Induction ofent-kaurene biosynthesis by low temperature in dwarf peas

Germinating pea (Pisum sativum L.) seeds of two dwarf cultivars, “Progress No. 9” and “Green Arrow”, and two tall cultivars, “Alaska” and “Alderman”, were treated with low temperature (3–5°C) for 14 days and then transferred to normal growing conditions (19–21°C for 16 h/14.5–16.5°C for 8 h) for an additional 10 days. Biosynthesis of [¹⁴C]ent-kaurene from [¹⁴C]2-mevalonic acid (2-MVA) was assayed in cell-free enzyme extracts prepared from shoot tips 10 days after cold treatment and was compared with activity in enzyme extracts prepared from noncold-treated, 10-day-old control plants. Shoot lengths of cold-treated plants were measured throughout a 35-day period and compared with shoot lengths of plants grown without cold treatment for 25–35 days. Low temperature induced a five-to 10-fold enhancement ofent-kaurene, hence potentially gibberellin (GA), biosynthesis in seedlings of the two dwarf cultivars but not in the tall cultivars. However, the lack of an increase in growth rate in the cold-treated dwarfs indicated that endogenous GA biosynthesis remained blocked at some point beyondent-kaurene in the biosynthetic pathway. Since the late-flowering “Alderman” cultivar did not exhibit enhanced biosynthesis ofent-kaurene, it appears that if vernalization in late-flowering cultivars of peas is correlated with enhanced GA biosynthesis, it is not the early part of the biosynthetic pathway which is affected.     

Inheritance of legumin and albumin contents in a cross between round and wrinkled peas

Summary Legumin and albumin are the fractions of pea seed proteins preferred to vicilin because of their high sulfur amino acid contents. The joint inheritance of legumin and albumin contents was studied in a cross between to contrasting lines of peas — one with high legumin and low albumin, and the other with low legumin and medium to high albumin. Single seed determinations were made in the parental, F₁; F₂ and backcross generations using rocket immunoelectrophoresis. In the non-segregating generations (P₁, P₂ and F₁), legumin and albumin contents were negatively correlated (r=–0.50). The estimates of correlation coefficients in the segregating generations (F₂, BC₁ and BC₂) were also about –0.5. However, the two estimates based on the round and on the wrinkled seeds separately in the F₂ generation were not significantly different from zero. At least four individual round F₂ seeds showed the desired recombination of high legumin with high albumin indicating that the unfavorable correlation can be broken. In this cross legumin content showed predominantly additive genetic variation whereas the dominance variance was the largest component for albumin content. A combined relative sulfur index, proposed as a convenient measure for selection, showed a narrow sense heritability of 47%. In general these results support the view that sulfur amino acid content of peas can be improved by breeding, but that the required selection regime must take both legumin and albumin content into account.     

Induction ofent-kaurene biosynthesis by low temperature in dwarf peas

Germinating pea (Pisum sativum L.) seeds of two dwarf cultivars, Progress No. 9 and Green Arrow, and two tall cultivars, Alaska and Alderman, were treated with low temperature (3–5°C) for 14 days and then transferred to normal growing conditions (19–21°C for 16 h/14.5–16.5°C for 8 h) for an additional 10 days. Biosynthesis of [¹⁴C]ent-kaurene from [¹⁴C]2-mevalonic acid (2-MVA) was assayed in cell-free enzyme extracts prepared from shoot tips 10 days after cold treatment and was compared with activity in enzyme extracts prepared from noncold-treated, 10-day-old control plants. Shoot lengths of cold-treated plants were measured throughout a 35-day period and compared with shoot lengths of plants grown without cold treatment for 25–35 days. Low temperature induced a five-to 10-fold enhancement ofent-kaurene, hence potentially gibberellin (GA), biosynthesis in seedlings of the two dwarf cultivars but not in the tall cultivars. However, the lack of an increase in growth rate in the cold-treated dwarfs indicated that endogenous GA biosynthesis remained blocked at some point beyondent-kaurene in the biosynthetic pathway. Since the late-flowering Alderman cultivar did not exhibit enhanced biosynthesis ofent-kaurene, it appears that if vernalization in late-flowering cultivars of peas is correlated with enhanced GA biosynthesis, it is not the early part of the biosynthetic pathway which is affected.     

The complexities of starch biosynthesis in cereal endosperms

Starch serves not only as an energy source for plants, animals, and humans but also as an environmentally friendly alternative for fossil fuels. Here, we describe recent findings concerning the synthesis of this important molecule in the cereal endosperm. Results from six separate transgenic reports point to the importance of adenosine diphosphate glucose pyrophosphorylase in controlling the amount of starch synthesized. The unexpected cause underlying the contrast in sequence divergence of its two subunits is also described. A major unresolved question concerning the synthesis of starch is the origin of nonrandom or clustered alpha-1,6 branch-points within the major component of starch, amylopectin. Developing evidence that several of the starch biosynthetic enzymes involved in amylopectin synthesis occur in complexes is reviewed. These complexes may provide the specificity for the formation of nonrandom branch-points.     

Promotion by gibberellic Acid of polyamine biosynthesis in internodes of light-grown dwarf peas

When gibberellic acid (GA₃; 5-35 micrograms per milliliter) is sprayed on 9-day-old light-grown dwarf Progress pea (Pisum sativum) seedlings, it causes a marked increase in the activity of arginine decarboxylase (ADC; EC 4.1.1.9) in the fourth internodes. The titer of putrescine and spermidine, polyamines produced indirectly as a result of ADC action, also rises markedly, paralleling the effect of GA₃ on internode growth. Ammonium (5-hydroxycarvacryl) trimethyl chloride piperidine carboxylate (AMO-1618; 100-200 micrograms per milliliter) causes changes in the reverse direction for enzyme activity, polyamine content, and growth. GA₃ also reverses the red-light-induced inhibition of ADC activity in etiolated Alaska pea epicotyls; this is additional evidence for gibberellin-light interaction in the control of polyamine biosynthesis. The enzyme ornithine decarboxylase (ODC; EC 4.1.1.17), an alternate source of putrescine arising from arginine, is not increased by GA₃ or by AMO-1618.     

Phosphate positioning and availability in the starch granule matrix as studied by EPR

Cu(2+) was introduced as an EPR probe into the starch granules isolated from different starch crop genotypes including transgenically modified potatoes generated for extreme amylose and starch phosphate monoester concentrations. Several discrete copper adducts bound to the starch matrix with different strength was revealed. It was found that phosphate has a significant influence on the type of these species, their number, location in the structure, and strength of binding. Well dispersed Cu(2+) complexes with axial symmetry are formed in the semicrystalline part of the starch linked through O-P- bonds in the phosphorylated starches. In the amorphous part of the starch, freely rotating hexaaqua complexes of Cu(2+) and complexes coupled antiferromagnetically are formed. The amount of the former increases with content of phosphate indicating enhanced binding of water in the granules. The results complement previous experimental data and molecular models for the starch granule with respect to the location and effects of phosphate and crystalline matter.     

The biosynthesis of flavonoid glycosides and their importance in chemosystematics

The present article reviews flavonoid O-glycosyltransferases with respect to the historical background, isolation and purification methods, properties of the enzymes involved (especially substrate specificities) and genetic control. The possible biosynthetic pathway leading to the formation of C-glycosides is also discussed. The second part of the article is an attempt to indicate the importance of glycosylation patterns in the field of chemosystematics, especially on the intra- and infra-specific levels. The position and nature of glycosylation are first discussed, and this is followed by examples indicating the importance of glycosylation patterns.     

A quantitative assessment of the importance of barley seed alpha-amylase, beta-amylase, debranching enzyme, and alpha-glucosidase in starch degradation.

Extracts of germinated barley (Hordeum vulgare L.) seeds of 41 different genotypes were analyzed for their activities of alpha-amylase, beta-amylase, alpha-glucosidase, and debranching enzyme and for their abilities to hydrolyze boiled soluble starch, nonboiled soluble starch, and starch granules extracted from barley seeds with water. Linear correlation analysis, used to quantitate the interactions between the seven parameters, revealed that boiled soluble starch was not a good substrate for predicting activities of enzymes functioning in in vivo starch hydrolysis as the extracts' abilities to hydrolyze boiled soluble starch was not correlated with their abilities to hydrolyze native starch granules. Activities of alpha-amylase and alpha-glucosidase were positively and significantly correlated with the seed extracts' abilities to hydrolyze all three starches. beta-Amylase was only significantly correlated with hydrolysis of boiled soluble starch. No significant correlations existed between debranching enzyme activity and hydrolysis of any of the three starches. Interactions between the four enzymes as they functioned together to hydrolyze the three types of starch were evaluated by path coefficient analysis. alpha-Amylase contributed to hydrolyses of all three starches primarily by its direct effect (noninteractive component). This direct contribution increased as the substrate progressed from the completely artificial boiled soluble starch, to the most physiologically significant substrate, native starch granules. alpha-Glucosidase contributed to the hydrolysis of boiled soluble starch primarily by its direct effect (noninteractive) yet contributed to starch granule hydrolysis primarily via its interaction with alpha-amylase (indirect effect). The contribution of beta-amylase to hydrolysis of boiled soluble starch was direct and it did not contribute significantly to hydrolysis of native starch granules.     

Effects of reaction conditions on the physicochemical properties of cationic starch studied by RSM

Granular cationic starches were prepared in aqueous phase without the addition of swelling inhibiting salts. Response surface methodology (RSM) was performed to analyze the effects of reaction conditions on physicochemical properties of the products. As the reaction time was prolonged from 2 to 5 and to 24 h, the relative contribution of the temperature to degree of substitution (DS) turned from minor to prominent. Good correlations were observed between the DS and the pasting temperature of the 2, 5, and 24 h cationized starches. By contrast, variation in the correlations between DS and the other physicochemical properties, with respect to reaction durations, revealed the processing pattern of cationization in starch granules along extending reaction times. This deduced pattern was confirmed by the granular and molecular characterizations using confocal laser scanning microscopy and high performance anion exchange chromatography, respectively.     

Light-induced degradation of starch granules in turions of Spirodela polyrhiza studied by electron microscopy

Spirodela polyrhiza forms turions, starch-storing perennial organs. The light-induced process of starch degradation starts with an erosion of the surface of starch grains. The grain size decreases over a period of red irradiation and the surface becomes rougher. The existence of funnel-shaped erosion structures demonstrates that starch degradation is also possible inside the grains. Neither etioplasts nor clues as to their transition into chloroplasts were found in the storage tissue by transmission electron microscopy. Juvenile chloroplasts always contained the starch grains which remained from amyloplasts. No chloroplasts were found which developed independently of starch grains. Amyloplasts are therefore the only source of chloroplasts in the cells of irradiated turions. The intactness of amyloplast envelope membranes could not be directly proved by electron microscopy. However, the light-induced transition of amyloplasts into chloroplasts provides indirect evidence for the integrity of the envelope membranes throughout the whole process. The starch grains are sequestered from the cytosolic enzymes, and only plastid-localized enzymes, which have access to the starch grains, can carry out starch degradation. In this respect the turion system resembles transitory starch degradation as known from Arabidopsis leaves. On the other hand, with α-amylase playing the dominant role, it resembles the mechanism operating in the endosperm of cereals. Thus, turions appear to possess a unique system of starch degradation in plants combining elements from both known starch-storing systems.