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In this study, we investigated the expression and functions of the transcription factor gene RDD4 (rice Dof daily fluctuations 4), which has sequence similarity to RDD1 that controls nutrient ion accumulation in rice. RDD4 protein was highly accumulated in leaf sheaths and localized to vascular bundles. RDD4-overexpressing plants (RDD4-OX) improved the accumulation of various nutrient ions, irrespective of nutrient concentration in a hydroponic solution. K+ and Cl- deficiencies induced the accumulation of other cations and anions, respectively. Interestingly, in RDD4-OX plants K+ and Cl- deficiencies increased PO4 3- and Mg2+ contents, respectively, despite opposite electric charges. Furthermore, PO4 3- deficiency induced NO3 - and Mg2+ accumulation in RDD4-OX plants. These data show that RDD4 is associated with the control of nutrient ion contents within plants. Also, photosynthetic CO2 assimilation in RDD4-OX plants was higher than in wild-type (WT) plants, although the sizes of shoots and panicles decreased in RDD4-OX plants. Subsequent microarray analysis indicated that OsFWL7, similar to maize CNR1 that negatively regulates plant size, showed the most significant difference in its expression levels between WT and RDD4-OX plants. Based on these results, it is hypothesized that a prominent increase in the OsFWL7 expression reduces plant size in RDD4-OX plants. This article is protected by copyright. All rights reserved.

To better understand the coordination between dark and light reactions during the transition from C3 to C4 photosynthesis, we optimized a method for separating thylakoids from mesophyll (MC) and bundle sheath cells (BSCs) across different plant species. We grew six Paniceae grasses including representatives from the C3 , C3 -C4 and C4 photosynthetic types and all three C4 biochemical subtypes [nicotinamide adenine dinucleotide phosphate-dependent malic enzyme (NADP-ME), nicotinamide adenine dinucleotide-dependent malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PEPCK)] in addition to Zea mays under control conditions (1000 μmol quanta m-2 s-1 and 400 ppm of CO2 ). Proteomics analysis of thylakoids under native conditions, using blue native polyacrylamide gel electrophoresis followed by liquid chromatography-mass spectrometry (LC-MS), demonstrated the presence of subunits of all light-reaction-related complexes in all species and cell types. C4 NADP-ME species showed a higher photosystems I/II ratio and a clear accumulation of the NADH dehydrogenase-like complexes in BSCs, while Cytb6 f was more abundant in BSCs of C4 NAD-ME species. The C4 PEPCK species showed no clear differences between cell types. Our study presents, for the first time, a good separation between BSC and MC for a C3 -C4 intermediate grass which did not show noticeable differences in the distribution of the thylakoid complexes. For the NADP-ME species Panicum antidotale, growth at glacial CO2 (180 ppm of CO2 ) had no effect on the distribution of the light-reaction complexes, while growth at low light (200 μmol quanta m-2 s-1 ) promoted the accumulation of light-harvesting proteins in both cell types. These results add to our understanding of thylakoid distribution across photosynthetic types and subtypes, and introduce thylakoid distribution between the MC and BSC of a C3 -C4 intermediate species.

A barley (Hordeum vulgare) plasma membrane type aquaporin, HvPIP2;5, was identified as an H2 O2 permeable aquaporin among 21 barley and rice PIPs examined in the heterologous expression system using Saccharomyces cerevisiae. Four TIPs were also detected as H2 O2 -transporting aquaporins among 15 barley and rice TIPs. Influx of H2 O2 into yeast cells expressing HvPIP2;5 was determined with a florescent-dye-based assay. Indirect immunofluorescence indicated that the expression of HvPIP2;5 protein was ubiquitous in root tissues, and was also weakly observed in leaf epidermal cells and cells in the vascular bundle. Point mutated variants of HvPIP2;5 were generated by the site-directed mutagenesis. Growth assays of yeast cells expressing these mutated HvPIP2;5 proteins suggested that Ser-126 in HvPIP2;5 has a large impact on H2 O2 transport with a minor influence on the HvPIP2;5-mediated water transport.

ARGONAUTE7 (AGO7), a key regulator of the trans-acting small interfering RNAs (ta-siRNA) pathway, plays a conserved role in controlling leaf pattern among species. However, little is known about the ta-siRNA pathway in regulating inflorescence architecture and fruit yield. In this study, we characterized the expression pattern, subcellular localization and developmental functions of SlAGO7 in tomato (Solanum lycopersicum). Overexpressing SlAGO7 in tomato exhibited pleiotropic phenotypes, including improved axillary bud formation, altered leaf morphology and inflorescence architecture, and increased fruit yield. Cross-sectioning of leaves showed that the number of vascular bundles was significantly increased in 35:SlAGO7 lines. Overexpression of SlAGO7 increased the production of ta-siRNA, and repressed the expression ta-siRNA-targeted genes (SlARF2a, SlARF2b, SlARF3 and SlARF4). Further analysis showed that overexpression of SlAGO7 alters the expression of key genes implicated in leaf morphology, inflorescence architecture, auxin transport and signaling. In addition, the altered auxin response of 35:SlAGO7 lines were also investigated. These results suggested that SlAGO7 plays a positive role in determining inflorescence architecture and fruit yield though the ta-siRNA pathway. Therefore, SlAGO7 represents a useful gene that can be incorporated in tomato breeding programs for developing cultivars with yield potential.

The membranes of Zea mays (maize) mesophyll cell (MC) chloroplasts are more vulnerable to salinity stress than are those of bundle sheath cell (BSC) chloroplasts. To clarify the mechanism underlying this difference in salt sensitivity, we monitored changes in the glycerolipid and fatty acid compositions of both types of chloroplast upon exposure to salinity stress. The monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) contents were higher in MC chloroplasts than in BSC chloroplasts, in both the presence and absence of salt treatment. Under salt conditions, the MGDG level in MC chloroplasts was significantly lower than under normal conditions, while it was unchanged in BSC chloroplasts. In both types of chloroplast, the contents of DGDG, phosphatidylglycerol and phosphatidylinositol remained at the same levels in control and salt-treated plants, whereas sulfoquinovosyldiacylglycerol and phosphatidylcholine were significantly lower and higher, respectively, upon salt treatment. In addition, the fatty acid composition and double bond index of individual lipid classes were changed by salt treatment in both BSC and MC chloroplasts, although these factors had no effect on glycerolipid content. These findings suggest that the difference in salt sensitivity of MC and BSC chloroplast membranes is related to differences in MGDG responses to salinity. Thus, we propose that the low MGDG content and the low sensitivity of MGDG to salinity in BSC chloroplasts render them more tolerant than MC chloroplasts to salinity stress.

Plants in the field are commonly exposed to fluctuating light intensity, caused by variable cloud cover, self-shading of leaves in the canopy and/or leaf movement due to turbulence. In contrast to C3 plant species, only little is known about the effects of dynamic light (DL) on photosynthesis and growth in C4 plants. Two C4 and two C3 monocot and eudicot species were grown under steady light or DL conditions with equal sum of daily incident photon flux. We measured leaf gas exchange, plant growth and dry matter carbon isotope discrimination to infer CO2 bundle sheath leakiness in C4 plants. The growth of all species was reduced by DL, despite only small changes in steady-state gas exchange characteristics, and this effect was more pronounced in C4 than C3 species due to lower assimilation at light transitions. This was partially attributed to increased bundle sheath leakiness in C4 plants under the simulated lightfleck conditions. We hypothesize that DL leads to imbalances in the coordination of C4 and C3 cycles and increasing leakiness, thereby decreasing the quantum efficiency of photosynthesis. In addition to their other constraints, the inability of C4 plants to efficiently utilize fluctuating light likely contributes to their absence in such environments as forest understoreys.

Understanding peroxidase function in plants is difficult because of the lack of substrate specificity, the high number of genes and their diversity in structure. In the present study, the relative expression of 22 genes coding putative peroxidases (E.C 1.11.1.x) in Arabidopsis was studied. The relative expression of AtPrx37 showed a correlation with the cessation of growth in rosette leaves as well as with the growth capacity along the flower stem. Using AtPrx37::GUS construction, its expression was associated with the vascular bundles. Furthermore, the overexpression of AtPrx37 under the control of CaMV 35S promoter rendered a dwarf phenotype with smaller plants and delayed development. The plants overexpressing AtPrx37 also showed an increase in the amount of esterified phenolic material associated with their walls. A role in the growth cessation and phenolic cross-linking during lignin deposition is postulated.

Phenylalanine ammonia-lyase (PAL) catalyses the first step controlling the rate of phenylpropanoid metabolism. Wounding is a ubiquitous stress in nature and in the harvesting and preparation of fruits and vegetables that induces an increase in PAL activity, an accumulation of phenolic compounds and subsequent tissue browning. A wound-inducible PAL gene (LsPAL1) was isolated from Romaine lettuce by RT-PCR. The putative protein encoded by LsPAL1 is similar to predictive polypeptides sequences for other PALs. The kinetics of PAL mRNA accumulation is similar to those of induced PAL enzyme activity, with enzyme activity following mRNA accumulation by 12 h. Wound-induced PAL transcripts accumulated in cells close to the wound sites. Tissue printing showed that PAL mRNA was associated with tissue next to the epidermis and vascular bundles. A heterologous PAL protein was expressed in E. coli and was found to show significant PAL activity.

Verticillium dahliae is a prominent generator of plant vascular wilting disease and sulfur (S)-enhanced defense (SED) mechanisms contribute to its in-planta elimination. The accumulation of S-containing defense compounds (SDCs) including elemental S (S(0) ) has been described based on the comparison of two near-isogenic tomato (Solanum lycopersicum) lines differing in fungal susceptibility. To better understand the effect of S nutrition on V. dahliae resistance both lines were supplied with low, optimal or supraoptimal sulfate-S. An absolute quantification demonstrated a most effective fungal elimination due to luxury plant S nutrition. High-pressure liquid chromatography (HPLC) showed a strong regulation of Cys levels and an S-responsive GSH pool rise in the bulk hypocotyl. High-frequency S peak accumulations were detected in vascular bundles of resistant tomato plants after fungal colonization by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). Global transcriptomic analysis suggested that early steps of the primary S metabolism did not promote the SDCs synthesis in the whole hypocotyl as gene expression was downregulated after infection. Enhanced S fertilization mostly alleviated the repressive fungal effect but did not reverse it. Upregulation of glutathione (GSH)-associated genes in bulk hypocotyls but not in vascular bundles indicated a global antioxidative role of GSH. To finally assign the contribution of S metabolism-associated genes to high S(0) accumulations exclusively found in the resistant tomato line, a spatial gene expression approach was applied. Laser microdissection of infected vascular bundles revealed a switch toward transcription of genes connected with cysteine (Cys) synthesis. The upregulation of LeOASTLp1 suggests a role for Cys as key precursor for local S accumulations (possibly S(0) ) in the vascular bundles of the V. dahliae-resistant tomato line.

Cell extension in the mesocotyl elongation zone (MEZ) of maize (Zea mays L.) seedlings is inhibited by light. The growth inhibition by blue light in the MEZ was reversible upon transfer to darkness. This experimental system was used for investigating the modification of mechanical cell-wall properties and the role of cell-wall lignification in cell elongation. The occurrence of lignin in the cortex and vascular bundle tissues of the MEZ was demonstrated by the isolation of diagnostic monomers released after thioacidolysis of the cell walls. Concomitantly with the inhibition of growth, blue light induces an increase in cell-wall stiffness (tensile modulus) as well as an increase in extractable lignin in the outer MEZ tissues (cortex + epidermis). Both effects are reversed when growth is resumed in the MEZ in darkness after a period of growth inhibition induced by 3 h light. In the vascular bundle light produces no comparable change in lignin content. Appearance and disappearance of phenylpropanoid material in MEZ cell walls in the light, or in darkness following a brief light treatment, respectively, can be visualized under the fluorescence microscope by characteristic changes in autofluorescence of tissue sections upon excitation with UV radiation. It is concluded from these results that light-induced lignification of primary walls is involved in cell-wall stiffening and thus inhibition of elongation growth in the MEZ of maize seedlings. Resumption of growth upon redarkening may be initiated by wall loosening in the uppermost MEZ region which displaces the lignified cell walls towards the lower mesocotyl region.

Four ecotypes of Phragmites australis from different habitats in northwest China were examined to compare their photosynthetic characteristics. In a swamp ecotype, the δ 13 C value of leaf materials was -34.0‰, and bundle sheath cells contained a small amount of organelles and round-shaped chloroplasts, as being similar to typical C 3 plants. In a dune ecotype, the δ 13 C value was - 20.9‰ and bundle sheath cells contained oval-shaped chloroplasts with poorly-developed grana. In light and heavy salt meadow ecotypes, δ 13 C values were - 30.6‰ and - 35.6‰, respectively. The shape of bundle sheath chloroplasts in the light salt meadow ecotype was intermediate between those of the swamp and dune ecotypes. Abundance of bundle sheath organelles in the heavy salt meadow ecotype was intermediate. The swamp ecotype had photosynthetic enzyme activities typical of C 3 type plants, whereas the dune ecotype had an increased activity of phosphoenolpyruvate carboxylase (PEPC), a key C 4 enzyme, and a decreased ribulose 1,5-bisphosphate carboxylase (Rubisco) activity. The light salt meadow and heavy salt meadow ecotypes had substantial activities of PEPC, which indicates potential for C 4 photosynthesis. These data suggest that this species evolved the C 3 -like ecotype in swamp environments and the C 4 -like C 3 -C 4 intermediate in dune desert environments, and C 3 -like C 3 -C 4 intermediates in salt environments.

Recent work has suggested that the photosynthetic rate of certain C 4 species can be stimulated by increasing CO 2 concentration, [CO 2 ], even under optimal water and nutrients. To determine the basis for the observed photosynthetic stimulation, we tested the hypothesis that the CO 2 leak rate from the bundle sheath would be directly related to any observed stimulation in single leaf photosynthesis at double the current [CO 2 ]. Three C 4 species that differed in the reported degree of bundle sheath leakiness to CO 2 , Flaveria trinervia, Panicum miliaceum, and Panicum maximum, were grown for 31-48 days after sowing at a [CO 2 ] of 350 μl I 1 (ambient) or 700 μl l -1 (elevated). Assimilation as a function of increasing [CO 2 ] at high photosynthetic photon flux density (PPFD, 1600 μmol m -2 S ') indicated that leaf photosynthesis was not saturated under current ambient [CO 2 ] for any of the three C 4 species. Assimilation as a function of increasing PPFD also indicated that the response of leaf photosynthesis to elevated [CO 2 ] was light dependent for all three C 4 species. The stimulation of leaf photosynthesis at elevated [CO 2 ] was not associated with previously published values of CO 2 leak rates from the bundle sheath, changes in the ratio of activities of PEP-carboxylase to RuBP carboxylase/oxgenase, or any improvement in daytime leaf water potential for the species tested in this experiment. In spite of the simulation of leaf photosynthesis, a significant increase in growth at elevated [CO 2 ] was only observed for one species, F. trinervia, Results from this study indicate that leaf photosynthetic rates of certain C 4 species can respond directly to increased [CO 2 ] under optimal growth conditions, but that the stimulation of whole plant growth at elevated carbon dioxide cannot be predicted solely on the response of individual leaves.

We isolated and characterized an Alnus glutinosa cDNA clone, pAg13, which corresponds to a gene expressed at higher levels in nodules induced by Frankia than in roots. The deduced polypeptide sequence is rich in glutamic acid and proline and contains a putative signal peptide indicating an extracellular location of Ag13. In situ hybridization showed that ag13 is expressed in the pericycle of the nodule vascular bundle and in infected cells that exhibited degradation of the endosymbiont.

We investigated the phloem loading pathway in barley, by determining plasmodesmatal frequencies at the electron microscope level for both intermediate and small blade bundles of mature barley leaves. Lucifer yellow was injected intercellularly into bundle sheath, vascular parenchyma, and thin-walled sieve tubes. Passage of this symplastically transported dye was monitored with an epifluorescence microscope under blue light. Low plasmodesmatal frequencies endarch to the bundle sheath cells are relatively low for most interfaces terminating at the thin- and thick-walled sieve tubes within this C3 species. Lack of connections between vascular parenchyma and sieve tubes, and low frequencies (0.5% plasmodesmata per μm cell wall interface) of connections between vascular parenchyma and companion cells, as well as the very low frequency of pore-plasmodesmatal connections between companion cells and sieve tubes in small bundles (0.2% plasmodesmata per μm cell wall interface), suggest that the companion cell-sieve tube complex is symplastically isolated from other vascular parenchyma cells in small bundles. The degree of cellular connectivity and the potential isolation of the companion cell-sieve tube complex was determined electrophysiologically, using an electrometer coupled to microcapillary electrodes. The less negative cell potential (average –52 mV) from mesophyll to the vascular parenchyma cells contrasted sharply with the more negative potential (–122.5 mV) recorded for the companion cell-thin-walled sieve tube complex. Although intercellular injection of lucifer yellow clearly demonstrated rapid (0.75 μm s-1) longitudinal and radial transport in the bundle sheath-vascular parenchyma complex, as well as from the bundle sheath through transverse veins to adjacent longitudinal veins, we were neither able to detect nor present unequivocal evidence in support of the symplastic connectivity of the sieve tubes to the vascular parenchyma. Injection of the companion cell-sieve tube complex, did not demonstrate backward connectivity to the bundle sheath. We conclude that the low plasmodesmatal frequencies, coupled with a two-domain electropotential zonation configuration, and the negative transport experiments using lucifer yellow, precludes symplastic phloem loading in barley leaves.

A genotype 'Sujata' developed earlier at CSIR-CIMAP from its parent 'Sampada' is considered to be the latex-less variety of Papaver somniferum. These two genotypes are contrasting in terms of latex and stem strength. Earlier we have carried out microarray analysis to identify differentially expressing genes from the capsules of the two genotypes. In this study, the peduncles of the two genotypes were compared for the anatomy revealing less number of laticifers in the cortex and vascular bundles. One of the important cell wall-related genes (for laccase) from the microarray analysis showing significantly higher expression in 'Sampada' capsule was taken up for further characterization in the peduncle here. It was functionally characterized through transient overexpression and RNAi suppression in 'Sujata' and 'Sampada'. The increase in acid insoluble lignin and total lignin in overexpressed tissue of 'Sujata', and comparable decrease in suppressed tissue of 'Sampada', along with corresponding increase and decrease in the transcript abundance of laccase confirm the involvement of laccase in lignin biosynthesis. Negligible transcript in phloem compared to the xylem tissue localized its expression in xylem tissue. This demonstrates the involvement of P. somniferum laccase in lignin biosynthesis of xylem, providing strength to the peduncle/stem and preventing lodging.

To assess genotypic variability in nutrient supply of shoot branches, the distribution of 32 P and 45 Ca exported from a source nodal root (24-h uptake period) was measured within a genotype of a large-leaved (Kopu) and a small-leaved (Tahora) cultivar of Trifolium repens. Source-sink relationships of plants were modified by root severance, defoliation, and shade treatments. In control plants of both genotypes distribution of 32 P and 45 Ca closely followed the pathways that could be predicted from the known phyllotactic constraints on the vascular system. As such there was little allocation of radioisotopes (3.1% and 2.5% of exported 32 P and 45 Ca, respectively) from the source root to branches on the opposite side of the parent axis (far-side branches). However, genotypic differences in nutrient allocation were apparent, when treatments were imposed to alter intra-plant source-sink relationships. In the large-leaved genotype, the imposed treatments had minor effects on the allocation to far-side branches; whereas, in the small-leaved genotype, root severance and defoliation treatments increased lateral transport to far-side branches to 30% ( 32 P) and 10% ( 45 Ca) of exported radioisotopes. Genotypes with low (8-9) and high (12-13) numbers of vascular bundles were selected from within the large-leaved cultivar. Distribution of 32 P was then measured after plants had been pre-treated by removal of all far-side roots two days prior to labelling. Genotypes with low vascular bundle number allocated 20% and those with high vascular bundle number 3.2% of exported 32 P to far-side branches. It was concluded (1) that genotypic variation exists within T repens for potential to alter intra-plant allocation of mineral nutrients in response to treatments that modify source-sink relationships within plants; and (2) that this variation is correlated with differences among genotypes in the organisation of the vasculature of their stolons.

Four cell wall proteins of seedling cotyledons of Prosopis chilensis were characterized by SDS-polyacrylamide gel electrophoresis. The molecular masses of these proteins were 180, 126, 107 and 63 kDa. All of them immuno-crossreacted with polyclonal antibodies raised against extensin from soybean seed coats. Immuno-dot blot analysis demonstrated that the minimum expression of cotyledonary cell wall proteins was 48 h after seed imbibition, while 24 h after wound stress the expression of these wall proteins increased four-fold. Tissue immuno-prints and immuno-histochemistry showed that the proteins are expressed in the cell wall of all tissues. However, the epidermis and vascular bundles of cotyledons, hypocotyls and roots, and the living cells surrounding the wounded areas highly expressed the wall proteins. When the primary roots of the seedlings were injured by performing cuts with razor blades, the seedlings achieved a growth three times faster than control seedlings and secondary roots developed in sites close to the injuries. Immuno-histochemistry of secondary roots revealed that the root tips and the area of the cortical tissue of the primary roots being pressed by the the emerging root tip, highly expressed the cell wall proteins.

Mesophyll and bundle sheath cells of maize leaves (Zea mays L.) both contain the enzymes ascorbate peroxidase (AP; EC 1.11.1.11) and glutathione reductase (GR; EC 1.6.4.2) which are involved in hydrogen peroxide detoxification. Since bundle sheath cells of maize are deficient in photosystem II and have high CO 2 levels, oxidative stress may be less severe in these cells than in mesophyll cells. The present study was conducted to determine if AP and GR activity levels preferentially increase in mesophyll cells relative to bundle sheath cells when plants are subjected to moderate drought. Although drought inhibited the growth of greenhouse-grown plants, it did not affect the levels of protein, chlorophyll or AP. GR was unaffected by drought in whole leaf tissue and mesophyll cells, but did increase slightly in bundle sheath cells. This slight increase is of questionable biological importance. AP and GR activity levels were similar in mesophyll cells, bundle sheath cells and in whole leaf tissue. The data suggest that moderate drought has little effect on enzymes of the hydrogen peroxide scavenging system and that mesophyll and bundle sheath cells may be exposed to similar levels of hydrogen peroxide.

In maize, the structure of bundle sheath cell (BSC) chloroplasts is less subject to salinity stress than that of mesophyll cell (MC) chloroplasts. To elucidate the difference in sensitivity to salinity, antioxidant capacities and localization of reactive oxygen species were investigated in both chloroplasts. Transmission electron microscopic observation showed that O2 (-) localization was found in both chloroplasts under salinity, but the accumulation was much greater in MC chloroplasts. H2 O2 localization was observed only in MC chloroplasts of salt-treated plants. In isolated chloroplasts, the activities of superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11) and dehydroascorbate reductase (DHAR, EC 1.8.5.1) were increased by salinity. While the enhancement of SOD activity was similar in both chloroplasts, the increase of APX and DHAR activities were more pronounced in BSC chloroplasts than in MC chloroplasts. Monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) and glutathione reductase (GR, EC 1.6.4.2) were undetectable in BSC chloroplasts, while they increased in MC chloroplasts under salinity. Although ascorbate content increased by salinity only in BSC chloroplasts, glutathione content increased significantly in both chloroplasts, and was higher in MC chloroplasts than in BSC chloroplasts. The content of thiobarbituric acid-reactive substances, which is an indicator of lipid peroxidation, was significantly increased by salinity in both chloroplasts. These results suggested O2 (-) -scavenging capacity was comparable between both chloroplasts, whereas H2 O2 -scavenging capacity was lower in MC chloroplasts than in BSC chloroplasts. Moreover, the increased lipid peroxidation under salinity was associated with the structural alteration in MC chloroplasts, while it had less impact on the structure of BSC chloroplasts.

The aqueous phase of cell walls in stems of Kalanchoe daigremontiana Hamet et Perrier de la Bâthie (apoplast) contained ascorbic acid (AA) and dehydroascorbic acid (DHA). Ratios of AA/(AA + DHA) were 0.31 ± 0.12 (SD, n = 4), whereas those of whole stems (tissues plus apoplast) were >0.9. The amounts of (AA + DHA) in the stems were 1970 ± 190 (SD, n = 4) nmol g−1 fresh weight and those in the apoplast were 14 ± 2 (SD, n = 4) nmol g−1 fresh weight of stems. Ratios of AA/(AA + DHA) differed in different tissues of the stems. The ratios of AA/(AA + DHA) of apoplast plus symplast were in the following order: pith ⋍ epidermis plus cortex > vascular bundle system, and those of apoplast were: pith > epidermis plus cortex > vascular bundle system. Ratios of AA/(AA + DHA) in the apoplast of the different tissues decreased to about 1/3 of the original values after wounding, while the amounts of (AA + DHA) remained largely unaffected. In contrast, soluble apoplastic peroxidase activities increased 30- to 70-fold on wounding. Hydrogen peroxide infiltrated into stems caused a rapid oxidation of AA. Coniferyl alcohol was oxidized by peroxidase in intercellular washing fluid and by cell wall-bound peroxidase. The oxidation of coniferyl alcohol by peroxidase in intercellular washing fluid was completely inhibited as long as AA was present in reaction mixtures. The oxidation of the coniferyl alcohol by cell wall-bound peroxidase was partially inihibited by AA and the degree of inhibition was dependent upon the concentration of AA. The possible functions of AA in the apoplast are discussed in relation to the control of peroxidase-dependent oxidation of phenolics.

A hypothesis is presented that the availability of water for export of nitrogenous products from legume nodules is a major factor limiting the efficiency of symbiotic nitrogen fixation. Water for export of solutes in the xylem probably depends largely on the import of water and reduced carbon in the phloeum, and one function of respiration may be to dispose of reduced carbon in order to increase the supply of water. A second hypothesis presented is that control of gas diffusion in soybean nodules is largely restricted to the cortex nearby the vascular bundles, thus making possible the linkage of solute balances in xylem and phloem with resistance to diffusion. These concepts are used in a re-examination of literature on manipulations of nodules and nodulated plants such as lowering of light levels, water stress, defoliation, stem girdling, and alteration of oxygen supply. The concept of translocation as a major factor limiting efficiency of symbiotic fixation is consistent with the failure of superior rhizobial isolates to improve N input significantly, and this limitation could also prevent exploitation of superior bacterial symbionts in the future

Stem non-structural carbohydrates (NSCs) and its relationship with yield formation was investigated under low nitrogen (LN) and normal nitrogen (NN) treatments, using 46 recombinant inbred lines from Zhenshan 97 × Minghui 63 (Oryza sativa). Apparent contribution of transferred NSC to grain yield (AC(NSC) ) ranged from approximately 1 to 28% under LN and from 1 to 15% under NN. Concentration and total mass of NSC in stem (TM(NSC) ) at heading, apparent transferred mass of NSC (ATM(NSC) ) and AC(NSC) were larger under LN compared with NN. However, there was no significant difference in the apparent ratio of transferred NSC from stems to grain (AR(NSC) ). ATM(NSC) was positively correlated with grain yield, 1000-grain weight and AC(NSC) under both nitrogen levels, whereas AR(NSC) was highly correlated with harvest index and AC(NSC) . Leaf area contributed more strongly to grain yield compared with ATM(NSC) under both LN and NN. ATM(NSC) showed larger direct effects on grain yield under LN compared with NN. TM(NSC) at heading, small vascular bundles (SVBs) and spikelets per m(2) under LN had positive direct effects on ATM(NSC) . SVB and spikelets per m(2) under LN had larger and positive direct effects, and large vascular bundles had negative direct effects on AR(NSC) . TM(NSC) at heading and SVB under LN had positive direct effects on AC(NSC) . In brief, LN supply increased stem NSC accumulation and translocation to developing grain. Components of the source-sink-flow system showed different effects on NSC translocation and contribution to yield formation, depending on genotype and nitrogen level.

Awns are long, stiff filamentous extensions of glumes in many grasses. In wheat, awns contribute up to 40% of the grain's photosynthetic assimilates, and assist in seed dispersal. Awns accumulate silica in epidermal hairs and papillae, and silica has been positively associated with yield and environmental stress tolerance. Here, the awns of a set of domesticated wheat genotypes and their direct progenitor, Triticum turgidum ssp. dicoccoides were characterized. In addition, the silica concentration in awns was genetically dissected in a tetraploid wheat population of recombinant inbred lines (RILs) derived from a cross between durum wheat (cv. Langdon) and wild emmer (accession G18-16). Scanning electron micrographs revealed a continuous silica layer under the cuticle. Extended silicification was identified in the epidermis cell wall and in sclerenchyma cells near the vascular bundles, but not in the stomata, suggesting that an active process directs the soluble silica away from the water evaporation stream. The number of silicified cells was linearly correlated to silica concentration in dry weight (DW), suggesting cellular control over silicification. Domesticated wheat awns contained up to 19% silica per DW, as compared with 7% in the wild accessions, suggesting selection pressure associated with the domestication process. Six quantitative trait loci (QTLs) for silica were identified in the awns, with a LOD score of 3.7-6.3, three of which overlapped genomic regions that contribute to high grain protein. Localization of silica in the awns and identification of QTLs help illuminate mechanisms associated with silica metabolism in wheat.

Gibberellin A4/7 (GA4/7) was applied twice weekly to 2-year-old Pinus sylvestris (L.) seedlings in each of two years, starting close to budbreak and ending after shoot elongation, but before cambial activity ceased. In 1988, the GA4/7 was injected into the 1987 terminal shoot (0, 0.2 or 2 mg seedling−1 application−1), while in 1990 it was applied as a soil drench (0, 10 or 50 mg seedling−1 application−1). In the 1988 experiment, GA4/7 treatment promoted diameter growth, and tended to increase both longitudinal growth and the indole-3-acetic acid (IAA) level in the 1988 terminal. In the 1990 experiment, GA4/7 treatment increased tracheid production, longitudinal growth, and the cambial region IAA concentration in the 1990 terminal, but did not affect its pith diameter, needle number, needle dry weight, or needle IAA level. Tracheid production in the previous-year's terminal was also promoted in both experiments. The 50 mg GA4/7 soil drench markedly elevated the concentrations of GA4, GA7 and GA9 in the needles and cambial region of the 1990 terminal, while the 10 mg treatment raised the GA4 level in the cambial region, providing evidence that GA4 and GA7 applied to the roots reaches the shoot system. The results support the hypothesis that the exogenous GA4/7-induced stimulation of tracheid production in the terminal shoot of intact plants is mediated through an increase in the IAA level in the cambial region. However, per se activity of GA4, GA7 or their metabolites cannot be ruled out.

C 4 species of the genus Aristida (Poaceae) have 3 distinct types of photosynthetic cells in their leaves, the mesophyll (M) cells, the outer bundle sheath (BS) cells, and the inner BS cells, and exhibit a unique Kranz-type leaf anatomy. The cellular localization of C 3 and C 4 photosynthetic enzymes was investigated in leaves of Aristida latifolia Domin by the protein A-gold immunocytochemical technique. The outerBS cells contained centripetally located small chloroplasts, which were structurally similar to those of the M cells

Various techniques were used to extract solutes from the free space of intact soybean [Glycine max (L.) Merr.] nodules. A variety of solutes (carbohydrates, amino acids, organic acids, ions) was found, but the major solute obtained with all methods was allantoic acid. Most work was done with a technique involving vacuum infiltration of intact detached nodules with water. This approach provided rapid sampling of the apoplastic solutes, and the results indicated that solutes were not derived from the xylem and phloem of ruptured vascular bundles. Infiltration of intact nodules with Fast Green showed dye penetration only to the barrier in the inner cortex, indicating that infected tissues did not contribute to solute composition. Although allantoic acid was the only ureide which could be detected in solute samples, no evidence was obtained for the presence of allantoinase in the cortical apoplast. The results suggest the transport of allantoic acid by an apoplastic route in nodules or the release of allantoic acid to the cortical apoplast in response to treatments which disrupt ureide export. Calculated values for solute concentrations in the cortical apoplast were in the hundred millimolar range, suggesting that apoplastic solutes may represent a significant osmotic component in the nodule cortex.

The difference spectroscopy technique has been utilized to investigate the temperature-induced spectral changes in mesophyll and bundle sheath chloroplasts of maize (Zea mays L. cv. Ganga-5) in order to assess the role of different pigment-protein complexes in the manifestation of temperature effect on the chloroplast membranes. Cooling and heating of both mesophyll and bundle sheath chloroplasts resulted in absorbance difference (AA) bands at similar wavelengths but the degree of absorb-ance changes were significantly higher in bundle sheath chloroplasts. For example, upon cooling to 7-8°C, positive AA bands were observed at 440, 490 and 680 nm in mesophyll chloroplasts and at 440, 495–500 and 680 nm in bundle sheath chloroplasts but the absorbance change at 680 nm was ca 2% in mesophyll chloroplasts, whereas it was ca 5% in bundle sheath chloroplasts, which have a lower content of light-harvesting pigment-protein complex. The role of chlorophyll-protein complexes was further investigated by monitoring the temperature-induced spectral changes of mesophyll and bundle sheath chloroplasts isolated from lincomycin-treated maize plants where lincomycin selectively inhibits the biosynthesis of specific chlorophyll-protein complexes. Results indicated that depletion of certain pigment-protein complexes in mesophyll chloroplasts made them more susceptible (a ca 4% vs ca 2% absorbance change upon cooling and a ca 6% vs ca 4% absorbance change upon heating) and less tolerant to temperature variation (a 76% vs 39% reversibility during ambientCoolingambient temperature cycle). The data indicate that pigment-protein complexes play a significant role in protecting the chloroplast membranes against temperature variation.

Regulatory ACT domains serve as amino acid-binding sites in certain amino acid metabolic enzymes and transcriptional regulators in bacteria. The ACT domain repeat protein (ACR) family in plants is primarily composed of four copies of the domain homologous to those of the bacteria Gln sensor GLND. In the current study, to evaluate the possible involvement of the protein OsACR9 in the Gln-sensing system related to nitrogen (N) metabolism in rice (Oryza sativa L.), subcellular localization of OsACR9 and its accumulation and cellular distribution in various rice organs were examined by transient expression analysis and immunological methods using a monospecific antibody, respectively. Transient expression analysis of OsACR9 fused with a synthetic green fluorescent protein in cultured rice cells suggested nuclear localization of OsACR9. In rice roots, OsACR9 protein was distributed in epidermis, exodermis, sclerenchyma and vascular parenchyma cells, and its accumulation markedly increased after supply of NH + 4 . In rice leaf samples, OsACR9 protein was abundant in the vascular parenchyma and mestome-sheath cells of young leaf blades at the early stage of development and in the vascular parenchyma and phloem-companion cells of mature leaf sheaths. OsACR9 protein also showed a high level of accumulation in vascular parenchyma cells of dorsal vascular bundles and aleurone cells in young rice grains at the early stage of ripening. The possibility of the nuclear protein OsACR9 acting as a Gln sensor in rice is subsequently discussed through comparison of its spatiotemporal expression with that of Gln-responsive N-assimilatory genes.

Portions of stems from the base of asparagus spears (Asparagus officinalis L. cv. Connovor Collossus) were dissected to give the following tissues: (1) pith, which was free of vascular bundles, (2) two surrounding layers, parenchyma and fibre I and II (PFI and PFII), containing parenchyma and vascular bundles, (3) sclerenchyma sheath, (4) epidermis and sub-epidermal layers and (5) asparagus vascular fibre (AVF). The alcohol-insoluble residues (AIRs) from these tissues were shown to be free of starch. They were analysed for moisture and protein, and the component sugars were released by two hydrolytic procedures, which helped to distinguish the sugars from non-cellulosic polysaccharides and cellulose. The AIRs from pith and epidermal tissues were relatively low in xylose, but were rich in cellulosic glucose, and sugars associated with pectic polysaccharides such as galacturonic acid, galactose and arabinose. Their major component polysaccharides (in decreasing amounts) were inferred to be pectic polysaccharides, cellulose, and hemicelluloses. AIR from sclerenchyma was rich in glucose and xylose, suggesting the presence of much cellulose and (acidic) xylans. The AIRs of PFI, PFII and AVF contained significant amounts of xylose in addition tn other sugars, and the major polysaccharides inferred to be present were pectic polysaccharides, cellulose and hemicelluloses, a significant proportion of which may be acidic xylans. Methylation analysis of the AIRs confirmed the above inferences. The bulk of the glucosyl residues were (1–4)-linked, and there were small but significant amounts of (1–4, 6)-linked glucosyl residues (the linkage characteristic of xyloglucans) in all the preparations. The presence of (1–4)-linked galactosyl, (1–5)-linked arabinosyl, terminal galactosyl, terminal arabinosyl, (1–2)- and (1–2, 4)-linked rhamnosyl residues in all the AIRs except that from sclerenchyma, confirmed the presence of significant levels of pectic polysaccharides in all the parenchyma tissues. All the preparations containing vascular tissues contained significant amounts of (1–4)-linked xylosyl residues, probably derived from acidic xylans. Even in the AIR of pith, a significant amount of (1–4)-linked xylosyl residues were detected. This may be due to the ability of these cells and the parenchyma cells associated with the vascular bundles, to undergo lignification in mature asparagus plants.

In order to estimate whether cytosolic glutamine synthetase (GS1; EC 6.3.1.2) is partly coupled to the reaction of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in developing organs of rice (Oryza sativa L.), we compared the expression pattern of transcripts and proteins for GS1 and PAL in the tissue sections from leaf blades at various stages of development. In immature vascular bundles of unexpanded leaf blades, GS1 mRNA was mainly detected in xylem parenchyma cells, mestome-sheath cells, and sclerenchyma cells. PAL transcripts were also accumulated in these cell types. Vascular bundles in midribs of immature leaf blades contained mRNAs and proteins for both GS1 and PAL abundantly in sclerenchyma cells, although distribution of these two proteins was not completely overlapped. In immature vascular bundles in midribs, lignin deposition was observed in cell walls of xylem parenchyma cells, mestome-sheath cells and sclerenchyma cells. These results implied that a part of GS1 in unexpanded leaf blades is possibly involved in reassimilation of ammonia released from PAL reaction during the lignin production.

Barley, like most other grasses that have been studied, contains two kinds of sieve tube. The first formed are called thin-walled sieve tubes because of their thin wall compared to the late-formed, and are associated with companion cells. The late-formed are thick-walled sieve tubes, which differentiate next to the metaxylem vessels and lack companion cells. Aphid (Sitobion yakini (Eastop) feeding was studied using light microscopy to determine if they preferentially feed from thin- or thick-walled sieve tubes in the barley leaf. Penetration of the stylets through the leaf epidermis and mesophyll was largely intercellular, becoming partly intercellular and, partly, intracellular inside the vascular bundle. Sixteen of 19 pairs of stylets (84%), and 293 of 317 (92%) stylet tracks terminated at the thin-walled sieve tubes, suggesting that Sitobion yakini feeds preferentially on the thin-walled sieve tubes which seem to be more attractive to the aphid. These thin-walled sieve tubes are thus probably the most functional in terms of phloem loading and transport.

The lignification process and lignin heterogeneity of fibre, vessel and parenchyma cell walls for various age classes of bamboo stems of Phyllostachys pubescens Mazel were investigated. It was shown that protoxylem vessels lignified in the early stage of vascular bundle differentiation, metaxylem vessel and fibre walls initiated lignification from the middle lamella and cell corners after the completion of vascular bundle differentiation. Most of the parenchyma cell walls lignified after the stem reached its full height, while a few parenchyma cells remained non-lignified even in the mature culm. The cell walls of fibres and most parenchyma cells thickened further during the stem growth to form polylamellate structure and the lignification process of these cells may last even up to 7 years. The fibre walls were rich in guaiacyl lignin in the early stage of lignification, and lignin rich in syringyl units were deposited in the later stage. Vessel walls mainly contained guaiacyl lignin, while both guaiacyl and syringyl lignin were present in the fibre and parenchyma cell walls.

The subcellular compartmentation of beta-glucosidase was studied in rye, maize and wheat seedlings by immunocytochemical methods. For detection, we used a 10 nm gold-labeled secondary antibody, and results were observed using transmission electron microscopy. In all three species, beta-glucosidase was found in plastids, cytoplasm and cell walls. In rye, gold particles were seen on cell walls and cytoplasm in epidermal cells of the root tip and shoot, in bundle sheath cells of the shoot and in all cells, except the vascular bundle cells of the coleoptile. Gold labeling was also observed in plastids of the bundle sheath cells of rye shoot tips and in cortical cells of root tips. In wheat, gold labeling was observed on cell walls and cytoplasm of epidermal cells in the shoot base and coleoptile, and on cell walls and plastids in epidermal cells of the root tip. In maize, gold labeling was mainly found in plastids or proplastids in vascular bundle cells and bundle sheath cells of the shoot, in bundle sheath cells of the coleoptile and in epidermal cells of the root. Some gold particles were also found in cell walls and cytoplasm of stomatal guard cells of the shoot base and vascular bundle cells of the shoot tip and in the cell walls of bundle sheath cells of the shoot tip and root tip epidermal cells. Results are discussed in relation to the role of beta-glucosidase in hydroxamic acid release and overall defense mechanism of monocotyledons.

Chloride transport in sheath and blade tissue and the cellular distribution of Cl- were investigated in an attempt to determine the physiological basis of the preferential accumulation of Cl- in sheaths of salt-stressed sorghum (Sorghum bicolor L.). Import and export of 36Cl- in leaf sheaths and blades of intact sorghum were followed over a 2 week period. X-ray microanalysis of frozen-hydrated bulk tissue samples was used to determine the accumulation of Cl- and other elements in the vacuoles of sheath and blade cells. Sheath tissue accumulated Cl- despite a relatively high Cl- turnover rate. Chloride was shown to accumulate in most cell types of the sheath, particularly in adaxial epidermal cells. After an initial increase in the concentration of Cl-, blade tissue regulated Cl- levels within certain limits. Chloride levels in blades were greater in the abaxial and adaxial epidermal cells than in other cell types. The epidermal cells of blades accumulated Cl- to approximately the same concentration as sheath epidermal cells. The Cl- concentration in the photosynthetically active mesophyll and bundle sheath cells, however, remained low. Thus, the partitioning of Cl- previously observed in the leaves of salinized sorghum apparently results from the ability of bundle sheath and mesophyll cells to maintain concentrations of Cl- at lower levels than do epidermal cells. In addition, the relatively large sheath parenchyma cells tend to serve as reservoirs for the storage of Cl-.

Three inbred lines of maize (Zea mays L.) from cool temperate regions and three from warm regions were grown at 14, 22, 30 and 38°C up to the same physiological age, the full expansion of the third leaf. The laminae of the second leaf were studied for anatomical traits of the chlorenchyma. The genotypic variation was considerable for all properties. Nevertheless, general temperature response curves existed for many properties with few deviations of single lines. The cross-sectional area of mesophyll cells and cross-sectional area and number of chloroplasts declined from 14 to 22°C, increased up to 30°C and declined again up to 38°C. The ratio of shortest to longest diameter of chloroplasts was low in mesophyll cells at intermediate temperatures, and maximum in bundle sheath cells at 30°C. Temperature response curves for other properties of the bundle sheath cells were predominantly genotype specific. Chloroplasts in bundle sheath cells were sometimes smaller than those of mesophyll cells, dependent on temperature and genotype. Values of lines from cool temperate and warm regions were mostly overlapping, but the cross-sectional area of mesophyll cells was larger in the latter than in the former lines at low temperature. The results are discussed in relation to the expression of photosynthetic properties.

The distribution of nitrite reductase (EC 1.7.7.1) and sulfite reductase (EC 1.8.7.1) between mesophyll ceils and bundle sheath cells of maize (Zea mays L. cv. Seneca 60) leaves was examined. This examination was complicated by the fact that both of these enzymes can reduce both NO-2 and SO2-3 In crude extracts from whole leaves, nitrite reductase activity was 6 to 10 times higher than sulfite reductase activity. Heat treatment (10 min at 55°C) caused a 55% decrease in salfite reductase activity in extracts from bundle sheath cells and mesophyll cells, whereas the loss in nitrite reductase activity was 58 and 82% in bundle sheath cells and mesophyll cell extracts, respectively. This result was explained, together with results from the literature, by the hypothesis that sulfite reductase is present in both bundle sheath cells and mesophyll cells, and that nitrite reductase is restricted to the mesophyll cells. This hypothesis was tested i) by comparing the distribution of nitrite reductase activity and sulfite reductase activity between bundle sheath and mesophyll cells with the presence of the marker enzymes ribulose-l, 5-bisphosphate carboxylase (EC 4.1.1.39) and phosphoe-nolpyruvate carboxylase (EC 4.1.1.32), ii) by examining the effect of cultivation of maize plants in the dark without a nitrogen source on nitrite reductase activity and sulfite reductase activity in the two types of cells, and iii) by studying the action of S2-on the two enzyme activities in extracts from bundle sheath and mesophyll cells. The results from these experiments are consistent with the above hypothesis.

Variable factors affecting the enzymatic isolation of mesophyll protoplasts from Triticum aestivum (wheat), a C3 gras, and mesophyll protoplasts and bundle sheath strands from Digitaria sanguinalis (crabgrass), a C4 grass, have been examined with respect to yields and also photosynthetic capacity after isolation. Preparations with high yields and high photosynthetic capacity were obtained when small transverse leaf segments were incubated in enzyme medium in the light at 30°C, without mechanical shaking and without prior vacuum infiltration. Best results were obtained with an enzyme medium that included 0.5 M sorbitol, 1 mM MgCl2, 1 mM KH2PO4, 2% cellulase and 0.1% pectinase at pH 5.5. In gerneral, leaf age and leaf segment size were important factors, with highest yields and photosynthetic capacities obtained from young leaves cut into segments less than 0.8 mm. To facilitate the cutting of such small segments, a mechanical leaf cutter is described that uniformly (± 0.05 mm) cuts leaf tissue into transverse segments of variable size (0.4–2 mm). Isolations that required more than roughly 4 h gave poor yields with reduced photosynthetic capacity; however, using the optimum conditions described, functional preparations could be roughly 2 h. High rates of light dependent CO2 fixation by the C4 mesophyll protoplasts required the addition of pyruvate and low levels of oxalacetate, while isolated bundle sheath strands and C3 mesophyll protoplasts supported CO2 fixation without added substrates. Rates of CO2 fixation by isolated wheat protoplasts generally exceeded the reported rates of whole leaf photosynthesis. Wheat mesophyll protoplasts and crabgrass bundle sheath strands were stable when stored at 4°C while C4 mesophyll protoplasts were stable when stored at 25°C.

Alder root nodules are capable of atmospheric nitrogen fixation. A differential treatment with kinetin and 2IP (2-isopentenyl adenine) was applied to the rooting medium of alder plants growing in test tubes containing Crone solution. The experiment led to the formation of a fair number of pseudonodules scattered on the root system of the plants treated with cytokinin levels higher than 0.6 μg/ml. Kinetin had a lowering effect on the overall growth of the plant, whereas the plants treated with 2IP showed no differences in growth as compared to the non-treated control plants. Histological sections of the pseudonodules show an unorganized tissue originating from the parental root cortex, and the presence of groups of tracheids giving the aspect of unorganized vascular bundles.

Corn (Zea mays L. cv. OP Golden Bantum) was grown under various low irradiances of red light (Pfr/Ptot∼ 0.8) and under high irradiance far-red light containing low amounts of red light (Pfr/Ptot∼ 0.05–0.15). Parameters of chloroplast development such as pigments, membrane polypeptides and infrastructure were compared among the various light sources. Results indicate that the requirement for phytochrome is saturated at low ratios of Pfr/Ptot (

The intracellular distribution of serine hydroxymethyltransferase (EC 2.1.2.1) was studied in young wheat (Triticum aestivum L. cv. Starke II) leaves by fractionation of protoplasts and further purification of peroxisomes and chloroplasts. Essentially all of the activity in wheat leaves was located in the mitochondria. Within the mitochondria the enzyme was mainly in the matrix as shown by centrifugation of sonicated wheat mitochondria. In the C4 plants, Zea mays (L. cv. Earliking), Panicum miliaceum and Panicum maximum (cv. Australia) belonging to different C4 types, serine hydroxymethyltransferase was almost exclusively found in bundle sheath cells. The location of this enzyme in leaves is consistent with its role relative to glycine decarboxylation during photorespiration.

In Sorghum vulgare L. (cv. INRA 450) leaves, two isoforms of glutamine synthetase GS1 and GS2 were identified by DEAE Sephacel chromatography and polyac-rylamide gel electrophoresis. GS1 was present both in etiolated and green leaves and its activity remained constant during the greening process. In green leaves another isoform GS2 was identified that was responsible for a 33% increase in total glutamine synthetase activity after light exposure. The two isoforms differed in their heat stability but exhibited similar pH optima and Km values for L-glutamate. The total glutamine synthetase activity was equally distributed between mesophyll and bundle sheath cells; moreover in the two types of cells both GS1 and GS2 were present in the proportions of 67% and 33% of the total respectively. In mesophyll cells the two isoforms had a different subcellular distribution: GS2 was localized in the chloroplast and GS1 in the cytosol.

A procedure was developed to obtain intact and purified mitochondria from mesophyll and bundle sheath tissues of Zea mays L. cv. I.N.R.A. 180, an NADP+-malic enzyme type C4 plant. There was little cross-contamination between the two mitochondrial fractions. Both types of mitochondria oxidized NADH, succinate and malate with respiratory control. In mesophyll mitochondria malate oxidation was highly sensitive to KCN (85–90% inhibition of first state 3) and showed good respiratory control. In bundle sheath mitochondria malate oxidation was less sensitive to cyanide (75-80% inhibition) and showed poor respiratory control. Malate and NADH appeared to be the best substrates for respiratory activity. Mesophyil mitochondria could not oxidize glycine, whereas bundle sheath mitochondria could. The results indicate that mesophyll and bundle sheath mitochondria of Zea mays are differentiated, not only with respect to the decarboxylation of malate but also with respect to the decarboxylation phase of photorespiration.

After application of [3H]-auxin (0.8 nmol) to a young leaf of Vicia faba L. cv. Aguadulce. about 6% (1.1 × 10-2 nmol) of applied IAA enters the stem during the first 6 h of transport. This corresponds to a [3H]-auxin flux which is probably not very different from the endogenous flux. A wave of [3H]- auxin moves down to the roots mainly among preferential pathways situated in the vascular bundle. This movement is accompanied and followed by certain events: (I) In the upper part of the stem, some radioactive molecules leave the pathways of polar transport and enter the young leaves near the donor leaf. (2) In other parts of the stem, the auxin transport is highly polar. As the peak of the wave approaches and passes a node with an axillary bud. and for a few hours afterwards, there is no clearly detectable radioactivity in this bud, although the nodal tissues are very radioactive. (3) A retention of labelled molecules often occurs in the nodes. (4) Retention of label is regularly seen in the basal part of the first internode and in the hypocotyl, which together form that part of the axis where Ifle highly inhibited cotyledonary buds are found. This retention is still manifest a week after the downward transport of [3H]-auxin. (5) After 48 h. a high proportion (about 45%) of the [3H]-auxin exported by the donor leaf is found in the roots. (6) Subsequently, a part of the label returns to the upper parts of the plant, and especially to the leaves, where it normally appears to be immobilized. (7) As time goes on some labelled molecules, probably coming from different areas, enter the axillary buds.

Numerous lateral branches of the concealed aerial roots of the grasstree Kingia australis ramify through the persistent leaf bases, suggesting a role in uptake of water and nutrients localed up to 8 m above ground. These leaf bases were shown to hold up to more than three times their weight in water. Water and nutrients (N, P and K) available in the leaf bases may greatly exceed that in an equal volume of surface soil, especially during the dry summer months. At this time, 32P injected# among the leaf bases is strongly absorbed by the aerial roots and translocated to the meristematic regions of other laterals beneath the point of application, but mostly to the stern and leaves above it. Scattered vascular bundles in the pith are the major routes for the rapid distribution of “P up and down the trunk following its departure from the aerial roots. Water and nutrients stored in the leaf bases and their subsequent uptake by the associated aerial roots may therefore contribute significantly to this species’ tolerance of long summer droughts and extremely impoverished soils.

The fluorescent probe sulphorhodamine G (SR) has been previously used as an indicator of low extra-cellular pH and, by inference, of proton extrusion activity in living leaves. In legumes the SR uptake and proton extrusion was characteristic of the extended bundle sheath system (EBS) or paraveinal mesophyll, composed of bundle sheath cells and the related network of bridging cells between veins. This system has been identified as a site of temporary storage of amino carbon in soybean. A tree species. Populus deltoides Bartr. ex Marsh, was known both to have the EBS system in its leaves and to carry organic nitrogen in its xylem sap. It is now shown that P. deltoides also accumulates the SR probe in the EBS system. This association has been explored in 8 other broad-leaved tree species. Seven of the 8 species have EBS systems and accumulate SR in them in early summer. The 8th species, Tilia americana L. has no EBS system and shows weak SR accumulation. The capacity to accumulate SR (and by inference to scavenge solutes from the transpiration stream) disappeared in all species at various stages in late summer. In two species, in addition, SR accumulation is interrupted for several weeks during fruit growth. It is proposed that EBS systems will be found in many dicotyledonous leaves, and will be found to scavenge solutes, especially organic nitrogen, from the xylem sap.

Mesophyll and bundle sheath chloroplasts were isolated by differential grinding from the leaves of two NADP-ME C4 plants, Setaria italica Beauv. cv. H-1, Pennisetum typhoides S & H. cv. AKP-2, and a NAD-ME C4 species Amaranthus paniculatus L. The mesophyll chloroplasts of C4 plants possessed slightly lower Km for ADP and Pi than those of bundle sheath chloroplasts. The Hill reaction activities and noncyclic photophosphorylation rates of the bundle sheath chloropiasts from S. italica and P. typhoides were less than one-fifth of those by the mesophyll chloroplasts. But the bundle sheath chloroplasts of A. paniculatus exhibited high rates of Hill reaction, cyclic as well as noncyclic photophosphorylation. The pigment- and eyiochrome composition suggested a relative enrichment of PS 1 in bundle sheath chloroplasts of S. italica and P. typhoides. The chain exists in both mesophyll and bundle sheath chloroplasts. As much as 35–52% of leaf chlorophyll was located in the bundle sheath chloroplasts. The photochemical activities of bundle sheath chloroplasts are significant though a major part of leaf photochemical potential is associated with the mesophyll chloroplasts.

Dimorphic chloroplasts of Zea mays L. cv. GH5004 from bundle sheath and mesophyll cells contained similar amounts of DNA, while bundle sheath chloroplasts contained twice the number of nucleoids compared to mesophyll chloroplasts. On average bundle sheath nucleoids were half the size of mesophyll nucleoids and contained half as much DNA. Electron microscope autoradiography of the chloroplasts showed that the nucleoid DNA is associated with the thylakoids and in the case of mesophyll chloroplasts preferentially with the grana. These observations suggest that the differences in nucleoid distribution may be due to differences in membrane morphology, with the small nucleoids of agranal bundle sheath chloroplasts being widely dispersed.

Basipetal transport of [5-3H] IAA in the wheat coleoptile 3H-IAA was applied to the tips of intact wheat coleoptiles (Triticum sativum L., var. Capitole) for 30, 60 or 120 min. The quantity of label per segment (1.5 mm), determined by liquid scintillation counting, was a decreasing exponential function of distance from the source of radioactivity. Chromatographic analysis showed that 3H-IAA was transported more rapidly than its tritiated metabolites. Antoradiographs of semi-thin sections were performed after application of 3H-IAA during 2 h and after treatment by DCC [1-(3-dimethyl-aminopropyl)-3-ethycarbodiimide hydrochloride]. The quantity of label per tissue and per cell was determined at 1.5, 3.0 and 4.5 mm from the tip. Tritiated IAA was estimated to account for 70 to 100% of the label, depending on the distance from the tip. The amount of auxin transported basipetally was greatest in the parenchyma, with lesser amounts transported in the inner and outer epidermis. Tissues of the vascular bundles did not contain more than 10% of total radioactivity of the coleoptile sections. The rate of auxin transport was greater in xylem and parenchyma than in epidermis and bundle sheath. Movement was very slow in procambium and phloem. In parenchyma and epidermis the quantity of label per cell as a function of distance from the tip agreed with the model of exponential regression. These results support the theory of polar diffusion of auxin.