Co-purification,co-imniunoprecipitation,and coordinate expression of acetyl-coenzyme A carboxylase activity,biotin carboxylase,and biotin carboxyl carrier protein of higher plants

Acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) is a regulatory enzyme of fatty acid synthesis, and in some higher-plant plastids is a multi-subunit complex consisting of biotin carboxylase (BC), biotin-carboxyl carrier protein (BCCP), and carboxyl transferase (CT). We recently described a Nicotiana tabacum L. (tobacco) cDNA with a deduced amino acid sequence similar to that of prokaryotic BC. We here provide further biochemical and immunological evidence that this higher-plant polypeptide is an authentic BC component of ACCase. The BC protein co-purified with ACCase activity and with BCCP during gel permeation chromatography of Pisum sativum L. (pea) chloroplast proteins. Antibodies to the Ricinus communis L. (castor) BC co-precipitated ACCase activity and BCCP. During castor seed development, ACCase activity and the levels of BC and BCCP increased and subsequently decreased in parallel, indicating their coordinate regulation. The BC protein comprised about 0.8% of the soluble protein in developing castor seed, and less than 0.05% of the protein in young leaf or root. Polypeptides cross-reacting with antibodies to castor BC were detected in several dicotyledons and in the monocotyledons Hemerocallis fulva L. (day lily), Iris L., and Allium cepa L. (onion), but not in the Gramineae species Hordeum vulgare L. (barley) and Panicum virgatum L. (switchgrass). The castor endosperm and pea chloroplast ACCases were not significantly inhibited by long-chain acyl-acyl carrier protein, free fatty acids or acyl carrier protein. The BC polypeptide was detected throughout Brassica napus L. (rapeseed) embryo development, in contrast to the multi-functional ACCase isoenzyme which was only detected early in development. These results firmly establish the identity of the BC polypeptide in plants and provide insight into the structure, regulation and roles of higherplant ACCases.Abbreviations ACCase acetyl-CoA carboxylase - ACP acyl carrier protein - BC biotin carboxylase - BCCP biotin carboxyl carrier protein - CT carboxyl transferase - MF multi-functional - MS multi-subunit We thank our colleagues Nicki Engeseth and Vicki Eccleston for advice on fatty acid analysis and Sarah Hunter for providing the developing Iris seed. This work was supported in part by grant MCB 9406466 from NSF. Acknowledgement is also made to the Michigan Agriculture Experiment Station for its support of this research.     

Effect of ammonium on the regulation of nitrate and nitrite transport systems in roots of intact barley (Hordeum vulgare L.) seedlings

The effect of NH ₄ ⁺ on the regulation of NO ₃ ^– and NO ₂ ^– transport systems in roots of intact barley (Hordeum vulgareL.) seedlings grown in NO ₃ ^– or NO ₂ ^– was studied. Ammonium partially inhibited induction of both transport systems. The inhibition was less severe in NO ₂ ^– -fed than in NO ₃ ^– -fed seedlings, presumably due to lower uptake of NH ₄ ⁺ in the presence of NO ₂ ^– . In seedlings pretreated with NH ₄ ⁺ subsequent induction was inhibited only when NH ₄ ⁺ was also present during induction, even though pretreated roots accumulated high levels of NH ₄ ⁺ . This indicates that inhibition may be regulated by NH ₄ ⁺ concentration in the cytoplasm rather than its total accumulation in roots. L-Methionine sulfoximine did not relieve the inhibition by NH ₄ ⁺ , suggesting that inhibition is caused by NH ₄ ⁺ itself rather than by its assimilation product(s). Ammonium inhibited subsequent expression of NO ₃ ^– transport activity similarly in roots grown in 0.1, 1.0, or 10 mM NO ₃ ^– for 24 h (steady-state phase) or 4 d (decline phase), indicating that it has a direct, rather than general feedback effect. Induction of the NO ₃ ^– transport system was about twice as sensitive to NH ₄ ⁺ as compared to the NO ₂ ^– transport system. This may relate to higher turnover rates of membraneassociated NO ₃ ^– -transport proteins.Abbreviations Mes 2(N-morpholino)ethanesulfonic acid - MSO L-methionine sulfoximine     

The roles of malate and aspartate in C4 photosynthetic metabolism of Flaveria bidentis (L.)

In C₄ grasses belonging to the NADP-malic enzyme-type subgroup, malate is considered to be the predominant C₄ acid metabolized during C₄ photosynthesis, and the bundle sheath cell chloroplasts contain very little photosystem-II (PSII) activity. The present studies showed that Flaveria bidentis (L.), an NADP-malic enzyme-type C₄ dicotyledon, had substantial PSII activity in bundle sheath cells and that malate and aspartate apparently contributed about equally to the transfer of CO₂ to bundle sheath cells. Preparations of bundle sheath cells and chloroplasts isolated from these cells evolved O₂ at rates between 1.5 and 2 mol · min^–1 · mg^–1 chlorophyll (Chl) in the light in response to adding either 3-phosphoglycerate plus HCO ₃ ^– or aspartate plus 2-oxoglutarate. Rates of more than 2 mol O₂ · min^–1 · mg^–1 Chl were recorded for cells provided with both sets of these substrates. With bundle sheath cell preparations the maximum rates of light-dependent CO₂ fixation and malate decarboxylation to pyruvate recorded were about 1.7 mol · min^–1 · mg^–1 Chl. Compared with NADP-malic enzyme-type grass species, F. bidentis bundle sheath cells contained much higher activities of NADP-malate dehydrogenase and of aspartate and alanine aminotransferases. Time-course and pulse-chase studies following the kinetics of radiolabelling of the C-4 carboxyl of C₄ acids from ¹⁴CO₂ indicated that the photosynthetically active pool of malate was about twice the size of the aspartate pool. However, there was strong evidence for a rapid flux of carbon through both these pools. Possible routes of aspartate metabolism and the relationship between this metabolism and PSII activity in bundle sheath cells are considered.Abbreviations DHAP dihydroxyacetone phosphate - NADP-ME(-type) NADP-malic enzyme (type) - NADP-MDH NADP-malate dehydrogenase - OAA oxaloacetic acid - 2-OG 2-oxoglutarate - PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - Pi orthophosphate - Ru5P ribulose 5-phosphate     

Characterization of a calcium/calmodulin-dependent protein kinase homolog from maize roots showing light-regulated gravitropism

Roots of many species respond to gravity (gravitropism) and grow downward only if illuminated. This light-regulated root gravitropism is phytochrome-dependent, mediated by calcium, and inhibited by KN-93, a specific inhibitor of calcium/calmodulin-dependent protein kinase II (CaMK II). A cDNA encoding MCK1, a maize homolog of mammalian CaMK, has been isolated from roots of maize (Zea mays L.). The MCK1 gene is expressed in root tips, the site of perception for both light and gravity. Using the [³⁵S]CaM gel-overlay assay we showed that calmodulin-binding activity of the MCK1 is abolished by 50 M KN-93, but binding is not affected by 5 M KN-93, paralleling physiological findings that light-regulated root gravitropism is inhibited by 50 M KN-93, but not by 5 M KN-93. KN-93 inhibits light-regulated gravitropism by interrupting transduction of the light signal, not light perception, suggesting that MCK1 may play a role in transducing light. This is the first report suggesting a physiological function for a CaMK homolog in light signal transduction.Abbreviations CaM calmodulin - CaMK (II) Ca²⁺/calmodulin-dependent protein kinase (II) - CBP CaM-binding protein - CDPK Ca²⁺-dependent protein kinase - MCK1 maize homolog of mamalian CaMK This work is supported by the National Aeronautics and Space Administration grant No: NAGW 238.     

Hexose metabolism in discs excised from developing potato (Solanum tuberosum L.) tubers

Metabolism of radiolabelled hexoses by discs excised from developing potato (Solanum tuberosum L.) tubers was been investigated in the presence of acid invertase to prevent accumulation of labelled sucrose in the bathing medium (Viola, 1996, Planta 198: 179–185). When the discs were incubated with either [U-¹⁴C]glucose or [U-¹⁴C]fructose without unlabelled hexoses, the unidirectional rate of sucrose synthesis was insignificant compared with that of sucrose breakdown. The inclusion of unlabelled fructose in the medium induced a dramatic increase in the unidirectional rate of sucroses synthesis in the tuber discs. Indeed, the decline in the sucrose content observed when discs were incubated without exogenous sugars could be completely prevented by including 300 mM fructose in the bathing medium. On the other hand, the inclusion of unlabelled glucose in the medium did not significantly affect the relative incorporation of [U-¹⁴C]glucose to starch, sucrose or glycolytic products. Substantial differences in the intramolecular distribution of ¹³C enrichment in the hexosyl moieties of sucrose were observed when the discs were incubated with either [2-¹³C]fructose or [2-¹³C]glucose. The pattern of ¹³C enrichment distribution in sucrose suggested that incoming glucose was converted into sucrose via the sucrose-phosphate synthase pathway whilst fructose was incorporated directly into sucrose via sucrose synthase. Quantitative estimations of metabolic fluxes in vivo in the discs were also provided. The apparent maximal rate of glucose phosphorylation was close to the extractable maximum catalytic activity of glucokinase. On the other hand, the apparent maximal rate of fructose phosphorylation was much lower than the maximum catalytic activity of fructokinase, suggesting that the activity of the enzyme (unlike that of glucokinase) was regulated in vivo. Although in the discs incubated with or without fructose the rates of starch synthesis or glycolysis were similar, the relative partitioning of metabolic intermediates into sucrose was much higher in discs incubated with fructose (0.6% and 32.6%, respectively). It is hypothesised that the equilibrium of the reaction catalysed by sucrose synthase in vivo is affected in discs incubated with fructose as a result of the accumulation of the sugar in the tissue. This results in the onset of sucrose cycling. Incubation with glucose enhanced all metabolic fluxes. In particular, the net rate of starch synthesis increased from 2.0 mol · hexose · g FW^–1 · h^–1 in the absence of exogenous glucose to 3.7 mol · hexose · g FW^–1 · h^–1 in the presence of 300 mM glucose. These data are taken as an indication that the regulation of fructokinase in vivo may represent a limiting factor in the utilisation of sucrose for biosynthetic processes in developing potato tubers.Abbreviations ADPGlc adenosine 5-diphosphoglucose - Glc6P glucose-6-phosphate - hexose-P hexose phosphate - NMR nuclear magnetic resonance - UDPGlc uridine 5-diphosphoglucose Many thanks to L. Sommerville for skillfull assistance and to J. Crawford and J. Liu for useful discussions on flux analysis. The research was funded by the Scottish Office Agriculture and Fisheries Department.     

Water and solute transport along developing maize roots

Hydraulic and osmotic properties were measured along developing maize (Zea mays L.) roots at distances between 15 and 465 mm from the root tip to quantify the effects of changes in root structure on the radial and longitudinal movement of water and solutes (ions). Root development generated regions of different hydraulic and osmotic properties. Close to the root tip, passive solute permeability (root permeability coefficient, P_sr) was high and selectivity (root reflection coefficient, _sr) low, indicative of an imperfect semipermeable root structure. Within the apical 100–150 mm, P_sr decreased by an order of magnitude and _sr increased significantly. Root hydraulic conductivity (Lp_r) depended on the nature of the force (hydrostatic and osmotic). Osmotic Lp_r was smaller by an order of magnitude than hydrostatic Lp_r and decreased with increasing distance from the root tip. Throughout the root, responses in turgor of cortical cells and late metaxylem to step changes in xylem pressure applied to the base of excised roots were measured at high spatial resolution. The resulting profiles of radial and longitudinal propagation of pressure showed that the endodermis had become the major hydraulic barrier in older parts of the root, i.e. at distances from the apex ä 150 mm. Other than at the endodermis, no significant radial hydraulic resistance could be detected. The results permit a detailed analysis of the root's composite structure which is important for its function in collecting and translocating water and nutrients.Abbreviations and Symbols CPP cell pressure probe - IT root segments with intact tips; - Lp_r root hydraulic conductivity - Lp_rh hydrostatic hydraulic conductivity of root - Lp_ro osmotic hydraulic conductivity of root - P_app hydrostatic pressure applied to cut end of root - P_c cell turgor - P_{c, cor} turgor of cortical cell - P_c,xyl turgor of late metaxylem vessel - P_ro stationary root pressure - P_r0,seal stationary root pressure of sealed root segment - P_sr solute permeability coefficient of root - RPP root pressure probe - TR root segments with tip removed - _sr reflection coefficient of root Dedicated to Professor Andreas Sievers on the occasion of his retirement     

The raz1 mutant of Arabidopsis thaliana lacks the activity of a high-affinity amino acid transporter

The raz1 mutant of Arabidopsis thaliana (L.) Heynh. has been selected as resistant to the toxic proline analogue, azetidine-2-carboxylic acid (2AZ). Seedlings of the mutant tolerated fivefold higher concentrations of 2AZ (ED₅₀ = 0.25 mM) than the wild-type seedlings (ED⁵⁰ = 0.05 mM). The mutant gene was found to be semi-dominant and the corresponding RAZ1 locus was mapped on chromosome 5 at 69.6±1.8 cM. The resistance to 2AZ could be fully and exclusively accounted for by the lower uptake rate of the proline analogue in the mutant. The influx of L-proline in roots of wild-type seedlings could be dissected into two components: (i) a component with a high affinity and a low capacity for l-proline (K _m≈20 gmM, V _max≈60 nmol·(g FW)^-1·h^-1) and also a high affinity for L-2AZ (K _i≈40 μM) and (ii) a low-affinity, high-capacity component (K _m≈5 mM: V _max = 1300 nmol·(g FW)^-1·h^-1). Clearly, the raz1 mutation affects the activity of a high-affinity transporter, because the high-affinity uptake of proline in the mutant was at least fivefold lower than in the wild-type, whereas the low-affinity uptake was unchanged.     

Immunochemical comparison of membrane-associated and secreted arabinogalactan-proteins in rice and carrot

Arabinogalactan-proteins (AGPs) occurring in suspension-cultured rice (Oryza saliva L.) cells, their conditioned medium and at the rice root apex were investigated using monoclonal antibodies and the AGP-binding -glucosyl Yariv reagent ( GlcY). A monoclonal antibody, LM2, was generated that recognized an acidic carbohydrate epitope common to two soluble AGPs occurring in the conditioned medium of proliferating rice cells, membrane-associated AGPs (rmAGP) in the cultured cells and two AGPs at the rice root apex. In addition, LM2 recognized AGPs secreted by suspensioncultured carrot (Daucus carota L.) cells. The two AGPs of the rice culture medium, srAGP1 and srAGP2, were discriminated by their mobilities during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reaction with GlcY, the presence of arabinogalactan epitopes and anion-exchange chromatography. The association of rmAGP with the plasma membrane was investigated by Triton-X-114/aqueous partitioning of both microsomal and plasma-membrane preparations and rmAGP was found to partition into the detergent phase, indicating that AGPs are hydrophobic plasma-membrane proteins in rice. This was in contrast to plasma-membrane AGPs of suspension-cultured carrot cells that partitioned into the aqueous phase. At the rice root apex most of the AGP was associated with the microsomal fraction and also partitioned into the detergent phase, although a distinct highmolecular-mass AGP entered the aqueous phase.Abbreviations AGP arabinogalactan-protein - GlcY -glucosyl Yariv reagent - ELISA enzyme-linked immunosorbent assay We gratefully acknowledge support from the Leverhulme Trust, the UK Biotechnology and Biological Sciences Research Council and the Royal Society.     

Induction and ionic basis of slow wave potentials in seedlings of Pisum sativum L.

Slow wave potentials (SWPs) are transient depolarizations which propagate substantial distances from their point of origin. They were induced in the epidermal cells of pea epicotyls by injurious methods such as root excision and heat treatment, as well as by externally applied defined steps in xylem pressure (Px) in the absence of wounding. The common principle of induction was a rapid increase in Px. Such a stimulus appeared under natural conditions after (i) bending of the epicotyl, (ii) wounding of the epidermis, (iii) rewatering of dehydrated roots, and (iv) embolism. The induced depolarization was not associated with a change in cell input resistance. This result and the ineffectiveness of ion channel blockers point to H(+)-pumps rather than ion channels as the ionic basis of the SWP. Stimuli such as excision, heat treatment and pressure steps, which generate SWPs, caused a transient increase in the fluorescence intensity of epicotyls loaded with the pH-indicator DM-NERF, a 2',7'-dimethyl derivative of rhodol, but not of those loaded with the pH indicator 2',7'bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Matching kinetics of depolarization and pH response identify a transient inactivation of proton pumps in the plasma membrane as the causal mechanism of the SWP. Feeding pump inhibitors to the cut surface of excised epicotyls failed to chemically simulate a SWP; cyanide, azide and 2,4-dinitrophenol caused sustained, local depolarizations which did not propagate. Of all tested substances, only sodium cholate caused a transient and propagating depolarization whose arrival in the growing region of the epicotyl coincided with a transient growth rate reduction.