Development of the Xylem Ureide Assay for the Measurement of Nitrogen Fixation by Pigeonpea (Cajanus cajan (L.) Millsp.)

Quantification of N₂ fixation by pigeonpea (Cajanus cajan (L.)Millsp.) in the field has proved difficult using techniquessuch as ¹⁵N isotope dilution, acetylene reduction and N difference.We report experiments to develop the ureide assay of N₂ fixationbased on extraction and analysis of xylem exudate. Plants ofpigeonpea cv. Quantum, inoculated with effective Rhizobium spp.CB756, were grown in a temperature-controlled glasshouse inlarge pots filled with a sand: vermiculite mixture, in waterculture and in a slightly acidic, red-brown earth in replicatedfield plots. Xylem exudate was collected as bleeding sap fromboth nodulated and unnodulated roots, and from detached nodules.Exudate was extracted also from detached shoots and stems ofpigeonpea using a mild vacuum (60–70 kN m^–2). Largedifferences in the composition of N solutes exported from rootsof N₂-dependent and nitrate-dependent plants suggested thatshifts in plant dependence on N₂ fixation may be reflected byconcomitant changes in N solutes. Thus, nodulated plants weresupplied throughout growth with either N-free nutrients or nutrientssupplemented with 1, 2, 5, 5, 10, or 20 mol m^{–3 15}. Plants were harvested at regular intervals fordry matter and vacuum-extracted exudate. The relative abundanceof ureides ([ureide-N/ureide-N + nitrate-N + -amino-N] ? 100)in the exudate was highly correlated with the proportion ofplant N (calculated using a ¹⁵N isotope dilution technique)derived from N₂ fixation. Two distinct phases of plant growthwere recognized and standard curves were prepared for each.The relationship between proportional dependence of plants onN₂ and xylem relative ureides was unaffected by mineral-N source,i.e. nitrate or ammonium. This result is discussed in relationto interpretation of material from field-grown plants. The effectsof plant genotype, strain of rhizobia, section of stem extracted,removal of leaves, time delay between shoot detachment and extraction,and diurnal characteristics were examined in order to identifypotential sources of error and to optimize sampling procedures. Key words: Ureides, allantoin, allantoic acid, N₂ fixation, pigeonpea, Cajanus cajan     

Identification of Amylase Inhibitor Deficient Mutants in Pigeonpea (Cajanus cajan (L.) Millisp.)

We have developed and analyzed several mutant lines (M6 generation) of pigeonpea (Cajanus cajan (L.) Millsp.) for the content of defensive proteins and antinutritional factors. Inhibitors of proteinase and of amylase, lectins, and raffinose family oligosaccharides were analyzed in mature seeds of different pigeonpea accessions (untreated) and compared with mutant lines. Proteinase inhibitor profiles were similar in terms of number and intensities of activity bands but they differ marginally in the activity units in pigeonpea accessions and mutants. Pigeonpea mutants showed significant differences in amylase inhibitor profiles as well as activity units from those of pigeonpea accessions. Interestingly, two mutants (A6-5-1 and A7-3-2) were identified to have absence of amylase inhibitor isoforms. Hemagglutinating activity and raffinose family oligosaccharides content were found to be significantly higher in mutants than in accessions. It is evident from the results that proteinase inhibitors of pigeonpea are stable while amylase inhibitors, lectins, and raffinose family oligosaccharides show altered expression upon mutagen treatments. These mutants will be ideal candidates for further evaluation.     

The Influence of Some Phenolic Compounds on Nodulation in Pigeonpea (Cajanus cajan(L.) Millsp.)

Studies on exogenous application of phenolic compoundsviz: p-hydroxybenzoic acid, resorcinol and chlorogenic acid each with concentration of 10^-4 M are done on the legume (Cajanus cajan (L.)Millsp.) AL-15. The effect of applied phenolic compounds as well as of structural differences in phenols indicate a marked influence of phenolic compounds in regulating growth processes in plants. Fresh and dry mass of various plant parts increased after foliar spray with phenols resulting in an improved harvest index. It is seen that phenols also play an important role in the initiation and development of nodules.     

Insights into Salt Stress-Induced Biochemical,Molecular and Epigenetic Regulation of Spatial Responses in Pigeonpea (Cajanus cajan L.)

Pigeonpea (Cajanus cajan L.) is a rich source of nutritionally good quality proteins, carbohydrates, minerals, and vitamins. However, environmental stresses adversely affect its productivity. Only limited reports are available on biochemical/physiological responses of pigeonpea under salt stress. The objectives of the present study were to screen pigeonpea germplasm accessions for salt stress tolerance, followed by understanding their biochemical, epigenetic and molecular responses. Based on germination, growth, and vigor of seedlings under salt stress, the most contrasting pair of salt-responsive genotypes (ICP1071- most salt-sensitive, and ICP7- most salt-tolerant) were selected. Three-week-old seedlings subjected to 250 mM NaCl stress for 7 days showed a significant increase in proline and reducing sugar contents in the case of ICP7, whereas a considerable increase in cell wall-degrading enzyme activity and protein oxidation was observed in ICP1071. Superoxide dismutase, peroxidase, and glutathione reductase activity increased considerably in shoots of ICP7. We observed the CcCYP gene to be upregulated in root, whereas CcCDR was upregulated in shoots of the salt-tolerant genotype to provide protection against the stress. The extent of DNA hypomethylation in the contrasting pigeonpea genotypes under salt stress was correlated with their salt tolerance level. Bisulfite sequencing of CcCDR revealed that methylation of three cytosine residues in CHH context in shoots of the ICP7 genotype due to salt stress results in 2.6-fold upregulated expression of the gene. With a 6.8% increase in methylation of the coding region of CcCDR, its expression level increased by 22%. To the best of our knowledge, this is the first report on a comprehensive study of salt-induced biochemical, epigenetic and molecular responses of pigeonpea, which might be useful in the development of improved salt-tolerant variety.

     

Pigeonpea genomics initiative (PGI): an international effort to improve crop productivity of pigeonpea (Cajanus cajan L.)

Pigeonpea (Cajanus cajan), an important food legume crop in the semi-arid regions of the world and the second most important pulse crop in India, has an average crop productivity of 780 kg/ha. The relatively low crop yields may be attributed to non-availability of improved cultivars, poor crop husbandry and exposure to a number of biotic and abiotic stresses in pigeonpea growing regions. Narrow genetic diversity in cultivated germplasm has further hampered the effective utilization of conventional breeding as well as development and utilization of genomic tools, resulting in pigeonpea being often referred to as an ‘orphan crop legume’. To enable genomics-assisted breeding in this crop, the pigeonpea genomics initiative (PGI) was initiated in late 2006 with funding from Indian Council of Agricultural Research under the umbrella of Indo-US agricultural knowledge initiative, which was further expanded with financial support from the US National Science Foundation’s Plant Genome Research Program and the Generation Challenge Program. As a result of the PGI, the last 3 years have witnessed significant progress in development of both genetic as well as genomic resources in this crop through effective collaborations and coordination of genomics activities across several institutes and countries. For instance, 25 mapping populations segregating for a number of biotic and abiotic stresses have been developed or are under development. An 11X-genome coverage bacterial artificial chromosome (BAC) library comprising of 69,120 clones have been developed of which 50,000 clones were end sequenced to generate 87,590 BAC-end sequences (BESs). About 10,000 expressed sequence tags (ESTs) from Sanger sequencing and ca. 2 million short ESTs by 454/FLX sequencing have been generated. A variety of molecular markers have been developed from BESs, microsatellite or simple sequence repeat (SSR)-enriched libraries and mining of ESTs and genomic amplicon sequencing. Of about 21,000 SSRs identified, 6,698 SSRs are under analysis along with 670 orthologous genes using a GoldenGate SNP (single nucleotide polymorphism) genotyping platform, with large scale SNP discovery using Solexa, a next generation sequencing technology, is in progress. Similarly a diversity array technology array comprising of ca. 15,000 features has been developed. In addition, >600 unique nucleotide binding site (NBS) domain containing members of the NBS-leucine rich repeat disease resistance homologs were cloned in pigeonpea; 960 BACs containing these sequences were identified by filter hybridization, BES physical maps developed using high information content fingerprinting. To enrich the genomic resources further, sequenced soybean genome is being analyzed to establish the anchor points between pigeonpea and soybean genomes. In addition, Solexa sequencing is being used to explore the feasibility of generating whole genome sequence. In summary, the collaborative efforts of several research groups under the umbrella of PGI are making significant progress in improving molecular tools in pigeonpea and should significantly benefit pigeonpea genetics and breeding. As these efforts come to fruition, and expanded (depending on funding), pigeonpea would move from an ‘orphan legume crop’ to one where genomics-assisted breeding approaches for a sustainable crop improvement are routine.     

Genetic patterns of domestication in pigeonpea (Cajanus cajan (L.) Millsp.) and wild Cajanus relatives

Pigeonpea (Cajanus cajan) is an annual or short-lived perennial food legume of acute regional importance, providing significant protein to the human diet in less developed regions of Asia and Africa. Due to its narrow genetic base, pigeonpea improvement is increasingly reliant on introgression of valuable traits from wild forms, a practice that would benefit from knowledge of its domestication history and relationships to wild species. Here we use 752 single nucleotide polymorphisms (SNPs) derived from 670 low copy orthologous genes to clarify the evolutionary history of pigeonpea (79 accessions) and its wild relatives (31 accessions). We identified three well-supported lineages that are geographically clustered and congruent with previous nuclear and plastid sequence-based phylogenies. Among all species analyzed Cajanus cajanifolius is the most probable progenitor of cultivated pigeonpea. Multiple lines of evidence suggest recent gene flow between cultivated and non-cultivated forms, as well as historical gene flow between diverged but sympatric species. Evidence supports that primary domestication occurred in India, with a second and more recent nested population bottleneck focused in tropical regions that is the likely consequence of pigeonpea breeding. We find abundant allelic variation and genetic diversity among the wild relatives, with the exception of wild species from Australia for which we report a third bottleneck unrelated to domestication within India. Domesticated C. cajan possess 75% less allelic diversity than the progenitor clade of wild Indian species, indicating a severe "domestication bottleneck" during pigeonpea domestication.     

Carbohydrate Metabolism in Drought-Stressed Leaves of Pigeonpea (Cajanus cajan)

Pigeonpea is a tropical grain-legume, which is highly dehydrationtolerant. The effect of drought stress on the carbohydrate metabolismin mature pigeonpea leaves was investigated by withholding waterfrom plants grown in very large pots (50 kg of soil). The moststriking feature of drought-stressed plants was the pronouncedaccumulation of D-pinitol (1D-3-methyl-chiro-inositol), whichincreased from 14 to 85 mg g^–1 dry weight during a 27d stress period. Concomitantly, the levels of starch, sucroseand the pinitol precursors myo-inositol and ononitol all decreasedrapidly to zero or near-zero in response to drought. The levelsof glucose and fructose increased moderately. Drought stressinduced a pronounced increase of the activities of enzymes hydrolysingsoluble starch (amylases) and sucrose (invertase and sucrosesynthase). The two anabolic enzymes sucrose phosphate synthase(sucrose synthetic pathway) and myo-inositol methyl transferase(pinitol synthetic pathway) also showed an increase of activityduring stress. These results indicate that pinitol accumulatedin pigeonpea leaves, because the carbon flux was diverted fromstarch and sucrose into polyols. Key words: Drought, polyols, pinitol, sucrose, starch, pigeonpea     

Thidiazuron-induced shoot regeneration in pigeonpea (Cajanus cajan L.)

Thidiazuron either alone or in combination with IAA induced high frequency shoot regeneration from primary leaf segments of three pigeonpea cultivars. Transfer of the cultures to medium with reduced concentration of thidiazuron resulted in further development of the shoots. The regenerated shoots were subsequently transferred to medium supplemented with BA, IAA and gibberellic acid where 5-10% of the shoots elongated further. Rooting of shoots could be obtained on half strength MS medium supplemented with NAA. Histological studies confirmed the mode of regeneration as shoot organogenesis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of NADP-Isocitrate Dehydrogenase from Nodules of Pigeonpea (Cajanus cajan)

NADP-isocitrate dehydrogenase from nodules of pigeonpea (Cajanus cajan L. cv UPAS-120) was partially purified to about 57 folds and its properties were studied. The enzyme showed an absolute requirement for a divalent cation which was fulfilled either by Mn⁺² or Mg⁺² and to a smaller extent by Co⁺². The enzyme exhibited a sigmoidal response to increasing concentrations of Mn²⁺ (S_0.5=0.3mM). The apparent Km values for isocitrate, NADP and Mg²⁺ were 21, 23 and 280 μM, respectively. It had an optimum pH of 8.0–8.2. The enzyme activity was not affected by various organic acids, amino acids and amides. NADH inhibited the activity non-competitively with respect to NADP. An apparent inhibition by ATP and ADP was due to chelation of divalent cation. NADPH acted competitively against NADP and non-competitively against isocitrate. Glutamate caused uncompetitive inhibition with respect to NADP and competitive against isocitrate. Kinetic studies suggested the reaction mechanism to be probably random sequential. Possible regulation of the enzyme activity in the nodules via cellular redox state and the levels of reaction products is discussed.     

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.     

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     

Inheritance of dwarfness in pigeon pea (Cajanus cajan (L.) millsp.)

Summary A mutant pigeon pea, showing dwarf and bushy growth, very late maturity, poor yield and abnormal flowers was isolated from the tall variety, Brazil P/2. It is proposed that the mutant be called dwarf.A single recessive gene appears to be responsible for dwarfness and seems to have pleiotropic effect on maturation. The symbol proposed for the dwarfness gene is d.

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Zusammenfassung Aus einer hochwüchsigen Straucherbse (Cajanus cajan (L.) Millsp.) der Sorte Brazil P/2 wurde eine Mutante mit verzwergtem und buschigem Wuchstyp, sehr später Reife, geringer Ertragsfähigkeit und abweichender Blütenform isoliert, für die der Name dwarf vorgeschlagen wird.Nur ein rezessives Gen scheint für den Zwergwuchs verantwortlich zu sein, das einen pleiotropen Effekt auf die Reife ausübt. Für dieses Gen wird das Symbol d vorgeschlagen.

     

Comparisons of Earlier- and Later-formed Pods of Pigeonpeas (Cajanus cajan (L.) Millsp.)

On branches of indeterminate cultivars of pigeonpea, floweringbegins at the basal nodes and proceeds acropetally; in morphologicallydeterminate cultivars, flowering begins on the apical racemesand proceeds basipetally. In cultivars of both types, withinthe racemes flowering proceeds acropetally. Under normal conditionsmore pods are set from earlier-formed flowers than from later-formedflowers, many of which are shed. Consequently the earlier-formedpods are found at the more basal nodes of racemes, and in indeterminatecultivars at the more basal nodes on the branches. The averageweight of earlier and later-formed pods, collected from thebasal and apical nodes of the racemes or of the branches, wassimilar; so was the number of seeds per pod, the weight perseed and the nitrogen content of the seeds. This pattern differsfrom that found in most herbaceous legumes, where later-formedpods are smaller, and indicates that pigeonpeas set fewer podsthan they are capable of filling. This behaviour may be relatedto the intrinsically perennial nature of pigeonpeas. The comparisonof the weights of earlier- and later-formed pods could providea simple screening procedure for identifying plants with anannual nature among existing cultivars or in breeders' lines. Cajanus cajan (L.) Millsp., pigeonpea, pod development, seed number, seed weight, nitrogen content     

Organogenesis and embryogenesis from diverse explants in pigeonpea (Cajanus cajan L.)

Seed and seedling expiants of pigeonpea were evaluated for organogenesis and somatic embryogenesis. De novo plant regeneration through organogenesis was obtained from mature cotyledons, primary leaves and roots of seedlings. Production of multiple shoots from the cotyledonary node was observed in cultures of whole seeds on 6-benzylaminopurine enriched medium. Somatic embryos were induced from immature cotyledons and embryonal axes, however, well-developed plants could not be derived from these embryos. The regenerants obtained through organogenesis were transferred to the field and grown to maturity.     

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.     

The Effects of Salinity on Nitrogen Fixation and Trehalose Metabolism in Mycorrhizal Cajanus cajan (L.) Millsp. Plants

Arbuscular mycorrhizal (AM) fungi exist widely in natural ecosystems as well as in salt-affected soils and are considered suitable candidates for bio-amelioration of saline soils. Plants respond to salinity by accumulating sugars and other low-molecular-weight compatible solutes. One such compound is trehalose, which has been found to play an important role as a stress protectant. The aim of the present investigation was to study interactions between an AM fungus and salinity stress on growth, nitrogen fixation, and trehalose metabolism in Cajanus cajan (L.) Millsp. (pigeonpea). Two genotypes [Sel 85N (salt-tolerant) and ICP 13997 (salt-sensitive)] were subjected to saline treatments with and without mycorrhizal inoculations. Salinity reduced plant biomass (shoot and root) in both genotypes and resulted in a decline in shoot-to-root ratio (SRR); however, a smaller decline was observed in Sel 85N than in ICP 13997. AM colonization was reduced with increasing salinity levels but mycorrhizal responsiveness (MR) increased. Genotypic variability in nitrogen fixation and trehalose metabolism in response to salinity and mycorrhization was observed. An increment in nodule number was accompanied by a reduction in dry mass. Subsequently, nodular activity (leghemoglobin, acetylene-reduction activity [ARA], nitrogen content) was reduced under soil salinity, which was more profound in ICP 13997 than in Sel 85N. The symbiotic association with Glomus mosseae led to significant improvement in plant dry mass and nitrogen-fixing potential of nodules under salt stress. Salinity led to an increase in trehalose-6-P synthetase (TPS) and trehalose-6-P phosphatase (TPP) activities resulting in increased trehalose content in nodules, which was accompanied by inhibition of trehalose catabolism (trehalase activity). AM plants had lower trehalase activity under saline and nonsaline conditions. Thus, a symbiotic relationship between plant roots and G. mosseae might have resulted in salinity tolerance in a genotype-dependent manner.