). However, the total nitrate-reducing capacity in a plantsystem depends on three important factors (a) the availability of nitrate (the substrate) inthe cytoplasm (b) the level of nitrate reductase and (c) its activity level (CAMPBELL1999).The increase in the level of CO2 in the stroma is also associated with an accelerated rateof its reduction which is evident from the increased carboxylation efficiency (CE), in re-sponse to HBR, over the control (Tab. 2). The leaves of the plants treated with HBR notonly photosynthesized at a faster rate, with higher chlorophyll content but also possessedan extended period of metabolic activity because HBR delayed senescence (data not in-cluded), as compared to the control. The overall effect of HBR was reflected by the higherdry mass of the plants and increased pod bearing capacity, resulting in improved seed yieldat harvest time.ACTA BOT. CROAT. 65 (1), 2006 2128-HOMOBRASSINOLIDE AND ENZYME ACTIVITYTab. 1. Net photosynthetic rate (PN) [µmol (CO2) m–2 s–1], stomatal conductance (gs) [mol m–2 s–1],carbonic anhydrase (CA) activity [mol (CO2) kg–1s–1], nitrate reductase (NR) activity [n mol(NO2) h–1 g–1] and nitrogen (N) content [%of DM] in Vigna radiata plants, sprayed with wa-ter (control), 28-homobrassinolide (HBR: 10–10, 10–8 or 10–6 M) at 15 d, after sowing (DAS)and sampled at 30 and 50 DAS. LSD for p = 0.05PN gs CAActivity NR Activity Nitrogen30DAS50DAS30DAS50DAS30DAS50DAS30DAS50DAS30DAS50DASControl 14.50 12.00 0.367 0.325 1.80 1.55 412 300 3.14 2.0910–10 17.80 13.75 0.445 0.400 2.19 1.82 495 345 3.55 2.2510–8 19.31 15.16 0.471 0.438 2.36 1.96 535 376 3.82 2.3910–6 17.00 13.18 0.417 0.371 2.07 1.70 470 351 3.57 2.26LSD 0.90 1.00 0.017 0.016 0.09 0.15 28 15 0.12 0.10Tab. 2. Carboxylation efficiency (CE) [mol m–2s–1], leaf chlorophyll content (Chl) [g kg–1 of FM],dry mass (g) of shoot, pod number plant–1 and seed yield (g) plant–1 in Vigna radiata plants,sprayed with water (control), 28-homobrassinolide (HBR: 10–10, 10–8 or 10–6 M) at 15 d, af-ter sowing (DAS) and sampled at 30 and 50 DAS. LSD for p = 0.05CE Chl DM PodnumberSeedyield30 DAS 50 DAS 30 DAS 50 DAS 30 DAS 50 DASControl 0.041 0.033 1.31 1.41 2.30 6.13 26.00 3.6510–10 0.049 0.040 1.52 1.66 2.51 6.63 31.60 3.9510–8 0.053 0.042 1.67 1.71 2.95 7.00 33.60 4.6410–6 0.047 0.036 1.43 1.65 2.56 6.64 30.14 3.90LSD 0.003 0.002 0.09 0.10 0.15 0.19 1.55 0.30AcknowledgementsAuthors are thankful to Dr. B. N. Vyas (Godrej Agrovet Ltd., Mumbai, India) forgenerous supply of 28-homobrassinolide. The financial support (Grant No. 9/11 (328)/2001-EMR-1) to the first author furnished by the Council of Scientific and IndustrialResearch, New Delhi, is gratefully acknowledged.ReferencesBISHOP, G. J., YOKOTA, T., 2001: Plant steroid hormones, brassinosteroids: current high-lights of molecular aspects on their synthesis/metabolism, transport, perception and re-sponse. Plant Cell Physiol. 42, 114–120.BRAUN, P.,WILD, A., 1984a: The influence of brassinosteroid on growth and parameters ofphotosynthesis of wheat and mustard plants. J. Plant Physiol. 116, 189–196.BRAUN, P., WILD, A., 1984b: The influence of brassinosteroid on growth promotingsteroidal lactone, on development and CO2-fixation capacity of intact wheat and mus-tard seedlings In Sybesma, C (ed,), Advances in photosynthesis research 3, 461–464.Martinus Nijhoff/ Dr. W Junk Publishers, The Hague.CAMPBELL, H. W., 1999: Nitrate reductase structure, function and regulation: bridging thegap between biochemistry and physiology. Ann. Rev. Plant Physiol. Plant Mol. Biol.50, 277–303.CLOUSE, S. D., SASSE, J. M., 1998: Brassinosteroids: essential regulators of plant growthand development. Ann. Rev. Plant Physiol. Plant Mol. Biol. 49, 427–451.DWIVEDI, R. S., RANDHAWA N. S., 1974: Evaluation of a rapid test for hidden hunger ofzinc in plants. Plant Soil 40, 445–451.HAYAT, S., AHMAD, A., FARIDUDDIN, Q., 2003: Brassinosteroids. A regulator of 21st cen-tury, In HAYAT, S., AHMAD, A. (eds.), Brassinosteroids: bioactivity and crop productiv-ity,231 – 246. 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Effect of 28-homobrassinolide on the nitrate reductase, carbonic anhydrase activities and net photosynthetic rate in Vigna radiata

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