Alicyclic acid metabolism in plants 3. Fate of 14C-shikimate and 14C-quinate in mung bean plants

Young mung bean plants (Phaseolus mungo) were exposed to ¹⁴C-shikimateor ¹⁴C-quinate in the light. After 8 or 23.5 hr of incubationat 25°C, radioactivities in free and bound amino acids,organic acids, soluble and insoluble carbohydrates, ether-solublefraction and lignin were determined. Shikimic and quinic acidswere separated by the combined use of paper-chromatography andcolumn chromatography. Specific activity of formed quinate orshikimate was only slightly lower than that of fed shikimateor quinate. Specific activities of phenylalanine, tyrosine andbound tryptophan were high as compared with those of non-aromaticamino acids. Discussion is focused upon the interconversionbetween shikimate and quinate, and their roles in the biosynthesisof aromatic amino acids. (Received November 15, 1968; )     

Multiple Forms of Acid Phosphatase in Cotyledons of Vigna mungo Seedlings

Acid phosphatase from cotyledons of dark-grown Vigna mungo seedlingswas separated into four forms (la, Ib, Ha and lib) by ion-exchangercolumn chromatographies. Each form of the acid phosphatase wascharacterized for its pH dependency, substrate specificity,thermal stability, activation energy, approximate molecularweight, and the effect of metal ions and other substances onits activity. Each form of the enzyme exhibited high activitytowards ATP and ADP relative to their activity towards p-nitrophenylphosphate,but showed very low activity towards phytate, a major organicphosphate reserve in cotyledons. Among the four forms, lib wasmost distinguishable by its low molecular weight and the thermalenhancement of activity.     

2. Alicyclic acid metabolism in plants Enzymes related to the shikimate pathway in developing mung bean seedlings

A study was made of chages in the activities of enzymes relatedto the biosynthesis of aromatic compounds in etiolated mungbean seedlings during their growth. Shikimate: NADP oxidoreductaseactivity in the root-shoot axes increased rapidly to attainits highest activity the 4th day after sowing, and remainedat that level over the experimental period of 7 days. 5-Dehydroquinatehydro-lyase activity continuously increased for at least 7 days.In the cotyledons, a gradual decrease in the activities of theseenzymes occurred. Phenylalanine ammonia-lyase activity in root-shootaxes gradually increased showing a maximum on the 6th day. Thehighest specific activity, on a protein basis, of this enzymewas seen in the initial stage of growth. In the cotyledons,a rise in total activity appeared on the 2nd day. Tyrosine ammonia-lyaseactivity was very low as compared with phenylalanine ammonia-lyase.The enzyme activities of light-germinated seedlings were comparedto those of dark-germinated seedlings on the 7th day. Lighthad practically no significant effect on the appearance of shikimate:NADP oxidoreductase and 5-dehydroquinate hydro-lyase activities.On the other hand, a marked effect from the light on the riseof phenylalanine ammonia-lyase and tyrosine ammonia-lyase activitieswas found, especially in the epicotyl-plumules. The results are discussed with respect to the metabolism ofalicyclic acids such as shikimic acid in the developing mungbean seedlings. ¹This work was partly supported by a grant-in-aid from the Ministryof Education.     

Some properties of shikimate: NADP oxidoreductase produced in sweet potato root tissue after slicing

The activity of shikimate: NADP oxidoreductase [EC 1. 1. 1.25] in sweet potato root tissue increased soon after slicing.Enzyme preparations obtained from both sliced tissue and fromfresh tissue probably contained a single enzyme component, andthey showed identical chromatographical behaviour. K_m values of the enzyme for NADP and shikimate were 1.0x10^–4Mand 1.3 x 10^–3M, respectively. Enzyme activity was potentlyinhibited by SH-inhibitors such as p-chloromercuribenzoate andoxidized glutathione. Enzyme activity was affected neither by mononucleotides suchas ATP, ADP and AMP, divalent cations, Mg⁺⁺, Ca⁺⁺ and Mn⁺⁺,nor by metabolites such as tryptophan, phenylalanine, tyrosineand t-cinnamic acid which are involved in aromatic compoundsyntheses. The enzyme rapidly lost its activity. This inactivation reactionshowed a time course consisting of two steps of the first-orderreaction. The inactivated enzyme preparation was not reactivatedby thiol compounds such as cysteine, 2-mercaptoethanol and glutathione,although these reagents, to a certain extent, protected theenzyme from inactivation. The results suggest that denaturationof the enzyme protein was involved in inactivation of the enzyme. ¹Part 74 of the phytopathological chemistry of sweet potatowith black rot and injury. ²Present address: Department of Biology, Faculty of Science,Tokyo Metropolitan University, Setagaya-ku, Tokyo. (Received August 5, 1968; )     

MECHANISM OF COLD INJURY IN SWEET POTATOES II. BIOCHEMICAL MECHANISM OF COLD INJURY WITH SPECIAL REFERENCE TO MITOCHONDRIAL ACTIVITIES

1. Respiratory activity of sweet potato root tissues, stored at0° (chilled) and 20° (control), was measured. The activityof the control sample remained approximately constant, whilethat of the chilled one declined sharply after about ten daysof the treatment. Just before this time, DNP-stimulated respiratoryactivity began to drop in the chilled sample, but remained constantin the control.
2. Oxidative and phosphorylative activities ofmitochondria fromthe chilled tissues were compared with thosefrom the control.Exactly as in respiration of the intact roottissues, a sharpdecline in mitochondrial oxidative activitywas found in thechilled tissue after about ten days of thetreatment, but notin the control. In spite of the decreasein oxidative activityof mitochondria, the P/O ratio remainedfairly constant throughoutthe storage. Protein nitrogen ofmitochondria in root tissuesalso remained constant.
3. Contentof polyphenolic substances did not show any significantdifferencebetween the chilled and control tissues.

(Received April 20, 1961; )     

CHANGES IN ACTIVITIES OF 5-DEHYDKOQUINATE HYDRO-LYASE AND SHIKIMATE-NADP OXIDOREDUCTASE IN SLICED SWEET POTATO ROOTS

Sweet potato roots of variety Norin No. 1 were cut into discs(319 mm), which were incubated at 28–29 for 4 days.At 24-hour intervals, activities of 5-dehydroquinate hydro-lyaseand shikimate-NADP oxidoreductase in the buffer extracts ofdiscs were estimated. The activities of both enzymes were significantlydetected in the fresh root tissues. 5-Dehydroquinate hydro-lyaseactivity per fresh weight increased by three to four times within2 days after slicing, and decreased gradually. Shikimate-NADPoxidoreductase activity at the lst-day incubation period wasfound to be about three times as much as the activity foundin the fresh roots, and it remained for 4 days after slicing.The role of these enzymes in polyphenol biosynthesis in thesliced root tissues is discussed. An attempt to detect the enzymicconversion of dehydroquinic acid to quinic acid is also described. ¹This paper constitutes Part 55 of the Phytopathological Chemistryof Sweet Potato with Black Rot. ²Present address: Department of Biology, Tokyo MetropolitanUniversity, Tokyo. Nagoya     

A STUDY ON 3-DEOXY-D-ARABINO-HEPTULOSONIC ACID 7-PHOSPHATE SYNTHASE IN HIGHER PLANTS

Several plant tissues were assayed for DAHP synthase activity.The activity was found in most of the plants examined, and thehighest activity was detected in developing mung bean plants.During the growth of mung bean shoots, the highest level ofthe activity on protein basis was seen 6 to 8 days after sowing. Some properties of DAHP synthase were investigated using theenzyme preparation mainly from mung bean shoots. The productof the reaction was identified as DAHP by paper chromatographyof the reaction mixture and by the absorption spectrum of thederivative formed by periodate-TBA treatment. Mg⁺⁺ at 1–5mM concentration increased the rate of DAHP formation threeto four times, although the enzyme was still active in the absenceof Mg⁺⁺. Optimal pH was found to be between pH 7.2 and 7.9.Relationship between the rate of DAHP formation and concomitantacid phosphate activity in enzyme preparations was discussed. (Received July 20, 1967; )     

Incorporation of some possible radioactive intermediates into chlorogenic acid in sliced sweet potato tissue

t-Cinnamic acid-2-¹⁴C, p-coumaric acid-2-¹⁴C and caffeic acid-2-¹⁴Cwere administered to discs of sweet potato roots and incorporationof each radioactive compound into chlorogenic acid was compared.The data suggest that chlorogenic acid is synthesized througheither or both of two major pathways, phenylalanine t-cinnamate t-cinnamoyl derivative p-coumaroyl derivative chlorogenicacid and phenylalanine t-cinnamate p-coumarate p-coumaroylderivative chlorogenic acid. ¹Part 75 of the phytopathological chemistry of sweet potatowith black rot and injury. ²Present address : Department of Biology, Tokyo MetropolitanUniversity, Setagaya-ku, Tokyo. (Received December 23, 1968; )     

Annual emissions of dissolved CO2, CH4, and N2O in the subsurface drainage from three cropping systems

Indirect emission of nitrous oxide (N₂O), associated with nitrogen (N) leaching and runoff from agricultural lands is a major source of atmospheric N₂O. Recent studies have shown that carbon dioxide (CO₂) and methane (CH₄) are also emitted via these pathways. We measured the concentrations of three dissolved greenhouse gases (GHGs) in the subsurface drainage from field lysimeter that had a shallow groundwater table. Aboveground fluxes of CH₄ and N₂O were monitored using an automated closed‐chamber system. The annual total emissions of dissolved and aboveground GHGs were compared among three cropping systems; paddy rice, soybean and wheat, and upland rice. The annual drainage in the paddy rice, the soybean and wheat, and the upland rice plots was 1435, 782, and 1010 mm yr^?1, respectively. Dissolved CO₂ emissions were highest in the paddy rice plots, and were equivalent to 1.05–1.16% of the carbon storage in the topsoil. Dissolved CH₄ emissions were also higher in the paddy rice plots, but were only 0.03–0.05% of the aboveground emissions. Dissolved N₂O emissions were highest in the upland rice plots, where leached N was greatest due to small crop biomass. In the soybean and wheat plots, large crop biomass, due to double cropping, decreased the drainage volume, and thus decreased dissolved GHG emissions. Dissolved N₂O emissions from both the soybean and wheat plots and the upland rice plots were equivalent to 50.3–67.3% of the aboveground emissions. The results indicate that crop type and rotation are important factors in determining dissolved GHG emissions in the drainage from a crop field.