RNA Binding by Plant Serpins in vitro.

Serpins constitute a large family of protease inhibitors with regulatory functions found in all living organisms. Most plant serpins have not been functionally characterized, with the exception of Arabidopsis thaliana AtSerpin1, an inhibitor of pro-apoptotic proteases, which is involved in the regulation of the programmed cell death induction, and Cucurbita maxima CmPS1, a phloem protein, which presumably inhibits insect digestive proteases and binds RNA. CmPS1 interacts most efficiently with highly structured RNA; in particular, it forms a specific complex with tRNA. Here, we demonstrated that AtSerpin1 also forms a complex with tRNA. Analysis of tRNA species bound by AtSerpin1 and CmPS1 in the presence of tRNA excess revealed that both proteins have no strict selectivity for individual tRNAs, suggesting specific interaction of AtSerpin1 and CmPS1 proteins with elements of the secondary/tertiary structure universal for all tRNAs. Analysis of CmPS1 binding of the microRNA precursor pre-miR390 and its mutants demonstrated that the pre-miR390 mutant with a perfect duplex in the hairpin stem lost the ability to form a discrete complex with CmPS1, whereas another variant of pre-miR390 with the native unpaired nucleotide residues in the stem retained this ability. These data indicate that specific interactions of plant serpins with structured RNA are based on the recognition of structurally unique spatial motifs formed with the participation of unpaired nucleotide residues in the RNA duplexes.

Experimental evidence suggesting that H2O2 is produced within the thylakoid membrane in a reaction between plastoquinol and singlet oxygen.

Plastoquinol (PQH2-9) and plastoquinone (PQ-9) mediate photosynthetic electron transfer. We isolated PQH2-9 from thylakoid membranes, purified it with HPLC, subjected the purified PQH2-9 to singlet oxygen ((1)O2) and analyzed the products. The main reaction of (1)O2 with PQH2-9 in methanol was found to result in formation of PQ-9 and H2O2, and the amount of H2O2 produced was essentially the same as the amount of oxidized PQH2-9. Formation of H2O2 in the reaction between (1)O2 and PQH2-9 may be an important source of H2O2 within the lipophilic thylakoid membrane.

The Podosphaera xanthii haustorium, the fungal Trojan horse of cucurbit-powdery mildew interactions.

The powdery mildew fungi are obligate biotrophic plant pathogens that develop a specialized structure for parasitism termed haustorium, which is responsible for nutrient uptake and factor exchange with the plant. In this work, we present a detailed microscopy analysis of the haustoria of the cucurbit powdery mildew fungus Podosphaera xanthii, a major limiting factor for cucurbit production worldwide. Despite being located inside plant epidermal cells, transmission electron microscopy (TEM) analysis showed the characteristic highly irregular outline of the extrahaustorial membrane that separates the extrahaustorial matrix of haustoria from the cytoplasm of the plant cell. TEM analysis also revealed the presence of some vesicles and electron-dense plaques of material surrounding the haustoria. In confocal microscopy analysis and aniline blue staining we found a positive correlation between haustorial development and deposition of callose, which is distributed as plaques around haustorial complex. In this study, a method for the isolation of P. xanthii haustoria was also adapted, which permitted the analysis of the formation of haustorial lobes and the visualization of vacuoles and the pool of vesicles inside the haustorial complex. Our findings suggested that the haustorial lobes were responsible for vesicular trafficking and most likely act as the main mediators of the fungus-plant dialogue. All of these findings were integrated into a model of the P. xanthii-host cellular interactions.

Histochemical visualization of ROS and antioxidant response to viral infections of vegetable crops grown in Azerbaijan.

Extremes of environmental conditions, such as biotic stresses, strongly affect plant growth and development and may adversely affect photosynthetic process. Virus infection is especially problematic in crops, because unlike other diseases, its impact cannot be reduced by phytosanitary treatments. The vegetable crops (Solanum lycopеrsicum L, Cucurbita melo L., Cucumis sativus L., Piper longum L., Solánum melongéna L., Vicia faba L.) showing virus-like symptoms were collected from fields located in the main crop production provinces of Azerbaijan. Infection of the plants were confirmed by Enzyme-linked immunosorbent assay using commercial kits for the following viruses: Tomato yellow leaf curl virus, Tomato mosaic virus, Tomato chlorosis virus, Melon necrotic spot virus and Cucumber mosaic virus, Bean common mosaic virus and Bean yellow mosaic virus. Generation sites of superoxide and hydrogen peroxide radicals and activities of enzymes involved in the detoxification of reactive oxygen species (catalase, glutathione reductase, ascorbate peroxidase, guaiacol peroxidase and superoxide dismutase) were examined in uninfected leaves and in leaves infected with viruses. High accumulation of superoxide and hydrogen peroxide radicals was visualized in infected leaves as a purple discoloration of nitro blue tetrazolium and 3,3'-diaminobenzidine tetrahydrochloride. It was found that the activities of APX and CAT significantly increased in all infected samples compared with non-infected ones. Dynamics of GR and Cu/Zn-SOD activities differed from those of CAT and APX, and slightly increased in stressed samples. Electrophoretic mobility profiling of APX, GPX and CAT isoenzymes was also studied.

Effects of dietary milled seed mixture on fatty acid status and inflammatory markers in patients on hemodialysis.

BACKGROUND: Plant seeds have gained interest for their health benefits due to their fatty acid content. The objective of this study was to determine the effects of dietary consumption of milled sesame/pumpkin/flax seed mixture on glycemic control, serum lipids, phospholipid fatty acid status, and inflammatory factors in patients on hemodialysis. METHODS: Thirty patients with well nutrition status (18 male, 12 female) were enrolled in the study. Participants consumed 30 g of milled sesame/pumpkin/flax (6 g/6 g/18 g, resp.) seeds mixture added to their habitual diet. RESULTS: Total n-6 and n-3 polyunsaturated fatty acids and levels of linoleic, dihomo-gamma-linolenic (DGLA), arachidonic, alpha-linolenic (ALA), eicosapentaenoic, docosapentaenoic, and docosahexaenoic (DHA) acid were increased after 12 weeks of supplementation. A significant decrease of the serum triglyceride level (P < 0.001), glucose, insulin, calculated IR HOMA (P < 0.05), and inflammatory markers (TNF-alpha, IL-6, and hs-CRP, P < 0.001) was observed after seed mixture treatment. The serum levels of CRP and TNF-alpha negative correlate with ALA, DHA, and DGLA. CONCLUSION: Results of this study indicated that dietary milled sesame/pumpkin/flax seed mixture added to a habitual diet lowered triglyceride and CRP, TNF-alpha, IL-6 levels, affect glycemic control and improved fatty acid profile and pruritus symptoms in hemodialysis patients.

A novel 3α-p-Nitrobenzoylmultiflora-7:9(11)-diene-29-benzoate and two new triterpenoids from the seeds of zucchini (Cucurbita pepo L).

Three novel multiflorane-type triterpenoids, 3α-p-nitrobenzoylmultiflora-7:9(11)-diene-29-benzoate (1), 3α-acetoxymultiflora-7:9(11)-diene-29-benzoate (2), and 3α-acetoxymultiflora-5(6):7:9(11)-triene-29-benzoate (3), along with two known related compounds 4 and 5 were isolated from the seeds of zucchini (Cucurbita pepo L). Their structures were determined on the basis of 1D and 2D NMR spectroscopy and HREIMS. Triterpenoids possessing a nitro group were not isolated previously.

Comparative analysis of cells and proteins of pumpkin plants for the control of fruit size.

Common pumpkin plants (Cucurbita maxima) produce fruits of 1-2 kg size on the average, while special varieties of the same species called Atlantic Giant are known to produce a huge fruit up to several hundred kilograms. As an approach to determine the factors controlling the fruit size in C. maxima, we cultivated both AG and control common plants, and found that both the cell number and cell sizes were increased in a large fruit while DNA content of the cell did not change significantly. We also compared protein patterns in the leaves, stems, ripe and young fruits by two-dimensional (2D) gel electrophoresis, and identified those differentially expressed between them with mass spectroscopy. Based on these results, we suggest that factors in photosynthesis such as ribulose-bisphosphate carboxylase, glycolysis pathway enzymes, heat-shock proteins and ATP synthase play positive or negative roles in the growth of a pumpkin fruit. These results provide a step toward the development of plant biotechnology to control fruit size in the future.

Complementarity in root architecture for nutrient uptake in ancient maize/bean and maize/bean/squash polycultures.

BACKGROUND AND AIMS: During their domestication, maize, bean and squash evolved in polycultures grown by small-scale farmers in the Americas. Polycultures often overyield on low-fertility soils, which are a primary production constraint in low-input agriculture. We hypothesized that root architectural differences among these crops causes niche complementarity and thereby greater nutrient acquisition than corresponding monocultures. METHODS: A functional-structural plant model, SimRoot, was used to simulate the first 40 d of growth of these crops in monoculture and polyculture and to determine the effects of root competition on nutrient uptake and biomass production of each plant on low-nitrogen, -phosphorus and -potassium soils. KEY RESULTS: Squash, the earliest domesticated crop, was most sensitive to low soil fertility, while bean, the most recently domesticated crop, was least sensitive to low soil fertility. Nitrate uptake and biomass production were up to 7 % greater in the polycultures than in the monocultures, but only when root architecture was taken into account. Enhanced nitrogen capture in polycultures was independent of nitrogen fixation by bean. Root competition had negligible effects on phosphorus or potassium uptake or biomass production. CONCLUSIONS: We conclude that spatial niche differentiation caused by differences in root architecture allows polycultures to overyield when plants are competing for mobile soil resources. However, direct competition for immobile resources might be negligible in agricultural systems. Interspecies root spacing may also be too large to allow maize to benefit from root exudates of bean or squash. Above-ground competition for light, however, may have strong feedbacks on root foraging for immobile nutrients, which may increase cereal growth more than it will decrease the growth of the other crops. We note that the order of domestication of crops correlates with increasing nutrient efficiency, rather than production potential.

The origin and composition of cucurbit "phloem" exudate.

Cucurbits exude profusely when stems or petioles are cut. We conducted studies on pumpkin (Cucurbita maxima) and cucumber (Cucumis sativus) to determine the origin and composition of the exudate. Morphometric analysis indicated that the exudate is too voluminous to derive exclusively from the phloem. Cold, which inhibits phloem transport, did not interfere with exudation. However, ice water applied to the roots, which reduces root pressure, rapidly diminished exudation rate. Sap was seen by microscopic examination to flow primarily from the fascicular phloem in cucumber, and several other cucurbit species, but primarily from the extrafascicular phloem in pumpkin. Following exposure of leaves to 14CO2, radiolabeled stachyose and other sugars were detected in the exudate in proportions expected of authentic phloem sap. Most of this radiolabel was released during the first 20 s. Sugars in exudate were dilute. The sugar composition of exudate from extrafascicular phloem near the edge of the stem differed from that of other sources in that it was high in hexose and low in stachyose. We conclude that sap is released from cucurbit phloem upon wounding but contributes negligibly to total exudate volume. The sap is diluted by water from cut cells, the apoplast, and the xylem. Small amounts of dilute, mobile sap from sieve elements can be obtained, although there is evidence that it is contaminated by the contents of other cell types. The function of P-proteins may be to prevent water loss from the xylem as well as nutrient loss from the phloem.

Quantification of plant cell coupling with three-dimensional photoactivation microscopy.

Plant cells are directly connected by plasmodesmata that form channels through the cell wall and enable the intercellular movement of cytosolic solutes, membrane lipids and signalling molecules. Transport through plasmodesmata is regulated not only by a fixed size-exclusion limit, but also by physiological and pathological adaptation. To understand plant cell communication, carbon allocation and pathogen attack, the capacities for a specific molecule to pass a specific cell-wall interface is an essential parameter. So far, the degree of cell coupling was derived from frequency and diameter of plasmodesmata in relevant tissues as assessed by electron microscopy of fixed material. However, plasmodesmata functionality and capacity can only be determined in live material, not from electron microscopy, which is static and prone to fixation artefacts. Plasmodesmata functionality was a few times assessed using fluorescent tracers with diffusion properties similar to cytosolic solutes. Here, we used three-dimensional photoactivation microscopy to quantify plasmodesmata-mediated cell-wall permeability between living Cucurbita maxima leaf mesophyll cells with caged fluorescein as tracer. For the first time, all necessary functional and anatomical data were gathered for each individual cell from three-dimensional time series. This approach utilized a confocal microscope equipped with resonant scanner, which provides the high acquisition speed necessary to record optical sections of whole cells and offers time resolution high enough to follow the kinetics of photoactivation. The results were compared to two-dimensional measurements, which are shown to give a good estimate of cell coupling adequate for homogenous tissues. The two-dimensional approach is limited whenever tissues interfaces are studied that couple different cell types with diverse cell geometries.

Quantifying immunogold localization on electron microscopic thin sections: a compendium of new approaches for plant cell biologists.

A review is presented of recently developed methods for quantifying electron microscopical thin sections on which colloidal gold-labelled markers are used to identify and localize interesting molecules. These efficient methods rely on sound principles of random sampling, event counting, and statistical evaluation. Distributions of immunogold particles across cellular compartments can be compared within and between experimental groups. They can also be used to test for co-localization in multilabelling studies involving two or more sizes of gold particle. To test for preferential labelling of compartments, observed and expected gold particle distributions are compared by χ(2) analysis. Efficient estimators of gold labelling intensity [labelling density (LD) and/or relative labelling index (RLI)] are used to analyse volume-occupying compartments (e.g. Golgi vesicles) and/or surface-occupying compartments (e.g. cell membranes). Compartment size is estimated by counting chance events after randomly superimposing test lattices of points and/or line probes. RLI=1 when there is random labelling and RLI >1 when there is preferential labelling. Between-group comparisons do not require information about compartment size but, instead, raw gold particle counts in different groups are compared by combining χ(2) and contingency table analyses. These tests may also be used to assess co-distribution of different sized gold particles in compartments. Testing for co-labelling involves identifying sets of compartmental profiles that are unlabelled and labelled for one or both of two gold marker sizes. Numbers of profiles in each labelling set are compared by contingency table analysis and χ(2) analysis or Fisher's exact probability test. The various methods are illustrated with worked examples based on empirical and synthetic data and will be of practical benefit to those applying single or multiple immunogold labelling in their research.

Differential spatial expression of A- and B-type CDKs, and distribution of auxins and cytokinins in the open transverse root apical meristem of Cucurbita maxima.

BACKGROUND AND AIMS: Aside from those on Arabidopsis, very few studies have focused on spatial expression of cyclin-dependent kinases (CDKs) in root apical meristems (RAMs), and, indeed, none has been undertaken for open meristems. The extent of interfacing between cell cycle genes and plant growth regulators is also an increasingly important issue in plant cell cycle studies. Here spatial expression/localization of an A-type and B-type CDK, auxin and cytokinins are reported in relation to the hitherto unexplored anatomy of RAMs of Cucurbita maxima. METHODS: Median longitudinal sections were cut from 1-cm-long primary root tips of C. maxima. Full-length A-type CDKs and a B-type CDK were cloned from C. maxima using degenerate primers, probes of which were localized on sections of RAMs using in situ hybridization. Isopentenyladenine (iPA), trans-zeatin (t-Z) and indole-3yl-acetic acid (IAA) were identified on sections by immunolocalization. KEY RESULTS: The C. cucurbita RAM conformed to an open transverse (OT) meristem typified by an absence of a clear boundary between the eumeristem and root cap columella, but with a distinctive longitudinally thickened epidermis. Cucma;CDKA;1 expression was detected strongly in the longitudinally thickened epidermis, a tissue with mitotic competence that contributes cells radially to the root cap of OT meristems. Cucma;CDKB2 was expressed mainly in proliferative regions of the RAM and in lateral root primordia. iPA and t-Z were mainly distributed in differentiated cells whilst IAA was distributed more uniformly in all tissues of the RAM. CONCLUSIONS: Cucma;CDKA;1 was expressed most strongly in cells that have proliferative competence whereas Cucma;CDKB2 was confined mainly to mitotic cells. iPA and t-Z marked differentiated cells in the RAM, consistent with the known effect of cytokinins in promoting differentiation in root systems. iPA/t-Z were distributed in a converse pattern to Cucma;CDKB2 expression whereas IAA was detected in most cells in the RAM regardless of their proliferative potential.

Light-induced transpiration alters cell water relations in figleaf gourd (Cucurbita ficifolia) seedlings exposed to low root temperatures.

Water relation parameters including elastic modulus (epsilon), half-times of water exchange (T(w)(1/2)), hydraulic conductivity and turgor pressure (P) were measured in individual root cortical and cotyledon midrib cells in intact figleaf gourd (Cucurbita ficifolia) seedlings, using a cell pressure probe. Transpiration rates (E) of cotyledons were also measured using a steady-state porometer. The seedlings were exposed to low ambient (approximately 10 micromol m(-2) s(-1)) or high supplemental irradiance (approximately 300 micromol m(-2) s(-1) PPF density) at low (8 degrees C) or warm (22 degrees C) root temperatures. When exposed to low irradiance, all the water relation parameters of cortical cells remained similar at both root temperatures. The exposure of cotyledons to supplemental light at warm root temperatures, however, resulted in a two- to three-fold increase in T(w)(1/2) values accompanied with the reduced hydraulic conductivity in both root cortical (Lp) and cotyledon midrib cells (Lp(c)). Low root temperature (LRT) further reduced Lp(c) and E, whether it was measured under low or high irradiance levels. The reductions of Lp as the result of respective light and LRT treatments were prevented by the application of 1 microM ABA. Midrib cells required higher concentrations of ABA (2 microM) in order to prevent the reduction in Lp(c). When the exposure of cotyledons to light was accompanied by LRT, however, ABA proved ineffective in reversing the inhibition of Lp. LRT combined with high irradiance triggered a drastic 10-fold reduction in water permeability of cortical and midrib cells and increased epsilon and T(w)(1/2) values. Measurement of E indicated that the increased water demand by the transpiring plants was fulfilled by an increase in the apoplastic pathway as principal water flow route. The importance of water transport regulation by transpiration affecting the hydraulic conductivity of the roots is discussed.

Nanoparticles as smart treatment-delivery systems in plants: assessment of different techniques of microscopy for their visualization in plant tissues.

BACKGROUND AND AIMS: The great potential of using nanodevices as delivery systems to specific targets in living organisms was first explored for medical uses. In plants, the same principles can be applied for a broad range of uses, in particular to tackle infections. Nanoparticles tagged to agrochemicals or other substances could reduce the damage to other plant tissues and the amount of chemicals released into the environment. To explore the benefits of applying nanotechnology to agriculture, the first stage is to work out the correct penetration and transport of the nanoparticles into plants. This research is aimed (a) to put forward a number of tools for the detection and analysis of core-shell magnetic nanoparticles introduced into plants and (b) to assess the use of such magnetic nanoparticles for their concentration in selected plant tissues by magnetic field gradients. METHODS: Cucurbita pepo plants were cultivated in vitro and treated with carbon-coated Fe nanoparticles. Different microscopy techniques were used for the detection and analysis of these magnetic nanoparticles, ranging from conventional light microscopy to confocal and electron microscopy. KEY RESULTS: Penetration and translocation of magnetic nanoparticles in whole living plants and into plant cells were determined. The magnetic character allowed nanoparticles to be positioned in the desired plant tissue by applying a magnetic field gradient there; also the graphitic shell made good visualization possible using different microscopy techniques. CONCLUSIONS: The results open a wide range of possibilities for using magnetic nanoparticles in general plant research and agronomy. The nanoparticles can be charged with different substances, introduced within the plants and, if necessary, concentrated into localized areas by using magnets. Also simple or more complex microscopical techniques can be used in localization studies.

Reciprocal phosphorylation and glycosylation recognition motifs control NCAPP1 interaction with pumpkin phloem proteins and their cell-to-cell movement.

In plants, cell-to-cell trafficking of non-cell-autonomous proteins (NCAPs) involves protein-protein interactions, and a role for posttranslational modification has been implicated. In this study, proteins contained in pumpkin (Cucurbita maxima cv Big Max) phloem sap were used as a source of NCAPs to further explore the molecular basis for selective NCAP trafficking. Protein overlay assays and coimmunoprecipitation experiments established that phosphorylation and glycosylation, on both Nicotiana tabacum NON-CELL-AUTONOMOUS PATHWAY PROTEIN1 (Nt-NCAPP1) and the phloem NCAPs, are essential for their interaction. Detailed molecular analysis of a representative phloem NCAP, Cm-PP16-1, identified the specific residues on which glycosylation and phosphorylation must occur for effective binding to NCAPP1. Microinjection studies confirmed that posttranslational modification on these residues is essential for cell-to-cell movement of Cm-PP16-1. Lastly, a glutathione S-transferase (GST)-Cm-PP16-1 fusion protein system was employed to test whether the peptide region spanning these residues was required for cell-to-cell movement. These studies established that a 36-amino acid peptide was sufficient to impart cell-to-cell movement capacity to GST, a normally cell-autonomous protein. These findings are consistent with the hypothesis that a phosphorylation-glycosylation recognition motif functions to control the binding of a specific subset of phloem NCAPs to NCAPP1 and their subsequent transport through plasmodesmata.

Membrane associated qualitative differences in cell ultrastructure of chemically and high pressure cryofixed plant cells.

Membrane contrast can sometimes be poor in biological samples after high pressure freezing (HPF) and freeze substitution (FS). The addition of water to the FS-medium has been shown to improve membrane contrast in animal tissue and yeast. In the present study we tested the effects of 1% and 5% water added to the FS-medium (2% osmium with 0.2% uranyl acetate in anhydrous acetone) on the quality and visibility of membranes in high pressure frozen leaf samples of Cucurbita pepo L. plants and compared them to chemically fixed cells (3% glutaraldehyde post-fixed with 1% osmium tetroxide). The addition of water to the FS-medium drastically decreased the amounts of well preserved cells and did not significantly improve the quality nor visibility of membranes. In samples that were freeze substituted in FS-media containing 1% and 5% water the width of thylakoid membranes was found to be significantly increased of about 20% and the perinuclear space was up to 76% wider in comparison to what was found in samples which were freeze substituted without water. No differences were found in the thickness of membranes between chemically and cryofixed cells that were freeze substituted in the FS-medium without water. Nevertheless, in chemically fixed cells the intrathylakoidal space was about 120% wider than in cryofixed cells that were freeze substituted with or without water. The present results demonstrate that the addition of water to the FS-medium does not improve membrane contrast but changes the width of thylakoid membranes and the perinuclear space in the present plant material. The addition of water to the FS-medium is therefore not as essential for improved membrane contrast in the investigated plant samples as it was observed in cells of animal tissues and yeast cells.

Ascorbic acid oxidase is dynamically regulated by light and oxygen. A tool for oxygen management in plants?

Ascorbic acid oxidase (AAO) is a plant blue-copper protein catalyzing dioxygen reduction to water using ascorbic acid as the electron donor. In spite of extensive molecular characterization the physiological role of AAO is still uncertain. Abundant mRNA, protein and activity of AAO were observed in illuminated leaves of Cucurbita pepo. AAO activity was found to be proportional to light intensity. The light effect was rapidly reversed in dark and activity remained low throughout the dark period. Activity was elicited in dark by increased oxygen concentration. AAO activity increased in the facultative CAM Kalanchoë blossfeldiana upon induction of the CAM cycle and decreased during germination of C. pepo and Zea mays under hypoxic conditions. These results strongly suggest that AAO activity could be part of a dynamic system for oxygen management in plants.

Nectar resorption and translocation in Cucurbita pepo L. and Platanthera chlorantha Custer (Rchb.).

Nectar resorption and sugar translocation were studied in Cucurbita pepo (Cucurbitaceae) and Platanthera chlorantha (Orchidaceae) by micro-autoradiography. In both species, nectar was resorbed in pollinated and unpollinated flowers and ovules developing into seeds were found to be the main sugar sink. In C. pepo, the mobility of resorbed sugars in pollinated female flowers was higher than in unpollinated ones; male flowers showed lower mobility of resorbed sugar. In P. chlorantha, radioactivity was detected in pollinated flowers below and above labelled unpollinated ones: the nearer the flower, the stronger the accumulation of label in developing fruits.

Control by cytokinins of the cellular behavior in the plate meristem of zucchini cotyledons.

The temporal and spatial effects of exogenous cytokinins on both cell expansion and division activity in the plate meristem of cultured zucchini cotyledons were studied. N6-benzylaminopurine (1-100 microM) and N-(2-chloro-4pyridyl)-N'-phenylurea (4PU-30) (0.1-100 microM) greatly stimulated the cell growth and division. They provoked multiple cell cycles, formation of larger clusters of daughter cells and an increase of the final number of cells. Both cytokinins led to earlier achievement of final cotyledon size and shortened the cell doubling time. By contrast to the purine cytokinin, phenylurea cytokinin 4PU-30 enlarged the cotyledon predominantly in length. Zeatin and kinetin were less effective, particularly in stimulating cell expansion. In low concentrations, all cytokinins were more effective in stimulating division activity rather than expansion. The cells in the cotyledon margins displayed a higher division activity, especially when treated with exogenous cytokinins. The final cotyledon and cluster areas were not of the strict proportional dependence upon the number of their cells. These results provide a novel example where stimulated cell division fails to evoke a respective increase in the final organ size.

Isolation of glyoxysomes from pumpkin cotyledons.

Peroxisomes are single-membrane-bound organelles found in virtually all eukaryotes. In plants, there are several classes of peroxisomes. Glyoxysomes are found in germinating seedlings and contain enzymes specific for the glyoxylate cycle, including isocitrate lyase and malate synthase. After seedlings become photosynthetic, leaf peroxisomes participate in reactions of the photorespiration pathway and contain characteristic enzymes such as glycolate oxidase and hydroxypyruvate reductase. As leaves begin to senesce, leaf peroxisomes are transformed back into glyoxysomes. Root peroxisomes in the nodules of legumes, for example, sequester enzymes such as allantoinase and uricase, which contribute to nitrogen metabolism in these tissues. Thus, peroxisomes participate in many metabolic pathways and contain specific enzyme complements, depending on the tissue source. All peroxisomes contain catalase to degrade hydrogen peroxide and enzymes to accomplish beta-oxidation of fatty acids. Glyoxysomes can be isolated from pumpkin cotyledons by standard differential centrifugation and density separation, as described in this article.