Effect of Tribenzylphosphate on the Phosphate Translocator of Isolated Pea Chloroplasts

The effect of tribenzylphosphate on the activity of the phosphatetranslocator of intact pea chloroplasts was tested. The translocatoractivity was followed by O₂ evolution, ¹⁴CO₂ fixation and ³²Pback-exchange. The reagent inhibited 3-phosphoglycerate dependent-photosyntheticactivities probably through an interaction with the PGA translocator. (Received September 11, 1985; Accepted November 21, 1985)     

The Phosphate Transporter from Pea Mitochondria (Isolation and Characterization in Proteolipid Vesicles)

The phosphate transporter from mitochondria will exchange matrix phosphate for cytosolic phosphate and facilitate either phosphate/proton symport or phosphate/hydroxyl ion antiport. The phosphate transported into the matrix by this carrier is either used for ATP synthesis or exchanges back out to the cytosol on the dicarboxylate transporter, permitting entry of malate and succinate into the matrix. The phosphate transporter was solubilized from etiolated pea (Pisum sativum L. cv Alaska) mitochondrial membranes with Triton X-114, purified approximately 500-fold by hydroxylapatite chromatography, and reconstituted into azolectin vesicles that were preloaded with 0.1 or 10 mM phosphate. Phosphate transport was measured as the exchange of preloaded phosphate for external [32P]phosphate. Phosphate/phosphate exchange occurred for over 40 min at room temperature with an apparent K0.5 of 1.6 mM and a maximum velocity of over 700 nmol (mg protein)-1 min-1. Diethyl pyrocarbonate was used as an inhibitor-stop reagent. Transport was inhibited by p-hydroxyphenylglyoxal, p-hydroxymercuribenzoate, pyridoxal 5-phosphate, and dansyl chloride but was insensitive to sulfate, nitrate, and N-ethylmaleimide, the standard inhibitor for the mammalian phosphate transporter. Phosphate/hydroxyl exchange was stimulated when the proton gradient was collapsed with carbonyl cyanide m-chlorophenylhydrazone, but phosphate/phosphate exchange was unaffected by the uncoupler.     

Effect of Cadmium on Activities of Some Enzymes of Glycolysis and Pentose Phosphate Pathway in Pea

Activities of alcohol dehydrogenase, hexokinase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase were significantly inhibited by cadmium in germinating pea (Pisum sativum L. cv. Bonneville) seeds. The effect was concentration dependent in the range of 0.25 to 1.0 mM CdCl₂. The magnitude of detrimental effect on these enzymes was reduced during later stage of germination (9 d) largely because of fall in the activities of these enzymes in the control seeds germinated in water. In vitro, activities of hexokinase, glucose-6-phosphate dehydrogenase, and alcohol dehydrogenase were inhibited at 0.5 mM Cd²⁺ in the reaction mixture by 62, 67, and 36 %, respectively, however, 6-phosphogluconate dehydrogenase was insensitive to Cd²⁺. This revised version was published online in June 2006 with corrections to the Cover Date.     

Esterification of Inorganic Phosphate by Glycolysis in Extracts from Normal and Pentan-1-ol Treated Pea Cotyledons

Esterification of inorganic phosphate by mitochondria-free extractsfrom pea cotyledons was shown to be stimulated by D-fructose-1:6-diphosphate, and in the presence of NaF, though not in itsabsence, to be dependent on the addition of acetaldehyde. Therate of this glycolytic phosphorylation was determined by thelevel of both diphosphopyridine nucleotide and adenosine triphosphate(or adeno-sine diphosphate) in the extracts. Adenosine triphosphateadded alone (2 mM.) inhibited phosphorylation, but this inhibitionwas reversed by diphosphopyridine nucleotide though not by reducedglutathione or cysteine. Adenosine triphosphate inhibited glycolytic phosphorylationin extracts from pentan-1-ol treated seeds to a greater extentthan in extracts from normal seeds; this effect has been shownto be due to the lower content of diphosphopyridine nucleotidein these seeds compared with normal seeds.     

Phosphorus Nutrition and the Intracellular Distribution of Inorganic Phosphate in Pea Root Tips: A Quantitative Study using31P-NMR

Pea plants (Pisum sativum L.) were supplied with external phosphatefor differing periods of time, so that their phosphorus statusvaried, and the intracellular distribution of inorganic phosphate(P₁) in the roots was examined by ³¹P nuclear magnetic resonance.Over the range of phosphorus nutrition investigated, the quantityof vacuolar P₁ per unit fresh weight of root tip changed considerably,whereas the quantity of cytoplasmic P₁ per unit fresh weightof root tip did not alter. The relative volumes of the cytoplasmand the vacuole in pea root tips seemed to be little affectedby differences in phosphorus nutrition, and this implied thatthe concentration of P₁ in the cytoplasm was kept almost constant,at a level estimated to be 18 mM. The rate of absorption of ³²P-labelled phosphate was negativelycorrelated with the vacuolar P₁ concentration, but there wasno clear correlation with the concentration of P₁ in the cytoplasm. Key words: Compartmentation, Cytoplasm, Vacuole, Concentration, Absorption     

Appearance of Three Chloroplast Isoenzymes in Dark-grown Pea Plants and Pea Seeds

Activity peaks characteristic of the chloroplastic Calvin cycle enzymes triose-phosphate isomerase, ribose 5-phosphate isomerase, and fructose 1,6-diphosphate aldolase are found in isoelectric focusing patterns of dark-grown pea (Pisum sativum) seedlings and seeds. Apparently, in this higher plant these three chloroplastic isoenzymes can be formed in the absence of light and of chloroplast formation.     

Pea Xyloglucan and Cellulose : IV. Assembly of beta-Glucans by Pea Protoplasts

The synthesis and assembly of xyloglucan were examined during early stages of wall regeneration by protoplasts isolated from growing regions of etiolated peas. During early stages of cultivation, fluorescence microscopy showed that the protoplast surface bound Calcofluor and ammonium salt of 8-anilino-1-naphthalene sulfonic acid and, in time, it also bound fluorescent fucose-binding lectin. Based on chemical analysis, 1,3-β-glucan was the main polysaccharide formed by protoplasts and xyloglucan and cellulose were minor wall components. Binding between cellulose and xyloglucan was not as strong as that in tissues of intact pea plants, i.e. mild alkali could dissolve most xyloglucan from the protoplast. However, the addition of exogenous pea xyloglucan into the culture medium stimulated the deposition of new polysaccharides into the protoplast wall and enhanced the close association of newly formed xyloglucan with cellulose.     

Linkage Maps in Pea

We have analyzed segregation patterns of markers among the late generation progeny of several crosses of pea. From the patterns of association of these markers we have deduced linkage orders. Salient features of these linkages are discussed, as is the relationship between the data presented here and previously published genetic and cytogenetic data.     

Hexokinase II of Pea Seeds

A second hexokinase (EC 2.7.1.1) was obtained from pea seed (Pisum sativum L. var. Progress No. 9) extracts. The enzyme, termed hexokinase II, had a high affinity (K_m, 48 micromolar) for glucose and a relatively low affinity (K_m, 10 millimolar) for fructose. The K_m for MgATP was 86 micromolar. Mg²⁺ was required for activity, but excess Mg²⁺ was inhibitory. MgADP inhibited hexokinase II. The addition of salts of monovalent cations increased hexokinase II activity. Al³⁺ was a strong inhibitor of the enzyme at pH 6.6 but not at the optimum pH (8.2). Citrate and 3-phosphoglycerate activated pea seed hexokinase II at pH 6.6, probably by coordinating with aluminum present as a contaminant in commercial ATP. The properties of hexokinase II are compared with those of the other three hexose kinases obtained from pea seed extracts. The possible role of these enzymes in plant carbohydrate metabolism is discussed.     

Pea chloroplast carnitine acetyltransferase

The purpose of this study was to resolve the controversy as to whether or not chloroplasts possess the enzyme carnitine acetyltransferase (CAT) and whether the activity of this enzyme is sufficient to support previously reported rates of fatty acid synthesis from acetylcarnitine. CAT catalyses the freely reversible reaction: carnitine + short-chain acylCoA <--> short-chain acylcarnitine + CoASH. CAT activity was detected in thc chloroplasts of Pisum sativum L. With membrane-impermeable acetyl CoA as a substrate. activity was only detected in ruptured chloroplasts and not with intact chloroplasts, indicating that the enzyme was located on the stromal side of the envelope. In crude preparations, CAT could only be detected using a sensitive radioenzymatic assay due to competing reactions from other enzymes using acetyl CoA and large amounts of ultraviolet-absorbing materials. After partial purification of the enzyme, CAT was detected in both the forward and reverse directions using spectrophotometric assays. Rates of 100 nmol of product formed per minute per milligram of protein were obtained, which is sufficient to support reported fatty acid synthesis rates from acetylcarnitine. Chloroplastic CAT showed optimal activity at pH 8.5 and had a high substrate specificity, handling C2-C4 acyl CoAs only. We believe that CAT has been satisfactorily demonstrated in pea chloroplasts.     

Pea Xyloglucan and Cellulose : III. Metabolism during Lateral Expansion of Pea Epicotyl Cells

Lateral expansion of the third internodes of pea epicotyls was evoked by treatment with either 2,4-dichlorophenoxyacetic acid (2,4-D) or ethylene gas. During growth, 2,4-D enhanced and ethylene inhibited the deposition of xyloglucan and cellulose in the cell wall, with the result that the wall framework (ghost) from ethylene-treated swollen tissue was much thinner than that from 2,4-D-treated. The level of activity of xyloglucan synthase, alkali-insoluble β-glucan synthases, and endo-1,4-β-glucanases were all enhanced by 2,4-D treatment but not by ethylene. Both 2,4-D and ethylene treatments led to increased osmotic potential in the swelling tissues. Accordingly, swelling after 2,4-D treatment was accompanied by xyloglucan degradation, concomitant with substantial net synthesis, but swollen tissue as a result of ethylene treatment was characterized by walls whose integrity was weakened by relatively low levels of newly deposited polysaccharides rather than by the degradation.     

Isoxazolin-5-ones and Amino Acids in Root Exudates of Pea and Sweet Pea Seedlings

Seeds of Pisum sativum L. cv Finale and Lathyrus odoratus L. cv Spencer were germinated aseptically in moistened sand in the dark. At several stages, the amino acid composition of the exudate and of the corresponding roots was analyzed. A number of common amino acids, including homoserine, were exuded by the growing seedling root in an early stage and were partly reabsorbed later. A number of uncommon amino acids, including several isoxazolin-5-one derivatives, uracil alanines, l-γ-glutamyl-d-alanine, and α-aminoadipic acid were exuded at different rates.     

豌豆肌动蛋白异型体基因的特异性表达

将分属三类豌豆（Pisum sativum）卷须肌动蛋白异型体（PEAc）的基因3'端进行了亚克隆,并利用限制性内切酶从中切取了完整的3'非翻译区片段,将之做为特异性探针进行DIG标记,并对不同发育时间豌豆根、茎、叶及花粉中提取的总RNA进行点杂交分析。发现其中I类（PEAcI）和II类（PEAcII）在根、茎、叶中都有表达,但存在发育时间上的表达特异性,尤其在叶中的差异较明显。而III类（PEAcIII）与前二者的差别更大,仅发现其在2 d及28 d的根、7 d的茎及18 d的叶中有表达。在花粉中,只有PEAcII有少量的表达,说明三类异型体基因的表达还存在组织特异性。     

Induction of Systemic Resistance in Pea to Pea Powdery Mildew by Exogenous Application of Salicylic Acid

Exogenous application of salicylic acid (SA) solutions to pea leaves induced systemic resistance to Erysiphe pisi. reducing by 20–30% the percentages of fungal germlings that successfully infected untreated leaves of SA-treated plants. SA concentrations of 1.5 and 15 mM were similarly effective, but 0.15 mM had no detectable effect. While 15 mM SA solutions were phytotoxic. 1.5 mM solutions caused no apparent damage indicating that resistance induction was not due to tissue damage. The induced resistance persisted for at least 13 days after treatment, but excision of treated leaves 1 day after SA application prevented full induction of systemic resistance, and the resistance was not expressed if untreated leaves were inoculated fewer than 3 days after SA application. The effect of SA was transmitted to leaves at nodes both above and below treated leaves. Chemical induction of systemic resistance may provide an additional means for controlling pea diseases.     

Transport of Photoassimilate in Pea Ovules

Interpretation of tracer washout from an attached empty seedcoat depends on whether photoassimilate within the apoplastof the seed coat is absorbed by the seed coat tissues. Usingsucrose trapping procedures, we were unable to see any evidencefor sucrose uptake from the seed coat apoplast which would beneeded to provide the seed coat with its carbohydrate requirementsif phloem unloading were into the apoplast. Once released intothe apoplast photoassimilate is unavailable to the seed coattissue. Changes between equimolar solutions of sorbitol andsorbitol/sucrose mixes induced small transient responses inseed coat unloading which suggest that sorbitol and sucrosehad different reflection coefficients and gave water relationresponses with rapid, and fatiguable, osmoregulation withinthe seed coat. Immediate inhibition of seed coat unloading with PCMBS is reported,followed by inhibition of import into the entire pod. PCMBSappears to be xylem mobile, thereby quickly being dispersedthroughout the entire experimental pod. A complex CCCP responseis reported, which is consistent with immediate inhibition ofsymplastic transport followed by membrane disruption. AlthoughCCCP inhibited seed coat unloading, there was no effect on ovuleimport. This has been interpreted as evidence that the seedcoat has an active role in control of photoassimilate importinto ovules. Key words: Pisum sativum, phloem unloading, seed coat unloading     

Cell-free Synthesis of Pea Seed Proteins

Both polysomes and polysomal RNA, isolated from cotyledons of ripening pea (Pisum sativum) seeds and supplemented respectively with wheat germ S-100 and S-30 fractions, were used to program the cell-free synthesis of polypeptides. The relationship of these polypeptide products to seed storage proteins has been investigated. When fractionated on sucrose density gradients the translation products did not coincide with native storage proteins, nor were they exactly coincident with the subunits of storage proteins on dissociating gels. Treatment with antiserum prepared against storage proteins precipitated only a very small proportion of these products. Nonetheless, tryptic peptide mapping showed that a significant proportion (up to 65%) of the in vitro products from cell-free systems were related to the storage proteins. Alternative interpretations of these results are that either the translatable mRNAs for storage proteins make up a small proportion of the total template isolated from pea cotyledon polysomes, or that storage protein polypeptides are made in significant amounts in vitro but lack major antigenic determinants which in vivo may be acquired during chain completion or post-translational modification.