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Wrinkled-seeded pea mutants (Pisum sativum L., genotypes rrrbrb-, rrRbRb-, and RRrbrb-) have seeds with reduced, but different, starch content and modified starch properties. Analysis of these mutants revealed an enhanced capacity of root nodules for symbiotic nitrogen fixation and of host plant organs for assimilation of ammonium nitrogen. This observation was confirmed by morphological data on organization of symbiotic system, by elevated nitrogenase activity, high protein accumulation in plants due to nitrogen fixation, and by enhanced activity of glutamine synthase in leaves and glutamate dehydrogenase in roots of mutants, as compared with the organs of wild-type pea. It is supposed that the aforementioned advantages of mutants are related to accumulation in seeds of elevated protein reserves that satisfy their demand for nitrogen during formation of symbiotic systems.

We have used a combination of techniques to study the structure and properties of C-type starch from pea seeds. It was found that all C-type starch granules contain both types of polymorph; the B polymorphs are in the center of the granule and are surrounded by the A polymorphs. During heating in excess salt solution the A and B polymorphs within C-type granules melt independently, giving a double transition in heat capacity and a two-step swelling, compared with single transitions for A- and B-type starches. It was shown that B polymorphs gave a transition with a lower peak temperature than A. The disruption of crystallinity during gelatinization began from the hilum area and was propagated along the granule, accompanied by swelling of disrupted areas. It is proposed that the swelling of disrupted parts of the granule decreases the melting temperature of the neighboring crystallites resulting in the progressive disruption of crystalline areas. The gelatinization process is dependent on the arrangement of A and B polymorphs within the granule. © 1998 John Wiley & Sons, Inc. Biopoly 45: 323–332, 1998

A method for estimating the proportions of ‘A’ and ‘B’ polymorphs comprising a sample of ‘C’ type starch is proposed which uses established experimental techniques with commercially available spreadsheet and X-ray analysis software. Waxy maize, potato and smooth pea starches were used to provide X-ray diffraction patterns characteristic of the ‘A’, ‘B’ and ‘C’ starch polymorphs. Samples of amorphous starches were also prepared. The method initially involved subtraction of the amorphous phase and instrumental background from the X-ray diffraction patterns of each starch sample using the spreadsheet program, Lotus 1-2-3. The remainder of the pattern, representing the crystalline portion of the starch sample, was then analysed by profile fitting to elucidate the positions and areas of individual diffraction peaks. The ratio of the total peak area to the areas under peaks characteristic of ‘A’ and ‘B’ type starches, respectively, were used to calculate the relative proportions of these polymorphs in smooth pea starch. These proportions were found to be 56±3% ‘A’ polymorph to 44±3% ‘B’ polymorph. A ‘C’ type pattern was constructed by using Lotus 1-2-3 to combine diffraction patterns from the crystalline portions of ‘A’ and ‘B’ type starches in the proportions given above. Polymorph patterns were obtained by manipulation of the diffraction patterns from the crystalline portions of starches using Lotus 1-2-3. An ‘A’ type pattern was obtained by subtraction of a ‘B’ type pattern from that of a ‘C’ type. Similarly, a ‘B’ type pattern was obtained by subtraction of an ‘A’ type pattern from that of a ‘C’ type.

Pea flour from green and yellow mature pea seeds (Pisum sativum) was processed by adding emulsifiers (Glycerol-monostearate GMS, and Amidan 250B polar type monoglyceride, A250). Doughs obtained were cooked for 15 min and 30 min in order to investigate the effect of the presence of emulsifiers and cooking treatment. The quality of macaroni was examined, cooking properties were defined and sensory assessment established. Changes in protein structure were studied by solution fractioning and SDS-PAGE. Modification of the monosaccharide, disaccharide and α-galactoside contents and trypsin inhibitor activity (TIA) were also studied. The addition of emulsifiers improved dough quality. Emulsifiers fundamentally change the solubility of protein fractions, and they were able to promote the soluble fraction molecular weight distribution and the infiltration of low molecular weight fractions into the protein network. The soluble carbohydrate content was not modified by the addition of emulsifiers whilst cooking time affected its content. α-Galactoside content and TIA were reduced after the addition of emulsifiers and the cooking of peas., Research founded by Copernicus Network EU (CIPA-CT92-4020) and Spanish Comision Interministerial de Ciencia y Tecnologia (CICYT) ALI94-1419-E. J. Frias acknowledges support from the European Union through an individual bursary (AIR3-BM93-1118).

Lathyrus sativus, also known as Khesari dhal in India, is a good source of protein and also desirable from the agricultural point of view as it is drought-resistant and can be grown with low agriculture inputs of fertiliser, irrigation and pesticide. In common with other legume seeds, Lathyrus contains the flatulence causing alpha-galactosides. This is a study of the physico-chemical properties of four selected Lathyrus sativus lines (Red flower, a high beta-ODAP line; EC-242692, a medium beta-ODAP line; L-1276, a low beta-ODAP line; and Pusa-24, a very low beta-ODAP line) to determine the most effective way of removing the flatulence factors by using simple processes such as soaking, dehusking and germination. Physico-chemical properties like swelling and hydration capacity of Lathyrus seeds were higher than other legumes, which suggests higher consumer preference in terms of cooking time and fuel consumption over other beans. Germination was the most effective procedure for removing alpha-galatosides (85-92%). Dehusking of pre-soaked seeds also achieved a high alpha-galactoside reduction (61-80%). Soaking in alkaline medium and thereafter boiling for 9 min brought about a lower reduction (33-74%), but these losses were still higher than those obtained by soaking in drinking water or soaking in boiled water for 2 h (11-49% and 7-44%, respectively).

The effects of mutant alleles at the r and rb loci on starch grain size and the levels of starch and amylose in developing pea (Pisum sativum L.) seeds have been examined. Four lines, near-isogenic except for genes at these loci, have been used to show that both mutations reduce levels of starch throughout embryo development and reduce levels still further when combined in the 'double mutant'. The reduction in starch content was due, at least in part, to a reduction in starch granule size. Although the proportion of starch in mature embryos was similar in the rrRbRb and RRrbrb lines, the starch content differed between these two lines during development, as a percentage of embryo dry weight. This difference was due to a reduction in the absolute growth of the embryo caused by the rb mutation. Lines homozygous for the mutant r allele with either wild-type (RbRb) or mutant (rbrb) alleles at the rb locus contained increased proportions of amylose in their starch throughout development, due to a reduced production of amylopectin. The presence of the rb mutation, however, also reduced the amount of amylose in relation to the reduction in total starch levels. Mutant alleles at both loci also reduced starch levels in the testa during development, the reduction due to rb being more extreme. Reciprocal crosses showed a maternal effect of the rb mutation on final seed size and on the absolute amount of starch in the embryo.

Mutant genes at two loci, r and rb, known to encode genes affecting the starch biosynthetic pathway, were studied for their effect on the structure and gelatinization of pea seed starches. Comparisons were made using starches from four lines (RRRbRb, rrRbRb, RRrbrb, and rrrbrb), near-isogenic except for genes at these two loci. All the starches had C-type X-ray diffraction patterns, but different contents of 'A' and 'B' polymorphs. The presence of a mutation at either locus increased the 'B' polymorph content in the starches, although the influence of the r mutation was much greater than that of rb. Differences were discovered in the crystalline stucture of the rrRbRb starch which correlated with a high content of amorphous phase as well as with the changes in amylopectin structure. In addition, changes in the crystalline structure of this sample correlated with a lack of co-operative transition during starch gelatinization in excess water. The RRrbrb starch had a greatly increased enthalpy of gelatinization in excess water compared with the wild-type starch. It is proposed that this effect is connected with specific charge interactions between the molecules in the starch granule. The rrrbrb starch had parameters of crystalline structure and gelatinization which reflected the different influences of the two genes. With regard to gelatinization, this starch had relatively wide co-operative transition and low enthalpy and a very high peak temperature of transition.

The levels of the raffinose family of α-galactosides (raffinose, stachyose, verbascose and ciceritol) and of the nutritional carbohydrates (fructose, sucrose and starch) were determined in the seed of a range of lentil lines from germplasm collections and commercial Spanish cultivars. Significant levels of variation were found for all the measured characters, and particularly for the α-galactosides. The greatest variation was found in verbascose content, which ranged from about 1 % of the seed dry weight to undetectable amounts. The relationship between these chemical components and morphological seed characteristics was assessed. The only significant relationship between the different groups of characteristics was a positive correlation between the level of verbascose and the average seed weight for each genotype.

Following EMS mutagenesis of pea (Pisum sativum L.) seed, nine mutants have been isolated whose embryos are affected in their development, being defective either in organogenesis or in cell differentiation. Studies of their morphology at the whole organ level and their histology indicated at least four groups: those with single cotyledons (representing at least three loci), two blocked at the early cotyledonary stage, one with abnormal root development and two with cellular defects. Genetic analysis indicated the mutants were behaving as monogenic recessive mutations. These mutants represent the first of their type for legumes and should prove especially useful, therefore, for examining those aspects of development characteristic of these species

Total protein content and amounts of albumin, legumin and vicilin have been determined for pea seeds from lines near-isogenic except for genes at therugosusloci,randrb(RR/RbRb; rr/RbRb; RR/rbrb; rr/rbrb). Seeds with the wildtype, round-seeded phenotype (RR/RbRb) had less protein on a total seed dry-weight basis than any of the wrinkled-seeded lines and this protein had a lower proportion of albumin. The lines which had recessive alleles at bothrandrbloci had the highest proportion of protein and the highest proportion of albumin. The roundseeded peas possessed nearly two-fold more legumin than the double recessive line, with proportions for the two single recessive lines falling in between these extremes. Vicilin levels were similarfor all four near-isogenic lines. SDS-PAGE analysis of the isolated albumin, legumin and vicilin fractions revealed no significant differences between the four lines. Differential scanning calorimetry of protein extracts showed that all the wrinkled-seeded near-isolines possessed legumin fractions with diminished thermal stability relative to that from the roundseeded, wild-type line.Chemically-induced mutants were also analysed for protein content and composition. These mutants have previously been shown to display great variation in starch and lipid levels. Total protein varied from 20.3% to 37.9%; however, relative proportions of albumin, legumin and vicilin were similar in all mutant lines. SDS-PAGE analysis identified two mutant pea lines which possessed a legumin A-chain of 65 000 Mras well as the typical 45 000 Mrform. Differential scanning calorimetry of protein extracts indicated that the legumin in all mutants had lower enthalpies of denaturation than the legumin in the round-seeded parent.The mutant pea lines possess exceptional variation with respect to starch, lipid and protein which raises opportunities for their use in the food and animal feedstuff industries.

L'expression des genes des principales proteines de stockage (viciline, conviciline, legumine) a ete suvie au cours du developpement in vivo d'embryons de pois et comparee avec l'expression de ces genes dans des embryons cultives in vitro en utilisant des ADN complementaires. Les ARNm correspondant sont moins abondants in vitro. On suggere que les differences observees sont liees au passage de la division a l'elongation cellulaire, et au vieillissement premature de l'embryon

Analyses of seeds from a wide range of pea lines have revealed extensive variation for all of the major fatty acids normally found in pisum. Unlike total lipid content, this variation was not affected by the presence of mutant alleles at the r and rb loci. To allow chemically analysed seed to be used in crosses, which is of particular importance for F 2 seed, a method has been developed by which individual seeds can be analysed non-destructively. This system entails drilling a small hole in the seed, away from the embryonic axis. The small quantity of powder produced by this process can be used for the chemical analysis but does not prevent the seed from being grown to produce progeny. Reducing the sample size had no significant effect upon the compositional analysis of the fatty acids.

Pea (Pisum sativum L.) mutant near-isogenic lines (RRrbrb, rrRbRb, rrrbrb) with lower starch but higher lipid contents, brought about by lesions in the starch biosynthetic pathway, had seed moisture sorption isotherms displaced below that of the wild type (RRRbRb). The negative logarithmic relationship between seed longevity and seed storage moisture content (%, f.wt basis), determined in hermetic storage at 65 degrees C, also differed: longevity in the mutant near-isogenic lines was poorer and less sensitive to moisture content than in the wild type (i.e. C(W) was lower). The low-moisture-content limit (m(c)) to this relation also differed, being lower in the mutant near-isogenic lines (5.4-5.9%) than in the wild type (6.1%). In contrast, all four near-isogenic lines showed no difference (P0.25) in the negative semi-logarithmic relationship between equilibrium relative humidity (ERH) and seed longevity. It is concluded that the effect of these alleles at the r and rb loci on seed longevity was largely indirect; a consequence of their effect on seed composition and hence on moisture sorption isotherms. However, this explanation could not be invoked at moisture contents below m(c) where differences in longevity remained substantial (RRRbRb double that of rrrbrb). Hence, these mutant alleles affected seed longevity directly at very low moisture contents.

Twenty-four short-time soaking (STS) processes were carried out to study the influence of some experimental variables (lentil/water ratio, temperature, illumination, elimination of the soaking medium and milling of the seeds) on the glucose, fructose, sucrose, total available soluble sugars, starch, vitamin B1, vitamin B2, available niacin, raffinose, ciceritol, stachyose, total α-galactosides and trypsin inhibitor activities of lentil flours obtained. The soaking process produced, in general, a reduction in the total available soluble sugars content of 8-70%. Glucose was not found in the raw lentils, but was present in the lentil flours obtained after soaking processes carried out in presence of light. Fructose increased in most of the experiments (22-967%) and sucrose decreased significantly (6-73%). Regarding the starch content, the soaking process produced a significant reduction (11-31%). The hydrosoluble vitamin content of lentils, in general, decreased or did not change. However, vitamin B2 and available niacin increased significantly in some experiments, especially in those carried out with ground lentils. The α-galactoside content suffered a large reduction (12-58%) and TIA levels were reduced by 2-33%. From the results obtained, it can be established that STS affects the nutrients and antinutrients of lentils in a different way depending on the experimental conditions. Lentil flours with a low level of antinutritional factors and a high vitamin level are obtained with ground lentils at 42 °C., This work was supported by the Spanish Comision Interministerial de Ciencia y Tecnología AGL-2000–1609-C02–01, and it is part of the PhD of I. Sierra and C. Sotomayor.

Raffinose family oligosaccharides (RFOs) are synthesized by a set of galactosyltransferases, which sequentially add galactose units from galactinol to sucrose. The accumulation of RFOs was studied in maturing seeds of two pea (Pisum sativum) lines with contrasting RFO composition. Seeds of the line SD1 accumulated stachyose as the predominant RFO, whereas verbascose, the next higher homolog of stachyose, was almost absent. In seeds of the line RRRbRb, a high level of verbascose was accumulated alongside with stachyose. The increase in verbascose in developing RRRbRb seeds was associated with galactinol-dependent verbascose synthase activity. In addition, a galactinol-independent enzyme activity was detected, which catalyzed transfer of a galactose residue from one stachyose molecule to another. The two enzyme activities synthesizing verbascose showed an optimum at pH 7.0. Both activities were almost undetectable in SD1. Maximum activity of stachyose synthase was about 4-fold higher in RRRbRb compared with SD1, whereas the activities of galactinol synthase and raffinose synthase were only about 1.5-fold higher in RRRbRb. The levels of galactinol synthase and stachyose synthase activity were reflected by steady-state levels of corresponding mRNAs. We suggest that the accumulation of verbascose in RRRbRb was controlled by a coordinated up-regulation of the last steps of verbascose biosynthesis.

(13)C cross-polarization magic angle spinning NMR has been used to study the ordered and disordered structures of starches with different water contents. The amorphous regions of starch have been shown to produce NMR patterns only if they are in a glassy state, the widths, positions, and areas of the peaks to some extent being dependent on the temperature and the water content of the starch. In the amorphous region, the peaks were all Gaussian in shape, while the peaks in the ordered regions had Lorentz profiles. Water contents in the range 10-50% did not influence the proportion of double helices in the starch. Decreasing the water content to 1-3%, however, resulted in a significant decrease in the proportion of double helices, the effect being greater in B- than in A-type starches. It is suggested that short-range order structures in starches (double helices) are stabilized by becoming part of long-range order structures (crystallites).

We studied the curves of delayed luminescence induction in leaves of pea plants with mutations affecting the starch branching enzyme (r locus) and ADPG pyrophosphorylase (rb locus). In mutated pants, a 75% reduction of starch content in seeds was observed. The half-decay time of delayed luminescence intensity during induction for double mutants was shown to be longer than for wild type plants.

The granular structure and gelatinisation properties of starches from a range of pea seed mutants were studied. Genes which affect the supply of substrate during starch synthesis (rb, rug3, rug4) affected the total crystallinity and possibly increased the content of A polymorphs in the starch. Conversely, genes directly affecting the synthesis of starch polymers (r, rug5, lam) increased the content of B polymorphs, but had a minimal effect on total crystallinity. During gelatinisation, starches from the rb, rug3, rug4 and lam mutants had narrow endothermic peaks which were similar to starch from the wild-type, although all the starches had different peak temperatures and enthalpy changes. Starches from r and rug5 mutants were very different to all other starches, having a very wide transition during gelatinisation. In addition, the amylopectin in starch from these mutants had altered chain lengths for those parts of the polymer which form the ordered structures in the granule.

The raffinose family of oligosaccharides (RFO) is present in relatively high levels in grain legume seeds. They are considered to be antinutritional compounds because they are, at least in part, believed to be responsible for causing flatulence in humans, which is the single most important factor in deterring people from including more legume seeds in their diet. The RFO also have important functions within the plant. They serve as transport metabolites in many legumes and have been proposed to play a positive role in cold acclimatisation and in conferring desiccation tolerance during seed maturation. These responses to environmental stresses are believed to result from the RFO acting as protecting agents for membrane bound-proteins. We have screened 70 pea lines from the test array of the John Innes Pisum germplasm collection, and lines were selected which had unusual RFO composition. The soluble sugars within these lines were quantified using High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD), and variants were identified which were deficient in verbascose and one with a reduced level of raffinose. These selected lines are being used in a crossing programme designed to study the genetics of the RFO pathway and to produce new material to test the effect of specific RFO on the plant’s responses to the environment and on the diets of humans and animals., JF gratefully acknowledges financial support from the European Union through an individual bursary (AIR3-BM93-1118). The John Innes Centre and Institute of Food Research are supported via a grant-in-aid from the Biotechnology and Biological Research Council and by the Ministry of Agriculture, Fisheries and Food.

A capillary zone electrophoretic (CZE) method is described for the analysis of the raffinose family of oligosaccharides (RFO) in pea seeds. Extraction of RFO was carried out in 80% ethanol and the extract was passed through a Sep-Pak C18 cartridge. This proved to be an improvement on the currently accepted purification technique. High-quality electrophoregrams were obtained which allowed the separation and quantitation of sucrose, raffinose, stachyose and verbascose. The CZE results were compared with those obtained by anion-exchange high-performance liquid chromatography coupled to a triple-pulsed amperometric detection (HPAC-PAD). The samples were obtained from four pea strains which were near-isogenic except for genes at the r and rb loci. A high degree of precision and reproducibility was obtained for the RFO compositions of all the pea strains. No statistically significant differences (p ⩽ 0.05) were found between the two analytical techniques using paired Student-t tests., The authors gratefully acknowledge support from the EC PEA ECLAIR Programme (AGRE No. 0073). J. Frias acknowledges support from the European Union through an individual bursary (AIR3-BM93-1118).

The effect of light and seed rinsing during the germination of lentil seeds (Lens culinaris var. Vulgaris cv. Magda-20) on trypsin inhibitor activity (TLA) has been studied. TLA decreased progressively during germination, while hydration grade and light exposure had less influence on TIA. In general, there was no significant change in TLA after 3 days of germination, except when seeds were rinsed only once and the experiment was conducted in the dark, showing a 10% TLA decrease. Six-day germination produced TLA decreases from 7% to 18%. In this case, the number of rinses had no significant effect on the final TLA, but the activity of protease inhibitors was significantly higher in seeds illuminated for 6 h per day. Maximal TLA decreases were found in seeds germinated in darkness and rinsed daily. Under these optimum conditions, TLA first decreased slowly (12% loss of this activity after 6 days) followed by a faster decrease (up to 45% of control seeds values) after 10 days of germination.

The analysis of low molecular weight carbohydrates in single seed of lentils has been carried out using two different high performance liquid chromatography (HPLC) methods. One used a reversed-phase column coupled to a refractive index detector (HPLC-RI), while the other utilised an anion-exchange phase column coupled to a triple-pulsed amperometric detector (HPAC-PAD). The latter was found to he more sensitive and could be used for the analysis of very small samples, hence allowing parts of a seed to be analysed and then grown and used for genetic studies.

Following dark adaptation, the response to irradiance of chlorophyll (Chl) fluorescence, the light-induced absorbance change around 820 nm (to measure reaction center Chl of photosystem I [PSI] P-700 oxidation), and CO2 fixation were examined in pea (Pisum sativum L.) leaves under a range of conditions. Initially, P-700 oxidation is restricted by a lack of regeneration of PSI electron acceptors, and the increase of oxidized P-700 (P-700+) that occurs during approximately the first 60 s of irradiation is largely independent of the resistance to electron flow between the two photosystems. Under these conditions, the quantum efficiency for linear electron flow is directly positively related to P-700+ accumulation, which is in contrast to the direct negative correlation that is the most frequently reported relationship between P-700+ accumulation and the quantum efficiency for linear electron flow.

The water content and osmotic potential (ψ2) of the different parts of the pea fruit have been measured during development of the seed in two lines near-isogenic except for the r locus. During the early development of both genotypes, the testa possessed a more negative ψ2 than either embryo, endosperm or pod while, at stages when liquid endosperm was present, the embryo always maintained ψ2, more negative than the endosperm. A clear effect of the r locus on water content and ψ2 was only observed in embryo tissue — wrinkled (rr) embryos possessing more water and maintaining a more negative ψ2 than round (RR) for most of the developmental period studied. The more negative ψ2 of wrinkled embryos correlated with their greater uptake of water in vivo. When cultured in vitro, the embryos of both genotypes showed maximum growth (fresh or dry weight) if 10 per cent sucrose was added to the medium (equivalent to about — 1.2 MPa). Round embryos, however, increased in weight more than wrinkled at all sucrose concentrations examined. Cultured embryos maintained a similar or more negative ψ2 than their surrounding medium; wrinkled more negative than round.

SUMMARY Photosynthetic activity in the yellow pods of gp.gp lines has been compared with that in normal Gp.Gp pods. As expected from a previous structural study, the occurrence of aberrant plastids in the mesocarp resulted in reduced activity in the gp.gp lines. Illuminated green fruits, 5–16 d post-anthesis showed net uptake of CO2, from the atmosphere. Positive carbon gain was extended until d 28 in deseeded pods. Conversely, in yellow (gp.gp) fruits no net uptake of CO2, from the atmosphere was observed at any stage, even in deseeded pods, but carbon losses in the light were restricted compared with those in the dark, indicating some photosynthetic activity. Analysis of the photosynthetic machinery within the yellow pods showed considerable differences in the effect of the gp gene on the mesocarp and endocarp layers. Compared with those of green pods, concentrations of RuBP carboxylase in the mesocarp were depressed, and the Hill reaction activity of isolated chloroplasts was virtually undetectable. However, in the parenchyma layer of the endocarp, RuBP carboxylase reached higher concentrations than in Gp.Gp pods, and Hill reaction rates were comparable. There was little difference between the lines in their PEP carboxylase concentrations in either tissue.

The effects of nitrogen level on characters relating to the performance of four early and three late flowering cultivars of Antirrhinum majus were assessed. Biometrical techniques were used to distinguish the effects of nitrogen from those due to sowing time and to varietal differences. The usefulness of these techniques to the breeder and to the grower are outlined.

The effect of genotype and plant density, over the range from 100 to 277 plants m~2, on plant to plant variation in precision sown microplots has been assessed for three 'leafless' (« afafstst ) pea ( Pisum sativum) lines. This range of planting densities did not significantly affect the total above ground biological yield per unit area of two of the genotypes (BS5 and BS4) whereas the biological yield of the third (BS1 5 1) declined at densities above 1 56 plants m~2. The differences were due to changes in seed yield. The effect of planting density on the variation between plants for biological yield within the microplots differed between the genotypes. The distribution pattern of BS4 and BS5 changed from normal to skewed with increased density, while the distribution for BS1 51 remained skewed at all planting densities. The differences between the three genotypes in the proportion of biological yield partitioned into seed yield (harvest index) on a unit area basis was due almost entirely to the differences in structure of the plant populations. The maximum level of partitioning by individual plants was similar for all three lines. The difference between this maximum for an individual and the crop harvest index therefore represents the area for improvement of crop harvest index through breeding. It is suggested that improvements in dried pea yields will come, therefore, by selecting plants which form more uniform populations with regard to plant size and to the proportion of plant biomass partitioned into seed (plant harvest index).

The effect of the yellow-podded (gp) gene on pea fruits was studied with reference to changes in their plastids. The main effect of the gene was on the mesocarp. The chlorophyll concentration of this layer was depressed to less than 5 % of that in normal green pods, allowing considerably higher light transmission through the pod wall. Ultrastructural studies showed that the mesocarp of yellow pods contained plastids with an internal membrane system restricted to single and paired membranes and vesicles. Unlike the chloroplasts of green mesocarps they lacked grana. Large starch grains were found in the plastids of mesocarps from both yellow and green pods. WeIl-developed chloroplasts, lacking starch grains, were found in the thin parenchymatous lining of the endocarp in yellow and green pods. Here chlorophyll concentrations were comparable in the two pod types and considerably higher than those found in the normal green mesocarp. However, the higher light levels transmitted to this layer in yellow pods correlated with a higher chlorophyll a: b ratio compared with that in the green pod.

Studies have been made of the growth of three foliage phenotypes of pea during the early growth of the pea crop prior to the onset of plant-plant interaction. These phenotypes were conventional (AfAfStSt), ‘semi-leafless’ (afafStSt) and ‘leafless’ (afafstst). The ‘leafless’ type had a lower growth rate compared with the conventional type, after 24 days from sowing and had a reduced relative growth rate (RGR) during the sampling period. The growth of the ‘semi-leafless’ type, however, was similar to that of the conventional phenotype. The reduced RGR of the ‘leafless’ plant was due to a reduced RGR of the stem portion of the plant, whereas the RGR of the remainder of the plant was similar in all three phenotypes. For each phenotype, two seed weight classes were sown and their growth compared. RGR was similar for these pairs of seed weight classes in each phenotype but the heavier seed weight class had a significantly superior growth rate in two of the three phenotypes. This is discussed in relation to the difference in the size of the embryonic axis. Results are discussed with regard to the development of a new phenotype for the dried pea crop.

In an attempt to explain previously observed differences between the growth rates of ‘leafless’ and conventional plants, we have compared representatives of three pea phenotypes; conventional (AfAfStSt), ‘semileafless’ (afafStSt) and ‘leafless’ (afafstst) with regard to their embryonic axis/embryo weight ratio and their patterns of pre-emergent growth. Within each phenotype, comparisons were made between genotypes which differed for mean weight per seed and between seed weight classes within each genotype. Although significant differences were found between genotypes for embryonic axis/embryo weight ratios, these could not be attributed to differences between phenotypes. Within each seed population there was a consistent decrease in this weight ratio with increasing seed size irrespective of phenotype.

Wang, T. L., Cook, S. K., Francis, R. J., Ambrose, M. J. and Hedley, C. L. 1987. An analysis of seed development in Pisum sativum. VI. Abscisic acid accumulation.—J. exp. Bot. 38: 1921-1932. The abscisic acid (ABA) content of wrinkled (rr) pea seed tissues has been quantified during development using multiple-ion-monitoring combined gas chromatography-mass spectrometry and a deuterated internal standard. The level of ABA in the embryo generally increased with increasing cotyledon fresh weight while that in the testa showed a distinct maximum at the time of maximum endosperm volume and the slowing in the growth of the testa. Pods contained relatively little ABA on a fresh weight basis. The total seed ABA content showed a biphasic distribution, the first maximum following the maximum growth rate of the testa and the second that of the embryo. The biphasic distribution of ABA in the pea seed was confirmed using a second pea genotype, near-isogenic to the first except for the r locus, and by the analysis of individual seeds using a radioimmunoassay for ABA. The first maximum was composed mainly of a testa component and the second mainly of an embryo component. When plants were grown in different environments, wrinkled seeds were found to contain slightly more ABA than round (RR) but this was only significant late in development. Immature seeds were capable of metabolizing l'-deoxy ABA to ABA, as deter mined by incorporation of either 3H or 2H, and the metabolite was present mainly in the testa. The production of ABA in pea seeds is discussed in relation to the development of the different seed tissues. Key words—Abscisic acid, peas, seed development. Correspondence to: John Innes Institute, Colney Lane, Norwich NR4 7UH, U.K.

The effect of artificially extended daylengths on the growth and flowering responses of 7 cultivars of Antirrhinum was assessed for 9 sowing-times at 2-week intervals. As the sowings progressed from Autumn to Winter, there was a reduction in the flowering-time and leaf number of the early cultivars after an initial increase; for the late cultivars there was a decrease from the first sowing. Only early cultivars showed a significant reduction for these parameters when artificially extended days were superimposed on plants from each sowing. The extended photoperiod had no effect on flower number, leaf area ratio (LAR) or the maximum growth rate (MGR). The LAR of early flowering cultivars decreased with sowing-time, whilst the late-flowering types showed the greatest change in MGR with sowing-time. These data are discussed in terms of selecting new Antirrhinum cultivars which, because of their sensitivity to daylength, can be easily manipulated by the grower.

Three different culture media have been examined for their ability to support growth in culture of embryos of two pea lines near-isogenic except for the r-locus. Embryos showed a greater increase in fresh weight on a medium containing 10% sucrose and a high level of a mixture of amino acids than on either one containing an equivalent amount of glutamine as the sole nitrogen source or one containing both inorganic nitrogen and a low level of glutamine. Small embryos (up to 10 mg fresh weight) showed the greatest relative increase in fresh weight. Decreasing the osmotic pressure of an agar medium by lowering the sucrose content to 2% and reducing the concentration of amino acids induced precocious germination. Shoot growth was more sensitive than root growth to increasing sucrose concentrations and optimum development was obtained when embryos were cultured in liquid culture at a high osmotic pressure followed by growth on an agar medium at low osmotic pressure. Alternatively, precocious germination could be induced by removing the cotyledons. Embryos of all sizes and of both genotypes of pea responded in a similar manner.

Seven F1 hybrids of Antirrhinum majus were grown in a range of environments and measurements were made of the time from sowing to budding and of the time from budding to flowering. The time to budding of all cultivars varied with environment, but was always longer for late-flowering types. The time from budding to flowering changed with environment in the early-flowering cultivars. This period was shorter and apparently independent of environment in the late-flowering types. The possibility of selecting types which combine the short vegetative phase of the early-flowering cultivars with the lower time for inflorescence development of the late ones is discussed.

SummaryThe effects of day-length and light intensity on the growth rate and leaf number of an early (Pink Ice) and a late (Orchid Rocket) flowering variety of Antirrhinum majus L. have been investigated. Growth rate, as determined by plant height or dry-weight increase, was greater for Pink Ice in all instances. When the two varieties were grown under similar conditions, the leaf numbers prior to flowering attained by Orchid Rocket were always greater than those of Pink Ice. With high light intensity the long-day (LD) responses of both varieties were characterized by leaf numbers which were on average 30 leaves less than the numbers found on plants grown in short days (SD) at high light intensity. Pink Ice exhibited this photoperiodic response to LD irrespective of whether the light intensity was high (26,000 lux) or low (6000 lux), whereas the leaf number of Orchid Rocket only declined in LD when the intensity was high. Plants of this variety grown in low light LD had leaf numbers similar to plants maint...

Activity changes in alanine aminotransferase (glutamate-pyruvate transaminase, GPT) in expanded fifth leaves of Lolium temulentum L. were studied with plants exposed to 8-h and 16-h photoperiods at 20 °C. Two phases were detected. The first was daylength independent and characterized by a rising activity, either on a fresh-weight or protein basis, whilst the second phase consisted of a rapid fall to a much lower activity during the course of a few days. This effect was promoted by 16-h/20°C conditions. Using variable exposure to long days and light-break treatments it was shown that the GPT depression did not relate directly to floral induction but was a response to increased incident light energy. Comparison with aspartate aminotransferase, acid phosphatase, malate dehydrogenase, and peroxidase suggested that GPT activity could be used as a sensitive indicator of incipient senescence. INTRODUCTION It has been demonstrated (Jones and Stoddart, 1970) that the activity of alanine aminotransferase (glutamate-pyruvate transaminase, GPT) in isolated shoot apices of Trifolium pratense L. is rapidly depressed by exposure to inductive long-day conditions. This effect was shown to correlate closely with the onset of the apical enlargement phase of floral initiation and the enzyme changes were obtained in the presence of an unchanged relative rate of incorporation of L-alanine into apical protein. There was, therefore, the suggestion that GPT might be of some significance in the regulation of the changeover from the vegetative to the reproductive condition at the shoot apex. To explore this possibility further, it was pertinent to ask whether the GPT depression effect was peculiar to the shoot apex or was detectable in other tissues, such as leaves, exposed to inductive photoperiods. To examine this aspect a plant with a greater sensitivity to photoperiodic induction than Trifolium was required. Studies have, therefore, been based upon Lolium temu lentum L. Some lines of this species have been shown to undergo floral initiation after exposure to a single inductive long-day cycle (Evans, 1958). MATERIALS AND METHODS The summer annual form of Lolium temulentum L. (Ba 3081) was used for all experiments. This material normally produces five leaves before responding to inductive long-days This content downloaded from 157.55.39.58 on Tue, 15 Nov 2016 03:56:30 UTC All use subject to http://about.jstor.org/terms 240 Hedley and St odd-art (Cooper, 1960) and shows no quantitative response either to short-day or low-temperature pretreatments. Seed from homogeneous populations, inbred over a large number of generations, was initially supplied by Dr. J. P. Cooper of the Welsh Plant Breeding Station, Aberystwyth. Growing conditions Seedlings were grown in John Innes No. 1 potting compost in a heated glasshouse under natural short-days. At the second leaf stage the pots were transferred to a controlled environ ment room giving 8 h of fluorescent illumination at a constant temperature of 20 °C, where they were maintained until required for use. Inductive daylengths were provided by a controlled environment room giving 16 h of fluorescent illumination at 20 °C. Both rooms had a light intensity of 11 000 lx at plant level. Light-break treatments consisted of a 1-h period of fluorescent illumination given midway through the dark period. Sampling and extraction Only the youngest fully expanded leaves on each axis were used, care being taken to ensure that samples were highly comparable in terms of physiological age and also free from fungal or visible virus infection. Samples consisted of 1 g fresh weight of leaf material ground in a mortar with 1 ml of buffer (tris 0-04 M, EDTA 0-25 mM, GSH 2-0 mM, pH 7-5) and a small quantity of washed sand. Extracts were centrifuged at 20 000 g for 20 min and the super natant used as the enzyme extract. All enzyme determinations were initiated within 1 h of extraction. Enzyme assays Alanine aminotransferase (E.C. 2.6.1.2) or glutamate-pyruvate transaminase (GPT) was determined by incubation at 37 °C with 1-0 ml of substrate (0-2 M ra-alanine, 0-002 M a-oxoglutarate in 0-1 M tris buffer at pH 7-4). Keto-acid production was assessed by formation of the dinitrophenylhydrazones and measurement of optical density at 546 nm (Reitmann and Frankel, 1957). Aspartate aminotransferase (E.C. 2.6.1.1) or glutamate-oxalacetate transaminase (GOT) was determined in a similar fashion to GPT, the substrate solution consisting of 0-1 M L-aspartate, 0-002 M a-oxoglutarate in 0-1 M tris buffer at pH 7-4. Malate dehydrogenase (E.C. 1.1.1.37) was determined by following loss of absorption at 340 nm due to oxidation of NADH. The test mixture consisted of 200 pmoles of oxaloacetate, 0-75 p.mole NADH and 0-1 ml of enzyme solution made to a total volume of 3-0 ml with 0-1 M pH 7-5 phosphate buffer. Change in optical density was followed for a period of at least 3 min on the linear phase of the reaction curve. Acid phosphatase (E.C. 3.1.3.2) was estimated using p-nitrophenyl phosphate as substrate (0-02 M in 0-1 M acetate buffer at pH 4-9). Release of nitrophenol was estimated by following change in optical density at 400 nm after 30 min incubation at 37 °C. Activity units were expressed as pinoles min-11-1. Peroxidase (E.C. 1.11.1.7) estimations used guaiacol as a donor. The assay was carried out in a Unicam SP800 spectrophotometer using 20 mM guaiacol, 40 mM hydrogen peroxide, and 10 mM phosphate buffer at pH 6-0. Tetraguaiacol formation was followed at 470 nm, enzyme concentrations being adjusted to give a linear reaction rate over a period of 2-4 min. Activities were calculated as tetraguaiacol units using an extinction coefficient of 26-6 cm^1 mM-1. All enzyme methods were checked for linearity between enzyme concentration and activity. Soluble protein determinations. Soluble protein was determined by the method of Lowry, Rosebrough, Lewis-Farr, and Randall, 1951. Estimation of protein synthesis Median leaf sections, 1 cm in length, were incubated in groups of four in a solution con sisting of 4 pmoles ATP, 4 pCi 3H-alanine (100 pCi/mM), 2pCi 14C-leucine (55-2pCi/mM) made to a total volume of 0-5 ml with 0-1 tris/EDTA buffer at pH 7-5. After incubation for 2-5 h at 37 °C the sections were rinsed with water and ground in a glass homogenizer. The homo genate was centrifuged at 4000 g for 5 min and 50 pi samples of supernatant were pipetted This content downloaded from 157.55.39.58 on Tue, 15 Nov 2016 03:56:30 UTC All use subject to http://about.jstor.org/terms Alanine Aminotransferase Activity in Lolium Leaves. I 241 onto 2-1 cm Whatman GF/A glass-fibre discs. Protein precipitation and washing were per formed essentially as described by Bollum (1968) and the radioactivity assessed by counting in a Beckman LS 100 liquid scintillation counter using a dioxane-based scintillation fluid. Efficiencies of 48 and 75 per cent were obtained for 3H and 14C respectively.

The activity of alanine aminotransferase (=glutamate-pyruvate transaminase, GPT) in dark-grown first leaves of Lolium temulentum L. was increased, after an initial lag-phase of 4-6 hr, by more than 130% during the first 24 hr of light-exposure. In comparison, aspartate aminotransferase (=glutamateoxalacetate transaminase, GOT) activity rose by only 18%. Red light treatments of up to 60 min duration produced subsequent increases in GPT activity but the effects were too small to indicate a phytochrome-mediated response. The amounts of enzyme formed were equivalent to those obtained with similar incident intensities of white light. Retuern to darkness after light exposure resulted in an arrestation of the light-stimulated GPT increase. Pre-treatment with cycloheximide caused either stimulatory or inhibitory effects depending upon the concentration applied but, in general, chlorophyll formation and GPT activity responded in a similar manner, whilst GOT showed virtually no response. Chloramphenicol at 6x10(-3) M depressed chlorophyll and Fraction 1 protein synthesis but stimulated GPT activity.The data are discussed in relation to the possible roles of GPT in the leaf. It is suggested that the enzyme, as determined, may be a complex of forms and that at least part of the activity may be involved in the early stages of chlorophyll biosynthesis.

Pisum sativum has been put forward as a practical alternative to soybean (Glycine max) as a protein source that can be grown in temperate climates. In contrast to the cereals little attention has been paid to the contribution of the reproductive parts of Pisum sativum in the carbon economy of the developing seed. This information would be important in subsequent breeding programmes designed to exploit knowledge of the physiological components of yield.

The usefulness of in vivo absorbance changes caused by P-700 oxidation are discussed and the principles of the measurement of these changes described. The possible application of this technique to remote sensing of photosynthetic performance is described.

The half time (t1/2) of the reduction of P‐700+ in the millisecond time frame is known to be limited by the reaction between plastoquinol and the cytochrome cytb6f complex. This is considered to be the rate limiting reaction of thylakoid electron transport and measurements of it provide a means of analysing how thylakoid election transport is regulated in vivo. The half time for the reduction of photochemically oxidized P‐700 has been measured in vivo using absorbance changes around 820 nm. The results showed that t1/2 is independent of irradiance and decreases as photosynthetic induction progresses. Even with a constant t1/2 the quantum efficiency of PSI declined as irradiance increased. The significance of the concept of photosynthetic control of electron transport is discussed in the light of these observations.