Chloroplast Doublets and Differential Staining of the Cell Walls in Photosynthetic Tissues of Amaranthus dubius Mart

Ultrastructural studies of the leaves of Amaranthus dubius revealpresence of chloroplast doublets in mature leaf tissues. Theyalso highlight the difference in the structural organisationof the bundle sheath and the mesophyll cell walls, a featurewhich may have functional significance. Amaranthus dubius Mart., Calaloo, mesophyll, bundle sheath, chloroplast doublets, plastid fusion, plastid division, differential staining of the cell walls     

Differentiation of Photorespiratory Activity between Mesophyll and Bundle Sheath Cells of C4 Plants I. Glycine Oxidation by Mitochondria

Mitochondria were isolated from mesophyll protoplasts and bundlesheath protoplasts or strands which were obtained by enzymaticdigestion of six C₄ species: Zea mays, Sorghum bicolor, Panicummiliaceum, Panicum capillare, Panicum maximum and Chloris gayana,representative of three C₄ types. Photorespiratory glycine oxidationand related enzyme activities of mesophyll and bundle sheathmitochondria were compared. Mesophyll mitochondria showed good P/O ratios with malate andsuccinate as substrate but lacked the ability to oxidize glycine.On the other hand, mitochondria isolated from bundle sheathprotoplasts of P. miliaceum and bundle sheath strands of Z.mays possessed glycine oxidation activity similar to that ofmitochondria from C₃ plant leaves. The two enzymes involvedin glycine metabolism in mitochondria, serine hydroxymethyltransferaseand glycine decarboxylase, were also assayed in the mitochondriaof the two cell types. The activities of the two enzymes inbundle sheath mitochondria were in the range found in C₃ mitochondria.In contrast, the activities in mesophyll mitochondria were eithernot detectable or far lower than those in bundle sheath mitochondriaand ascribed to contaminating bundle sheath mitochondria. The present results indicate the deficiency of a complete glycineoxidation system in mesophyll mitochondria and also a differentiationbetween mesophyll and bundle sheath cells of C₄ plants withrespect to the photorespiratory activities of the mitochondria. (Received June 8, 1983; Accepted August 29, 1983)     

Some Factors Concerning the Centripetal Disposition of Bundle Sheath Chloroplasts during the Leaf Development of Eleusine coracana

Factors concerning the chloroplast disposition in bundle sheathcells were investigated in finger millet (Eleusine coracanaGaertn.), and NAD malic enzyme type C₄ plant with the centripetalarrangement of bundle sheath chloroplasts. Segments were cutfrom immature regions of emerging leaves in which the centripetalarrangement of bundle sheath chloroplasts had not yet been established.The leaf segments were floated on solutions with or withoutreagents. Sections were made of the segments at time intervalsand the distribution of bundle sheath chloroplasts was observedby light microscopy. The bundle sheath chloroplasts migratedto the vascular bundle and established a centripetal arrangementby 12-16 h in control solutions. Auxins, cycloheximide and cytochalasinB inhibited the disposition of bundle sheath chloroplasts whilechloramphenicol and colchicine had no effect. The inhibitoryeffect of auxins appeared only at early stages of chloroplastmigration while cycloheximide and cytochalasin B were effectiveeven at later stages. Cessation of elongation growth, cytoplasmicprotein synthesis and microfilaments seemed to be associatedwith the centripetal disposition of bundle sheath chloroplasts.Copyright1993, 1999 Academic Press Bundle sheath chloroplast, C₄ plant, chloroplast orientation, Eleusine coracana, finger millet     

A Comparative Study of the Structure-Function Relationship of Cross Veins in Leaves of Digitaria eriantha and Zea mays

Ultrastructural and functional differences between the crossveins of Digitaria eriantha and Zea mays were investigated.Cross veins of both genera possess similar conducting tissues,namely one tracheary element and one sieve cell. In Digitariaeriantha these conducting elements are associated with onlytwo parenchyma cells, and, those in Zea mays are completelysurrounded by chJorenchyma cells. The protein ribulose 1,5-bisphosphatecarboxylase/oxygenase (rubisco) was used as a probe for CO₂fixation sites by comparing its distribution in the varioustissue types in the leaves of the two genera. The protein wasfound to be equally and uniformly distributed in the stromalregions of the chlorenchyma sheath cell chloroplasts of longitudinalveins of both genera. The chlorenchyma sheath cells in crossveins of Zea mays show a similar distribution of the enzymeas the longitudinal bundles. However, this enzyme was shownto be absent in the cross vein parenchyma cells of Digitariaeriantha and in the mesophyll cells of both genera. Cross veins, immuno-gold labelling, ribulose 1,5-bisphosphate carboxylase/oxygenase, Digitaria eriantha, Zea mays     

Comparative Leaf Epidermal Anatomy of Mutants of Barley (Hordeum vulgare L. 'Himalaya') which Differ in Leaf Length

We wished to determine the nature of differences in epidermalcell numbers and dimensions between leaves of different lengthin mutants of barley (Hordeum vulgare L. ‘Himalaya’).Three comparisons were made: leaf one (L1)vs. leaf four (L4);wild typevs. nine dwarf mutants and wild typevs. a slender mutant.L1 was shorter than L4, and for most lines this was associatedwith a change in epidermal cell number for the blade, and inboth cell number and length for the sheath. Compared to wildtype, the smaller leaves of dwarf plants generally had shorterand fewer cells in both blade and sheath. The blade of slenderplants was the same length (L1) or longer (L4) than wild type,while the sheath was longer than that of wild type for bothL1 and L4. Slender plants had longer but fewer cells than thewild type along the blade of L1, and shorter but more cellsfor the blade of L4. In the sheath, slender plants had longerand more (L1) or fewer (L4) cells than did the wild type. ForL1, variation in blade width amongst the barley lines was associatedwith a change in file width and file number. For L4, blade widthvaried only with file number, except for slender plants wherenarrow blades were associated with reduced file width. Hencethere was no consistent correlation between changes in cellsize or cell (or file) number with changes in leaf length orwidth. Differences depended on the leaf (L1vs. L4), leaf part(bladevs. sheath), and the nature of the mutation (dwarfvs.slender). Barley (Hordeum vulgare L. ‘Himalaya’); leaf epidermis; dwarf mutant; slender mutant     

The Effect of Light Quality on Shoot Extension Growth in Three Species of Grasses

The effect of light quality on the extension growth of vegetativeshoots and on the final size of their leaves was investigatedin plants of Lolium multiflorum, Sporobolus indicus and Paspalumdilatatum. Three experimental approaches were used, (a) redor far-red end-of-day irradiations of sunlight-grown plants,(b) different red/far-red ratios of white light in a growthroom and (c) sunlight enrichment with radiation of differentred/far-red ratios or with different amounts of far-red lightduring the photoperiod. Plants treated with end-of-day far-redor low red/far-red ratios throughout the photoperiod developedlonger leaves and, as a result, longer shoots. This effect wasmore marked in leaf sheaths than in blades. Tiller extensionand leaf sheath length increased with the amount of far-redadded to sunlight in a simple hyperbolic relationship. Theseresults show that vegetative grass shoots respond to light qualityin a way similar to internodes of dicotyledonous plants. Lolium multiflorum Lam., Sporobolus indicus (L.), Paspalum dilatatum (Poir.), leaf growth, tiller growth, photomorphogenesis     

Growth and Senescence of the Successive Leaves on a Cocksfoot Tiller. Effect of Nitrogen and Cutting Regime

The effects of nitrogen supply and cutting regime on the morphologicalcharacteristics (leaf appearance and expansion rates, leaf growthduration, leaf lifespan) of a cocksfoot sward were studied overthree growing seasons to gain a better understanding of thechanges in tiller characteristics (length and age of laminae,number of leaves per tiller) over time and in different seasons.We show that, for a given regrowth, the lamina expansion rateat the tiller level depended on herbage nitrogen status, butthe time course of its components differed according to nitrogensupply. When nitrogen was supplied, leaf appearance was fasterbut also decreased faster. In other words, the length of successivelaminae increased faster when nitrogen was supplied. The samewas true for the growth duration of the laminae and their lifespan.These changes resulted from the length of the sheaths from whichthe successive laminae emerged. As nitrogen increased cell number,it changed the ratio of lamina length_n+1/sheath length_nmorethan the ratio lamina length_n/sheath length_nat the same insertionlevel. Therefore sheath length increased faster and leaf appearancedecreased faster when nitrogen was supplied. This finding helpsto explain the effects of different heights and frequenciesof cutting in terms of their effects on sheath length. Copyright2000 Annals of Botany Company Nitrogen, defoliation, cocksfoot, sheath     

Impact of chloroplastic- and extracellular-sourced ROS on high light-responsive gene expression in Arabidopsis

The expression of 28 high light (HL)-responsive genes of Arabidopsiswas analysed in response to environmental and physiologicalfactors known to influence the expression of the HL-responsivegene, ASCORBATE PEROXIDASE2 (APX2). Most (81%) of the HL-responsivegenes, including APX2, required photosynthetic electron transportfor their expression, and were responsive to abscisic acid (ABA;68%), strengthening the impression that these two signals arecrucial in the expression of HL-responsive genes. Further, fromthe use of mutants altered in reactive oxygen species (ROS)metabolism, it was shown that 61% of these genes, includingAPX2, may be responsive to chloroplast-sourced ROS. In contrast,apoplastic/plasma membrane-sourced H₂O₂, in part directed bythe respiratory burst NADPH oxidases AtrbohD and AtrbohF, wasshown to be important only for APX2 expression. APX2 expressionin leaves is limited to bundle sheath parenchyma; however, forthe other genes in this study, information on their tissue specificityof expression is sparse. An analysis of expression in petioles,enriched for bundle sheath tissue compared with distal leafblade, in HL and control leaves showed that 25% of them had>10-fold higher expression in the petiole than in the leafblade. However, this did not mean that these petiole expressiongenes followed a pattern of regulation observed for APX2. Key words: Arabidopsis, chloroplast, excess light, gene expression, plasma membrane, reactive oxygen species, signalling     

Striga hermonthica decreases photosynthesis in Zea mays through effects on leaf cell structure

Maize seedlings were grown in pots either with or without preconditionedseeds of the parasitic weed, Striga hermonthica. After between4 and 8 weeks, net photosynthesis in the leaves of maize plantsinfected with Striga decreased compared to leaves of uninfectedcontrol plants. The activities of four enzymes of photosyntheticmetabolism were, however, little affected by infection. A pulse-chaseexperiment using ¹⁴CO₂ showed that C₄ acids were the main earlyproducts of assimilation even when the rate of photosynthesiswas much decreased by infection, but more radio-activity appearedin glycine and serine than in leaves of healthy maize plants.Leaves of infected maize required longer to reach a steady rateof photosynthesis upon enclosure in a leaf chamber than leavesof uninfected plants after similar treatment. Electron microscopy of transverse sections of the leaves ofinfected maize indicated that the cell walls in the bundle sheathand vascular tissue were less robust than in leaves of healthyplants. The results suggest that infection with Striga causesan increase in the permeability of cell walls in the bundlesheath, leakage of CO₂ from the bundle sheath cells and decreasedeffectiveness of C₄ photosynthesis in host leaves. Key words: Zea mays, Striga hermonthica, photosynthesis, photorespiration, enzyme activity     

Centripetal Disposition of Bundle Sheath Chloroplasts During the Leaf Development of Eleusine coracana

Distribution of chloroplasts in bundle sheath cells was examinedby light and electron microscopy during the leaf developmentof finger millet (Eleusine coracana Gaertn.), an NAD malic enzymetype C₄ plant with centripetal arrangement of bundle sheathchloroplasts. Young chloroplasts are almost evenly distributedalong the cell walls in bundle sheath cells of folded immatureleaves. In elongating leaves and above the elongation zone thebundle sheath chloroplasts tend to lie along the radial wallsand the walls adjacent to the vascular bundle. They furthermigrate near to the vascular bundle and finally establish acentripetal arrangement. Mitochondria, microbodies and nucleusmigrate along with the chloroplasts. Etioplasts and other organellesare centripetally located in the bundle sheath cells of etiolatedseedlings grown in the dark. Bundle sheath chloroplast, C₄ plant, chloroplast, chloroplast orientation, Eleusine coracana, finger millet     

An Application of the Prussian Blue Technique to a Light Microscope Study of Water Movement in Transpiring Leaves of Cotton (Gossypium hirsutum L.)

Prussian blue deposition in transpiring leaves of Gossypiumhirsutum L. indicated that water moved rapidly from the trachearyelements by a path along the bundle sheath parenchyma to thebundle sheath extension parenchyma and then laterally throughthe epidermis to the sub-stomatal cavities or to the tnchomes.A slower pathway of water movement to the substomatal cavitiesoccurred through the mesophyll. The path of Prussian blue depositionindicated that waterleft the leaf through the stomata and toa lesser extent through the trichomes. In the outer periclinalwall of epidermalcells Prussian blue was deposited below butnot within the cuticle.     

The Influence of Leaf Development on the Expression of C4 Metabolism in Flaveria trinervia, a C4 Dicot

The initial products of ¹⁴CO₂ assimilation were determined understeady state illumination of leaves of Flaveria trinervia, aC₄ dicot of the NADP-mialic enzyme subgroup. Leaf age influencedthe partitioning of ¹⁴CO₂ between the C₄ cycle and the reductivepentose phosphate (RPP) pathway. An estimated 10 to 12%of theCO₂ entered the RPP pathway directly in leaves about 20% fullyexpanded, whereas CO₂ was apparently fixed entirely throughthe C₄ pathway in leaves 75% or more expanded. This partitioningpattern was attributed to the bundle sheath compartment in youngleaves having a relatively high conductance to CO₂ (i.e., beingsomewhat leaky). Of the initially labelled C₄ acids, the proportion that wasmalate, relative to aspartate, increased continuously duringleaf expansion (from 60 : 40 to 87 : 13 at full expansion).Concurrently, there was an increase in the whole leaf activityof NADP malate dehydrogenase and a decrease in the activitiesof aspartate and alanine aminotransferases. Low chlorophylla/b values were observed in young leaves, which may coincidewith an enhanced capacity for non-cyclic electron transportin the bundle sheath chloroplasts of such tissue. Both enhancedaspartate metabolism and direct fixation of CO₂ in the bundlesheath could provide a greater sink for utilization of photochemicallyderived NADPH in the bundle sheath of young leaves. Such metabolicchanges are discussed in relation to a possible decrease inCO₂ conductance of the bundle sheath during leaf development. (Received March 4, 1986; Accepted June 25, 1986)     

Characterisation of Non-Uniform Photosynthesis Induced by Abscisic Acid in Leaves Having Different Mesophyll Anatomies

The effects of abscisic acid (ABA) on photosynthesis in leavesof Helianthus annuus L. were compared with those in leaves ofVicia faba L. After the ABA treatment, the response of photosyntheticCO₂ assimilation rate, A, to calculated intercellular partialpressure of CO₂, P_i, (A(p_i) relationship) was markedly depressedin H. annuus. A less marked depression was also observed inV.faba. However, when the abaxial epidermes were removed fromthese leaves, neither the maximum rate nor the CO₂ responseof photosynthetic oxygen evolution was affected by the applicationof ABA. Starch-iodine tests revealed that photosynthesis was not uniformover the leaves of H. annuus treated with ABA. The starch contentwas diffferent in each bundle sheath extension compartment (thesmallest subdivision of mesophyll by veins with bundle sheathextensions, having an area of ca. 0.25 mm² and ca. 50 stomata).In some compartments, no starch was detected. The distributionof open stomata, examined using the silicone rubber impressiontechniques, was similar to the pattern of starch accumulation.In V.faba leaves, which lack bundle sheath extensions, distributionof starch was more homogeneous. These results indicate that the apparent non-stomatal inhibitionof photosynthesis by ABA deduced from the depression of A(pⁱ)relationship is an artifact which can be attributed to the non-uniformdistribution of transpiration and photosynthesis over the leaf.Intercellular gaseous environment in the ABA-treated leavesis discussed in relation to mesophyll anatomy. ¹ Present address: Department of Botany, Duke University, Durham,NC 27706, U.S.A. (Received September 30, 1987; Accepted January 13, 1988)     

Distribution and Production of trans-Aconitic Acid in Barnyard Grass (Echinochloa crus-galli var. oryzicola) as Putative Antifeedant against Brown Planthoppers

A quantitative analysis of organic acid in leaves of barnyardgrass revealed that the contents of trans-aconitic acid werehigh, suggesting that this compound may act as an antifeedantagainst brown planthoppers. However, trans-aconitic acid couldnot be detected in the phloem sap which was considered to bethe main nutrient source for brown planthoppers. trans-Aconiticacid was formed in vitro from cis-aconitic acid through theaconitate isomerase activity which was detected only in theleaf sheath, but not in the leaf blade. (Received August 10, 1992; Accepted November 30, 1992)     

Diversity of Cellulose Microfibril Arrangement in the Cell Walls of Lygeum spartum Leaves

Extraction of matrix material, cytochemical methods and stereoanalysiswere used to study cell-wall architecture in leaves of Lygeumspartum, a grass species growing in sub-and regions of the Mediterraneanzone Most tissues were lignified with the exception of mesophyllcells Better results were obtained with NaOH extraction thanwith methylamine extraction The different types of fibres (sub-epidermal,axial and bundle sheath fibres), parenchyma and epidermal cellswere characterized by specific cell-wall organization The differentwall textures were variations of the basic helicoidal model Lygeum spartum L, lignified leaf fibres, cell wall, helicoidal texture, polysaccharide cytochemistry     

Activities of Principal Photosynthetic and Photorespiratory Enzymes in Leaf Mesophyll and Bundle Sheath Protoplasts from the C3-C4 Intermediate Flaveria ramosissima

Mesophyll and bundle sheath protoplasts were differentiallyisolated for the first time from leaves of a C₃-C₄ intermediate,Flaveria ramosissima. Protoplasts were partially purified fromleaf digests following differential centrifugation and flotationon dextran step-gradients. Two mesophyll and one bundle sheathfraction were obtained, with relative purities of the preparationsdetermined visually as >95% for mesophyll and >80% forbundle sheath. Representative C₃ and C₄ photosynthetic enzymes had substantialactivities, on a chlorophyll basis, in all three protoplastpreparations. The activity of phosphoenolpyruvate carboxylasewas highest in the lower density mesophyll fraction and lowestin the bundle sheath fraction. Conversely, the activity of NADP-malicenzyme was highest in the bundle sheath, and lowest in the lightermesophyll preparation. Ribulose 1,5-bisphosphate carboxylase/oxygenasehad similar activity in all three preparations, as did glycolateoxidase. However, glycine decarboxylase was about 3-fold enrichedin the bundle sheath fraction. The data indicate that the partialcompartmentation of photorespiratory metabolism may contributealong with limited C₄ photosynthesis to reducing photorespirationin this intermediate species. (Received April 27, 1988; Accepted June 17, 1988)     

Distribution of enzymes related to C3 and C4 pathway of photosynthesis between mesophyll and bundle sheath cells of Panicum hians and Panicum milioides

Enzymes of the C₄, C₃ pathway and photorespiration have beenanalyzed for P. hians and P. milioides, which have chlorenchymatousbundle sheath cells in the leaves. On whole leaf extracts thelevels of PEP carboxylase are relatively low compared to C₄species, RuDP carboxylase is typical of C₃ species, and enzymesof photorespiratory metabolism appear somewhat intermediatebetween C₃ and C₄. Substantial levels of PEP carboxylase, RuDPcarboxylase, and photorespiratory enzymes were found in bothmesophyll and bundle sheath cells. Low levels of C₄-acid decarboxylatingenzymes may limit the capacity for C₄ photosynthesis in P. hiansand P. milioides. The results on enzyme activity and distributionbetween mesophyll and bundle sheath cells are consistent withCO₂ fixation via C₃ pathway in these two species. ¹ This research was supported by the College of Agriculturaland Life Sciences, University of Wisconsin, Madison; and bythe University of Wisconsin Research Committee with funds fromthe Wisconsin Alumni Research Foundation; and by the NationalScience Foundation Grant BMS 74-09611. (Received September 16, 1975; )     

NADP-malic enzyme: immunolocalization in different tissues34 plant maize and the C3 plant wheat

In situimmunolocalization and Western blot analysis of separatedcellular and subcellular fractions, were used to determine thelocalization of different isoforms of NADP-malic enzyme in bothwheat (C₃) and maize (C₄) plants. In both techniques, an affinitypurified anti-(maize 62 kDa NADP-ME) lgG from the maize greenleaf isoform also reacted with a 72 kDa protein in tissues ofC4 plants as well as C3 plants. The light- inducible 62 kDaisofomi is located in bundle sheath chioroplasts of maize leaves.In etiolated leaves and in roots of maize there is evidencefor the occurrence of a 72 kDa isoform which co-migrates on2-D (SDS and isoelectric focusing) PAGE. The 72 kDa isoformis also present in low levels in green leaves. This form mayoccur in multiple intracellular compartments; but in situ immunolocalizationexperiments and Western blot and activity assays on fractionatedprotoplasts indicate that a significant amount of this isoformoccurs in plastids. With regards to C³ plants such as wheat,a 72 kDa isoform in leaves is largely confined to the chloroplastsbased on in situ immunolocalization and Western blots and enzymeactivity assays with fractionated protoplasts. In maize, itappears that the constitutive expression pattern of a possibleC₃ ancestral gene for NADP-malic enzyme has been maintained,and a high level expression of a light-inducible isoform locatedin bundle sheath chloroplasts (62 kDa) has been acquired duringits evolution. Key words: NADP-malic enzyme, Triticum aestivum, Zea mays     

Localization of Starch in the Base of Narcissus Leaves

The localization of starch in the leaves and flower stalk ofNarcissus tazetta is related to stages of development includingleaf abscission. Starch begins to appear in the newly vacuolatedparenchyma of the leaf primordia when primordia are about 0.5mm in length, and becomes localized in the leaf bases at theonset of differentiation into sheath and blade. The part playedby the intercalary meristems as a physiological and anatomicalbarrier to the distribution of starch in these organs is stressed,and the possible mechanism is discussed.     

The regulation of leaf elongation and xyloglucan endotransglycosylase by gibberellin in 'Himalaya' barley (Hordeum vulgare L.)

The potential role of xyloglucan endotransglycosylase (XET)in GA-stimulated cell elongation was investigated during leafexpansion in barley (Hordeum vulgare L.). XET activity in aqueousextracts of leaves was detected in all segments along the elongatingblade of leaf 1 of seedlings, but was at highest levels in basalsegments. Leaf 1 elongation rates of gibberellin (GA)-responsivedwarf mutants were lower than the wild type, and accompaniedby reduced levels of XET activity. Leaf elongation rates ofthe dwarfs increased following treatment with gibberellic acid(GA₃) associated with higher levels of XET activity. The slendermutant, crossed into a dwarfing background, exhibited high ratesof leaf 1 elongation and high levels of XET activity withoutadded GA₃. The elongation of leaf 3 in a GA-responsive dwarfmutant was also studied. Treatment with GA₃ resulted in bladeand sheath lengths being 5-fold and 7-fold (respectively) thelengths of controls, and again there were increases in bladeand sheath XET activities. To investigate the basis for changesin XET activity levels two XET-related cDNA clones were isolated.RNAs detected by the two clones occurred at the highest levelsin basal segments of rapidly elongating leaves, but they haddifferent distribution patterns along the leaf. Overall, thedata indicate that an XET-like activity is detectable in barleyleaves, that the activity level and related. Key words: Gibberellin (GA), leaf elongation, Hordeum vulgare, xyloglucan endotransglycosylase (XET)