Michael J. Dalling

Nitrogen redistribution during grain growth in wheat (Triticum aestivum L.)

The technique of EDTA-enhanced phloem exudation (King and Zeevaart, 1974: Plant Physiol. 53, 96–103) was evaluated with respect to the collection and identification of amino acids exported from senescing wheat leaves. Whilst the characteristics of the exudate collected conform with many of the accepted properties of phloem exudate, unexpectedly high molar proportions of phenylalanine and tyrosine were observed. By comparing exudation into a range chelator solutions with exudation into water, the increased exudation of phenylalanine and tyrosine relative to the other amino acids occurring when ethylene-diaminetetracetic acid was used, was considered to an artefact.In plants thought to be relying heavily on mobilisation of protein reserves to satisfy the nitrogen requirements of the grain, the major amino acids present in flag-leaf phloem exudate were glutamate, aspartate, serine, alanine and glycine. Only small proportions of amides were present until late in senescence when glutamine became the major amino acid in phloem exudate (25 molar-%). Glutamine was always the major amino acid in xylem sap (50 molar-%).The activities of glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.7.1), glutamate dehydrogenase (EC 1.4.1.3) and asparagine synthetase (EC 5.3.5.4) were measured in the flag leaf throughout the grain-filling period. Glutamine synthetase and glutamate-synthase activities declined during this period. Glutamate-dehydrogenase activity was markedly unchanged despite variation in the number of multiple forms visualised after gel electrophoresis. The activity of the enzyme reached a peak only very late in the course of senescence of the flag leaf. No asparagine-synthetase activity could be detected in the flag leaf during the grain-filling period.

Degradation of ribulose-1,5-bisphosphate carboxylase by proteolytic enzymes from crude extracts of wheat leaves.

In crude extracts from the primary leaf of wheat seedlings, Triticum aestivum L., cv. Olympic, maximum proteinase activity, as determined by measuring the rate of release of amino nitrogen from ribulose-bisphosphate carboxylase (RuBPCase), was found to be obtained only when EDTA and L-cysteine were included in the extraction buffer. Highest proteinase activity was obtained by grinding at pH 6.8, although the level of activity was similar in the pH range 5.6 to 8.0; this range also coincided with maximum extractability of protein. The lower amount of RuBPCase degrading proteinase extracted at low pH was not due to an effect of pH on enzyme stability. The optimum temperature of reaction was 50° C and reaction rates were linear for at least 120 min at this temperature. In the absence of substrate the proteinase was found to be very sensitive to temperatures above 30° C, with even short exposures causing rapid loss of activity. The relation between assay pH and RuBPCase degradation indicated that degradation was restricted to the acid proteinase group of enzymes, with a pH optimum of 4.8, and no detectable activity at a pH greater than 6.4. The levels of extractable RuBPCase proteinase exhibited a distinct diurnal variation, with activity increasing during the latter part of the light period and then declining once the lights were turned off. The effect of leaf age on the level of RuBPCase, RuBPCase proteinase and total soluble protein was investigated. Maximum RuBPCase activity occurred 9 days after sowing as did soluble protein. After the maximum level was obtained, the pattern of total soluble protein was shown to be characterised by three distinct periods of protein loss: I (day 9–13) 125 ng leaf-1 day-1; II (day 15–27) 11 ng leaf-1 day-1; III (day 29–49) 22 ng leaf-1 day-1. Comparison of the pattern of RuBPCase activity and total protein suggest that the loss of RuBPCase may be largely responsible for the high rate of protein loss during period I. Proteinase activity increased sharply during the period of most rapid loss of RuBPCase activity, and because the specific activity of RuBPCase also declined, we concluded that RuBPCase was being degraded more rapidly than the other proteins. Once the majority of the RuBPCase was lost, there did not appear to be a direct relation between RuBPCase proteinase activity and rate of total soluble protein loss, since the proteinase exhibited maximum activity during the slowest period of protein loss (II), and was declining in activity while the rate of protein loss remained stable during the third and final period of total protein loss.

Nitrogen redistribution during grain growth in wheat (Triticum aestivum L.) : I. Peptide hydrolase activity and protein breakdown in the flag leaf, glumes and stem.

The activity of a range of endo- and exopeptidase enzymes have been measured in the glumes, flag leaf and stem during the period of grain development in wheat. The enzymes show a sequential pattern of appearance with activity peaks occurring at a number of intervals from anthesis until just prior to the cessation of grain growth. Of the enzymes studied only the haemoglobin- and casein-degrading activity and alanylglycine-dipeptidase activity increased during the period of rapid protein loss, while aminopeptidase, carboxypeptidase and leucyltyrosine dipeptidase reached maximum activity prior to this period.

A Galactolipase Activity Associated with the Thylakoids of Wheat Leaves (Triticum aestivum L.)

Abstract When chloroplasts prepared from the primary leaves of wheat are incubated at pH 6.0, a large proportion of the thylakoid galactolipids, particularly monogalactosyl diacylglycerol (MGDG), is rapidly degraded. The hydrolysis is incomplete, producing Iyso-MGDG, together with free fatty acids (predominately linolenic acid, LNA). Initial rates are extremely rapid (in excess of l/µmol LNA mgchl -1 min -1 ) but an endpoint is reached after 1–2 min, when as much as half the thylakoid MGDG has been partially hydrolysed. The «galactolipase» activity is associated with the thylakoid membranes, but is exposed to the stromal surface. It is inhibited by the sulfhydryl-enyzme inhibitors phenylmercuric acetate and p-chloromercuribenzoid acid. The enzyme is inactivated by exposure to temperatures > 30 °C, and is sensitive to detergent or sonication treatments that disturb the integrity of the thylakoid membranes. It has not yet been possible to isolate an active protein fraction.

The Effect of Sulfur Deficiency on the Organisation and Photosynthetic Capability of Wheat Leaves

Summary An examination of the effects of sulfur stress on wheat leaves and leaf metabolism have shown that sulfur deficiency interferes with the normal pattern of leaf development.The most obvious morphological symptoms of sulfur deficiency are a reduction in final leaf area, rate of leaf development, and leaf chlorophyll content.At the cellular level, sulfur deficiency reduces mesophyll cell number per cm 2 and chlorophyll content per chloroplast by 62% and 75 %, respectively.Chloroplast number per mesophyll cell was unaffected.The capacity of chloroplasts to undertake in vitro non-cyclic electron transport was also reduced by about 75 %, although the rate per unit chlorophyll was uneffected by plant sulfur status.

Endopeptidase and Carboxypeptidase Enzymes of Vacuoles Prepared from Mesophyll Protoplasts of the Primary Leaf of Wheat Seedlings

Summary Vacuoles prepared from mesophyll protoplasts of the primary leaf of wheat ( Triticum aestivum L. cv. Egret) contain carboxypeptidase (CP) and endopeptidase (EP) activity. CP activity was optimal at pH 5.2. The most preferred substrate was Z-Phe-Ala and activity against a wide range of other substrates was dependent on the C-terminal amino acid. Z-dipeptides with proline in any position were not hydrolysed. CP activity was inhibited by phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, sodium dodecyl sulfate (SDS) and 4-hydroxymercuric benzoic acid. Thus the CP enzyme from the wheat leaf is a Serine Carboxypeptidase (EC 3.4.16). EP activity, assessed by following the degradation of the Large Sub Unit (M r 53.1 kD) of Ribulose 1,5-Bisphosphate Carboxylase (RuBPCase) by SDS-PAGE, was optimal at pH 5.2 (-SDS) and 5.5 (+SDS). The overall rate of degradation of the Large Sub Unit (LSU) was increased about 2.6-fold by the addition of 0.2 % SDS to the assay medium. In the absence of SDS there were only three prominent polypeptide breakdown products, M r 52.1, 50.9 and 50.9 kD. The 50.9 and 50.1 kD products appeared resistant to further hydrolysis, whilst the 52.1 kD product was eventually degraded. The presence of SDS increased the heterogeneity of the breakdown products, polypeptides with M r 51.8, 44.4, 43.6, 30.3 and 29.5 kD were especially prominent. The major portion of the vacuolar EP activity was considered to belong to the Thiol proteinase group (EC 3.4.22), although indirect evidence was obtained for two other EP activities. There appeared to be little co-operation between the EP and CP activities in the degradation of RuBPCase.

Characterization of Peptide Hydrolase Activity Associated with Thylakoids of the Primary Leaves of Wheat

Summary Intact chloroplasts, free of vacuolar contamination, can be isolated from mesophyll protoplasts prepared from the primary leaves of 8–10 day old wheat seedlings ( Triticum aestivum L.). Thylakoids prepared from these chloroplasts exhibited peptide hydrolase activity. Activity was detected by following the degradation of the large sub-unit of surface-associated ribulose hisphosphate carboxylase (RuBPCase) using the technique of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The large subunit of RuBPCase was degraded with a pH optimum of 4.8 by an enzyme which was sensitive to sulfhydryl-group inhibitors. At pH 4.8 there was little activity against any of the thylakoid proteins. The surface-associated RuBPCase was easily removed by washing the thylakoids with sodium pyrophosphate. With repeated washing, the peptide hydrolase activity was also removed from the thylakoids. A second type of peptide hydrolase activity was detected only after treatment of the thylakoids with sodium dodecyl sulfate. In this instance, the α and β sub-units of coupling factor, a major thylakoid protein, were degraded. Degradation was optimal at pH 6.2 and was inhibited with sulfhydryl-group inhibitors.

The effect of a thylakoid-associated galactolipase on the morphology and photochemical activity of isolated wheat leaf chloroplasts

Summary This study investigated the physiological effects of a thylakoid-bound galactolipase activity, which may participate in thylakoid catabolism during foliar senescence. Electron transport through photosystem II was completely inhibited in thylakoids after only 30 sec of «galactolipase»activation. The thylakoids were completely uncoupled after 60sec. PS I was less sensitive, being reduced by about 60% in high light at the endpoint of «galactolipase» activity. However, light response curves for PS I revealed that the quantum efficiency was very sensitive to galactolipid hydrolysis, although normal rates of electron transport could be achieved by increasing intensities of light. The effects of galactolipid hydrolysis on electron transport were almost entirely eliminated in the presence of BSA, indicating that most, if not all of the disruption was attributable to the accumulation of free fatty acids in the membranes. Ultrastructural examination revealed that hydrolysis of thylakoid MGDG caused dissociation and degeneration of stromal lamellae, while granal stacks remained intact but greatly swollen. The effects were only slightly lessened in the presence of BSA. In contrast, thylakoids exposed to exogenous LNA were largely unstacked, but the lamellae retained their integrity and were more or less paired. Effects of LNA were completely mitigated in the presence of BSA. The structures resulting from lipid hydrolysis resemble those reported in naturally senescing leaves. The results did not indicate that galactolipid depletion adversely affected the photosynthetic function of the thylakoid membranes, but it may contribute to the morphological changes observed in thylakoids during senescence.

Mobilization of Nitrogen and Phosphorus from Endosperm

Publisher Summary This chapter discusses the mobilization of stored forms of nitrogen and phosphorus from the endosperm during seed germination. The conceptual model of nitrogen and phosphorus mobilization incorporates the important interrelation between the embryo, aleurone layer, starchy endosperm, and scutellum. Each has a specific function with respect to mobilization, transport, further processing, and transfer to the growing seedling. The sequence of events important to mobilization is as follows in which cell inhabitation is followed or coincident with an increase in the in situ activity of the latent peptide hydrolases. The endopeptidase appears to be a pepstatin-A sensitive acid proteinase and is associated with the aleurone layer protein bodies. It seems that the amino acids for the next phase of germination, the gibberellic acid-induced synthesis and secretion of a range of hydrolytic enzymes, arise as a result of the activity of those enzymes detected in the resting seed. Apart from phytase, which degrades the aleurone layer phytin, the other hydrolytic enzymes must be secreted into the endosperm. These enzymes are all acid peptide hydrolases, a property consistent with the low pH of the endosperm. The degradation of RNA and LPC occurs also by action of enzymes synthesized and secreted by the aleurone layer. Absorption is rapid and the measured in vitro rates appear more than adequate to cope with the in situ flux of nitrogen from the endosperm. The scutellum also functions as a secondary processing tissue, a function well defined for nitrogen. This tissue is a rich source of aminopeptidase and alkaline dipeptidase activity, the enzymes most likely to be responsible for the hydrolysis of the small peptides absorbed from the endosperm.

A Causal Link between «Galactolipase» and «Chlorophyll Oxidase» in Wheat Leaf Chloroplasts

Summary Isolated chloroplasts from wheat or barley leaves contain an oxidative chlorophyll-bleaching activity (Chlorophyll oxidase, or CHLOX) which is activated in the presence of free polyunsaturated fatty acids. We have shown that CHLOX can be activated by an endogenous «galactolipase » activity, which releases linolenic acid (LNA) from the abundant thylakoid lipid monogalactosyl diacylglycerol (MGDG). The galactolipase activity is a feature of intact thylakoid membranes and has a pH optimum at 6.0. The liberation of LNA is initially very rapid in vitro , but reaches an endpoint after 1-2 min. Thus a brief preincubation of thylakoids at pH 6.0 is sufficient to initiate chlorophyll-bleaching. While the dose of endogenously supplied LNA was relatively low, the response of CHLOX was approximately four times as great as to equivalent amounts of exogenous LNA.