Levels of Betaine and Betaine Aldehyde Dehydrogenase Activity in the Green Leaves, and Etiolated Leaves and Roots of Barley

The accumulation of betaine and the induction of betaine aldehydedehydrogenase, which catalyzes the last step in the synthesisof betaine, were analyzed in salt-stressed barley leaves. Whenhydroponically grown barley plants were transferred to a mediumthat contained 200 mM NaCl, the levels of both betaine and thetotal extractable betaine aldehyde dehydrogenase activity inthe leaves increased approximately 7-fold and 3-fold when calculatedon the basis of total leaf protein, respectively, over the courseof 7 days. Betaine aldehyde dehydrogenase activity was alsodetected in either etiolated leaves or roots of barley plantsgrown under aseptic conditions. Betaine was detected in bothetiolated leaves and roots at levels that were about 20% ofthat in green leaves when calculated on a fresh weight basis. ¹ This research was supported financially by a research grantfrom the Ministry of Education, Science and Culture (63560080) (Received March 9, 1990; Accepted May 29, 1990)     

Immunological Studies of Betaine Aldehyde Dehydrogenase in Barley

The changes in the level of the protein for betaine aldehydedehydrogenase, which catalyzes the last step in the synthesisof glycinebetaine, were analyzed with antiserum raised againstSDS-denatured betaine aldehyde dehydrogenase from spinach. Inbarley leaves, the levels of betaine aldehyde dehydrogenaseprotein were found to be enhanced by the addition of 200 mMNaCl to the growth medium. These changes in the level of theenzyme protein corresponded to those in the activity of theenzyme, as described in our previous study (Arakawa et al. 1990).The extent of this enhancement was reduced when barley plantswere relieved from salt stress. An increase in the level ofthe protein was also induced by water stress, such as the withholdingof water or the addition of polyethylene glycol 6000. Betainealdehyde dehydrogenase protein was detected in etiolated leavesand roots, as well as in green leaves. In etiolated leaves,the level of betaine aldehyde dehydrogenase protein was notaffected by salt stress. ¹ This work was supported by a grant from the Bio-Media Projectof the Japanese Ministry of Agriculture, Forestry and Fisheries(BMP92-III-l-1).     

Foliar Uptake and Movement of Chlormequat in Cereals: Influence of Dose and Addition of Related Chemicals

Increased amounts of chlormequat applied to leaves of barleyand wheat resulted in a greater proportion being taken up andmoving to the tips of treated leaves. Combined foliar applicationwith certain other amines also increased uptake and movementof chlormequat - choline and glycine betaine were particularlyeffective - but a few others had the reverse effect. Ammoniumand sodium salts of acetic, butyric and hydrochloric acids alsoincreased movement of ¹⁴C-chlormequat Hordeum vulgare L., barley, Triticum aestivum L., wheat, chlormequat, foliar uptake     

Radiotracer evidence implicating phosphoryl and phosphatidyl bases as intermediates in betaine synthesis by water-stressed barley leaves

In barley, glycine betaine is a metabolic end product accumulated by wilted leaves; betaine accumulation involves acceleration of de novo synthesis from serine, via ethanolamine, N-methylethanolamines, choline, and betaine aldehyde (Hanson, Scott 1980 Plant Physiol 66: 342-348). Because in animals and microorganisms the N-methylation of ethanolamine involves phosphatide intermediates, and because in barley, wilting markedly increases the rate of methylation of ethanolamine to choline, the labeling of phosphatides was followed after supplying [¹⁴C]ethanolamine to attached leaf blades of turgid and wilted barley plants. The kinetics of labeling of phosphatidylcholine and betaine showed that phosphatidylcholine became labeled 2.5-fold faster in wilted than in turgid leaves, and that after short incubations, phosphatidylcholine was always more heavily labeled than betaine. In pulse-chase experiments with wilted leaves, label from [¹⁴C]ethanolamine continued to accumulate in betaine as it was being lost from phosphatidylcholine. When [¹⁴C]monomethylethanolamine was supplied to wilted leaves, phosphatidylcholine was initially more heavily labeled than betaine. These results are qualitatively consistent with a precursor-to-product relationship between phosphatidylcholine and betaine.     

The Role of the Stem in the Partitioning of Na+ and K+ in Salt-Treated Barley

Hordeum vulgare cv. California Mariout was grown for 50 d insand culture at 100 mol m^–3 NaCl. Xylem sap was collectedthrough incisions at the base of individual leaves along thestem axis by applying pressure to the root system. K⁺ concentrationsin the xylem sap reaching individual leaves increased towardsthe apex, while concentrations of Na⁺, NO₃^–, and Cl^–declined. Phloem exudate was obtained by collecting into Li₂EDTAfrom the base of excised leaves. K/Na ratios of phloem exudatesincreased from older to younger leaves. K/Na ratios in xylem sap and phloem exudate were combined withchanges in ion content between two harvests (38 and 45 d aftergermination) and the direction of phloem export from individualleaves, to construct an empirical model of K⁺ and Na⁺ net flowswithin the xylem and phloem of the whole plant. This model indicatesthat in old leaves, phloem export of K⁺ greatly exceeded xylemimport. In contrast, Na⁺ export was small compared to importand Na⁺ once imported was retained within the leaf. The direction of export strongly depended on leaf age. Old,basal leaves preferentially supplied the root, and most of theK⁺ retranslocated to the roots was transferred to the xylemand subsequently became available to the shoot. Upper leavesexported to the apex. Young organs were supplied by xylem andphloem, with the xylem preferentially delivering Na⁺ , and thephloem most of the K⁺ . For the young ear, which was still coveredby the sheath of the flag leaf, our calculation predicts phloemimport of ions to such an extent that the surplus must havebeen removed by an outward flow in the xylem. Within the culm,indications for specific transfers of K⁺ and Na⁺ between xylemand phloem and release or absorption of these ions by the tissuewere obtained. The sum of these processes in stem internodes and leaves ledto a non-uniform distribution of Na⁺ and K⁺ within the shoot,Na⁺ being retained in old leaves and basal stem internodes,and K⁺ being available for growth and expansion of young tissues. Key words: Hordeum vulgare L., K⁺, Na⁺, stem, salt stress     

14C-Translocation in Kalanchoe pinnata at two Different Stages of Development

Mayoral, M. L. and Medina, E. 1985. ¹⁴C-translocation in Kalanchoepinnata at two different stages of development.—J. exp.Bot. 36: 1405–1413 Translocation of ¹⁴C-compounds from mature leaves was measuredin plants of Kalanchoe pinnata to determine the interactionbetween plant age and CAM phase when CO² is taken up. Matureleaves of 4 and 12 month old plants were fed with ¹⁴CO² eitherduring CAM phase 1 (midnight) or at the beginning of CAM phase4 (early afternoon). Export of ¹⁴C activity from source leaves,and distribution of ¹⁴C activity in soluble and insoluble compoundswas measured both in source leaves and sink organs. In 4 monthold plants 4 d were needed to export 76% of total ¹⁴C activityincorporated during CAM phase 1, while leaves labelled at thebeginning of CAM phase 4 exported 44% of total ¹⁴C activityafter 4 h, and 80% after 24 h. In both cases the major fractionof total radioactivity translocated was found in the roots inthe form of neutral sugars. Differences in translocation patternsare due to distribution of ¹⁴C in the source leaves, 96 % of¹⁴C taken up during CAM phase 1 is found in the insoluble fractionat the end of the subsequent phase 3, while 93 % of total radioactivitytaken up at the beginning of phase 4 is found in the solublefraction at the end of this phase. In 12 month old plants labelledduring phase 1 very little translocation could be detected atthe end of phase 3, while only 20% of total radioactivity wastranslocated from leaves labelled during phase 4 and measured4 h later. ¹⁴C activity in the older leaves had a similar distributionin soluble and insoluble fractions as the one determined inthe younger plants. Ability to translocate carbon compoundsfrom source leaves during phase 3 was shown by loading matureleaves at dawn with ¹⁴C-sucrose. Here again, mature leaves ofyounger plants showed faster translocation of radioactivitythan those of older plants Key words: Kalanchoe, crassulacean acid metabolism, translocation, sink, source relationships     

Some further studies on metabolism of acetate-l-14C in potassium deficient rice leaves

Acetate-1-¹⁴C was fed to excised leaves of normal and potassiumdeficient rice plants. The rate of respiratory evolution of¹⁴CO₂ was increased by a potassium deficiency. Malonate, glyoxylateor malate supplied enhanced the metabolism of acetate-l-¹⁴C.These results suggest an accelerated turn of the TCA cycle inpotassium deficient leaves. Malate synthetase activity was notrecognized in either normal or deficient leaves. However, condensingenzyme activity was higher in deficient leaves than in normalones. An addition of DNP to the leaves increased ¹⁴CO₂ productionfrom acetate-l-¹⁴C though its effect was smaller in deficientleaves than it was in normal ones. This result may suggest anincrease in the turnover rate of ATP or loose coupling of electrontransfer with oxidative phosphorylation in deficient leaves.Chromatographic separation of cold acidsoluble nucleotides hasshown that the ATP level was lowered by a potassium deficiency,though the ADP level was not affected. ¹Present address: Sericultural Experiment Station, Suginami-Ku,Tokyo     

Betaine Lipids in Lower Plants. Biosynthesis of DGTS and DGTA in Ochromonas danica (Chrysophyceae) and the Possible Role of DGTS in Lipid Metabolism

Membrane lipids and fatty acids of Ochromonas danica were analyzed.Of the two betaine lipids, the homoserine lipid DGTS mainlycontains 14:0 and 18:2 fatty acids, while the alanine lipidDGTA is enriched in 18:0, 18:2 and 22:5 fatty acids. Of thepolar moiety of DGTA, improved NMR data are presented. On incubationof cells with [3,4-¹⁴C]methionine, DGTS as well as DGTA werelabelled. With [1-¹⁴C]methionine as a substrate, the label appearedin DGTS only. If double labelled [³H](glycerol)/[¹⁴C](polarpart)DGTS was used as a precursor, radioactivity was incorporatedspecifically into DGTA in which the isotope ratio was unchangedcompared to the precursor. Thus, the glyceryltrimethylhomoserinepart of DGTS acts as the precursor of the polar group of DGTA.Labelling of cells with [1-¹⁴C]oleate in a pulse-chase mannerand subsequent analysis of the label in the fatty acids andmolecular species of different lipids including DGTS and DGTA,suggested a clearly different role of the two betaine lipids:DGTS acts as a i) primary acceptor for exogenous C₁₈ monoeneacid, ii) substrate for the desaturation of 18:1 to 18:2 acid,and iii) donor of mainly 18:2 fatty acid to be distributed amongPE and other membrane lipids. Into DGTA, in contrast, fattyacids are introduced only after elongation and desaturation.As a result, the biosynthesis of DGTA from DGTS involves a decarboxylationand recarboxylation of the polar part and a simultaneous deacylationand reacylation of the glycerol moiety. (Received January 28, 1992; Accepted March 11, 1992)     

The Translocation and Redistribution of Manganese in Avena

⁵⁴Mn present in the first two leaves of oat seedlings subsequentlydeprived of manganese was later redistributed to leaves 4 and5. ⁵⁴Mn was found in leaves 3 and 4 even when the roots of seedlingswere excised immediately after exposure to ⁵⁴Mn, but more wasdetected if the roots were left intact. ⁵⁴Mn applied as a drop to the 4th leaf of manganese-deficientoat plants was concentrated in the stem and translocated primarilyto the youngest developing leaf or to the grain if present.⁵⁴Mn was readily detected in the roots but almost none was translocatedto the first three leaves. More ⁵⁴Mn was translocated in 96hrs. than 24, but little or no more was translocated in 192hrs. Plants which were given 0.5 p.p.m. stable manganese until theyreached the 4th leaf stage, and were then exposed to ⁵⁴Mn, showeda fairly uniform distribution of ⁵⁴Mn throughout the plant.There was relatively slight concentration at active growth centres. It is concluded that physiologically significant redistributionof manganese occurs in the oat plant.     

Partitioning and redistribution of sulphur during S-stress in Macroptilium atropurpureum cv. Siratro

During the first 7 d of sulphate-deprivation stored SO₄^2- wasredistributed and assimilated into organic forms in the tropicallegume Macroptilium atropurpu-reum cv. Siratro. However, whilstthe sulphate content of all tissues declined after removingthe external SO₄^2- supply this was slowest in mature leaves.By contrast, the total S content of mature leaves declined markedlyin the absence of external sulphate whilst that of both youngleaves and roots increased. Furthermore, when radiolabelledSO₄^2- was applied to abraded surfaces of mature leaves, mostof the translocated label was recovered in the root following2 d SO₄^2- deprivation. By contrast, radiolabelled SO₄^2-appliedto young leaves was mostly retained in these tissues and nottranslocated. Within 3 d of removing the SO₄^2- supply there was a large increasein extractable APS-sulphotransferase activity in roots accompaniedby a decline in nitrate reductase activity, but these effectswere not seen in leaves. Five days after the removal of SO₄^2-there was a large increase in the content of asparagine in roots. The results are discussed in relation to the co-ordination ofNO₃^- and SO₄^2- uptake and assimilation and the partitioningof sulphur during S-stress. Key words: Sulphate supply, stomatal conductance, ATP-sulphurylase, APS-sulphotransferase, nitrate reductase     

Varietal differences in sodium uptake in barley cultivars exposed to soil salinity or salt spray

Barley varieties are known to differ in the extent of Na⁺ andCl^– accumulation in leaves when grown in saline soil orhydroponic culture. In particular, the cv. Chevron accumulatesmore Na⁺ than the more salt-tolerant cv. CM67, and has lowerleaf K⁺ concentrations. When salt was applied as a spray tothe leaves, CM67 accumulated more Na+ than Chevron, and theselection Sinis 27 (from a landrace collected on the Sinis Peninsulaof Sardinia) accumulated more Na⁺ than Sinis 28. In some casesleaf K⁺ concentrations decreased in response to high concentrationsof salt sprayed on to the leaves. Accumulation of Na⁺ was greaterin the 4th leaf than in the flag leaf. Added CaCI₂ had oppositeeffects when added to the salt applied to the soil or to thesaline spray. In the soil, CaCI₂ reduced Na⁺ uptake; appliedto the leaf it increased Na⁺ uptake. Pre-wetting the leavesbefore the salt spray, or washing the leaves with non-salinewater 1 h after the salt spray, reduced the uptake of Na⁺ andCl^–. It is clear that tolerance to salt applied as saltspray or in the soil are different characteristics. Key words: Barley, salt, foliar uptake, calcium     

Oxidative Damage Caused by an Excess of Copper in Oat Leaves

The relationship between the toxicity of Cu²⁺ ions and oxidativereactions in plant cells was studied. Segments of leaves from6- and 9-day-old oat seedlings were incubated in solutions thatcontained Cu²⁺ ions at various concentrations for 24 h in thelight. High concentrations of Cu²⁺ ions caused the breakdownof chlorophyll and carotenoid, as well as an increase in membranepermeability and rates of lipid peroxidation. These effectswere more pronounced in older leaves than in younger ones. Scavengersof free radicals, such as Tiron, sodium benzoate and mannitol,prevented the increases in these parameters of senescence. WhileTiron was more effective in this regard in younger leaves, sodiumbenzoate was more effective in older ones. The treatment withCu²⁺ ions enhanced the activity of superoxide dismutase, especiallyin the younger leaves. By contrast, Cu²⁺ ions decreased theactivities of catalase and ascorbate peroxidase in both oldand young leaves. Scavengers of free radicals protected theseenzymes against inactivation. These results indicate that an excess of Cu²⁺ ions causes rapidsenescence in plant leaves via oxidative reactions in the light. The reactions involve formation of ‘O^–₂, H₂O₂ andHO’ and a subsequent decrease in antioxidant defenceswhich in turn enhances the efectiveness of toxic species ofoxygen. (Received March 17, 1993; Accepted September 28, 1993)     

Translocation and metabolism of glycine betaine by barley plants in relation to water stress

The glycine betaine which accumulated in shoots of young barley plants (Hordeum vulgare L.) during an episode of water stress did not undergo net destruction upon relief of stress, but its distribution among plant organs changed. During stress, betaine accumulated primarily in mature leaves, whereas it was found mainly in young leaves after rewatering. Well-watered, stressed, and stressed-rewatered plants were supplied with [methyl-¹⁴C]betaine (8.5 nmol) via an abraded spot on the second leaf blade, and incubated for 3 d. In all three treatments the added ¹⁴C migrated more or less extensively from the second leaf blade, but was recovered quantitatively from various plant organs in the form of betaine; no labeled degradation products were found in any organ. When 0.5 mol of [methyl-¹⁴C]betaine was applied via an abraded spot to the second leaf blades of well-watered, mildly-stressed, and stressed-rewatered plants, ¹⁴C was translocated out of the blades at velocities of about 0.2–0.3 cm/min which were similar to velocities found for applied [¹⁴C]sucrose. Heat-girdling of the sheath prevented export of [¹⁴C]betaine from the blade. When 0.5 mol [³H]sucrose and 0.5 mol [¹⁴C]betaine were suppled simultaneously to second leaf blades, the ³H/¹⁴C ratio in the sheath tissue was the same as that of the supplied mixture. After supplying tracer [¹⁴C]betaine aldehyde (the immediate precursor of betaine) to the second leaf blade, the ¹⁴C which was translocated into the sheath was in the form of betaine. Thus, betaine synthesized by mature leaves during stress behaves as an inert end product and upon rewatering is translocated to the expanding leaves, most probably via the phloem. Accordingly, it is suggested that the level of betaine in a barley plant might serve as a useful cumulative index of the water stress experienced during growth.     

Exogenous glycine betaine and proline play a protective role in heat-stressed barley leaves (Hordeum vulgare L.): A chlorophyll a fluorescence study

Abstract

Effects of drought and exogenous glycine betaine and proline on Photosystem II (PSII) photochemistry were studied in barley leaves under heat stress induced by exposing them to 45°C for 10 min. Polyphasic fluorescence transient (OJIP) was used to evaluate PSII photochemistry in leaves treated with either glycine betaine or proline, combined or not with heat treatment. A distinct K step in the fluorescence transient OJIP appeared in control leaves, indicating an inactivation of the oxygen evolving complex (OEC). Drought stress and exogenous glycine betaine and proline modified the shape of the OJIP curve of leaves heated at 45°C and the K step was not as pronounced. Increased thermostability of PSII may be associated with the resistance of OEC and increased energy connectivity between PSII units. The thermostability of PSII was also reflected by a lower decrease in maximum quantum yield of primary photochemistry (?_Po = F _V/F _M) and performance index (PI). Exogenous application of glycine betaine or proline can play an important role in enhancing plant stress tolerance and may help reduce effects of environmental stresses.     

Leaf Development in Lolium temulentum: Photosynthesis and Photosynthetic Proteins in Leaves Senescing under Different Irradiances

Changes in carbon fixation rate and the levels of photosyntheticproteins were measured in fourth leaves of Lolium temulentumgrown until full expansion at 360 µmol quanta m^–2s^–1 and subsequently at the same irradiance or shadedto 90 µmol m^–2 s^–1. Ribulose-1,5-bisphosphatecarboxylase/oxygenase (Rubisco), light-harvesting chlorophylla/b protein of photosystem II (LHCII), 65 kDa protein of photosystemI (PSI), cytochrome f (Cytf) and coupling factor 1 (CF₁) declinedsteadily in amount throughout senescence in unshaded leaves.In shaded leaves, however, the decrease in LHCII and the 65kDa protein was delayed until later in senescence whereas theamount of Cyt f protein decreased rapidly following transferto shade and was lower than that of unshaded leaves at the earlyand middle stages of senescence. Decreases in the Rubisco andCF₁ of shaded leaves occurred at slightly reduced rates comparedwith unshaded leaves. These results indicate that chloroplastproteins in fully-expanded leaves are controlled individually,in a direction appropriate to acclimate photosynthesis to agiven irradiance during senescence. (Received August 20, 1992; Accepted January 5, 1993)     

Protein synthesis in Xanthium leaf development

Young, expanding Xanthium leaves had many soluble proteins;older leaves had progressively fewer. The leaves that grew themost rapidly incorporated the most ¹⁴CO₂ into their proteins.The relative intensity of ¹⁴CO₂ incorporation into the differentsoluble proteins changed with leaf development. (Received November 17, 1969; )     

Betaine Accumulation and [C]Formate Metabolism in Water-stressed Barley Leaves

Barley (Hordeum vulgare L.) plants at the three-leaf stage were water-stressed by flooding the rooting medium with polyethylene glycol 6000 with an osmotic potential of −19 bars, or by withholding water. While leaf water potential fell and leaf kill progressed, the betaine (trimethylglycine) content of the second leaf blade rose from about 0.4 micromole to about 1.5 micromoles in 4 days. The time course of betaine accumulation resembled that of proline accumulation. Choline levels in unstressed second leaf blades were low (<0.1 micromole per blade) and remained low during water stress. Upon relief of stress, betaine-like proline—remained at a high concentration in drought-killed leaf zones, but betaine did not disappear as rapidly as proline from viable leaf tissue during recovery.

When [methyl-¹⁴C]choline was applied to second leaf blades of intact plants in the growth chamber, water-stressed plants metabolized 5 to 10 times more ¹⁴C label to betaine than control plants during 22 hours. When infiltrated with tracer quantities of [¹⁴C]formate and incubated for various times in darkness or light, segments cut from water-stressed leaf blades incorporated about 2- to 10-fold more ¹⁴C into betaine than did segments from unstressed leaves. In segments from stressed leaves incubated with [¹⁴C]formate for about 18 hours in darkness, betaine was always the principal ¹⁴C-labeled soluble metabolite. This ¹⁴C label was located exclusively in the N-methyl groups of betaine, demonstrating that reducing equivalents were available in stressed leaves for the reductive steps of methyl group biosynthesis from formate. Incorporation of ¹⁴C from formate into choline was also increased in stressed leaf tissue, but choline was not a major product formed from [¹⁴C]formate.

These results are consistent with a net de novo synthesis of betaine from 1- and 2-carbon precursors during water stress, and indicate that the betaine so accumulated may be a metabolically inert end product.

     

The Influence of Plant Water Deficit on Distribution of 14C-labelled Assimilates in Cacao Seedlings

The relationship between plant water status and distributionof ¹⁴C-labelled assimilates in cacao (Theobroma cacao L.) wasevaluated after ¹⁴CO₂ pulse labelling leaves of seedlings subjectedto varying levels of water deficiency. The proportion of ¹⁴Cexported by source leaves was strongly affected by seedlingwater status. An increasing proportion of labelled assimilatesremained in source leaves at both 24-h and 72-h harvests aswater stress intensity increased. Water stress reduced the distributionof exported label to leaves and to the expanding flush in particularbut increased the proportion of label in stems and roots. Theresults suggest that current photoassimilates may be temporarilystored in source leaves and stems of cacao seedlings duringperiods of plant water deficit. The stress-induced changes inpartitioning of labelled carbon were in concordance with changesin shoot to root biomass ratios, which was likely due to greaterreduction in growth of above-ground organs to that of roots. Theobroma cacao L, assimilate partitioning, cacao, ¹⁴C-photoassimilate, water stress, water potential     

Betaine Synthesis from Radioactive Precursors in Attached, Water-stressed Barley Leaves

In wilted barley leaves, betaine accumulates at about 200 nanomoles per 10 centimeters leaf per day. Results with ¹⁴C-labeled precursors were qualitatively and quantitatively consistent with de novo synthesis of this betaine from serine via ethanolamine, choline, and betaine aldehyde and indicated that water stress may increase the activities of all steps in this pathway except the last.