Early degradation of photosynthetic membranes in carob and sunflower cotyledons

The assimilatory activity of cotyledons can play an essential role in the survival of seedlings with a slow and delayed development of primary leaves. Changes in the photosynthetic activity of the cotyledon, from the onset of greening through senescence, were studied in two such plants, carob and sunflower, in order to determine its efficiency and duration, also in connection with the achievement of assimilatory autonomy by the plantlet. Chlorophyll analyses showed that the cotyledon's chloroplasts reached maximal greening in plantlets with a pair of expanded leaves. In contrast, the cotyledon's photosynthetic activity, measured as the rate of oxygen release, started to decrease early, before expansion of primary leaves. The decrease was due to the inactivation of a number of photosystem II (PSII) units, as revealed by immunodetection of breackdown products of the reaction centre's D1 and D2 thylakoid proteins. No signals of PSII alteration were noticed in the primary leaf chloroplasts that differentiated under the same environmental conditions. The damage to the cotyledon PSII, occurring in a non-photoinhibitory situation, might be due to a slower rate of turnover of D1 polypeptide than in the leaf thylakoids. The differential turnover of this protein in cotyledons and in leaves might represent an organ-specific regulation of the photosynthetic activity. The peculiarity of the cotyledon thylakoids make these organs useful objects for studying the metabolic cycle of both D1 and D2 proteins in vivo, under non-photoinhibiting conditions.     

A reassessment of the intervention of calmodulin in the regulation of stomatal movement

Commelina cammunis L., a monocotyledonous plant whose stomata are highly sensitive to calcium ions, was used to study calmodulin (CaM) involvement in stomatal movements. CaM was detected and quantified in guard cell and mesophyll cell protoplasts by western blot and by ⁴⁵Ca²⁺-overlays. CaM was found to be 3- to 7-fold more abundant on a per protein basis in guard cell than in mesophyll cell protoplasts. Numerous guard cell proteins that bind CaM in a Ca²⁺-dependent manner were detected by gold-labelled CaM overlays. Using bioassays with epidermal strips, different CaM-antagonists were found to induce a net stimulation of stomatal opening in darkness or under illumination (trifluoperazine > compound 48/80 ∼ fluphenazine > W7 > W5). As CaM is frequently involved in the regulation of phosphorylation processes, the effects of different inhibitors of protein kinases on stomatal movements were studied. In red plus blue light, a promotion of the stomatal aperture was observed in the nanomolar range with K252a and KT5926 and in the micromolar range with KT5720 ≫ ML7 ∼ ML9 ≫ H7 > KN62. Only the inhibitors with a high specificity for Ca²⁺-CaM dependent protein kinases (K252a, KT5926, ML7, ML9) triggered a stomatal opening in darkness and increased stomatal aperture in red plus blue light. Taken together, these data strongly suggest that a Ca²⁺- or a Ca²⁺-CaM-dependent protein kinase plays a central role in the calcium transduction pathway leading to the maintaining of stomatal closure.     

Inoculation of soybean (Glycine max. (L.) Merr.) with genistein-preincubated Bradyrhizobium japonicum or genistein directly applied into soil increases soybean protein and dry matter yield under short season conditions

In short-season soybean production areas, low soil temperature is the major factor limiting plant growth and yield. The decreases in soybean yield at low temperatures are mainly due to nitrogen limitation. Genistein, the most effective plant-to-bacterium signal in the soybean (Glycine max (L.) Merr.) nitrogen fixation symbiosis, was used to pretreat Bradyrhizobium japonicum. We have previously reported that this increased soybean nodulation and nitrogen fixation in growth chamber studies. Two field experiments were conducted on two adjacent sites in 1994 to determine whether the incubation of B. japonicum with genistein, prior to application as an inoculant, or genistein, without B. japonicum, applied onto seeds in the furrow at the time of planting, increased soybean grain yield and protein yield in short season areas. The results of these experiments indicated that genistein-preincubated bradyrhizobia increased the grain yield and protein yield of AC Bravor, the later maturing of the two cultivars tested. Genistein without B. japonicum, applied onto seeds in the furrow at the time of planting also increased both grain and protein yield by stimulation of native soil B. japonicum. Interactions existed between genistein application and soybean cultivars, and indicated that the cultivar with the greatest yield potential responded more to genistein addition.     

Identification and localization of calreticulin in plant cells

Summary In the present study we have investigated the presence and distribution of calreticulin in plant protoplasts. Calreticulin was purified from plant homogenates using a selective ammonium sulfate precipitation procedure developed for the purification of mammalian calreticulins and shown to bind calcium in⁴⁵Ca²⁺ overlay assays. The protein was localized to plant cell endoplasmic reticulum by the indirect immunofluorescence staining of protoplasts with anti-calreticulin antibodies. No calreticulin was observed within large vacuoles. We conclude that calreticulin is present in the endoplasmic reticulum of plant cells, where, by analogy to the mammalian endoplasmic reticulum, it may play a major role in Ca²⁺ binding and storage.Abbreviations ER endoplasmic reticulum - SR sarcoplasmic reticulum - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - PBS phosphate-buffered saline     

An abscisic acid analog inhibits abscisic acid-induced freezing tolerance and protein accumulation, but not abscisic acid-induced sucrose uptake in a bromegrass (Bromus inermis Leyss) cell culture

The application of abscisic acid (ABA), either as a racemic mixture or as optically resolved isomers, increases freezing tolerance in a bromegrass (Bromus inermis Leyss) cell culture and induces the accumulation of several heat-stable proteins. Two stereoisomers of an ABA analog, 23 dihydroacetylenic abscisyl alcohol (DHA), were used to study the role of ABA-induced processes in the acquisition of freezing tolerance in these cells. Freezing tolerance was unchanged in the presence of (–) DHA (LT₅₀ -9°C), and no increase in heat-stable protein accumulation was detected; however, the (+) enantiomer increased the freezing tolerance (LT₅₀ -13°C) and induced the accumulation of these polypeptides. All three forms of ABA increased freezing tolerance in the bromegrass cells, although (–) ABA was less effective than either (+) or (±) ABA when added at equal concentrations. Cells pretreated with 20 or 50 M (–) DHA displayed lower levels of freezing tolerance following the addition of 2.5, 7.5 or 25 M (±) ABA. Full freezing tolerance could be restored by increasing the concentration of (±) ABA to > 25 M. Pretreatment of cells with (–) DHA (20 or 50 M) had no effect on freezing tolerance when 25 M (+) ABA was added. The induction of freezing tolerance by 25 M (–) ABA was completely inhibited by the presence of 20 M (–) DHA. The accumulation of ABA-responsive heat-stable proteins was inhibited by pretreatment with 20 M (–) DHA in cells treated with 2.5 or 7.5M (+) ABA, and in cells treated with 25 M (–) ABA. The accumulation of these polypeptides was restored when (±) or (+) ABA was added at a concentration of 25 M. The analysis of proteins which cross-reacted with a dehydrin antibody revealed a similar inhibitory pattern as seen with the other ABA-responsive proteins. The effects of the various isomers of ABA and DHA on cell osmolarity and sucrose uptake was also investigated. In both cases, (±) and (+) ABA had pronounced effects on the parameters measured, whereas (–) ABA treated cells gave substantially different results. In both sucrose uptake and cell osmolarity, DHA had no significant effect on the results obtained following (±) or (+) ABA treatment. Maximum freezing tolerance was only observed in cells when both heat-stable protein accumulation and sucrose uptake were observed.Abbreviations ABA abscisic acid - DHA 2,3 dihydroacetylenicabscisyl alcohols - DMSO dimethyl sulfoxide - LT₅₀ temperature at which 50% of cells are killed The authors would like to acknowledge the technical assistance of Angela Bollman, Bruce Ewan and Angela Shaw. This work was supported by grants from the Natural Science and Engineering Research Council of Canada to L.V.G. and N.H.L., and a grant from the University of Saskatchewan to R.W.W.     

Detection and characterization of a glutenin subunit with unusual high Mr at the Glu-A1 locus in hexaploid wheat

A hexaploid wheat landrace collected from the Baluchistan province of Pakistan was found to possess a novel high-molecular-weight glutenin subunit (HMW-GS). The subunit has a very slow electrophoretic mobility as revealed by SDS-PAGE, and its molecular weight is comparable to that of the highest molecular weight glutenin subunit (2.2 encoded in the D-genome) reported so far in hexaploid wheat varieties and landraces of Japanese origin. Evidence obtained from (PCR) gene amplification studies using the primers specific for Glu-1 loci proved that the gene coding for this novel subunit belongs to the Glu-A1 locus located on the long arm of chromosome 1A. Digestion of the amplified gene (PCR product) with restriction enzymes indicated that the novel gene differs from prevailing Glu-A1 alleles (null, 1 and 2^*) by an extra DNA fragment of approximately 600 base pairs. The results also indicated that the novel subunit is most probably a derivative of subunit 2^* that has very likely incorporated the 600-bp fragment following a process of unequal crossing over. The present findings were further substantiated by reserved phase high performance liquid chromatography (RP-HPLC) analysis.     

Immunohistochemical characteristics of the constituents of senile plaques and amyloid angiopathy in aged cynomolgus monkeys

Abstract: In this study, we immunohistochemically examined the several constituents of senile plaques (SPs) and cerebral amyloid angiopathy (CAA) in aged cynomolgus monkeys. Apolipoprotein E (apoE) deposited in all mature plaques and CAA, and in half of the diffuse plaques. Alpha-1-antichymotripsin (αACT) deposited in half of the mature plaques and in one third of the CAA. Amyloid precursor protein (APP), ubiquitin (Ub), and microtubule-associated protein-2 (MAP-2) accumulated in the swollen neurites of mature plaques. Glial fibrillary acidic protein (GFAP) was detected in the astrocytes and their processes surrounding the mature plaques. Tau was detected in neither the SPs nor CAA. Therefore, mature plaques involved extracellular Aβ, apoE, and αACT, and also astrocytes and swollen neurites. However, diffuse plaques involved only extracellular Aβ and apoE. Since these features, except for tau, were consistent with those in humans, this animal model will be useful for studying the pathogenesis of cerebral amyloid deposition.     

The effects of activators of G-proteins,mastoparan and compound 48/80, on the G-protein/adenylate cyclase system in cultured melanophores of the black-moor goldfish,Carassius auratus

 To investigate the functions of GTP-binding protein(s) in the melanosome-aggregating response in fish melanophores, the effects of activators of G-proteins, namely, mastoparan and compound 48/80, were examined in cultured melanophores of the balck-moor goldfish, Carassius auratus. Both mastoparan and compound 48/80 induced an approximately 40% increase in the GTP-hydrolyzing activity in the melanophore membranes compared to the basal level. In intact melanophores, these compounds inhibited the effect of 3-isobutyl-1-methylxanthine, which induced the accumulation of intracellular cAMP. Pretreatment of melanophores with pertussis toxin at 1 μg ⋅ ml^-1 for 15 h attenuated the inhibitory effect of mastoparan on the accumulation of cAMP. However, pretreatment with the toxin only slightly attenuated the inhibitory effect of compound 48/80 on the accumulation of cAMP. In addition, compound 48/80 at 1 mg ⋅ ml^-1 induced full aggregation of the melanosomes in melanophores, though mastoparan at 5 μmol ⋅ l^-1 induced only 10–20% aggregation of melanophores. These results suggest that mastoparan and compound 48/80 can each activate the inhibitory G-protein in goldfish melanophores, which results in inhibition of adenylate cyclase activity. This signal-transduction pathway is involved in the aggregation of melanosomes in these cells. Accepted: 3 June 1996