Ureide biogenesis and the enzymes of ammonia assimilation and ureide biosynthesis in nitrogen fixing pigeonpea (Cajanus cajan) nodules

Allantoic acid production from IMP, XMP, inosine, xanthosine, hypoxanthine, xanthine, uric acid and allantoin was investigated by incubating each of these substrates withCajanus cajan cytosol and bacteroid fractions separately in the presence and absence of NAD+ and allopurinol. Allantoic acid synthesis by bacteroid fraction could only be observed with uric acid and allantoin as substrates. Addition of NAD⁺ or allopurinol to the reaction mixtures had no effect. However, with cytosol fraction, allantoic acid was produced by each of these substrates, with maximum rate with allantoin. With NAD⁺ or with allopurinol, allantoic acid was produced only with uric acid and allantoin as substrates. NADH production with cytosol fraction could again be observed with all the substrates. Except with uric acid and allantoin, allopurinol completely inhibited NADH formation. Regardless of the presence or absence of allopurinol, none of the substrates exhibited significant activity with bacteroid fraction. Based on the activities of glutamine synthetase, glutamate synthase, glutamate dehydrogenase, aspartate aminotransferase, asparagine synthetase, nucleotidase, nucleosidase, xanthine de-hydrogenase, uricase and allantoinase and their intracellular localisation in various nodule fractions, a probable pathway for the biogenesis of ureides in pigeonpea nodules has been proposed     

Isolation and culture of protoplasts of pigeonpea (Cajanus cajan L.)

Summary High yields of protoplasts were obtained from leaves of aseptically grown plants and calli originated from different explants, in several cultivars of Cajanus cajan L. The protoplasts divided to form cell clusters in modified KM 8p medium and developed to protocolonies after dilution with liquid Caboche's medium within three to four weeks of culture. The protocolonies proliferated to form green calli on solid Caboche's medium. No shoots or plants were obtained.Abbreviations BAP 6-benzylaminopurine - NAA -napthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Kin kinetin - Zea zeatin - Adn S adenine sulphate - GA ₃ gibberellic acid     

Purification and characterization of urease from dehusked pigeonpea (Cajanus cajan L) seeds

Urease has been purified from the dehusked seeds of pigeonpea (Cajanus cajan L.) to apparent electrophoretic homogeneity with approximately 200 fold purification, with a specific activity of 6.24 x10(3) U mg(-1) protein. The enzyme was purified by the sequence of steps, namely, first acetone fractionation, acid step, a second acetone fractionation followed by gel filtration and anion-exchange chromatographies. Single band was observed in both native- and SDS-PAGE. The molecular mass estimated for the native enzyme was 540 kDa whereas subunit values of 90 kDa were determined. Hence, urease is a hexamer of identical subunits. Nickel was observed in the purified enzyme from atomic absorption spectroscopy with approximately 2 nickel ions per enzyme subunit. Both jack bean and soybean ureases are serologically related to pigeonpea urease. The amino acid composition of pigeonpea urease shows high acidic amino acid content. The N-terminal sequence of pigeonpea urease, determined up to the 20th residue, was homologous to that of jack bean and soybean seed ureases. The optimum pH was 7.3 in the pH range 5.0-8.5. Pigeonpea urease shows K(m) for urea of 3.0+/-0.2 mM in 0.05 M Tris-acetate buffer, pH 7.3, at 37 degrees C. The turnover number, k(cat), was observed to be 6.2 x 10(4) s(-1) and k(cat)/K(m) was 2.1 x 10(7) M(-1) s(-1). Pigeonpea urease shows high specificity for its primary substrate urea.     

Plant regeneration via somatic embryogenesis in pigeonpea (Cajanus cajan L. Millsp)

Efficient plant regeneration via somatic embryogenesis has been developed in pigeonpea. Cotyledon and leaf explants from 10-day-old seedlings produced embryogenic callus and somatic embryos when cultured on Murashige and Skoog (MS) medium supplemented with 10 μm thidiazuron (TDZ). Subsequent withdrawal of TDZ from the induction medium resulted in the maturation and growth of the embryos into plantlets on MS basal medium. The rooted plantlets were transferred and acclimatized on vermiculite where they showed normal morphological characters. Received: 23 December 1996 / Revision received: 22 July 1997 / Accepted: 2 August 1997     

Variability in nuclear DNA content within pigeonpea,Cajanus cajan (Fabaceae)

4C DNA values have been estimated in 16 cultivars ofCajanus cajan by cytophotometry. The values range between 6.19 pg to 7.97 pg, a 23.6% variation. The cultivars form four groups which differ significantly from each other but have insignificant difference within them. The implications of this variability with respect to the heterogeneity and origin of this legume crop are discussed.     

Nodulation,nitrogen fixation and nitrogen uptake in pigeonpea (Cajanus cajan (L.) Millsp) of different maturity groups

Summary The seasonal patterns of nodulation, acetylene reduction, nitrogen uptake and nitrogen fixation were studies for 11 pigeonpea cultivars belonging to different maturity groups grown on an Alfisol at ICRISAT Center, Patancheru, India. In all cultivars the nodule number and mass increased to a maximum around 60–80 days after sowing and then declined. The nodule number and mass of medium- and late-maturing cultivars was greater than that of early-maturing cultivars. The nitrogenase activity per plant increased to 60 days after sowing and declined thereafter, with little activity at 100 days when the crop was flowering. At later stages of plant growth nodules formed down to 90 cm below the soil surface but those at greater depth appeared less active than those near the surface. All the 11 cultivars continued to accumulate dry matter until 140 days, with most biomass production by the late-maturing cultivars (up to 11 t ha⁻¹) and least by the early-maturing determinate cultivars (4 t ha⁻¹). Total nitrogen uptake ranged from 69 to 134 kg ha⁻¹. Nitrogen fixation by pigeonpea was estimated as the difference in total nitrogen uptake between pigeonpea and sorghum and could amount to 69 kg N ha⁻¹ per season, or half the total nitrogen uptake. Fixation by pigeonpea increased with crop duration, but there were differences within each maturity group. The limitations of the methods used for estimating N₂ fixation by pigeonpea are discussed. Submitted as J.A. No. 552 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT).     

Cellular compartmentation of ureide biogenesis in root nodules of cowpea (Vigna unguiculata (L.) Walp.)

Cowpea (Vigna unguiculata (L.) Walp.) nodules have been investigated by means of cytochemical and immunocytochemical procedures at the ultrastructural level in order to assess the role of the uninfected cells in ureide biogenesis. Uricase activity in the nodules was shown by cytochemical methods to be localized exclusively in the numberous large peroxisomes confined to the uninfected cells; the small peroxisomes in the infected cells did not stain for uricase. Uricase was also localized in the peroxisomes of uninfected cells by immunogold techniques employing polyclonal antibodies against nodule-specific uricase of soybean. There was no labeling above background of any structures in the infected cells. The results indicate that the uninfected cells are essential for ureide biogenesis in cowpea. Although tubular endoplasmic reticulum, the presumptive site of allantoinase, increases greatly in the uninfected cells during nodule development, it virtually disappears as the nodules mature. The inconsistency between the disappearance of the tubular endoplasmic reticulum from older nodules and the high allantoinase activity reported for older plants remains to be explained.Abbreviations DAB 3,3-diaminobenzidine - ER endoplasmic reticulum - GARG goat anti-rabbit immunoglobulin G - IgG immunoglobulin G - kDa knodalton - Mr apparent molecular mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

In vitro regeneration and transformation of pigeonpea [Cajanus cajan (L.) Millsp]

A requirement for generating transgenic pigeonpea [Cajanuscajan (L.) Millsp] plants is the development of a highly efficientin vitro regeneration procedure. This goal was achieved byusing germinated seedlings grown on B5 medium supplemented with 10 mgl^–1 6-benzylaminopurine, which induced differentiatingcallus formation in the cotyledonary node region. The calli were transferred onB5 medium with 0.2 mg l^–1 6-benzylaminopurine toobtain shoot induction. Elongated shoots were then further cultured on a B5hormone-free medium for rooting. Using this regeneration system transgenicpigeonpea plants were obtained both by particle bombardment andAgrobacterium tumefaciens-mediated gene transfer. Thepresence of the transgenes in the pigeonpea genome was confirmed by GUS assays,PCR and Southern hybridisation. The transgenic rooted plants were successfullytransferred to soil in the greenhouse. GUS and PCR assays of T1 progeniesconfirmed that the transgenes were stably transmitted to the next generation.This is the first report of successful use ofAgrobacteriumas well as particle bombardment for production of transgenic pigeonpea plants.     

Characterization of a proteinase inhibitor from Cajanus cajan (L.)

A protein proteinase inhibitor (PI) has been purified from pigeonpea Cajanus cajan (L.) PUSA 33 variety by acetic-acid precipitation, salt fractionation and chromatography on a DEAE-Cellulose column. The content of inhibitor was found to be 15 mg/20 g dry weight of pulse. The molecular weight of the inhibitor as determined by SDS-PAGE under reducing conditions was found to be about 14,000. It showed inhibitory activity toward proteolytic enzymes belonging to the serine protease group, namely trypsin and alpha-chymotrypsin. The inhibitory activity was stable over a wide range of pH and temperatures. Estimation of sulfhydryl groups yielded one free cysteine and at least two disulfide linkages. N-terminal sequence homology suggests that it belongs to the Kunitz inhibitor family. Structural analysis by circular dichroism shows that the inhibitor possesses a largely disordered structure.     

Sex allocation and mating systems in pigeonpea,Cajanus cajan (Fabaceae)

Sex allocation theory predicts that: (1) resources allocated to androecium should decrease with an increase in selfing, (2) a decrease in androecium biomass should be accompanied by an increase in the biomass of pistils, and (3) a decrease in androecium biomass should be coupled with a decrease in flower size, specifically corolla biomass. Another predicted change in reproductive traits associated with variation in selfing concerns seed to ovule ratios, but does not directly stem from sex allocation theory. It has been postulated that seed to ovule ratios should be positively correlated with the amount of selfing. These predictions were tested for six accessions of pigeonpea,Cajanus cajan L., that differed in selfing rates. The results were remarkably in accordance with the predictions. We conclude that sex allocation theory provides a powerful tool to understand the evolution of many reproductive traits in plants.     

Measurement of N2-fixation in field-grown pigeonpea [Cajanus cajan (L.) Millsp.] using15N-labelled fertilizer

Two experiments were carried out from 1981 to 1983 in Vertisol field at ICRISAT Center, Patancheru, India to measure N₂-fixation of pigeonpea [Cajanus cajan (L.) Millsp.] using the¹⁵N isotope dilution technique. One experiment examined the effect of control of a nodule-eating insect on fixation while another in vestigated the effect of intercroping with cereals on fixation and the residual effect of pigeonpea on a succeeding cereal crop. Although both experiments indicated that at least 88% of the N in pigeonpea was fixed from the atmosphere, one result is considered fortuitous in view of the differential rates of growth of the legume and the control, sorghum [Sorghum bicolor (L.) Moench]. The difference method of calculation in dieated negative fixation and the results emphasized the problem of finding a suitable nonfixing control. In a second experiment, when all plants were confined to a known volume of soil to which¹⁵N fertilizer was added in the field, these problems were overcome, and isotope dilution and difference methods gave similar results of N₂-fixation of about 90%. In intercropped pigeonpea 96% of the total N was derived from the atmosphere. This estimate might be an artifact. There was no evidence of benefit from N fixed by pigeonpea to intercropped sorghum plants. Plant tissue¹⁵N enrichments of cereal crops grown after pigeonpea indicated that the cereal derived some N fixed by the previous pigeonpea. Thus residual benefits to cereals are not only an effect of ‘sparing’ of soil N.     

Improved growth of Cajanus cajan (L.) Millsp. in an unsterile tropical soil by three mycorrhizal fungi

Under glasshouse conditions Cajanus cajan plants grown in a dark red latosol were fertilized with soluble simple superphosphate and hardly soluble rock phosphate and inoculated with three VA mycorrhizal fungi (M₁, Gigaspora margarita; M₂, Scutellospora verrucosa; M₃, Acaulospora rehmii) from the Cerrado ecosystem, Brazil. Only with rock phosphate plant growth was significantly increased by all fungi. Enhanced P uptake corresponded with higher yields and proved to be a characteristic of the VA myccorhizae. A definite relationship between infection intensity and efficiency of VA mycorrhizae was not detected. Spore production was generally more pronounced in the treatment with rock phosphate, especially with M₁ and M₂. Nodulation of Cajanus cajan was greatly improved by all fungi in the treatment with rock phosphate. It is suggested that the increased plant development and nodulation was due to improved uptake of P by mycorrhiza.     

Plant regeneration in pigeonpea [Cajanus cajan (L.) Millsp.] by organogenesis

A method for regenerating pigeonpea [Cajanus cajan (L.) Millsp.] plants has been developed using distal cotyledonary segments of mature seeds as explants. A large number of shoot buds were induced directly from explants of genotypes T-15-15 and GAUT-82-90 when cultured on six different basal media fortified with 22.2 μm N⁶-benzylaminopurine, 2.3 μm kinetin, and 271 μm adenine sulfate. The shoot buds developed into shoots when they were subcultured on the same medium but with one-tenth concentrations of cytokinins and adenine sulfate. The shoots elongated by subculturing first two to three times on Murashige and Skoog (MS) basal medium supplemented with 2.22 μm N⁶-benzylaminopurine and 0.54 μm α-naphthaleneacetic acid or on half-strength MS medium containing 2.89 μm gibberellic acid, and then once on the same medium without growth regulators. Elongated shoots were rooted with 80–85% efficiency on MS medium with 4.92 μm indole-3-butyric acid and the plantlets were transferred for hardening. Plants survival in pots was 70–75%. This method may be useful for improving the crop through genetic manipulations. Received: 11 August 1997 / Revision received: 12 January 1998 / Accepted: 30 January 1998     

Genotype-Dependent morphogenetic potentiality of various explants of a food legume,the pigeon pea (Cajanus cajan L.)

Summary The morphogenetic response of various explants of seven different cultivars of a food legume, the pigeon pea (Cajanus cajan L.), has been studied. The stimulation and elongation of shoot buds into shoots derived from the mature embryo axis and intact seed on Murashige and Skoog’s medium supplemented with 2.32µM kinetin and 22.2µM benzyladenine was found to be optimum in Murashige and Skoog’s medium supplemented with 0.46µM kinetin, 0.53µM naphthalene acetic acid, and 0.29µM gibberellic acid. Even though the response of these two explants for formation of shoot buds in all the genotypes is 30–100% depending on media composition, subsequent growth and elongation of these shoot buds into plants is genotype dependent and is restricted to two genotypes. Cotyledon and epicotyl explants of pigeon pea cultivars on the other hand differentiated directly into four to eight and two to four shoots, respectively, depending on the media composition and genotype. In vitro rhizogenesis of regenerated shoots was 80% and the survival of these plantlets in the field was 70–80%. NCL Communication no.: 5667.     

Genetic variation of trypsin and chymotrypsin inhibitors in pigeonpea [Cajanus cajan (L.) Millsp.] and its wild relatives

Variation in the trypsin inhibitors (TIs) and the chymotrypsin inhibitors (CIs) among 69 pigeonpea [Cajanus cajan (L.) Millsp.] strains from a wide geographical distribution and among 17 accessions representing seven wild Cajanus species was studied by electrophoretic banding pattern comparisons and by spectrophotometric activity assays. The TI and CI electrophoretic migration patterns among the pigeonpea strains were highly uniform but varied in the inhibitor band intensities. The migration patterns of the inhibitors in the wild Cajanus species were highly species specific. The mean TI activity of pigeonpea strains (2279 units) was significantly higher than that of the wild Cajanus species (1407 units). However, the mean CI activity in the pigeonpea strains (62 units) was much lower than that in the wild species (162 units). Kenya 2 and ICP 9151 were the lowest and the highest, respectively, in both the TI and CI activities among all the pigeonpea strains used in this study. A highly-significant positive correlation was observed between the TI and CI activities. The Bowman-Birk type inhibitors with both TI and CI activities were identified in all the pigeonpea strains and also in the accessions of all the wild species except C. volubilis (Blanco) Blanco. The C. volubilis accession ICPW 169 was found to be null for both CI bands and CI activity. Environment, strain, and environment x strain interaction showed highly-significant effects on both the TI and CI activities. Growing the pigeonpea strains at a different environment from their area of adaptation increased TI and CI activities and also altered the maturity period.     

Seedling emergence of chickpea (Cicer arietinum L.), pigeonpea (Cajanus cajan L.) and pearl millet (Pennisetum typhoides L.) Effect of differential soil crusting,as induced by raindrop size,and depth of sowing

The effects of differential soil crusting, as achieved by varying the raindrop size, and depth of sowing on seedling emergence of chickpea (Cicer arietinum L.), pigeonpea (Cajanus cajan L.) and pearl millet (Pennisetum typhoides L.) were investigated. Three drop diameters: 2.75, 1.65 and 0.93 mm and four depths of sowing: 2, 4, 6, and 8 cm were studied. Mean time of emergence increased with increase in raindrop size and depth of sowing. Crops differed in their emergence capacity under adverse soil conditions from delay in the emergence of chickpea to complete failure in the case of pigeonpea. Pearl millet was not significantly affected by drop size but was found to be sensitive to depths beyond 4 cm. Within the chickpea crop, the variety H 208 was observed to have better emergence qualities than Pusa 209. Other parameters like epicotyl diameter, swelling index, and the area of crust broken by the emerging seedling were measured.     

Biosystematic relationships among Cajanus,Atylosia, and Rhynchosia species and evolution of pigeonpea (Cajanus cajan (L.) Millsp.)

Summary Biosystematic studies encompassing morphocytological and electrophoretic analyses of Cajanus cajan, seven species of Atylosia and one of Rhynchosia revealed that A. cajanifolia is closest to C. cajan, followed by A. lineata, A. scarabaeoides, A. sericea, A. albicans, A. volubilis, A. platycarpa and R. rothii, in that order. A revision has been suggested for the taxonomic placement of the seven Atylosia species. Regarding the evolution of cultivated C. cajan, three possible alternatives have been suggested. Firstly, C. cajan could have evolved through gene mutation in A. cajanifolia; secondly, some of the Atylosia species and pigeonpea probably evolved from the same source; and thirdly, the pigeonpea might have developed from naturally occurring interspecific crosses of A. lineata and A. scarabaeoides.     

Enzymes of ammonia assimilation in root nodules of Trigonella foenum-graecum

The main pathway of ammonia assimilation in the root nodules of Trigonella foenum-graecum is via nodule cytosol glutamine synthetase-glutamate synthase.     

Genetic fingerprinting of pigeonpea [Cajanus cajan (L.) Millsp.] and its wild relatives using RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were used for the identification of pigeonpea [Cajanus cajan (L.) Millsp.] cultivars and their related wild species. The use of single primers of arbitrary nucleotide sequence resulted in the selective amplification of DNA fragments that were unique to individual accessions. The level of polymorphism among the wild species was extremely high, while little polymorphism was detected within Cajanus cajan accessions. All of the cultivars and wild species under study could be easily distinguished with the help of different primers, thereby indicating the immense potential of RAPD in the genetic fingerprinting of pigeonpea. On the basis of our data the genetic relationship between pigeonpea cultivars and its wild species could be established.NCL Communication No. 6062