Young Cotyledons Research Articles

Advances in pectin and pectinase research. * Voragen F, Schols H and Visser R. eds. 2003. The Netherlands: Kluwer Academic Publishers. 145 (hardback). 491 pp.

Advances in pectin and pectinase research. * Voragen F, Schols H and Visser R. eds. 2003. The Netherlands: Kluwer Academic Publishers. 145 (hardback). 491 pp.

A“bulb”subregion in the vacuolar membrane

Complex, mobile spherical structures continuous with the vacuolar limiting membrane were identified in young cotyledons of Arabidopsis by expression of a vacuolar membrane marker(r-TIP-GFP)and by electron microscopy, and were named“bulbs”. These structures are cytoplasm wrapped by two layers of intricately folded membrane; both layers are continuous with the vacuolar limiting membrane. When visualized using r-TIP-GFP, the fluorescence intensity of bulbs is about3-fold higher than that of the vacuolar limiting membrane. However, bulbs were negative for GFP-AtRab75, another vacuolar marker. These results suggest that bulbs form a subregion in the continuous vacuolar membrane that concentrate some proteins and segregate others.

A complex and mobile structure forms a distinct subregion within the continuous vacuolar membrane in young cotyledons of Arabidopsis.

The plant vacuole is a multifunctional organelle which is essential for growth and development. To visualize the dynamics of plant vacuolar membranes, gamma-TIP (tonoplast intrinsic protein) was fused to GFP and expressed in Arabidopsis thaliana. The marker molecule was targeted to the vacuolar membranes in most tissues, as expected. In rapidly expanding cells, some additional spherical structures were often observed within the lumen of vacuoles, which emitted strong fluorescence. To confirm their normal presence, we examined wild-type Arabidopsis cotyledons by transmission electron microscopy. The metal-contact rapid-freezing method revealed that the vacuolar lumen of epidermal cells contained many cytoplasmic projections, which often formed spherical structures (1-3 microm diameter) consisting of double membranes. Thus we concluded that these structures are authentic and named them 'bulbs'. Three-dimensional reconstruction from serial electron microscopic images demonstrates that bulbs are very intricately folded, but are continuous with the limiting vacuolar membrane. The fluorescence intensity of bulbs is about threefold higher than that of vacuolar membrane. GFP-AtRab75c, another marker of the vacuole, did not give fluorescent signals of bulbs in transgenic plants, but the existence of bulbs was still confirmed by electron microscopy. These results suggest that bulbs define a subregion in the continuous vacuolar membrane, where some proteins are concentrated and others segregated.

Changes in the glucosinolate-myrosinase defense system in Brassica juncea cotyledons during seedling development.

Optimal defense theory (ODT) predicts that plant defenses will be allocated to plant organs and tissues in proportion to their relative fitness values and susceptibilities to attack. This study was designed to test ODT predictions on the myrosinase-glucosinolate defense system in Brassica juncea by examining the relationships between the fitness value of B. juncea cotyledons and the levels and effectiveness of cotyledon defenses. Specifically, we estimated fitness value of cotyledons during plant development by measuring plant growth and seed production after cotyledon damage or removal at successive seedling ages. Cotyledon removal within five days of emergence had a significant impact on growth and seed production, but cotyledon removal at later stages did not. Consistent with ODT, glucosinolate and myrosinase levels in cotyledons also declined with seedling age, as did relative defenses against a generalist herbivore, Spodoptera eridania, as estimated by bioassay. Declines in glucosinolates were as predicted by a passive, allometric dilution model based on cotyledon expansion. Declines in myrosinase activity were significantly more gradual than predicted by allometric dilution, suggesting active retention of myrosinase activity as young cotyledons expand.

Nonspecific intercellular protein trafficking probed by green-fluorescent protein in plants

Plasmodesmata mediate intercellular transport of proteins, nucleic acids, and small molecules in plants. We show that transiently produced green-fluorescent protein (GFP) trafficked intercellularly in the epidermis of sink leaves, but not of source leaves, in tobacco and cucumber. In contrast, the protein did not traffic in either sink or source leaves of tomato. On the other hand, the protein spread extensively from cell to cell in the epidermis of all leaves and stems ofArabidopsis thaliana as well as in young hypocotyls and cotyledons of tomato and cucumber. GFP could traffic from epidermis to ground tissues in hypocotyls but not in cotyledons of cucumber. GFP fused to a number of mutant forms of the cucumber mosaic virus 3a movement protein (CMV 3a MP) failed to traffic from cell to cell, suggesting that GFP does not have a specific motif for plasmodesmal trafficking. Our data, together with previous findings, indicate that plasmodesmata can mediate both specific and nonspecific intercellular trafficking of proteins. Furthermore, our data suggest that nonspecific protein trafficking is controlled by species-, development-, organ-, and tissue-specific factors. Since GFP can readily traffic from cell to cell, it raises the questions of how metabolites are compartmentalized intercellularly in a plant and of whether some endogenous plant proteins traffic nonspecifically from cell to cell to perform physiological functions yet to be elucidated.

Altered lignin composition in phenylalanine ammonia-lyase-inhibited radish seedlings: implications for seed-derived sinapoyl esters as lignin precursors

We earlier reported that when phenylalanine ammonia-lyase (PAL) activity in radish seedlings was inhibited by the competitive inhibitor 2-aminoindan-2-phosphonic acid (AIP), soluble sinapoyl esters carried over from the seed were converted to wall-bound esters in young cotyledons. We now report that these soluble sinapoyl esters may also be converted into lignin in the cotyledons. When radish seedlings were grown in the presence of 100 μM AIP, lignin formation (determined as lignothioglycolic acid) was inhibited ca. 74% in the cotyledons and ca. 80% in hypocotyls plus roots. The syringyl to guaiacyl (S/G) ratio in the lignin of AIP-grown plants, as determined by alkaline cupric oxidation and from Fourier-transform infrared (FT-IR) spectra, was higher in cotyledons, but lower in hypocotyls plus roots, as compared to plants grown on distilled water. These results support the view that soluble sinapoyl esters preformed in seeds may contribute to the syringyl moiety of lignin in cotyledons during early seedling development and that there is no appreciable transport of soluble sinapoyl esters from cotyledons to the hypocotyls and roots.

Identification of three kinds of mutually related composite elements conferring S phase-specific transcriptional activation.

Conservation of the Oct motif (CGCGGATC) is a remarkable feature of plant histone gene promoters. Many of the Oct motifs are paired with a distinct motif, Hex, TCA or CCAAT-box, constituting the type I element (CCACGTCANCGATCCGCG), type II element (TCACGCGGATC) and type III element (GATCCGCG-N14-ACCAATCA). To clarify the roles of these Oct-containing composite elements (OCEs) in cell cycle-dependent and tissue-specific expression, we performed gain-of-function experiments with transgenic tobacco cell lines and plants harboring a derivative of the 35S core promoter/beta-glucuronidase fusion gene in which three or four copies of an OCE had been placed upstream. Although their activities were slightly different, results showed that each of the three types of OCEs could confer the ability to direct S phase-specific expression on a heterologous promoter. In transgenic plants, the type I and III elements exhibited a similar activity, directing expression in meristematic tissues, whereas the activity of the type II element appeared to be restricted to young cotyledons and maturating guard cells. Mutational analyses demonstrated that the co-operation of Oct with another module (Hex, TCA or CCAAT-box) was absolutely required for both temporal and spatial regulation. Thus, OCEs play a pivotal role in regulation of the expression of plant histone genes.

Dark-Induced Expression of Genes for Asparagine Synthetase and Cytosolic Glutamine Synthetase in Radish Cotyledons is Dependent on the Growth Stage

In radish, the level of transcripts from the cytosolic glutamine synthetase (GS1) gene (Glnl;l) increased as the cotyledons aged and senesced. After the transfer to darkness, the level of transcripts from Glnl;l in senescing (3 weeks after germination) cotyledons which was abundant before transfer, increased, but that in young (1 week after germination) cotyledons which was very low before transfer, did not. On the contrary, transcripts from the asparagine synthetase (AS) gene accumulated after the transfer to darkness in young cotyledons but not in senescing cotyledons. The amount of free Gin in the phloem exudates collected from the cotyledons increased during senescence when the Glnl;l transcripts accumulated in the cotyledons. The accumulation of AS transcripts, however, led to the increase in the level of Asn in the cotyledons rather than in the phloem exudates from the cotyledons. These data suggest that the synthesis of Gin is related to the translocation of nitrogen from senescing cotyledons, whereas that of Asn is involved in the transient nitrogen storage in the tissue, and also that the synthesis of these amino acids is dependent on the expression of genes for synthetases of these amino acids.

The first step of gibberellin biosynthesis in pumpkin is catalyzed by at least two copalyl diphosphate synthases encoded by differentially regulated genes.

The first step in gibberellin biosynthesis is catalyzed by copalyl diphosphate synthase (CPS) and ent-kaurene synthase. We have cloned from pumpkin (Cucurbita maxima L.) two cDNAs, CmCPS1 and CmCPS2, that each encode a CPS. Both recombinant fusion CmCPS proteins were active in vitro. CPS are translocated into plastids and processed by cleavage of transit peptides. For CmCPS1 and CmCPS2, the putative transit peptides cannot exceed the first 99 and 107 amino acids, respectively, because longer N-terminal deletions abolished activity. Levels of both CmCPS transcripts were strictly regulated in an organ-specific and developmental manner. Both transcripts were almost undetectable in leaves and were abundant in petioles. CmCPS1 transcript levels were high in young cotyledons and low in roots. In contrast, CmCPS2 transcripts were undetectable in cotyledons but present at significant levels in roots. In hypocotyls, apices, and petioles, CmCPS1 transcript levels decreased with age much more rapidly than those of CmCPS2. We speculate that CmCPS1 expression is correlated with the early stages of organ development, whereas CmCPS2 expression is correlated with subsequent growth. In contrast, C. maxima ent-kaurene synthase transcripts were detected in every organ at almost constant levels. Thus, ent-kaurene biosynthesis may be regulated through control of CPS expression.

Fucosyltransferase and the biosynthesis of storage and structural xyloglucan in developing nasturtium fruits

Young, developing fruits of nasturtium (Tropaeolum majus L.) accumulate large deposits of nonfucosylated xyloglucan (XG) in periplasmic spaces of cotyledon cells. This "storage" XG can be fucosylated by a nasturtium transferase in vitro, but this does not happen in vivo, even as a transitory signal for secretion. The only XG that is clearly fucosylated in these fruits is the structural fraction (approximately 1% total) that is bound to cellulose in growing primary walls. The two fucosylated subunits that are formed in vitro are identical to those found in structural XG in vivo. The yield of XG-fucosyltransferase activity from membrane fractions is highest per unit fresh weight in the youngest fruits, especially in dissected cotyledons, but declines when storage XG is forming. A block appears to develop in the secretory machinery of young cotyledon cells between sites that galactosylate and those that fucosylate nascent XG. After extensive galactosylation, XG traffic is diverted to the periplasm without fucosylation. The primary walls buried beneath accretions of storage XG eventually swell and lose cohesion, probably because they continue to extend without incorporating components such as fucosylated XG that are needed to maintain wall integrity.

Observation and isolation of the amyloplasts ribosomes in cotyledon cells of lotus

Ribosome-like particles have been found in the proplastids in young cotyledon cells of lotus (Nelumbo nucifera Gaertn L.) Following the development of young embryo, some lamellar structures and tubular complex occurred in the plastids in young cotyledon cells, and some ribosomlike particles appeared in the loose region of these membrane system and stroma. About 15–20 d after fertilization, with the further development of plastid, a large number of starch and DNA were synthesized in the plastids, and the plastids contained abundant and clear morphologically ribosomes, some of which presented spiral structure. About 16–18 d after fertilization, amyloplasts were isolated and purified from cotyledon of lotus, and ribosomes bands were obtained by use of sucrose density gradient centrifugation of ribosomes isolated from amyloplasts. RNA and protein contents of ribosomes have also been determined.

Polarized microtubule deposition coincides with wall ingrowth formation in transfer cells ofVicia faba L. cotyledons

The epidermal transfer cells in developingVicia faba L. cotyledons are highly polarized. Extensive wall ingrowths occur on their outer periclinal walls and extend part way down both anticlinal walls. This ingrowth development serves to increase the surface area of the plasma membrane and thus maximize porter-dependent uptake of sugars from the seed apoplasm. In contrast, the inner periclinal walls of these transfer cells do not form wall ingrowths. We have commenced a study of the mechanisms responsible for establishing this polarity by first analysing the microtubule (MT) cytoskeleton in developing transfer cells. Thin sections of fixed cotyledons embedded in methacrylate resin were processed for immunofluorescence microscopy using monoclonal anti-β-tubulin and counterstained with Calcofluor White to visualize wall ingrowths. In epidermal cells of young cotyledons where wall ingrowths were yet to develop, MT labelling was detected around all cortical regions of the cell. However, in cells where wall ingrowths were clearly established, MT labelling was detected almost exclusively in cortical regions adjacent to the wall ingrowths. Little, if any, MT labelling was detected on the anticlinal or inner periclinal walls of these cells. This distribution of MTs was most prominent in cells with well developed wall ingrowths. In these cells, a subpopulation of MTs were also detected emanating from the subcortex and extending towards the wall ingrowth region. The possible role of MT distribution in establishing transfer cell polarity and wall ingrowth formation is discussed.

Cell cycle events in developing neem (Azadirachta indica) seeds: are they related to intermediate storage behaviour?

AbstractSeeds of neem (Azadirachta indicaA. Juss.), a multipurpose tropical tree, have limited desiccation tolerance. Whether their intermediate storage behaviour might be caused by an elevated percentage of 4C nuclei in the embryo at maturity was investigated. Morphological development of neem seeds was monitored on selected trees in Ouagadougou, Burkina Faso. Developing seeds were tested for germinability, and cell cycle events were determined by using flow cytometry and analysing the level of β-tubulin. Germination could occur after 8 weeks of seed development, but normal seedlings resulted only after 10 weeks or more. The change in fruit colour from green to yellow began after approximately 12 weeks of seed development. Immature, 4-week-old embryos about 2 mm in length had 15% of cells in the G2phase and 60% in the G1phase of the cell cycle, as indicated by 4C and 2C nuclear DNA levels, respectively. During maturation, the proportion of G2cells declined to 3% at full maturity and slightly further after drying, and the proportion of G1cells increased to approximately 90%. A strong β-tubulin signal was observed in tips of young embryonic radicles and cotyledons, but a weak or non-detectable signal was found in 9-week-old ones and in those from green-mature and yellow fruits. Because DNA replication and β-tubulin level were almost negligible at seed maturity, as in orthodox tomato seeds, it is suggested that these factors are not involved in the intermediate storage behaviour of neem seeds.

Control of Flea Beetles on Canola with Insecticides Applied as Seed Treatments, 1992

Abstract This test was designed to evaluate fungicide and insecticide seed treatments against naturally occurring populations of plant pathogens and insects. The treated seed was planted at the MSU Post experiment farm near Bozeman on 4 May. Four row plots measuring 1.3 m x 6 m and seeded at 160 seeds/m2 were arranged in a randomized block design with 4 replicates. Flea beetle damage was assessed on young cotyledons 24 d after seeding. Stand density counts were made after the 4 true leaf stage. Three applications of Sevin insecticide were applied at approximately 2 wk intervals, starting at the time the plots were rated for flea beetle damage. Data collected were analyzed using analysis of variance. The statistical relationship between means was determined with Student-Newman-Keuls multiple comparison test at P = 0.05. In this test, the fungicides were chosen to control a wide spectrum of disease organisms.

Differences in ion uptake among roots of various types

Abstract Ion uptake by tap and by lateral roots of peas (Pisum sativum L.) was investigated. The roots were either of intact seedlings or of seedlings whose young shoots or cotyledons had been removed. Tap roots seem to before responsive to temperature, and more susceptible to ion competition, than lateral roots. This indicates that the contribution of the metabolic uptake system, in tap roots, is dominant over that of the non‐metabolic one. The uptake capability of both types of roots was lowered by removal of the shoots. Apparently, under such treatments, the contribution of the metabolic component, to the total uptake, was reduced. Studies of plant mineral nutrition should take into account such differences in the physiological and morphological characteristics that exist among the various root types.

Developmental profile of sinalbin (p-hydroxybenzyl glucosinolate) in mustard seedlings,Sinapis alba L., and its relationship to insect resistance

Sinalbin was identified as a chemical component of insect anti-xenosis and antibiosis resistance mechanisms in seedlings ofSinapis alba by DEAE-Sephadex chromatography, HPLC, treatment with sulfatase and myrosinase, various feeding tests using artificial and natural substrates, and by measuring sinalbin concentrations in cotyledons and leaves during seedling development. The effects of sinaibin on feeding were dependent upon the insect species and upon the rapidly changing profile of sinaibin concentrations in the developing seedling. The high concentrations of sinalbin found in young cotyledons (up to 20 mM) and leaves (up to 10 mM) deterred the feeding of the flea beetle,Phyllotreta cruciferae Goeze and larvae of the bertha armyworm,Mamestra configurata Walker. The protection that sinalbin confers upon the vulnerable, newly emerged seedling (and upon tiny, young leaves) appears critical for the first few days of survival ofS. alba under feeding pressure from flea beetles in the field. The lower concentrations of sinaibin found in older cotyledons and leaves (2-3 mM) offer little or no protection againstP. cruciferae and may actually stimulate the feeding of this crucifer specialist. These concentrations of sinaibin, however, are still effective in reducing the level of feeding by larvae of the more generalist feederM. configurata.

Identification and characterization of nonsedimentable lipid-protein microvesicles.

Previously uncharacterized lipid-protein microvesicles have been isolated from young and senescing bean cotyledon tissue. The microvesicles are nonsedimentable and enriched in phospholipid degradation products (free fatty acids, long-chain aldehydes, and long-chain hydrocarbons). They range from 70 to 170 nm (radius) with a mean radius of 132 nm, and it is clear from freeze-fracture electron micrographs that they are bilayered in nature. Nonsedimentable lipid-protein microvesicles containing the same products of phospholipid degradation but smaller were also formed in vitro when smooth microsomal membranes from young cotyledon tissue were treated with Ca2+ to stimulate enzymatic degradation of phospholipids. The data suggest that these microvesicles comprise an intermediate stage of membrane lipid deterioration. They appear to serve as a vehicle for moving phospholipid degradation products out of membranes into the cytosol during senescence and perhaps also during normal membrane lipid turnover.

Alterations in membrane protein conformation in response to senescence-related changes

Measurements of diphenylhexatriene fluorescence polarization in smooth microsomal membranes isolated from senescing bean cotyledons have indicated that membrane bulk lipid fluidity decreases by ca two-fold as the cotyledons age. This change in fluidity can be largely attributed to an increase in relative sterol concentration (from ca 7 to ca 45 mol% with respect to phospholipid) and is accompanied by a change in the average conformation of membrane proteins detectable by spin labelling with 3-maleimido-PROXYL. When the sterol concentration of smooth microsomal membranes from young cotyledons was increased in vitro by treatment with cholesteryl hemisuccinate, the membrane lipid fluidity was reduced to a value comparable to that for corresponding membranes from senescent cotyledons. The cholesteryl hemisuccinate treatment also induced conformational changes in the membrane proteins, but did not alter protein composition as shown by SDS-PAGE. The observations collectively indicate that the sterol-induced decrease in bulk lipid fluidity of smooth microsomal membranes accompanying senescence is of sufficient magnitude to alter membrane protein conformation. These changes in protein conformation may also reflect sterol-induced protein aggregation, which is known to occur at relative sterol concentrations in excess of ca 27 mol% with respect to phospholipid.

Lipid Breakdown in Smooth Microsomal Membranes from Bean Cotyledons Alters Membrane Proteins and Induces Proteolysis

The effects of lipid degradation on proteins of smooth microsomal membranes isolated from young bean cotyledons have been examined by three techniques, viz. fluorescence energy transfer from tryptophan to cis-parinaric acid; protein spin-labelling with 3-maleimido PROXYL; and SDS-PAGE. Lipid degradation was induced in isolated membranes by activiting phospholipase D and phosphatidic acid phosphatase through the addition of Ca 2+ , by treatment with exogenous phospholipase C to simulate the concerted actions of phospholipase D and phosphatidic acid phosphatase or by treatment with exogenous phospholipase A 2 to generate endogenous substrate for lipoxygenase. All of the treatments induced time-dependent changes in lipid-protein interaction and in protein conformation, and the treatment with phospholipase A 2 also engendered proteolysis

Relationship of Alternative Respiratory Capacity and Alternative Oxidase Amount during Soybean Seedling Growth

In soybean (Glycine max [L.] Merr.) axis tissue and light- and dark-grown cotyledons 3 to 9 days of age, increasing amount of alternative oxidase corresponded with the increasing alternative pathway capacity of isolated mitochondria from these tissues. From 9 to 12 days of age, however, little or no change in amount of alternative oxidase occurred in cotyledons even though there were substantial changes in capacity. This suggests that, whereas alternative oxidase content may be important in determining the capacity of the alternative pathway in young cotyledons and in axis tissue, other factors regulate capacity in older cotyledons