D. E. Briggs

Changes in cell wall polysaccharides of germinating barley grains

Abstract Decorticated barley grains were germinated at 25° for 6 days, until the endosperm reserves were nearly exhausted. The neutral monosaccharide components of the hydrolysates of the cell walls and gums from the embryo, aleurone layer and starchy endosperm and the endospermic starch were determined at daily intervals. The amount of embryo cell wall polysaccharide increased 40 times and glucose became the major component, followed in abundance by xylose and arabinose. The cell wall and gum polysaccharides of the aleurone layer (plus testa) and the starchy endosperm declined during germination and their compositions altered. The endospermic starch also decreased. In the early stages of germination the apparent composition of the cell walls of the aleurone layer and starchy endosperm depended upon how they had been prepared. After 6 days the cell walls and gums had provided a significant carbohydrate supply to the living tissues, equivalent to 18.5% of the endospermic polysaccharide degraded during growth, starch having provided the remaining 81.5%.

Gibberellins and α-amylase formation in germinating barley

Abstract Gibberellin-like material was estimated in barley corns “malted” at 14·4° or germinated on moistened filter paper at 25°, using a lettuce hypocotyl elongation assay. α-Amylase was measured ( a ) in these samples, ( b ) in grains (endosperms) deembryonated after various malting times then incubated further, and ( c ) in endosperms dosed individually with known amounts of GA 3 (gibberellic acid), and incubated at 14.·4°. The dosing technique provides the basis for a method of detecting and estimating 0·05 mμg or less GA 3 . A relationship existed between the “gibberellin” content and the rate of α-amylase synthesis in malting grain. Time lags were detected between the appearance of “gibberellin” in the whole grain, the arrival of the “gibberellin” in the endosperm and the appearance of α-amylase. For the first time the quantitative relations between GA 3 added to individual endosperms and consequent α-amylase production were investigated. These quantitative results strongly supported the concept that “gibberellin”, originating from the embryo, is of overwhelming importance in regulating the synthesis of α-amylase in the aleurone layer in malting grain, at least when the endosperm has been hydrated and sensitized by the postulated “embryo factor”. The endogenous gibberellin behaved more like GA 3 than GA 1 , or a mixture of varying proportions. Grain germinated on moistened filter papers at 25° gave apparently anomalous results in that the maximum value of α-amylase occurred before the greatest level of “gibberellin”.

Hydrocarbons, phenols and sterols of the testa and pigment strand in the grain of Hordeum distichon

Abstract Material from the testa of decorticated barley grains contained hydrocarbons, esters, triglycerides, free sterols, 5- n -alkylresorcinols, and traces of free alcohols, carbonyl compounds, and various polar, acidic materials. The hydrocarbon fraction was mainly a series of n -alkanes, extending at least from C 11 to C 36 , in which the C 29 and C 31 components were prominent. Two minor series of alkanes were also present. Sometimes a trace of an unsaturated hydrocarbon was detected. The ester fraction contained sterols and alkanols esterified by fatty acids, which differed in relative amounts from the fatty acids found in the triglycerides. The triglycerides were thought to have leached from within the grain. At least five free sterols were present, including sitosterol and campesterol. The 5- n -alkylresorcinols were at least twelve members of a homologous series, of which four, C 25 , C 27 , C 29 , and C 31 , made 98% of the total. Members of the series with even numbers of carbon atoms were also present. It is suggested that they are partly responsible for excluding microorganisms from the interior of the grain. The testa membrane, with the associated pigment strand, contained an estolide of fatty acids and various hydroxyacids, a polysaccharide component, and uncharacterized material.

α-Amylase in germinating, decorticated barley—I : α-Amylase, conditions of growth, and grain constituents

Abstract In germinating barley α-amylase is largely confined to the scutellum, starchy endosperm and aleurone. The levels attained are markedly altered by variations in the quantity of water available and the temperature. Added gibberellic acid, GA 3 , accelerates enzyme formation and breakdown, and increases the maximum quantity of enzyme. Alterations in the level of α-amylase do not seem to be regulated directly by levels of endogenous amino acids or simple carbohydrates. Continuing proteolysis, the removal of starch and calcium ions and falling pH may all contribute to falling enzyme activity. A hypothesis is advanced relating the effects of GA 3 and the destruction of α-amylase.

Enzyme formation and release by isolated barley aleurone layers

Abstract Aleurone layers, with testa attached, were prepared from degermed, decorticated barley with the aid of a fungal enzyme preparation. The preparations appeared intact under the scanning electron microscope. By using antibiotics only in an early stage preparations were obtained uncontaminated by micro-organisms and which, when incubated under optimal conditions with gibberellic acid, GA3, produced near-maximal amounts of α-amylase. The enzyme accumulated in the tissue before it was released into the incubation medium. Daily replacement of the incubation medium, containing GA3, depressed the quantity of α-amylase produced. α-Amylase was also produced in response to gibberellins GA1, GA4 and GA7 and, to a much lesser extent, helminthosporol and helminthosporic acid. A range of other substances, reported elsewhere to induce α-amylase formation, failed to do so in these trials. At some concentrations, glutamine marginally enhanced the quantity of enzyme formed during prolonged incubations. It is confirmed that α-glucosidase occurs in the aleurone layer and embryo of ungerminated barley, and increases in amount during germination. GA3 is shown to enhance this increase. When embryos arc burnt, to prevent gibberellin formation, no rise in α-glucosidase levels occurs unless GA3 is supplied to the grains. As the activity of α-glucosidase and other enzymes have been determined as ‘α-amylase’ by some assay methods, their alterations in activity in response to GA3 necessitates a re-evaluation of the evidence for de novo) synthesis of α-amylase in aleurone tissue.

α-amylase in germinating, decorticated barley—III : Effects of adding CCC and other chemical substances

Abstract Chlorocholine chloride, CCC, has been shown to inhibit growth and the formation of α-amylase in germinating barley. The addition of gibberellic acid, GA 3 , did not fully reverse the inhibition of growth caused by CCC, but α-amylase synthesis increased to the level found in grains cultured with GA 3 only. Thus CCC had a toxic effect on the embryo in addition to suppressing the endogenous formation of gibberellins. In the presence of GA 3 and large quantities of CCC α-amylase synthesis in barley was slow relative to the rate of enzyme formation with GA 3 only, but the rate of decline in enzyme was also retarded. Choline gave results generally similar to those produced by CCC, when applied to germinating barley. High levels of potassium sulphate slowed enzyme degradation but not synthesis in grain germinated with or without GA 3 . Some twenty-three compounds were added to germinating barley in the presence and absence of gibberellic acid, and the results were interpreted in terms of varying damaging effects towards the endogenous production of gibberellins, embryo growth, and the system synthesizing α-amylase in the aleurone layer. The results suggest that choline, hordenine and other naturally occurring basic substances may regulate the endogenous synthesis of gibberellins in germinating barley.

α-Amylase in germinating, decorticated barley-II. Effects of physically damaging the grain

Abstract α-Amylase was measured in barley grains, incubated with or without gibberellic acid, after the grains had been burnt or punctured in different ways. Conditions were found such that α-amylase production, and by inference the production of endogenous gibberellins, was inhibited independently of the effect of treatments on coleoptile growth. Damage to a small area of the aleurone results in an unexpectedly large reduction in α-amylase synthesis, suggesting that to some extent the aleurone functions as one unit rather than as a collection of independent cells. α-Amylase, induced by exogenous GA 3 in grains where the embryo had been burnt, was destroyed more slowly than in grains where embryo growth was normal, favouring the hypothesis that withdrawal of substances by the growing embryo from the endosperm favours the breakdown of α-amylase. The pH of endosperm homogenates also favoured this view. The method by which the grains were sterilized before burning the embryos influenced the rate of formation of α-amylase relative to whole grains, each in the presence of GA 3 .

Metabolism of ent-kaurenol-[17-14C], ent-kaurenal-[17-14C] and ent-kaurenoic acid-[17-14C] by germinating Hordeum distichon grains

Abstract Subcellular fractions from germinated barley embryos, chloroplast preparations and whole germinating barley grains are able to carry out the conversions ent -kaurenol → ent -kaurenal → ent -kaurenoic acid → ent -hydroxykaurenoic acid, the initial steps of the biosynthetic pathway to gibberellins. Whole grains, and chloroplasts to a slight extent, incorporate radioactivity from ent -kaurenol-[17- 14 C] and ent -kaurenoic acid-[17- 14 C] into materials with similar but distinct properties from the gibberellins GA 1 , GA 3 , GA 4 and GA 7 .

Phosphate mobilization in grains of Hordeum distichon

Abstract When decorticated grains were germinated at 14·5°, inorganic substances moved from the endosperm, mainly the aleurone layer, to the embryo. The level of Pi rose in the embryo and endosperm, and the embryo appeared to accumulate Pi against a concentration gradient. The level of organic-P declined in the endosperm, particularly in the alcurone layer. Separated aleurone layers, incubated at 25°, released only small amounts of organic or inorganic phosphate. However, when incubated with gibberellic acid (GA 3 ), a massive release of Pi occurred at the expense of organic phosphates within the tissue. This release followed a sigmoid pattern with time following a lag and was virtually complete in 6 days. Allowing the aleurone layer to dry before incubating with GA 3 reduced or abolished the lag period of Pi release and only marginally depressed the total amount ultimately freed. In contrast, α-amylase production was depressed by the longer periods of drying. The major phosphate of the aleurone was phytate ( meso -inositol hexaphosphate, IP 6 ), but traces of 1P 4 , IP 3 , IP 2 , IP 1 , Pi and unidentified phosphates were detected. During incubation with GA 3 the IP 6 content fell, and the lower esters of inositol rose slightly and then fell in a pattern indicating that the phosphate groups of each IP 6 molecule were being sequentially hydrolysed. After 6 days incubation, the tissue phosphates were reduced to a very low level. Attempts to isolate aleurone grains containing phytate were unsuccessful.

Externally applied gibberellic acid and α-amylase formation in grains of barley (Hordeum distichon)

Abstract During germination the aleurone layer of barley grains becomes progressively less able to form more α-amylase in response to a dose of gibberellic acid (GA3). This decline appears to be linked to the presence of a growing embryo. In whole grains the embryo ‘modulates’ the response (α-amylase formation) to controlled external applications of GA3 in a dose-dependent manner. Sugars, and some other metabolites, repress α-amylase formation in transected grains, apparently by reducing levels of endogenously produced gibberellins. This effect is partly, but not completely, reversed by additions of GA3. External applications of GA3 augment the levels of several gibberellin fractions within the grain. The nature of the gibberellin material remaining on the surface of the grains alters with time. Grains treated with GA3 contain a conjugate of low biological activity, possibly a glycoside, that is hydrolysed by a mixed glycosidase preparation to release a biologically-active gibberell in resembling GA3.