Carrot Storage Roots Research Articles

Evidence for an interaction between cytosolic aldolase and the ATP- and pyrophosphate-dependent phosphofructokinases in carrot storage roots

Immunoaffinity chromatography was employed to identify potential plant cytosolic aldolase (ALD c) binding proteins. A clarified homogenate of carrot storage root was chromatographed on a column of protein-A—Sepharose that had been covalently coupled to anti-(carrot root ALD,) immunoglobulin G. The column was washed with phosphate-buffered saline (PBS), followed by step-wise elution with increasing concentrations of NaCl in PBS. Several proteins were eluted following application of the salt gradient. Western blotting identified the major eluting proteins to be the PP i-dependent phosphofructokinase (PFP) and the cytosolic form of the ATP-dependent phosphofructokinase (PFK c), enzymes that are metabolically sequential to ALD c. The results suggest that ALD c may specifically interact with PFP and PFK c in carrots.

CDNA cloning of carrot extracellular beta-fructosidase and its expression in response to wounding and bacterial infection.

We isolated a full-length cDNA for apoplastic (extracellular or cell wall-bound) beta-fructosidase (invertase), determined its nucleotide sequence, and used it as a probe to measure changes in mRNA as a result of wounding of carrot storage roots and infection of carrot plants with the bacterial pathogen Erwinia carotovora. The derived amino acid sequence of extracellular beta-fructosidase shows that it is a basic protein (pl 9.9) with a signal sequence for entry into the endoplasmic reticulum and a propeptide at the N terminus that is not present in the mature protein. Amino acid sequence comparison with yeast and bacterial invertases shows that the overall homology is only about 28%, but that there are short conserved motifs, one of which is at the active site. Maturing carrot storage roots contain barely detectable levels of mRNA for extracellular beta-fructosidase and these levels rise slowly but dramatically after wounding with maximal expression after 12 hours. Infection of roots and leaves of carrot plants with E. carotovora results in a very fast increase in the mRNA levels with maximal expression after 1 hour. These results indicate that apoplastic beta-fructosidase is probably a new and hitherto unrecognized pathogenesis-related protein [Van Loon, L.C. (1985). Plant Mol. Biol. 4, 111-116]. Suspension-cultured carrot cells contain high levels of mRNA for extracellular beta-fructosidase and these levels remain the same whether the cells are grown on sucrose, glucose, or fructose.

Purification and characterization of cytosolic aldolase from carrot storage root

A single fructose-1,6-bisphosphate (FBP) aldolase has been detected in extracts from carrot storage roots (Daucus carota L.). The enzyme was purified 850-fold to electrophoretic homogeneity and a final specific activity of 26.3 mumols of FBP utilized/min per mg of protein. SDS/PAGE of the final preparation revealed a single protein-staining band of 40 kDa. The native molecular mass was determined by analytical gel filtration to be 159 kDa, indicating that the enzyme is a homotetramer. Denaturing isoelectric focusing revealed two predominant protein-staining bands, with pI values of 5.6 and 5.7. The enzyme is a class I aldolase, since EDTA or metal ions had no effect on its activity. The enzyme was relatively heat-stable, had an activation energy (Ea) of 68.3 kJ.mol-1, and had an absorption coefficient of 8.08 x 10(4) M-1.cm-1 at 280 nm. Km values for FBP and sedoheptulose 1,7-bisphosphate (SBP) were both determined to be 6 microM (pH optima 7.4). The specificity constant with FBP was 2.6 times that obtained with SBP. Ribose 5-phosphate, 6-phosphogluconate, MgAMP, glucose 1-phosphate and phosphoenolpyruvate (PEP) were inhibitors. PEP was a mixed-type inhibitor with respect to FBP (Ki = 3.2 mM, K'i = 5.1 mM). No activators were found. Rabbit anti-(carrot aldolase) polyclonal antibodies immunoprecipitated the activity of both carrot root aldolase and spinach leaf cytosolic aldolase, but not that of spinach leaf plastid aldolase. Western-blot analysis also revealed cross-reactivity with cytosolic, but not plastid, spinach leaf aldolase, indicating that the single carrot root aldolase is cytosolic.

Antibody localization of extensin in cell walls of carrot storage roots

The accumulation and cross-linking of hydroxyproline-rich glycoproteins (HRGPs) in cell walls of dicotyledonous plants has been correlated with a number of wall-strengthening phenomena. Polyclonal antibodies raised against glycosylated extensin-1, the most abundant HRGP in carrot (Daucus carota L.) cell walls, recognize this antigen on gel and dot blots and on thin sections of epoxy-embedded carrot-root cell walls. Since wall labeling can be largely reduced by preincubating the antibodies with purified extensin-1, most labeling can be attributed to recognition of this antigen. The remaining label may be the result of recognition of extensin-2, a second carrot HRGP, or other wall components (cellulose, hemicellulose and pectin are not recognized). Extensin-1 label was distributed quite uniformly across the cell wall but was absent from the expanded middle lamella at the intersection of three or more cells and was reduced in the narrow middle lamella between two cells. This distribution is essentially the same as that of cellulose. Because of limitations of this labeling technique, it is not possible to construct a complete model of the structure of the cross-linked extensin matrix. Nonetheless, short, linear arrays of gold particles may represent small portions of the extensin matrix or of individual extensin molecules as they are exposed on the surface of sections. These and other results presented here indicate that: a) newly synthesized extensin is added to the wall by intussusception; b) extensin cannot cross the middle lamella separating the walls of adjacent cells; and c) incorporation of extensin is a late event in the development of phloem-parenchyma cell walls in carrot.

Analysis of the expression of extensin and p33-related cell wall proteins in carrot and soybean

We have identified p33 as a cell wall protein in carrot storage roots that increases in the cell wall after wounding, using antibodies raised against a synthetic peptide derived from the p33 cDNA sequence. The mRNA encoding p33 increases markedly within 1 h after wounding in carrots, indicating that p33 may be involved in one of the early cellular responses to physical damage. In contrast with extensin, p33 mRNA does not accumulate in carrot roots in response to ethylene or in carrot suspension culture cells incubated in the presence of a crude endogenous elicitor fraction. These results indicate that the wound-induced signals responsible for the synthesis of p33 and extensin after wounding are different. Protein immunologically related to carrot p33 and RNA transcripts homologous to p33 cDNA sequences were detected in the apical hook of the soybean seedling indicating that a cell wall protein related to p33 is also synthesized during plant development.

13C NMR determination of sugar levels in storage roots of carrot (Daucus carota)

13C NMR was used to detect sugars in detached fibrous and storage roots of carrots (Daucus carota L. cv. Chantenay Red‐Cored). The relative amounts of sucrose, glucose and fructose in storage roots of two ages and in fibrous roots were similar to those detected by a destructive, enzymatic sugar determination method. In tap roots all three sugars were present with glucose being marginally predominant in 8‐week‐old roots and sucrose being the major sugar present in 12‐week‐old roots. Glucose was the major sugar present in fibrous roots with small amounts of sucrose and no fructose detected. The results indicate that with some instrumental modification, 13C NMR could be used as a non‐destructive method for measuring sugar levels in intact storage root systems during development.

Mannitol transport and suitability as an osmoticum in root cells

Fluxes of mannitol across plasmalemma and tonoplast of excised carrot storage root tissue were measured using compartmental analysis of 14C tracer exchange. Mannitol metabolism and the contribution of [14C]‐labelled metabolites to efflux was shown to be small. Similar but less extensive measurements were made on red beet (Beta vulgaris L.), barley (Hordeum vulgare L.) and maize (Zea mays L.) roots. Calculated values of the reflection coefficient for mannitol were close to one, but, despite this, the inflow of mannitol was sufficient to dissipate the mannitol concentration gradient between inside and outside the cells within the time it takes them to adjust vacuolar concentrations. Thus mannitol is not suitable as an osmoticum in osmotic adjustment experiments in these root tissues. Mannitol flows appear to be passive. Permeability to mannitol (about 10−10 m s−1 is greater at the plasmalemma than at the tonoplast in carrot, and this would tend to cause the cytoplasm to swell. The implications for the control of cytoplasmic volume are discussed.

Characteristics of Sulfate Transport Across Plasmalemma and Tonoplast of Carrot Root Cells

Compartmental analysis of (35)SO(4) (2-) exchange kinetics is used to obtain SO(4) (2-) fluxes and compartment contents in carrot (Daucus carota L.) storage root cells, where 2 to 5% of the SO(4) (2-) taken up is reduced to organic form. The necessary curve fitting is verified by (a) consistency between ;content versus time' and ;rate versus time' plots of washout data; (b) agreement between loading and washout kinetics; and (c) correct identification of the fastest exchange phase as being from extracellular spaces.Sulfate is actively transported up an electrochemical potential gradient at both plasmalemma and tonoplast. The plasmalemma influx is from 2 to 10 times higher than the tonoplast influx, is much greater than the SO(4) (2-) reduction rate, and would not limit the rate of either. This is consistent with the finding that the plasmalemma influx is not regulated by internal SO(4) (2-) or cysteine (Cram 1982 Plant Sci Lett, in press).Both SO(4) (2-) influxes rise with only limited saturation as the external SO(4) (2-) concentration increases up to 50 millimolarity. Both effluxes appear to be passive, with extensive recycling in the plasmalemma influx pump. SO(4) (2-) permeability is about 10(-11) meter per second at both membranes.The high, nonlimiting fluxes of SO(4) (2-) at the plasmalemma relative to the tonoplast (found also in Lemna; Thoiron, Thoiron, Demarty, Thellier 1981 Biochim Biophys Acta 644: 24-35) contrasts with SO(4) (2-) fluxes in bacteria and with Cl(-) fluxes in plant cells. Their implications for work on characteristics and regulation of SO(4) (2-) uptake in roots and tissue cultures are discussed.

Chloride accumulation in plant cells as a homeostatic system: Energy supply as a dependent variable

During the accumulation of KCl by excised tissue of the carrot storage root, Cl−, influx falls by 90% or more. The possibility that this reduction is caused by depletion of respiratory substrate and consequent limitation of Cl− influx by energy supply is disproved. If energy were limiting, then after accumulating KCl (i) the rate of O2 uptake would be less; (ii) the ATP content might be less; (iii) external glucose and/or sucrose would stimulate O2 uptake and Cl− influx; and (iv) respiratory uncouplers would stimulate O2 uptake less. In each case the opposite was found. The ratio of O2 uptake in KCl-loaded tissue to that in nonloaded tissue was 1.25±0.21 (7); the ATP concentration was the same after KCl loading; glucose did not stimulate O2 uptake (except indirectly in the same way as a nonmetabilized glucose analogue) nor did glucose or sucrose stimulate Cl− influx; and the maximal O2 uptake in the presence of the uncoupler ClCCP was the same after KCl loading.

A model to predict the effects of complex row spacings on the yields of root crops

SUMMARYA mathematical plant competition model developed by Currah (1975), and subsequently modified by the author, is briefly described. The model has two useful properties: (a) it can cope with complex irregular row spacing systems such as occur in commercial practice and (b) it can be ‘calibrated’ to the yield values at any site by using data from previous crops.Parameter values of the modified model are calculated for both carrot and red beet crops. At least 70% of the variation in these data could be accounted for by fitting the model, a performance which was similar to, but never as good as, fitting analysis of ariance models. There were no systematic deviations between the fitted and observed values.Previously estimated parameter values were used to predict mean root weights of carrot and red beet storage roots. Overall agreement between predicted and observed data was good, although some systematic deviations occurred.The practical value of the modified version of the model and its strengths and weaknesses are discussed.

Pinwheel inclusions in morphogenesis: A possible alternative to induction by viruses

Pinwheel inclusions (PWs) were found in cells of callus tissue derived from explants of secondary phloem parenchyma of carrot (Daucus carota) storage root and grown on a basal medium containing zeatin and indoleacetic acid or coconut milk, naphthalene acetic acid, or combinations of these. Preliminary attempts to demonstrate the presence of viruses in the callus tissue failed. The possibility that the tissles were infected by a low titer or unstable conventional virus or by a defective mutant has not been ruled out. However, two lines of evidence suggest that the PWs in these tissues may be a result of culture conditions and not of virus infection. First, no PWs or other cytoplasmic inclusions were found in cells of otherwise similar tissue cultured on basal medium alone, and multifibrillar bundles (MFBs) but not PWs were found when the tissues were cultured on a medium that stimulates differentiation and morphogenesis. Second, culture stimulated to differentiate and containing MFBs only were returned to the supplemented basal medium and subsequently found to contain both PWs and MFBs.

Is Decreased Activity of Nitrate Reductase in Roots of Carrot Infested with Northern Root-Knot Nematode Related to the Increased Level of Chlorogenic Acid?

In carrot inlested with northern root-knot nematode nitrate reductase (NR) activity can either be decreased (plants grown in a garden) or increased (nitrate starved plants grown in a glasshouse) as compared with healthy plants. Tissues of secondary phloem and secondary xylem of carrot storage root of both healthy and infested plants respond similarly to induction with KNO 3 in darkness. In the induced leaves and the main non-storage root of infested plants NR activity is halved in comparison with healthy plants. Chlorogenic acid and phenols accumulate in galls and galled fibrous roots. Chlorogenic acid at p H 3.5 increases NR activity in tissues of storage root, and at p H 6.0 decreases it. The latter effect may be due to oxidation of chlorogenic acid to o -quinone. Light enhances NR activity in secondary phloem and secondary xylem induced with KNO 3 . This effect does not occur when [1], either chlorogenic acid at p H 6.0 is added to the induction medium, or [2], corresponding tissues from nematode infested plants are taken into analyses. It is suggested that northern root-knot nematode has no a direct effect on NR activity in carrot. However, polyphenols accumulated in the tissues penetrated by the nematode may exert dual effect on NR. They may increase NR activity at limited supply of nitrate or markedly decrease it in presence of nitrate and in light.

Cell Wall and Protoplast Isoperoxidases in Relation to Injury, Indoleacetic Acid, and Ethylene Effects

In tobacco (Nicotiana Tabacum) pith, sweet potato (Ipomoea batatas), and carrot (Daucus carota) storage roots, differences were found between cell wall and protoplast peroxidases in their isoenzyme patterns, activity, and reaction to tissue injury.In the pith of elongating tobacco internodes, 90% of total activity was associated with the walls, 80% of which was due to the ionically and covalently bound fractions. With senescence, the increase in activity occurred mainly in the protoplast and the free fraction in the walls. The major protoplast isoperoxidases formed the free wall fraction, and the minor ones formed the wall bound fractions. The minor isoperoxidases were the only ones whose activities were affected by cut injury. Neither senescence nor injury caused qualitative changes in the cell isoperoxidase pattern. In the potato root cells, the isoperoxidases were also distinctive in their distribution and in their reactions to injury as well as to ethylene.In potato and carrot roots, the wall fractions contributed 30 and 95%, respectively, to total peroxidase activity. The injury-induced increase in peroxidase activity occurred mainly in the protoplast of tobacco pith and potato roots and almost entirely in the ionically bound fraction of the walls in carrot roots. Ethylene stimulated peroxidase development in potato roots, especially in the protoplast, but had no effect in either tobacco pith or carrot roots. Ethylene did not affect the inhibiting action of indoleacetic acid on peroxidase activity in tobacco pith, nor did indoleacetic acid significantly affect the stimulating action of ethylene in potato roots.Actinomycin D inhibited the injury-dependent development of peroxidase in tobacco pith, stimulated the injury- and ethylene-dependent peroxidase development in potato root, and had no effect on peroxidase development in carrot root. Cycloheximide prevented the increase in peroxidase activity in tobacco pith and potato root, but not in the carrot root. Thus, the enhancement of peroxidase activity resulted from either enzyme activation exclusively or from enzyme synthesis. RNA synthesis seems to be only indirectly related to isoperoxidase synthesis caused by injury or ethylene.

Fluxes of a Nonelectrolyte and Compartmentation in Cells of Carrot Root Tissue

Permeation of thiourea into cells of carrot (Daucus carota L.) storage root discs was found to be governed by its concentration gradient only. The analysis of the course of thiourea efflux from preloaded discs shows two distinct exponential curves, apart from an initial very fast component. Reasons are given for relating the first exponential part of the curve to efflux from the cytoplasm and the second exponential part to efflux from the vacuole.The rate constant for thiourea efflux through the tonoplast is markedly temperature-dependent (activation energy 13.2 kcal mole(-)) and is lowered by 2,4-dinitrophenol and by phenyl mercuric acetate treatments. The rate constant for its efflux through the plasmalemma, on the other hand, has a low activation energy (4.2 kcal mole(-)), which is equal to that of free diffusion of thiourea in aqueous solution, and is not affected by the metabolic inhibitors. Possible reasons for the difference in the properties of the two membranes are discussed.