Development of Microbody Membrane Proteins during the Transformation of Glyoxysomes to Leaf Peroxisomes in Pumpkin Cotyledons

The microbody transition observed in the cotyledons of somefatty seedlings involves the conversion of glyoxysomes to leafperoxisomes. To clarify the molecular mechanisms underlyingthe microbody transition, we established a method for the preparationof highly purified microbodies. SDS-PAGE and immunoblot analysisof isolated microbodies from pumpkin cotyledons at various stagesshowed that glyoxysomal enzymes are replaced by leaf-peroxisomalenzymes during the microbody transition. Two proteins in glyoxysomalmembranes, with molecular masses of 31 kDa and 28 kDa, werenot solubilized from the membranes with 0.2 M KCl, an indicationthat these proteins are bound tightly with glyoxysomal membranes.Their polyclonal antibodies were raised against the respectivepurified protein. Immunoblot analysis of subcellular fractionsand immunogold analysis confirmed that these proteins were specificallylocalized on glyoxysomal membranes. Analysis of these membraneproteins during development revealed that the amounts of thesemembrane proteins decreased during the microbody transitionand that the large one was retained in leaf peroxisomes, whereasthe small one could not be found in leaf peroxisomes after completionof the microbody transition. The results clearly showed thatmembrane proteins in glyoxysomes change dramatically duringthe microbody transition, as do the enzymes in the matrix. ¹Present address: School of Agriculture, Nagoya University Chikusa,Nagoya, 464-01 Japan.     

Immunocytochemical Localization of Uricase in Peroxisomes of Soybean Cotyledons

Antibodies specific for nodule uricase were used for immunocytochemistryto demonstrate the presence of uricase in cotyledons of soybean(Glycine max) during germination and early seedling growth.The enzyme was localized exclusively in peroxisomes. ¹Permanent address: Department of Plant Cytology and Cytochemistry,University of Lodz, Lodz, Poland ²Current address: Department of Plant Science, University ofArizona, Tucson, AZ 85721, U.S.A.     

Development of Enzymes of the Glyoxylate Cycle during Senescence of Pumpkin Cotyledons

The presence and activities of isocitrate lyase (EC 4.1.3.1 [EC] )and malate synthase (EC 4.1.3.2 [EC] ) were studied during senescenceof pumpkin cotyledons (Cucurbita sp. Amakuri Nankin). Afterincubation of detached cotyledons in permanent darkness, theactivities appeared and increased up to the eighth day and thendeclined, while the activities of catalase (EC 1.11.1.6 [EC] ), glycolateox-idase (EC 1.1.3.1 [EC] ), and hydroxypyruvate reductase (EC 1.1.1.81 [EC] )decreased dramatically. After fractionation of cell organellesby sucrose density gradient, we detected isocitrate lyase andmalate synthase activities in peroxisomal fractions. The activityof the two key enzymes of the glyoxylate cycle also increasedduring senescence in vivo and we confirmed the presence of thetwo enzymes in the peroxisomal fractions after sucrose gradientcentrifugation. At every point examined, the level of malatesynthase was demonstrated by immunoblotting. It is concludedthat the development of isocitrate lyase and malate synthaseactivities represents the transition from leaf peroxisomes toglyoxysomes and that such a phenomenon is associated with senescence. (Received January 25, 1991; Accepted March 22, 1991)     

A Study of Formate Production and Oxidation in Leaf Peroxisomes during Photorespiration

When glycolate was metabolized in peroxisomes isolated from leaves of spinach beet (Beta vulgaris L., var. vulgaris) formate was produced. Although the reaction mixture contained glutamate to facilitate conversion of glycolate to glycine, the rate at which H₂O₂ became “available” during the oxidation of [1-¹⁴C]glycolate was sufficient to account for the breakdown of the intermediate [1-¹⁴C]glyoxylate to formate (C₁ unit) and ¹⁴CO₂. Under aerobic conditions formate production closely paralleled ¹⁴CO₂ release from [1-¹⁴C]glycolate which was optimal between pH 8.0 and pH 9.0 and was increased 3-fold when the temperature was raised from 25 to 35 C, or when the rate of H₂O₂ production was increased artificially by addition of an active preparation of fungal glucose oxidase.     

Glyoxylate Cycle Enzymes in Peroxisomes Isolated from Petals of Pumpkin (Cucurbita sp.) during Senescence

The activities of the two unique enzymes of the glyoxylate cycle,isocitrate lyase (EC 4.1.3.1 [EC] ) and malate synthase (EC 4.1.3.2 [EC] ),were undetectable in petals of pumpkin (Cucurbita sp. AmakuriNankin) until the end of blooming, but they appeared duringsenescence. The activity of catalase (EC 1.11.1.6 [EC] ) increased,glycolate oxidase (EC 1.1.3.1 [EC] ) activity did not change, whilehydroxypyruvate reductase (EC 1.1.1.81 [EC] ) activity peaked at fullblooming stage and declined thereafter. After fractionationof cellular organelles on a sucrose density gradient, we detectedisocitrate lyase and malate synthase activities in peroxisomalfractions only from petals at the senescing stage. Northernblot analysis revealed that malate synthase mRNA increased duringpetal senescence. Citrate synthase (EC 4.1.3.7 [EC] ) and malate dehydrogenase(EC 1.1.1.37 [EC] ) activities were also present, while aconitase(EC 4.2.1.3 [EC] ) was not detectable in peroxisomal fractions. Moreoverthe presence of 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35 [EC] )and urate oxidase (EC 1.7.3.3 [EC] ) in the peroxisomal fractionsfrom senescing petals indicates that peroxisomes could be involvedboth in the ß-oxidation pathway and in the purinecatabolism during petal senescence. (Received May 25, 1991; Accepted September 25, 1991)     

Catalase Degradation in Sunflower Cotyledons during Peroxisome Transition from Glyoxysomal to Leaf Peroxisomal Function

First order rate constants for the degradation (degradation constants) of catalase in the cotyledons of sunflower (Helianthus annuus L.) were determined by measuring the loss of catalase containing ¹⁴C-labeled heme. During greening of the cotyledons, a period when peroxisomes change from glyoxysomal to leaf peroxisomal function, the degradation of glyoxysomal catalase is significantly (P = 0.05) slower than during all other stages of cotyledon development in light or darkness. The degradation constant during the transition stage of peroxisome function amounts to 0.205 day⁻¹ in contrast to the constants ranging from 0.304 day⁻¹ to 0.515 day⁻¹ during the other developmental stages. Density labeling experiments comprising labeling of catalase with ²H₂O and its isopycnic centrifugation on CsCl gradients demonstrated that the determinations of the degradation constants were not substantially affected by reutilization of ¹⁴C-labeled compounds for catalase synthesis. The degradation constants for both glyoxysomal catalase and catalase synthesized during the transition of peroxisome function do not differ. This was shown by labeling the catalases with different isotopes and measuring the isotope ratio during the development of the cotyledons. The results are inconsistent with the concept that an accelerated and selective degradation of glyoxysomes underlies the change in peroxisome function. The data suggest that catalase degradation is at least partially due to an individual turnover of catalase and does not only result from a turnover of the whole peroxisomes.     

Effects of Cytokinin and Light on Polyamines during the Greening Response of Cucumber Cotyledons

The cucumber cotyledon greening test was used as a model systemto study possible relationships between cytokinins and polyaminesduring the greening process. When cytokinin was applied to dark-growncotyledons, large increases in chlorophyll and putrescine levelswere observed. However, inhibition of putrescine biosynthesiswith D-arginine and difluoromethylarginine did not affect chlorophyllproduction, and applied polyamines proved inhibitory to greening.Addition of 50 mM K^$ to the cytokinin treatments increased chlorophyllsynthesis but caused a marked reduction in putrescine levels.These results indicate that the large increase in putrescinecontent that derives from cytokinin treatment of the cotyledonsis not essential for the cytokinin-induced greening response. ¹Present address: Crop Science Department, University of Guelph,Guelph, Ontario, Canada, NIG 2W1. ²Present address: Agrogen Biotechnologies Inc., 520W. 6th Ave.,Vancouver, B.C., Canada, V5Z 4H5. (Received June 29, 1987; Accepted October 9, 1987)     

Autophagy-Related Proteins Are Required for Degradation of Peroxisomes in Arabidopsis Hypocotyls during Seedling Growth

Plant peroxisomes play a pivotal role during postgerminative growth by breaking down fatty acids to provide fixed carbons for seedlings before the onset of photosynthesis. The enzyme composition of peroxisomes changes during the transition of the seedling from a heterotrophic to an autotrophic state; however, the mechanisms for the degradation of obsolete peroxisomal proteins remain elusive. One candidate mechanism is autophagy, a bulk degradation pathway targeting cytoplasmic constituents to the lytic vacuole. We present evidence supporting the autophagy of peroxisomes in Arabidopsis thaliana hypocotyls during seedling growth. Mutants defective in autophagy appeared to accumulate excess peroxisomes in hypocotyl cells. When degradation in the vacuole was pharmacologically compromised, both autophagic bodies and peroxisomal markers were detected in the wild-type vacuole but not in that of the autophagy-incompetent mutants. On the basis of the genetic and cell biological data we obtained, we propose that autophagy is important for the maintenance of peroxisome number and cell remodeling in Arabidopsis hypocotyls.     

Nucleic Acid Metabolism during Greening and Unrolling of Barley Leaf Segments

Barley (Hordeum vulgare) leaf segments unroll and green when illuminated. Illuminated segments also have an increased capacity for RNA synthesis. Part of this increased RNA synthesis may be attributed to an increased RNA polymerase activity. In addition, following illumination there is an increased formation of polysomes.     

In-Depth Proteome Analysis of Arabidopsis Leaf Peroxisomes Combined with in Vivo Subcellular Targeting Verification Indicates Novel Metabolic and Regulatory Functions of Peroxisomes

Peroxisomes are metabolically diverse organelles with essential roles in plant development. The major protein constituents of plant peroxisomes are well characterized, whereas only a few low-abundance and regulatory proteins have been reported to date. We performed an in-depth proteome analysis of Arabidopsis (Arabidopsis thaliana) leaf peroxisomes using one-dimensional gel electrophoresis followed by liquid chromatography and tandem mass spectrometry. We detected 65 established plant peroxisomal proteins, 30 proteins whose association with Arabidopsis peroxisomes had been previously demonstrated only by proteomic data, and 55 putative novel proteins of peroxisomes. We subsequently tested the subcellular targeting of yellow fluorescent protein fusions for selected proteins and confirmed the peroxisomal localization for 12 proteins containing predicted peroxisome targeting signals type 1 or 2 (PTS1/2), three proteins carrying PTS-related peptides, and four proteins that lack conventional targeting signals. We thereby established the tripeptides SLM> and SKV> (where > indicates the stop codon) as new PTS1s and the nonapeptide RVx₅HF as a putative new PTS2. The 19 peroxisomal proteins conclusively identified from this study potentially carry out novel metabolic and regulatory functions of peroxisomes. Thus, this study represents an important step toward defining the complete plant peroxisomal proteome.     

Chloroplast Biogenesis: XX. Accumulation of Porphyrin and Phorbin Pigments in Cucumber Cotyledons during Photoperiodic Greening

A study of greening in cucumber (Cucumis sativus L.) cotyledons grown under a light (14-hour) dark (10-hour) photoperiodic regime was undertaken. The pools of protoporphyrin IX, Mg-protoporphyrin IX monoester, protochlorophyllide, and protochlorophyllide ester were determined spectrofluorometrically. Chlorophyll a and b were monitored spectrophotometrically. Pigments were extracted during the 3rd hour of each light period and at the end of each subsequent dark period during the first seven growth cycles. Protoporphyrin IX did not accumulate during greening. Mg-protoporphyrin IX monoester and longer wavelength metalloporphyrins accumulated during the light cycles and disappeared in the dark. Their disappearance was accompanied by the accumulation of protochlorophyll. Higher levels of protochlorophyll were observed in the dark than in the light, and the greatest accumulation occurred during the third and fourth dark cycles. Protochlorophyllide was present in 3- to 10-fold excess over protochlorophyllide ester; it was detectable during the period of net chlorophyll accumulation as well as afterward. In contrast, protochlorophyllide ester was observable only during the first four photoperiodic cycles, suggesting that it was a metabolic intermediate only during the early stages of chlorophyll accumulation. Between the third and fourth growth cycles, a rapid increase in area and fresh weight per cotyledon began. This was accompanied by a 250-fold increase in the level of chlorophyll a + b during the three subsequent growth cycles. No lag period in the accumulation of chlorophyll b was observed, and at all stages of greening, the chlorophyll a/b ratio was approximately 3.     

南瓜茎、叶、花的营养成分分析

对南瓜茎、叶、花等部位进行了粗脂肪、粗蛋白、粗纤维、总糖、Vc等主要营养成分和K、Na、Ca、Mg、Zn等矿质元素的测定。结果表明,南瓜茎、叶、花营养丰富,各种营养成分较为全面。南瓜叶中粗脂肪、粗蛋白、Vc、K、Ca、Mg、Mn、Fe、Zn等成分含量均高于南瓜茎和南瓜花。与其他8种常见蔬菜相比,主要营养成分和矿质元素含量也远远高于这8种蔬菜。为南瓜茎、叶、花的菜用价值提供了科学依据,也为增加人们的膳食营养、改变餐桌食品花样、开发蔬菜资源及其利用价值提供了参考。     

Photocontrol of the Accumulation of Plastid Polypeptides during Greening of Tomato Cotyledons : Potentiation by a Pulse of Red Light

A pulse of red light acting through phytochrome accelerates the formation of chlorophyll upon subsequent transfer of dark-grown seedlings to continuous white light. Specific antibodies were used to follow the accumulation of representative subunits of the major photosynthetic complexes during greening of seedlings of tomato (Lycopersicon esculentum). The time course for accumulation of the various subunits was compared in seedlings that received a red light pulse 4 h prior to transfer to continuous white light and parallel controls that did not receive a red light pulse. The light-harvesting chlorophyll-binding proteins of photosystem II (LHC II), the 33-kD extrinsic polypeptide of the oxygen-evolving complex (OEC1), and subunit II of photosystem I (psaD gene product) all increased in the light, and did so much faster in seedlings that received the inductive red light pulse. The red light pulse had no significant effect on the abundance of the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), nor on several plastid-encoded polypeptides: the large subunit of Rubisco, the β subunit of the CF₁ complex of plastid ATPase, and the 43- and 47-kD subunits of photosystem II (CP43, CP47). Subunits I (cytochrome b₆f) and III (Rieske Fe-S protein) of the cytochrome b₆f complex showed a small or no increase as a result of the red pulse. The potentiation of greening by a pulse of red light, therefore, is not expressed uniformly in the abundance of all the photosynthetic complexes and their subunits.     

CO2-Fixation, Glycolate Formation, and Enzyme Activities of Photorespiration and Photosynthesis during Greening and Senescence of Sunflower Cotyledons

Time-courses of ¹⁴CO₂-fixation and of enzyme activities involvedin photorespiration and photosynthesis were determined duringthe life span of cotyledons from sunflower seedlings (Helianthusannuus L.). Glycolate formation in vivo was estimated from theresults of combined labelling and inhibitor experiments. NADPH-glyceraldehyde-3-phosphatedehydrogenase, NADPH-glyoxylate reductase and chlorophyll werewell correlated with the time-course of ¹⁴CO₂-fixation (photosynthesis).There was, however, a considerable discrepancy between the developmentalsequence of photosynthesis and that of both ribulose-l,5-bisphosphatecarboxylase and glycolate oxidase. Furthermore, time-coursesof glycolate oxidase activity in vitro and of glycolate formationin vivo differed significantly. Therefore, the use of glycolateoxidase as a marker for the activity of photorespiration ingreening sunflower cotyledons may be questionable. Results from¹⁴CO₂-labelling experiments with cotyledons treated with theglycolate oxidase inhibitor 2-hydroxy butynoic acid suggestthat glycolate formation relative to CO₂-fixation is reducedin senescent cotyledons. Key words: Development, glycolate oxidase, photorespiration, ribulose-l,5-bisphosphate carboxylase, oxygenase     

Characterisation of Cyanobacterial Bicarbonate Transporters in E. coli Shows that SbtA Homologs Are Functional in This Heterologous Expression System

Cyanobacterial HCO₃ ^- transporters BCT1, SbtA and BicA are important components of cyanobacterial CO₂-concentration mechanisms. They also show potential in applications aimed at improving photosynthetic rates and yield when expressed in the chloroplasts of C3 crop species. The present study investigated the feasibility of using Escherichia coli to assess function of a range of SbtA and BicA transporters in a heterologous expression system, ultimately for selection of transporters suitable for chloroplast expression. Here, we demonstrate that six β-forms of SbtA are active in E. coli, although other tested bicarbonate transporters were inactive. The sbtA clones were derived from Synechococcus sp. WH5701, Cyanobium sp. PCC7001, Cyanobium sp. PCC6307, Synechococcus elongatus PCC7942, Synechocystis sp. PCC6803, and Synechococcus sp. PCC7002. The six SbtA homologs varied in bicarbonate uptake kinetics and sodium requirements in E. coli. In particular, SbtA from PCC7001 showed the lowest uptake affinity and highest flux rate and was capable of increasing the internal inorganic carbon pool by more than 8 mM relative to controls lacking transporters. Importantly, we were able to show that the SbtB protein (encoded by a companion gene near sbtA) binds to SbtA and suppresses bicarbonate uptake function of SbtA in E. coli, suggesting a role in post-translational regulation of SbtA, possibly as an inhibitor in the dark. This study established E. coli as a heterologous expression and analysis system for HCO₃ ^- transporters from cyanobacteria, and identified several SbtA transporters as useful for expression in the chloroplast inner envelope membranes of higher plants.     

Mig6 Is a Sensor of EGF Receptor Inactivation that Directly Activates c-Abl to Induce Apoptosis during Epithelial Homeostasis

A fundamental aspect of epithelial homeostasis is the dependence on specific growth factors for cell survival, yet the underlying mechanisms remain obscure. We found an "inverse" mode of receptor tyrosine kinase signaling that directly links ErbB receptor inactivation to the induction of apoptosis. Upon ligand deprivation Mig6 dissociates from the ErbB receptor and binds to and activates the tyrosine kinase c-Abl to trigger p73-dependent apoptosis in mammary epithelial cells. Deletion of Errfi1 (encoding Mig6) and inhibition or RNAi silencing of c-Abl causes impaired apoptosis and luminal filling of mammary ducts. Mig6 activates c-Abl by binding to the kinase domain, which is prevented in the presence of epidermal growth factor (EGF) by Src family kinase-mediated phosphorylation on c-Abl-Tyr488. These results reveal a receptor-proximal switch mechanism by which Mig6 actively senses EGF deprivation to directly activate proapoptotic c-Abl. Our findings challenge the common belief that deprivation of growth factors induces apoptosis passively by lack of mitogenic signaling.     

Comparative Effects of Abscisic Acid and Methyl Jasmonate in Greening Cucumber Cotyledons and Its Application to a Bioassay for Abscisic Acid

In the cucumber cotyledon greening system, abscisic acid (ABA)is more potent inhibitor of growth and chlorophyll productionand/or destruction than methyl jasmonate (MJ). The inhibitoryeffect of ABA is apparent within 5 h of exposure to light whereasMJ is ineffective at all concentrations tested (10^–6 to10^–3 M). With longer exposure of 24 h to light and inthe presence of 40 mM KC1, the inhibition of growth and chlorophyllproduction by ABA is more pronounced whereas MJ does not inhibitgrowth and inhibits chlorophyll levels only at the higher concentrations.Both benzyladenine and KC1 stimulate chlorophyll productionand increase the fresh weights of the cucumber cotyledons andeither one of these compounds reverse the inhibitory effectsof ABA. Inhibition of chlorophyll production by ABA is valuableas a simple and rapid bioassay for abscisic acid. Under similarconditions cytokinins increase chlorophyll production and hencethe cucumber cotyledon greening system is ideal for detectingboth ABA which inhibits and cytokinins which stimulate chlorophyllproduction. (Received December 6, 1982; Accepted June 9, 1983)