The development of isocitric lyase activity in germinating cotton seed

In cotyledons of germinating cotton (Gossypium hirsutum L. var. Stoneville 213) seedlings, in the dark, isocitric lyase (EC 4.1.3.1) activity peaks after 2 days and thereafter slowly declines to a negligible value after 8 days. The maximum activity of this enzyme in cotyledons of 2-day-old seedlings was 16.2 μmoles of glyoxylate formed/15 min·10 cotyledon pairs. Actinomycin D at a concentration of 10 μg/ml, if added to the imbibing solution, completely prevents the development of isocitric lyase activity in these germinating seed. In cotyledons of germinating cotton seedlings, in the light, isocitric lyase activity peaks after 2 to 3 days and sharply declines to a negligible value after 4 days. The maximum activity of this enzyme in cotyledons of 2- to 3-day-old seedlings was 13.2 μmoles of glyoxylate formed/15 min·10 cotyledon pairs. Actinomycin D at a concentration of 10 μg/ml, if added to the imbibing solution, severely inhibits the development of enzyme activity.     

COTYLEDON PHOTOSYNTHESIS DURING SEEDLING GROWTH OF COTTON,GOSSYPIUM HIRSUTUM L.

Respiration and net photosynthetic O₂ production by cotton cotyledons were determined from an early age through the senescent stage. Various treatments were applied to cotyledons to assess the importance of current photosynthesis as compared to translocation of reserves to seedling development. Rates of respiration and net photosynthesis per cm² were high on 1-day-old cotyledons, but the rates decreased sharply with rapid expansion to reach a fairly stable rate. Respiration per cotyledon decreased linearly with age until the onset of senescence, then exhibited a distinct climacteric rise followed by a sharp decrease. Net photosynthesis per cotyledon increased until expansion was completed and then decreased linearly and steeply with age. Excision of cotyledons, inhibition of photosynthesis either chemically or by covering, and removal of the terminal bud indicated that current photosynthesis is a potent force behind early epicotyl growth.     

Adenosylhomocysteinase and adenosine nucleosidase activities in Lupinus luteus cotyledons during seed formation and germination

The activities of adenosylhomocysteinase (EC 3.3.1.1) and adenosine nucleosidase (EC 3.2.2.7) were assayed in extracts from yellow lupin (Lupinus luteus L.) cotyledons at different stages of seed formation and seedling development. Adenosylhomocysteinase activity was demonstrated in all the cotyledon extracts examined. Its lowest level was found in the dry seeds and the highest, in 4-day-old seedling cotyledons. Extracts from the cotyledons of maturating seeds, dry seeds, and seedlings up to the second day of growth exhibited no adenosine nucleosidase activity. Adenosine nucleosidase activity appeared in the cotyledons of 2-day-old seedlings and its highest level was reached in 4-to 5-day-old seedlings. There is no inhibitor of adenosine nucleosidase in the maturating and dry yellow lupin seeds. No activator of a possible zymogen form of adenosine nucleosidase from maturating or dry seeds occurs in the growing seedlings.     

A development-dependent hemagglutinin from cucumber surfaces: I. Hemagglutinin activity of cotyledon surfaces

During studies on the chemistry of plant surfaces, we observed that concomitant with the development of cucumber (Cucumis sativus L. cv `ELEM') cotyledons an agglutinin that agglutinates human erythrocytes appeared on epicuticular surfaces. The agglutinin was released from cotyledon surfaces into distilled water by a 1-minute immersion (or even less). Homogenization of the washed cotyledons released residual agglutinating activity. The surface-located hemagglutinating activity was age-dependent and occurred in dark- and light-grown cotyledons. Agglutinating activities were present in light-grown cotyledons of 2- to 14-day-old seedlings and in dark-grown cotyledons of 3- to 17-day-old seedlings. Agglutinating activities were maximal in cotyledons of 3- to 4-day-old seedlings. No activity could be detected in dry seeds or in seedlings up to 2 days and after 17 days of germination. The hemagglutinating activity was specifically inhibited by N,N′,N″-triacetylchitotriose, chitin, and chitosan.     

In Vitro Studies of Nitrate Reductase Activity in Cotton Cotyledons: Effects of Dowex 1-Cl and BSA

Germinating cotton (Gossypium hirsutum L. cv. Deltapine 16) cotyledons developed two peaks of in vitro nitrate reductase activity; the first was stable in vitro and appeared 24 hours after imbibition; and the second, which was extremely labile in vitro, began to develop after the seedlings had emerged and developed chlorophyll. Nitrite reductase activity peaked only after the seedlings had emerged. Dowex 1-Cl (10%, w/v) and bovine serum albumin (3%, w/v) significantly improved the activity of extracted enzyme; greater improvement occurred as expansion of the cotyledons progressed. The major effect of bovine serum albumin on nitrate reductase activity in cotyledon extracts appeared to be that of making the extracted enzyme more active rather than increasing the amount of nitrate reductase extracted or improving the stability of the extracted enzyme.     

Mapping sugar beet pectin acetylation pattern

Homogalacturonan-derived partly methylated and/or acetylated oligogalacturonates were recovered after enzymatic hydrolysis (endo-polygalacturonase+pectin methyl esterase+side-chain degrading enzymes) of sugar beet pectin followed by anion-exchange and size exclusion chromatography. Around 90% of the GalA and 75% of the acetyl groups present in the initial sugar beet pectin were recovered as homogalacturonan-derived oligogalacturonates, the remaining GalA and acetyl belonging to rhamnogalacturonic regions. Around 50% of the acetyl groups present in sugar beet homogalacturonans were recovered as partly methylated and/or acetylated oligogalacturonates of degree of polymerisation 5 whose structures were determined by electrospray ionization ion trap mass spectrometry (ESI-IT-MSn). 2-O-acetyl- and 3-O-acetyl-GalA were detected in roughly similar amounts but 2,3-di-O-acetylation was absent. Methyl-esterified GalA residues occurred mainly upstream 2-O-acetyl GalA. Oligogalacturonates containing GalA residues that are at once methyl- and acetyl-esterified were recovered in very limited amounts. A tentative mapping of the distribution of acetyl and methyl esters within sugar beet homogalacturonans is proposed. Unsubstituted GalA residues are likely to be present in limited amounts (approximately 10% of total GalA residues), due to the fact that methyl and acetyl groups are assumed to be most often not carried by the same residues.     

Differences in cytokinin control on cellular dynamics of zucchini cotyledons cultivated in two experimental systems

The effect of endogenous cytokinins on the pattern of palisade cell division post-germination does not depend on the conditions of cotyledon development -in planta (attached to seedlings) or in vitro (isolated from dry zucchini seeds and cultured on water). In cotyledons originating from 4-day-old seedlings (experimental system 1), exogenous cytokinin temporarily (in the first 2 day of cultivation) enhanced post-mitotic cell enlargement of palisade cells, mainly due to enhanced water uptake and use of cell storage compounds, all of which lead to cotyledon senescence. Cytokinin is not able to resume the completed palisade cell division on day 5. As a result, the number of cells and the final areas of treated and control cotyledons are quite similar. By contrast, the effects of cytokinin on cotyledons isolated from dry seeds (experimental system 2) are better expressed, promoting an increase in number of palisade cells accompanied by additional cotyledon area enlargement. However, the prolonged post-mitotic cell expansion in control cotyledons compensates for the reduced speed of cell growth and division activity and decreases differences in final cotyledon area between treatments. The results define cell division as the primary target of cytokinin stimulation in cotyledon tissues competent for division, and determine the temporal patterns of palisade cell cycling related to cotyledon age. This knowledge permits a better choice of experimental system to study effects on cell proliferation and cell growth, as well as cell enlargement and senescence-related events using physiologically homogeneous material.     

THE EFFECT OF COTYLEDON EXCISION ON THE GROWTH OF PEA SEEDLINGS

Pea plants grown under controlled conditions for 28 days were sampled nine times during this growth period. On each sampling day 80 plants were harvested. Cotyledons were excised from 40 plants and the root-shoot axis allowed to continue growth. The other 40 plants were divided into roots, shoots, and cotyledons; the dry weights of alcohol-water extracts and residues from these organs were determined. Shoot lengths, numbers of nodes, buds, flowers, and fruits of 28-day-old plants, whose cotyledons were excised at different times, were determined. These data indicate that cotyledon excision after the 13th day has no apparent effect on seedling growth, whereas excision before the 13th day reduces growth. Glucose-U-C¹⁴ supplied to attached cotyledons of 6- and 16-day-old seedlings resulted in no major difference in the distribution pattern of recovered label throughout the plant even though 16-day-old cotyledons were depleted of their normal endogenous food reserve.     

Release of a Soluble Phytoalexin Elicitor from Mycelial Walls of Phytophthora megasperma var. sojae by Soybean Tissues

A soluble elicitor of glyceollin accumulation was released from insoluble mycelial walls of Phytophthora megasperma var. sojae after incubation with soybean cotyledon tissue for as little as 2 minutes. Various enzymic and chemical treatments of the released elicitor indicated that the activity resided in a carbohydrate moiety, and gel filtration disclosed the presence of at least two active molecular species. Cell-free extracts from soybean cotyledons or hypocotyls also released soluble elicitors from fungal cell walls that were similar to those released by living cotyledon tissue. These results may suggest that contact of fungal pathogens with host tissues is required to release fungal wall elicitors which then initiate phytoalexin accumulation in the plant.     

Cytokinins in Germinating Seeds of Phaseolus vulgaris L. I. Changes in Endogenous Levels Within the Cotyledons

Ethanolic extracts from the cotyledons of mature dry Phaseolusvulgaris L. seed yielded cytokinin-like activity which co-chromatographedwith zeatin and ribosylzeatin. Under conditions which stimulatedgermination and cotyledon expansion, the level of these cytokininsdecreased rapidly in both intact embryos and excised cotyledons.In the excised cotyledons the decrease was continuous, resultingin very low levels of cytokinin being detected after 4 daysof incubation. With the embryonic axis present, however, theinitial decrease was arrested and reversed after 3 days. Thissuggests that the cotyledons do not synthesize cytokinins butthat these hormones are imported from the embryonic axis, particularlyonce radicle growth is well under way. Phaseolus vulgaris, bean, cotyledons, cytokinins, germination     

Degenerative changes in properties of the mitochondrial inner membrane in pea cotyledons during germination

Mitochondria isolated from 5-day-old pea cotyledons had lowrespiration activity and did not respond to exogenous ADP, whilethose from 1-day-old cotyledons respired actively and respondedto ADP. The former mitochondria, but not the latter, were verysusceptible to destruction during extraction and purification.The mitochondrial inner membrane isolated from 5-day-old cotyledonswas less dense than that from 1-day-old cotyledons. The specificactivity of SDH in the former membrane was lower than that inthe latter, while both membranes were similar to each otherwith respect to the specific activity of Cyt ox. Disc electrophoresisof solubilized membrane on polyacrylamide gel containing SDSshowed that the mitochondrial inner membrane from 5-day-oldcotyledons contained lesser amounts of several polypeptidescompared with that from 1-day-old cotyledons. Such alterationsin the mitochondrial inner membrane were not observed with theexcised cotyledons cultivated for 5 days. (Received June 17, 1977; )     

DNA changes involving repeated sequences in senescing soybean (Glycine max) cotyledon nuclei

Age-related DNA changes in soybean ( Glycine max L. cv. Ransom) cotyledon nuclei were investigated by Feulgen cytophotometry and cloned DNA probes. The amount of nuclear DNA in 17-day-old senescing cotyledons was 23% lower than that of 5-day-old young cotyledons. In order to understand the nature of senescence-related DNA loss, fragments of repetitive DNA from young cotyledons were cloned into Escherichia coli HB101 cells by DNA recombination. The cloned DNA probed changes in specific repeated nucleotide sequences in senescing cotyledons. The colony hybridization between cloned DNA and [³²P]-labeled total DNA from 5- and 17-day cotyledons indicated loss of specific repeated sequences. The selected sequences of repeated DNA further showed a loss in their copy numbers. The study suggested that some repetitive DNA sequences were degraded selectively in the genome of senescing cotyledons.     

Changes in leucyl-tRNAs and aminoacyl tRNA synthetases in developing and aging soybean cotyledons

Leucine specific tRNA of soybean cotyledons was frationated into six peaks (1–6). The relative amounts of Leu-tRNA 5 and 6 are lower in developing cotyledons than in germinating cotyledons. Leu-tRNA synthetase from developing cotyledons is less active in aminoacylating Leu-tRNA 5 and 6 compared to enzyme from 5-day-old germinating cotyledons. Leu-tRNA synthetase from cotyledons of germinating seedlings and developing cotyledons can be fractionated into three peaks (1–3). Peak 1 in the developing cotyledon is about 36% less than peak 1 from 5-day-old germinating cotyledons. Peaks 2 and 3 from developing cotyledons are about 10 and 18% higher than from germinating cotyledons, respectively. Peak 1 from developing cotyledons acylates all six species of Leu-tRNA in contrast with peak 1 from germinating cotyledons, which essentially acylates only Leu-tRNA 5 and 6. The specificity of peaks 2 and 3 towards Leu-tRNA 1–4 is identical in both the organs.     

Diversity of Aeromonas sp. in Flemish drinking water production plants as determined by gas-liquid chromatographic analysis of cellular fatty acid methyl esters (FAMEs)

G. HUYS, I. KERSTERS, M. VANCANNEYT, R. COOPMAN, P. JANSSEN AND K. KERSTERS. 1995. Gas-liquid chromatography of cellular fatty acid methyl esters (FAMEs) was used to determine the phenotypic and genotypic diversity among 489 presumptive Aeromonas strains isolated from five Flemish drinking water production plants. FAME profiles were compared with the predetermined library profiles of a representative database, AER48C, which contains the mean FAME data of all 14 currently established hybridization groups (HGs) or genospecies within Aeromonas. Using AER48C, more than 93% (457 strains) of all presumptive aeromonads isolated on ampicillin-dextrin agar were unequivocally identified as belonging to this genus. Moreover, 85.5% and 73.5% of these strains could be assigned to a particular phenospecies or HG, respectively. Raw and treated surface water samples were dominated by members of the Aer. hydrophila complex (38.8%, comprising HGs 1–3), followed by the Aer. caviae complex (22.7%, comprising HGs 4–6) and the Aer. sobria complex (16.7%, comprising HGs 7–9). HGs 3, 5A/B and 8 were the most prominent genospecies in this type of water. On the other hand, it was found that raw and treated phreatic groundwater samples displayed a much more limited species diversity since these were almost entirely dominated (95.8%) by strains belonging to HGs 2 and 3 of the Aer. hydrophila complex. In general, flocculation-decantation and sand filtration were not shown to influence the overall species distribution in any of the plants examined.     

Auxin transport into cotyledons and cotyledon growth depend similarly on the ABCB19 Multidrug Resistance-like transporter

ABCB19 of Arabidopsis thaliana (formerly known as MDR1 and PGP19) belongs to the Multidrug Resistance-like ( MDR ) or B group of the ATP-binding cassette (ABC) transporter superfamily, and mediates polar auxin transport in stems and roots. Here we have investigated the role of ABCB19 and auxin distribution in cotyledon development. During embryogenesis, confocal microscopy showed ABCB19 protein to be present in cotyledons during their main growth phase, but not later. Analysis of ProDR5:GFP expression patterns showed a significantly diminished and restricted auxin distribution pattern in abcb19 cotyledons. Nonetheless, development of abcb19 embryonic cotyledons was very similar to that of wild-type. Post-germination, ABCB19 was present in the plasma membrane of cotyledon epidermal, mesophyll and petiole cells during blade expansion. Post-germination cotyledon blade expansion in abcb19 was 65% slower than in wild-type, although the epidermal cell area was reduced by only 17%. The growth rate reduction quantitatively correlated with reduced auxin levels rather than auxin sensitivity as indicated by quantitative ProDR5:GUS assays and direct auxin measurements, and may be explained by the 50% reduction in the import of auxin through the petioles of abcb19 cotyledons during the period of maximum expansion. Taken together, these data indicate that cotyledon expansion during the establishment of photoautotrophic growth depends on ABCB19-mediated auxin import.     

Analysis of pectic epitopes recognised by hybridoma and phage display monoclonal antibodies using defined oligosaccharides, polysaccharides, and enzymatic degradation

The structure of epitopes recognised by anti-pectin monoclonal antibodies (mAbs) has been investigated using a series of model lime-pectin samples with defined degrees and patterns of methyl esterification, a range of defined oligogalacturonides and enzymatic degradation of pectic polysaccharides. In immuno-dot-assays, the anti-homogalacturonan (HG) mAbs JIM5 and JIM7 both bound to samples with a wide range of degrees of methyl esterification in preference to fully de-esterified samples. In contrast, the anti-HG phage display mAb PAM1 bound most effectively to fully de-esterified pectin. In competitive inhibition ELISAs using fully methyl-esterified or fully de-esterified oligogalacturonides with 3-9 galacturonic acid residues, JIM5 bound weakly to a fully de-esterified nonagalacturonide but JIM7 did not bind to any of the oligogalacturonides tested. Therefore, optimal JIM5 and JIM7 binding occurs where specific but undefined methyl-esterification patterns are present on HG domains, although fully de-esterified HG samples contain sub-optimal JIM5 epitopes. The persistence of mAb binding to epitopes in pectic antigens, with 41% blockwise esterification (P41) and 43% random esterification (F43) subject to fragmentation by endo-polygalacturonase II (PG II) and endo-pectin lyase (PL), was also studied. Time course analysis of PG II digestion of P41 revealed that JIM5 epitopes were rapidly degraded, but a low level of PAM1 and JIM7 epitopes existed even after extensive digestion, indicating that some HG domains were more resistant to cleavage by PG II. The chromatographic separation of fragments produced by the complete digestion of P41 by pectin lyase indicated that a very restricted population of fragments contained the PAM1 epitope while a (1-->4)-beta-D-galactan epitope occurring on the side chains of pectic polysaccharides was recovered in a broad range of fractions.     

Fruit softening: evidence for pectate lyase action in vivo in date (Phoenix dactylifera) and rosaceous fruit cell walls

Background and AimsThe programmed softening occurring during fruit development requires scission of cell wall polysaccharides, especially pectin. Proposed mechanisms include the action of wall enzymes or hydroxyl radicals. Enzyme activities found in fruit extracts include pectate lyase (PL) and endo-polygalacturonase (EPG), which, in vitro, cleave de-esterified homogalacturonan in mid-chain by β-elimination and hydrolysis, respectively. However, the important biological question of whether PL exhibits action in vivo had not been tested.MethodsWe developed a method for specifically and sensitively detecting in-vivo PL products, based on Driselase digestion of cell wall polysaccharides and detection of the characteristic unsaturated product of PL action.Key ResultsIn model in-vitro experiments, pectic homogalacturonan that had been partially cleaved by commercial PL was digested to completion with Driselase, releasing an unsaturated disaccharide (‘ΔUA–GalA’), taken as diagnostic of PL action. ΔUA–GalA was separated from saturated oligogalacturonides (EPG products) by electrophoresis, then subjected to thin-layer chromatography (TLC), resolving ΔUA–GalA from higher homologues. The ΔUA–GalA was confirmed as 4-deoxy-β-l-threo-hex-4-enopyranuronosyl-(1→4)-d-galacturonic acid by NMR spectroscopy. Driselase digestion of cell walls from ripe fruits of date (Phoenix dactylifera), pear (Pyrus communis), rowan (Sorbus aucuparia) and apple (Malus pumila) yielded ΔUA–GalA, demonstrating that PL had been acting in vivo in these fruits prior to harvest. Date-derived ΔUA–GalA was verified by negative-mode mass spectrometry, including collision-induced dissociation (CID) fragmentation. The ΔUA–GalA:GalA ratio from ripe dates was roughly 1:20 (mol mol^–1), indicating that approx. 5 % of the bonds in endogenous homogalacturonan had been cleaved by in-vivo PL action.ConclusionsThe results provide the first demonstration that PL, previously known from studies of fruit gene expression, proteomic studies and in-vitro enzyme activity, exhibits enzyme action in the walls of soft fruits and may thus be proposed to contribute to fruit softening.     

Host-Pathogen Interactions : XXI. Extraction of a Heat-Labile Elicitor of Phytoalexin Accumulation from Frozen Soybean Stems

An extract of frozen and thawed soybean (Glycine max L. Merr. cv. Wayne) stems is active, in wounded soybean cotyledons, as a heat-labile elicitor of phytoalexins. The elicitor activity of the extract is destroyed by heating to 95°C for 10 minutes. The fraction that contains heat-labile elicitor activity releases heat-stable elicitor-active molecules from purified soybean cell walls. Heat-labile elicitor activity voids a Bio-Gel P-6 column and can be absorbed onto and eluted from a DEAE Sephadex ion exchange column. Using the cotyledon phytoalexin elicitor assay, maximum heatlabile elicitor activity was obtained when soybean stems were extracted with acetate buffer at pH 6.0. Addition of 1 millimolar CaCl₂ increased apparent heat-labile elicitor activity. The heat-labile elicitor stimulated maximum phytoalexin accumulation when applied to cotyledons immediately after the cotyledons were cut. Partially purified stem extracts lost heat-labile elicitor activity during storage for several days at 3°C. The possible role of a heat-labile elicitor in stimulation of phytoalexin accumulation by both abiotic and biotic elicitors is discussed.     

Factors affecting transient gene expression in cultured radiata pine cotyledons following particle bombardment

Transfer and expression of the β–glucuronidase gene ( gusA ) in cultured cotyledons of radiata pine ( Pinus radiata D. Don ) were obtained by particle bombardment. Conditions for optimum transient expression were established by using plasmid pB[/12], delivered by gold particles, 1.6 μm in diameter, into 8-day-old cultured cotyledons. Helium pressure of 7.6 MPa, bombardment distance between the stopping screen and the target tissues of 6 cm, and 0.8 μg of plasmid DNA per bombardment proved to be the best parameters for transient expression; using these parameters 79% of bombarded cotyledons showed GUS activity, with 4.3 blue spots per cotyledon. This system was used for studying the expression of several gus-driven promoters the expression of the sunflower ubiquitin gene promoter was higher (99% of positive cotyledons, with 14.2 blue spots per cotyledon) than that of the CaMV 35S promoter, whereas the rice actin and the maize alcohol dehydrogenase gene promoters gave lower gusA expression, as determined histochemically. These results were confirmed by using the gus fluorometric assay. Use of the sunflower ubiquitin gene promoter resulted in gusA expression up to 20 days after bombardment, with a significant level of gus expressing loci per bombarded cotyledon, whereas with the CaMV 35S promoter gusA expression was lost 12 days after bombardment.     

Metabolism of carbohydrate and lipid reserves in germinated cotton seeds

Utilization of reserve lipid and carbohydrates during germination (0–12 h) and postgerminative growth (12–48 h) was studied in cotton (Gossypium hirsutum L.) seedlings. Raffinose and stachyose were utilized during the germination period and early growth; mobilization was associated with -galactosidase (EC 3.2.1.22) activity. Results from pulse-chase experiments with [³H]raffinose supplied exogenously to 4-h soaked seeds indicated that raffinose-derived catabolites contributed to the coincident increase in cotyledon sucrose and starch, and to the small increase in axis dry weight. Starch appears to be an alternative sink for end products of hydrolysis of reserve carbohydrates prior to the onset of rapid axis growth and cotyledon expansion. Mobilization of neutral lipid commenced at about 16 h after soaking, concomitant with development of key glyoxylate-cycle and other gluconeogenesis-related enzyme activities. Axis dry weight increased three-fold between 24 and 48 h. Results from pulse-chase (3 h, 16 h) experiments in which [2-¹⁴C]acetate was supplied to cotyledons of intact 22-h-old seedlings showed that acetate-derived metabolites were not transported exclusively to the axes, but were partitioned between axes and cotyledons. Only 27% of total incorporated radioactivity was recovered in axes following the chase, 18% was evolved as CO₂, and the rest was recovered in water-soluble substances (20%) and polymers (31%) within the cotyledons. Of the polymers, 55% of the activity was in polysaccharides (Starch, pectic substances, hemicellulose, cellulose), 25% in protein, and 20% in unidentified neutral and acidic compounds. Considering these data, the amount of lipid mobilized, and various routes by which supplied [2-¹⁴C]acetate could be metabolized, it appears that lipidderived compounds contribute only 25–40% of axis dry-weight gain. Lipid-derived substances retained in the cotyledons likely are utilized for expansion and differentiation of the cotyledons into photosynthetic organs.