De novo synthesis of d-alanine in germinating Pisum sativum seedlings

The amounts of d-alanine derivatives, γ-l-glutamyl-d-alanine and N-malonyl-d-alanine, increase rapidly during the early growth of pea seeds. Pyruvate-[1?¹⁴C], l-alanine-[U?¹⁴C], d-alanine-[U?¹⁴C], l-alanine-[¹⁵N] and ¹⁵NH₄Cl were therefore fed to the seedlings and the incorporation investigated. Labelling results revealed that pea seedlings can utilize these erogenous compounds to form d-alanine and that labelled l-alanine is effectively converted to the d-enantiomer with retention of ¹⁴C and, largely, ¹⁵N label. Enzyme analyses in vitro provided additional evidence that the extract of pea seedlings catalyzes the direct conversion of l-alanine to d-alanine. The data suggest that the de novo synthesis of d-alanine in pea seedlings occurs by a racemase reaction.     

Occurrence and some properties of a novel γ-glutamyltransferase responsible for the syntheis of γ-L-glutamul-D-alanine in pea seedlings

Occurrence of a novel γ-glutamyltransferase responsible for the formation of γ-L-glutamyl-D-alanine was demonstrated in pea seedlings, and the enzyme was purified 600-fold. The enzyme preparation catalyzed the transfer of the γ-glutamyl moiety of L-glutamine and other γ-glutamyl compounds to D-amino acids. In the formation of γ-L-glutamyl peptides of D-amino acids, L-glutamine served as the most effective γ-glutamyl donor and D-alanine acted as a highly-specific acceptor. The maximum activity of the γ-glutamyl transfer reaction between L-glutamine and D-alanine was observed at pH 9.5 and the apparent K_m values for these amino acids were estimated to be 2.0 and 2.9mM, respectively. This unique γ-glutamyltransferase activity was always accompanied by the catalytic activities of the known γ-glutamyltransferases during the purification procedure.     

Protein bodies and vacuoles as lysosomes : investigations into the role of mannose-6-phosphate in intracellular transport of glycosidases in pea cotyledons

We have failed to detect the presence of mannose-6-phosphate in the oligosaccharide moiety of glycoproteins from pea (Pisum sativum L. cv Burpeeana) cotyledons using an assay system sensitive to 10 picomoles of mannose-6-phosphate. We were also unable to demonstrate any retention of glycosidase activity from pea seedlings and pea cotyledons on Sepharose-coupled phosphomannosyl receptor proteins isolated from bovine liver which were, however, able to retain phosphomannosylated hexosaminidase purified from Dictyostelium discoideum secretions. Furthermore, although Sepharose-coupled phosphomannosylated hexosaminidase from Dictyostelium was able to bind phosphomannosyl receptors from bovine liver we were unable to detect the retention of any protein from acetone powder extracts of pea seedlings or from endoplasmic reticulum-associated proteins of pea cotyledons.

Based on this collective evidence we conclude that mannose-6-phosphate does not appear to play a role in the targeting of hydrolytic enzymes from the endoplasmic reticulum to the protein bodies in pea cotyledons.

     

Oxaloacetate translocator in plant mitochondria

(1) Mitochondria were prepared from leaves of spinach, green and etiolated seedlings and roots of pea, potato tuber and rat liver and heart. In the case of leaf mitochondria, an improved isolation procedure resulted in high respiratory rates (460–510 nmol/mg protein per min) and good respiratory control ratio (6.8–9.8) with glycine as substrate. (2) In these mitochondria oxaloacetate transport was studied either by following the inhibitory effect of oxaloacetate on the respiration of NADH-linked substrates or by determining the consumption of [4-¹⁴C]oxaloacetate. (3) Studies of the competition by other carboxylates and effect of inhibitors on the oxaloacetate transport demonstrate that mitochondria from spinach leaves, green pea seedlings, etiolated pea seedlings and pea roots contain a specific translocator for oxaloacetate with a very high affinity to its substrate (K_m = 3–7 μM) and an even higher sensitivity to its competitive inhibitor phthalonate (K_i = 3–5 μM). The V_max values ranged from 150 to 180 nmol/mg protein per min for mitochondria from etiolated pea seedlings and pea roots and from 550 to 570 nmol/mg protein per min for mitochondria from spinach leaves and green pea seedlings. In mitochondria from potato tuber, the K_m was about one order of magnitude higher (V_max = 450 nmol/mg protein per min). In mitochondria from rat liver and rat heart, a specific translocator for oxaloacetate was not found. (4) The oxaloacetate translocator enables the functioning of a malate-oxaloacetate shuttle for the transfer of reducing equivalents across the inner mitochondrial membrane. (5) This malate-oxaloacetate shuttle appears to play a role in the photorespiratory cycle in catalyzing the transfer of reducing equivalents generated in the mitochondria during glycine oxydation to the peroxysomal compartment for the reduction of β-hydroxypyruvate. (6) Interaction between the mitochondrial and the chloroplastic malate oxaloacetate shuttles would make it possible for surplus-reducing equivalents, generated by photosynthetic electron transport, to be oxidized by mitochondrial electron transport.     

Phytochrome Regulation of Greening in Pisum: Chlorophyll Accumulation and Abundance of mRNA for the Light-Harvesting Chlorophyll a/b Binding Proteins

A brief pulse of red light eliminates or reduces the lag in chlorophyll accumulation that occurs when dark-grown pea seedlings are transferred to continuous white light. The red light pulse also induces the accumulation of specific mRNAs. We compared time courses, escape from reversal by far-red light, and fluence-response behavior for induction of mRNA for the light-harvesting chlorophyll a/b binding proteins (Cab mRNA) with those for induction of rapid chlorophyll accumulation in seedlings of Pisum sativum cv Alaska. In both cases the time courses of low fluence and very low fluence responses diverged from each other in a similar fashion: the low fluence responses continued to increase for at least 24 hours, while the very low fluence responses reached saturation by 8 to 16 hours. Both responses escaped from reversibility by far-red slowly, approaching the red control level after 16 hours. The fluence-response curve for the Cab mRNA increase, on the other hand, showed threshold and saturation at fluences 10-fold lower than threshold and saturation values for the greening response. Therefore, the level of Cab mRNA, as measured by the presence of sequences hybridizing to a cDNA probe, does not limit the rate of chlorophyll accumulation after transfer of pea seedlings to white light. The Cab mRNA level in the buds of seedlings grown under continuous red light remained high even when the red fluence rate was too low to allow significant greening. In this case also, abundance of Cab mRNA cannot be what limits chlorophyll accumulation.     

Hydrogen Peroxide Content and Catalase Activity on Inoculation with Root Nodule Bacteria of Pea Seedlings with Different Ability for Nodulation

Hydrogen peroxide (H₂O₂) content and catalase activity were studied in pea (Pisum sativum L.) seedlings with a normal (cultivar Marat) and disrupted (pea mutants) process of nodulation that were inoculated with the nitrogen-fixing bacterium Rhizobium leguminosarum strain CIAM 1026. Differences in hydrogen peroxide content and catalase activity in pea seedlings with different ability for nodulation that were inoculated with rhizobia were found. It was assumed that H₂O₂ and catalase are involved in defensive and regulatory mechanisms in the host plant.     

Interconversion of gibberellin A5 to gibberellin 3 in seedlings of dwarf Pisum sativum

[³H]-Gibberellin A₅ ([³H]-GA₅) applied to seedlings of dark-grown dwarf pea (Pisum sativum L. cv. Meteor), was converted to two acidic compounds, GA₃ and a chromatographically similar unknown. Identification of GA₃ was made by gas-liquid radiochromatography using three stationary phases.     

Loss of phytochrome photoreversibility in vitro: I. Extraction and partial purification of killer

Crude pea (Pisum sativum L. var. Alaska) phytochrome extracts contain a substance, “Killer,” which interacts with the far red-absorbing form of phytochrome causing a net loss of spectrophotometrically detectable phytochrome in vitro. Killer is absent from crude extracts of Avena phytochrome, is separable from pea phytochrome by gel filtration, and is alcohol-extractable from etiolated pea seedlings. Killer activity in alcohol extracts behaved, during partial purification, in a manner identical to that derived from pea phytochrome preparations. The mass extraction and partial purification of Killer are described.     

Free amino acids and γ-glutamyl peptides in fagaceae

Amino acids have been investigated in seeds and fresh parts of members of the Fagaceae. Seeds from the genus Fagus contain willardiine, 5-hydroxy-6-methylpipecolic acids, N-[N-(3-amino-3-carboxypropyl)-3-amino-3-carboxypropyl]azetidine-2-carboxylic acid and γ-glutamyl peptides, mainly γ-glutamylphenylalanine. These compounds are nearly or totally absent from leaves of F. silvatica and from seedlings and immature seeds of F. silvatica var. purpurea; instead, the seedlings contain large amounts of γ-l-glutamyl-l-isoleucine and γ-l-glutamyl-l-leucine. γ-l-Glutamyl-l-tryptophan and γ-l-glutamyl-γ-l-glutamyl-l-phenylalanine, not previously known from nature, have been isolated from seeds of F. silvatica var. purpurea. The structures have been confirmed by syntheses. 4-Hydroxypipecolic acid (with trans-configuration) has been identified in seeds of F. japonica Maxim. and F. sieboldii Endl. None of the above compounds was found in Quercus or Castanea species whereas argininosuccinic acid was identified in Castanea sativa.     

GH3 expression and IAA-amide synthetase activity in pea (Pisum sativum L.) seedlings are regulated by light,plant hormones and auxinic herbicides

The formation of auxin conjugates is one of the important regulatory mechanisms for modulating IAA action. Several auxin-responsive GH3 genes encode IAA-amide synthetases that are involved in the maintenance of hormonal homeostasis by conjugating excess IAA to amino acids. Recently, the data have revealed novel regulatory functions of several GH3 proteins in plant growth, organ development, fruit ripening, light signaling, abiotic stress tolerance and plant defense responses. Indole-3-acetyl-aspartate (IAA-Asp) synthetase catalyzing IAA conjugation to aspartic acid in immature seeds of pea (Pisum sativum L.) was purified and characterized during our previous investigations. In this study, we examined the effect of auxin and other plant hormones (ABA, GA, kinetin, JA, MeJA, SA), different light conditions (red, far-red, blue, white light), and auxinic herbicides (2,4-D, Dicamba, Picloram) on the expression of a putative GH3 gene and IAA-amide synthesizing activity in 10-d-old pea seedlings. Quantitative RT-PCR analysis indicated that the PsGH3-5 gene, weakly expressed in control sample, was visibly induced in response to all plant hormones, different light wavelengths and the auxinic herbicides tested. Protein A immunoprecipitation/gel blot analysis using anti-AtGH3.5 antibodies revealed a similar pattern of changes on the protein levels in response to all treatments. IAA-amide synthetase activity determined with aspartate as a substrate, not detectable in control seedlings, was positively affected by a majority of treatments. Based on these results, we suggest that PsGH3-5 may control the growth and development of pea plants in a way similar to the known GH3 genes from other plant species.     

Subcellular localization of flavonoid synthesizing enzymes in Pisum,Phaseolus, Brassica and Spinacia cultivars

Functionally-intact chloroplasts were obtained from 11-day-old pea (Pisum sativum cv Midfreezer) seedlings. Enzyme-distribution studies with ribulose bisphosphate carboxylase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase showed that ca 2.1% of the total tissue chloroplasts were present in the chloroplast preparation. The rate of intactness of chloroplast preparations was 34–82%. SAM:caffeic acid methyltransferase, flavanone synthase, UDPG:flavonoid-3-O-glucosyltransferase and SAM:quercetin methyltransferase activities were measured in the homogenate, supernatant and chloroplast lysate fractions. Significant activities of the above four enzymes could only be detected in the homogenate and supernatant fractions. Similar experiments with 11-day-old seedlings of green beans (Phaseolus vulgaris cv Early Gallatin), red cabbage (Brassica oleracea cv Red Danish) and 6-week-old plants of spinach (Spinacia oleracea cv Bloomsdale) showed a similar distribution of the flavonoid synthesizing enzymes. We conclude that under the reported conditions chloroplasts are not involved in flavonoid biosynthesis.     

Novel vitamin E forms in leaves of Kalanchoe daigremontiana and Phaseolus coccineus

In the present study, we isolated novel tocochromanols from green leaves of Kalanchoe daigremontiana and primary leaves of etiolated seedlings of Phaseolus coccineus that were identified as β-, γ-, and δ-tocomonoenols with unsaturation at the terminal isoprene unit of the side chain. The content of γ-tocomonoenol in leaves of etiolated bean increased gradually with the age of seedlings, reaching 50% of the γ-tocopherol level in 40-day-old plants. The content of this compound in leaves was increased by short illumination of etiolated plants and by addition of homogentisic acid, a biosynthetic precursor of tocopherols. These data indicated that γ-tocomonoenol is synthesized de novo from homogentisic acid and tetrahydro-geranylgeraniol diphosphate, a phytol precursor. Based on these results, a biosynthetic pathway of tocomonoenols is proposed.     

Loss of recovery capacity of plasmalemma k influx after cutting in chlorsulfuron pretreated maize roots

Active K⁺ influx was studied in apical segments from maize (Zea mays L., hybrid lines XL 342) and pea (Pisum sativum L. var Laxton superbo) seedlings pretreated with the herbicide chlorsulfuron (2-chloro-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl) aminocarbonyl]benzenesulfonamide).

Even though both plants were sensitive to chlorsulfuron, a strong inhibition of K⁺ uptake only was evident in maize root segments after 12 hours pretreatment with 10 micromolar chlorsulfuron. The inhibition was revealed only when maize root segments were washed for 2 hours before uptake measurements. This was done in order to recover K⁺ influx inhibited by cutting injury. Consequently, we demonstrated that roots from chlorsulfuron pretreated maize seedlings lost the capacity to recover from cutting injury by washing. By contrast, K⁺ influx in pea roots was not inhibited by chlorsulfuron because pea roots notoriously do not exhibit the `washing' effect.

     

Inhibitory oxidation products of indole-3-acetic Acid: 3-hydroxymethyloxindole and 3-methyleneoxindole as plant metabolites

Extracts of pea seedlings (Pisum sativum, variety Alaska) oxidize indole-3-acetic acid to a bacteriostatic compound which has been identified as 3-hydroxymethyloxindole. At physiological pH this compound is readily dehydrated to 3-methyleneoxindole, another bacteriostatic agent. The extracts of pea seedlings also contain a reduced triphosphopyridine nucleotide-linked enzyme which reduces 3-methyleneoxindole to 3-methyloxindole, a non-toxic compound.

These enzymatic reactions also take place in intact seedlings; thus, a pathway of indole-3-acetic acid degradation via oxindoles appears to be pertinent to plant metabolism.

The significance of such metabolism lies in the fact that a key intermediate of this pathway, 3-methyleneoxindole, is a sulfhydryl reagent capable of profound effects on metabolism and growth.

     

Synthesis and accumulation of pea plastocyanin in transgenic tobacco plants

The pea plastocyanin gene in a 3.5 kbp Eco RI fragment of pea nuclear DNA was introduced into tobacco by Agrobacterium-mediated transformation. Regenerated plants contained pea plastocyanin located within the chloroplast thylakoid membrane system. Analysis of seedlings from a self-pollinated transgenic plant containing a single copy of the pea plastocyanin gene indicated that seedlings homozygous for the pea gene contained almost twice as much pea plastocyanin as seedlings hemizygous for the pea gene. Homozygous seedlings contained approximately equal amounts of pea and tobacco plastocyanins. The amount of tobacco plastocyanin in leaves of transgenic plants was unaffected by the expression of the pea plastocyanin gene. The mRNA from the pea gene in tobacco was indistinguishable by northern blotting and S1 nuclease protection from the mRNA found in pea. In both pea and transgenic tobacco, expression of the pea plastocyanin gene was induced by light in leaves but was suppressed in roots. Pea plastocyanin free of contaminating tobacco plastocyanin was purified from transgenic tobacco plants and shown to be indistinguishable from natural pea plastocyanin by N-terminal protein sequencing and ¹H NMR spectroscopy.     

Behavior of Etiolated Peas (Pisum sativum cv Alaska) When Obstructed by a Mechanical Barrier

Etiolated pea (Pisum sativum cv Alaska) seedlings growing against a horizontal barrier in the soil will assume a horizontal orientation and continue to grow for prolonged periods of time. With removal of the barrier or after seedlings grow out from underneath the obstruction, seedlings immediately return to normal vertical growth. Ethylene production increased several hours after the seedlings began to grow horizontally and not at the first contact with a barrier. Increases in ethylene production from horizontally growing seedlings were associated with decreased rates of elongation and increased stem diameter. The data suggest that increased ethylene production does not play a mediating role in the horizontal growth of pea seedlings when obstructed during emergence. We conclude that seedlings follow a path of least resistance when they grow against a barrier in the soil.     

Level nitric oxide (NO) and growth of roots of etiolated pea seedlings

Data regarding the interrelation of nitric oxide (NO) content in roots of 3-day-old etiolated pea seedlings and their growth under different concentrations of N-containing compounds were obtained. The concentration of exogenous compounds (sodium nitroprusside SNP, KNO₃, NaNO₂, L-arginine) rendering an inhibiting effect on the growth of roots were established, and the NO content in roots was determined at these concentration. It was shown that the inhibition of growth and highest NO content in the roots was determined with SNP (4 mM) and NaNO₂ (2 mM) during 24 h exposition of seedlings. This dependence was not established in combinations with KNO₃ (20 mM) and L-arginine (4 mM). We established that a NO scavenger, hemoglobin (4 μM), fully or partially removed the toxic effect of SNP, nitrate, and nitrite on growth. The effect of NO on the growth and the participation of N-containing compounds in generation of NO in roots of pea seedlings is discussed.     

ent-Kaurene Biosynthesis in Cell-Free Extracts of Excised Parts of Tall and Dwarf Pea Seedlings

Investigations on the sites of ent-kaur-16-ene (ent-kaurene) biosynthesis were conducted with cell-free extracts from several excised parts of 10-, 13-, and 16-d-old tall and dwarf pea (Pisum sativum L.) seedlings. [¹⁴C]Mevalonic acid was incorporated into ent-kaurene in cell-free extracts from young developing leaves and elongating internodes of tall (`Alaska') and dwarf (`Progress No.9') pea seedlings at all three stages of development. ent-Kaurene biosynthesis also occurred readily in cell-free extracts from shoot tips, petioles, and stipules near the young elongating internodes. The ent-kaurene-synthesizing activity found in young developing tissues declined as tissues matured. Little or no activity was detectable in enzyme extracts from cotyledons and root tips at different stages. In light grown tall pea internodes ent-kaurene-synthesizing activity was low as they began to elongate, reached a maximum when the internodes reached about 2 cm in length and declined as they matured. Activity in extracts of dwarf shoot tips and internodes was generally lower than in equivalent tall plants, but the activity in dwarf leaves and stipules was somewhat higher than in tall plants. With the exception of root tips, there is a strong correlation between growth potential of a tissue and the rate of ent-kaurene biosynthesis in extracts from that tissue.     

Effects of Kinetin, IAA, and Gibberellin on Ethylene Production, and Their Interactions in Growth of Seedlings

Kinetin in concentrations of 10⁻⁸ to 10⁻⁴ m, stimulated ethylene production in 3 and 4-day old etiolated seedlings of Alaska pea (Pisum sativum L. var. Alaska). Seedlings of other species responded similarly. The response to kinetin depended on the age of the seedlings.