John A. Zwar

The effects of gibberellic acid and abscisic acid on α-amylase mRNA levels in barley aleurone layers studies using an α-amylase cDNA clone

SummaryTwo cDNA clones were characterized which correspond to different RNA species whose level is increased by gibberellic acid (GA3) in barley (Hordeum vulgare L.) aleurone layers. On the criteria of amino terminal sequencing, amino acid composition and DNA sequencing it is likely that one of these clones (pHV19) corresponds to the mRNA for α-amylase (1,4-α-D-glucan glucanohydrolase, EC 3.2.1.1.), in particular for the B family of α-amylase isozymes (Jacobsen JV, Higgins TJV: Plant Physiol 70:1647–1653, 1982). Sequence analysis of PHV19 revealed a probable 23 amino acid signal peptide. Southern hybridization of this clone to barley DNA digested with restriction endonucleases indicated approximately eight gene-equivalents per haploid genome.The identity of the other clone (pHV14) is unknown, but from hybridization studies and sequence analysis it is apparently unrelated to the α-amylase clone.Both clones hybridize to RNAs that are similar in size (∼1500b), but which accumulate to different extents following GA3 treatment: α-amylase mRNA increases approximately 50-fold in abundance over control levels, whereas the RNA hybridizing to pHV14 increases approximately 10-fold. In the presence of abscisic acid (ABA) the response to GA3 is largely, but not entirely, abolished. These results suggest that GA3 and ABA regulate synthesis of α-amylase in barley aleurone layers primarily through the accumulation of α-amylase mRNA.

Gibberellic acid and abscisic acid modulate protein synthesis and mRNA levels in barley aleurone layers

Using in vivo pulse labeling, changes in the pattern of protein synthesis were detected in isolated barley aleurone layers treated with fibberellic acid (GA3). GA3 greatly altered the relative rates of synthesis of many polypeptides, increasing some, notably α-amylase, and decreasing others. α-Amylase synthesis increased until it was the major product (over 60%) of protein synthesis after 24h. The pulse-labeled pattern of secreted polypeptides was also changed by GA3. There was the expected increase in α-amylase together with a number of other polypeptides but there was reduced secretion of several polypeptides also.Cell-free translation of RNA isolated from control and hormone-treated tissues was used to measure changes in mRNA levels. GA3 caused many changes, particularly in the level of mRNA for α-amylase. In vitro synthesized α-amylase, identified by immunoaffinity chromatography, had an Mr of 46 000. This polypeptide was partially processed to a polypeptide with Mr 44 000 by the addition of dog pancreas membranes to the in vivo translation mixture. The level of mRNA for α-amylase began to increase 2–4 h after GA3 was added and reached a maximum level of about 20% of total mRNA after 16 h. Thus after 16 h, the synthesis of α-amylase as a proportion of total protein synthesis, continued to increase while the level of its mRNA as a proportion of total mRNA remained constant. These results indicate that protein synthesis was modified more extensively than we can account for by changes in mRNA.Abscisic acid (ABA) reversed all of the effects of GA3 on protein synthesis and mRNA levels. It also promoted synthesis of a small number of new polypeptides and increased the level of some mRNAs. GA3 reversed the accumulation of ABA-promoted mRNAs. Although, ABA strongly suppressed the increase in the level of translatable mRNA for α-amylase, there was an even stronger inhibition of enzyme synthesis and accumulation.We conclude that both GA3 and ABA regulate protein synthesis both positively and negatively in aleurone cells largely by regulating levels of mRNA and in the case of α-amylase, possibly also by changing the efficiency of translation of its mRNA.

Gibberellic-acid-responsive protoplasts from mature aleurone of Himalaya barley

Gibberellic acid (GA3)-responsive protoplasts were prepared from mature aleurone layers of Himalaya barley. Protoplasts prepared in air (air-protoplasts) synthesized α-amylase (EC 3.2.1.1) in the presence of GA3 at a rate which was 4–5 times greater that in its absence. Protoplasts prepared in nitrogen (N2-protoplasts) took longer than air-protoplasts to respond to GA3 but α-amylase synthesis ultimately attained a rate which was similar to that for air-protoplasts and which was many times that occurring in the absence of the hormone. Many characteristics of the protoplast response were similar to those of intact aleurone layers. α-Amylase arose by new synthesis, its synthesis was inhibited by abscisic acid, it was isozymically similar to aleurone layer enzyme, most of it was secreted into the incubation medium and its synthesis was accompanied by accumulation of α-amylase mRNA. GA3-induced changes in protein synthesis and cell structure also resembled those of intact aleurone cells. We conclude that the response of the protoplasts to GA3 is normal and that they present a useful system for the study of GA3 action in barley aleurone.

α-Amylase production and leaf protein synthesis in a gibberellin-responsive dwarf mutant of 'Himalaya' barley (Hordeum vulgare L.)

A dwarf mutant, M117, was isolated following sodium-azide mutagenesis of barley (Hordeum vulgare L. ‘Himalaya’). Treatment of the mutant with gibberellic acid (GA3) restored growth to levels of the tall parent, α-Amylase production was examined in germinated grains of the dwarf mutant and in Himalaya plants treated with gibberellin (GA) biosynthesis inhibitors. The mutant showed reduced α-amylase activity relative to the parent when grains were germinated on water, but activities were equivalent to the parent following germination on GA3 solution. Germination of normal or mutant grains in the presence of GA biosynthesis inhibitors led to reduced α-amylase activity levels, but normal levels were restored if GA3 was included in the inhibitor solution. These data are consistent with a model in which α-amylase production in the germinated grain is regulated by the supply of active GAs. Treatment of M117 with GA3 increased the length, fresh weight, dry weight, volume, cell number, and protein content of the first leaf. Proteins being synthesized in the first leaf were labelled with [35S]methionine and fractionated by two-dimensional electrophoresis. No reproducible qualitative or quantitative differences in protein profiles were detected in response to GA3 treatment. In contrast, first leaves from seedlings exposed to dehydration stress had profiles clearly distinguishable from those of control seedlings. Stem sections from dwarf plants maintained on 10 μM GA3 in the presence of sucrose elongated significantly more than controls without GA3, but two-dimensional analysis of the [35S]methionine-labelled radioactive polypeptides again revealed no GA3-induced differences. It was concluded that enhanced elongation rates of leaves or stem segments were not associated with major changes in gene expression.

Cytokinin activities of phenylurea derivatives - Bud growth.

The induction of cell division in isolated stem-pith of tobacco, a characteristic of the class of plant growth regulators called cytokinins (Skoog, Strong and Milles, 1965), is caused by about 300 structural variants of phenylurea (Bbuce, Zwar and Kefford, 1965; Bruce and Zwar, 1965). Another biological activity of cytokinins is their accelera tion of the growth of buds (Wickson and Thimann, 1958; Miller, 1961). This paper reports the activities of some phenylurea derivatives in the growth of lateral pea buds retarded in their growth by the activity of the apical bud. The synthesis and purification of the phenylurea derivatives was described by Bruce and Zwar (1965). The methods used were similar to those of Sachs and Thimann (1964). Pea (Pisum sativum cv. Alaska1) plants were grown in soil in a glasshouse with 25° C day/200 C night temperature. Uniform plants about 14 days old were used and each treatment was applied to approximately 25 plants. The youngest buds that were exposed and therefore were accessible to treatment were at the fourth node above the cotyledons. These buds were treated directly with a solution of 7 per cent Car bo wax 1500 in 50 per cent (wjv) aqueous ethanol without or with the compound at 0.05 per cent (w/v) concentration. In some treatments the solution applied to the buds contained 10-5 M gibberellic acid. Each bud received 5 [xl of this solution and a similar dose 3 days later. The lengths of buds were measured 7 days after the first treatment, when control buds were 1 to 2 mm long. As an index of the effect of compounds upon bud growth, the mean length of treated buds is expressed as a percentage of the mean length of control buds which received only Carbowax in aqueous ethanol. The activities of the ureas in cell division, as determined by Bruce and Zwar (1965), are included for comparison. These cell division acti vities were obtained by culturing expiants of tobacco stem pith on a sucrose-salts medium containing a series of concentrations of the com pound under test. After 14 days of culture, nests of new cells could be