The developmental patterns of lysosomal enzyme activities during Ca2+-induced sporangium formation in Achlya bisexualis

The present paper describes intracellular changes in ribonuclease specific activity during Ca²⁺-induced sporangium formation in the water mold Achlya bisexualis. The enzymes undergo a decrease in activity prior to crosswall formation followed by an increase in activity during spore cleavage. As spore discharge occurs the RNase activity again decreases. A large percentage of the nuclease activity is associated with a lysosomal-like fraction of the cell, but there is also considerably activity associated with nuclear and microsomal fractions. Addition of cycloheximide or actinomycin D at various times during development prevents further decrease or increase in the enzyme activity. Mixing of cell extracts from different developmental stages provides evidence that inhibitors or activators of the enzyme activity are not responsible for the activity levels evident at the different stages. There is a change in the total levels of presumptive mRNA during Ca²⁺-induced sporangial formation which appears to be associated with the patterns of RNase activity. Utilizing total cellular RNA and Poly(A)⁺ RNA with the crude ribonuclease preparations, no substrate specificity could be ascertained.     

Patterned distribution of membrane-associated Ca2+ during pore formation inMicrasterias

Summary During the stage of pore formation developing cells ofMicrasterias denticulata show a patterned distribution of fluorescent dots on the plasma membrane after treatment with chlorotetracycline. The center-to-center spacing of these dots corresponds with the distances between the individual cell wall pores ofMicrasterias. Therefore it is supposed that the patterned distribution of pores and their formation which is mediated by special pore vesicles are related to local accumulations of membrane-associated Ca²⁺. Membrane-associated Ca²⁺ seems not only to be functional in tip growth but to be a general mediator for recognition and fusion processes between various vesicles and the plasma membrane.     

The role of carbon dioxide and oxygen in determining chlorophyll fluorescence quenching during leaf development

Chlorophyll fluorescence emission at 680 nm (F680) and the rate of CO₂ fixation were measured simultaneously in sections along the length of wheat and maize leaves. These leaves possess a basal meristem and show a gradation in development towards the leaf tip. The redox state of the primary electron acceptor, Q, of photosystem II was estimated using a non-invasive method. Distal mature leaf sections displayed typical F680 induction curves which were generally anti-parallel with CO₂ fixation and during which Q became gradually oxidised. In leaf-base sections net assimilation of CO₂ was not detectable, F680 quenched slowly and monotonously without displaying any of the oscillations typical of mature tissue and Q remained relatively reduced. Sections cut from mid-regions of the leaf showed intermediate characteristics. There were no major differences between the wheat and maize leaf in the parameters measured. The results support the hypothesis that generation of the transthylakoid proton gradient and associated ATP production is not a major limitation to photosynthesis during leaf development in either C₃ or C₄ plants. Removal of CO₂ from the mature leaf sections caused little change in steady-state F680 and produced about 50% reduction of Q. When O₂ was then removed, F680 rose sharply and Q became almost totally reduced. In immature tissue unable to assimilate CO₂, removal of O₂ alone caused a similar large rise in F680 and reduction of Q whilst removal of CO₂ had negligible effects on F680 and the redox state of Q. It is concluded that in leaf tissue unable to assimilate CO₂, either because CO₂ is absent or the tissue is immature, O₂ acts as an electron acceptor and maintains Q in a partially oxidised state. The important implication that O₂ may have a role in the prevention of photoinhibition of the photochemical apparatus in the developing leaf is discussed.Abbreviations F680 chlorophyll fluorescence emission at 680 nm - PSI photosystem I - PSII photosystem II - Q PSII primary electron acceptor - pH transthylakoid proton gradient     

Constancy of organellar genome copy numbers during leaf development and senescence in higher plants

In higher plants, plastid and mitochondrial genomes occur at high copy numbers per cell. Several recent publications have suggested that, in higher plants like Arabidopsis and maize, chloroplast DNA is virtually absent in mature and old leaves. This conclusion was mainly based on DAPI staining of isolated chloroplasts. If correct, the finding that chloroplasts in mature leaves lack DNA would change dramatically our understanding of gene expression, mRNA stability and protein stability in chloroplasts. In view of the wide implications that the disposal of chloroplast DNA during leaf development would have, we have reinvestigated the age dependency of genome copy numbers in chloroplasts and, in addition, tested for possible changes in mitochondrial genome copy number during plant development. Analyzing chloroplast and mitochondrial DNA amounts in Arabidopsis and tobacco plants, we find that organellar genome copy numbers remain remarkably constant during leaf development and are present in essentially unchanged numbers even in the senescing leaves. We conclude that, during leaf development, organellar gene expression in higher plants is not significantly regulated at the level of genome copy number and we discuss possible explanations for the failure to detect DNA in isolated chloroplasts stained with DAPI.     

Changes in the activities of chloroplast and cytosolic isoenzymes of glutamine synthetase during normal leaf growth and plastid development in wheat

Soluble protein extracts and chloroplasts from a serial sequence of transverse sections of a 7-d-old wheat leaf (Triticum aestivum cv. Maris Huntsman) were used to study changes in the activity of glutamine synthetase (GS; EC 6.3.1.2) during cell and chloroplast development. Glutamine synthetase activity increased more than 50-fold per cell from the base to the tip of the wheat leaf. Two isoenzymes of GS were separated using fast protein liquid chromatography (FPLC). Glutamine synthetase localized in the cytoplasm (GS1) eluted at about 0.21 M NaCl, and the isoenzyme localized in the chloroplast (GS2) eluted at about 0.33 M NaCl. The increase in GS activity during leaf development was found to be caused primarily by an increase in the activity of the chloroplast GS2. The activity of the cytoplasmic GS1 remained constant as the cells were displaced from the base to the tip of the leaf, whereas GS2 activity increased within the chloroplast throughout development. At the base of the leaf, 26% of total GS activity was cytoplasmic; the remaining 74% was in the chloroplast. At 10 cm from the base, only 4% of the activity was cytoplasmic, and 96% was in the chloroplast. The results indicate that the chloroplast GS2 is probably responsible for most of the ammonia assimilation in the mature wheat leaf, whereas cytoplasmic GS1 may serve a role in immature developing leaf cells.Abbreviations FPLC fast protein liquid chromatography - GS glutamine synthetase - GS1 cytoplasmic glutamine synthetase - GS2 chloroplast glutamine synthetase     

Changes in the density of microtubular networks in mesophyll cells and mesophyll derived protoplasts of Nicotiana and Triticum during leaf development

Changes in the density of microtubular mesh-works were analysed in mesophyll cells and mesophyll derived protoplasts of Nicotiana tabacum L. and Triticum aestivum L. during leaf development. The main purpose of this study was to test whether the low density, if not lack, of microtubular networks recently described in protoplasts that had been isolated from fully differentiated mesophyll cells happened during protoplast isolation or whether the loss of microtubules actually occurred during differentiation of the leaf tissue. Immunofluorescence microscopy showed that the density of the microtubular cytoskeleton in the leaf tissue decreased steadily after cessation of cell growth in both species. Nevertheless, in Triticum microtubule disappearance was swifter and occurred along a gradient from the base to tip of the leaf, a phenomenon reflecting the differences in the ontogeny between the dicotyledonous Nicotiana and the mono-cotyledonous Triticum leaves. Protein extraction from leaf tissues and Western blot analysis indicated that in both species the disappearance of microtubules was the result of a degradation of tubulin and not only due to a depolymerisation into tubulin subunits. When the cell walls were removed from live cells and the protoplasts released, the original patterns of the microtubules became obscured and, particularly in differentiated cells, the integrity and density of the microtubule strands deteriorated. The potential application of the density of the microtubular cytoskeleton as a marker in studies on differentiation and dedifferentiation in mesophyll cells and protoplasts is discussed.We wish to thank Silke Heichel for excellent technical assistance. We also express our thanks to the group of A.M. Lambert at CNRS, Strasbourg, France, for advice during establishment of our Western blot system. The work was supported by a grant of the German Ministry of Science and Technology (BMFT).     

Behavior of organelles and their nucleoids in the shoot apical meristem during leaf development in Arabidopsis thaliana L.

The behavior of organelle nucleoids and cell nuclei was studied in the shoot apical meristem and developing first foliage leaves of Arabidopsis thaliana. Samples were embedded in Technovit 7100 resin, cut into thin sections and stained with 4-6-diamidino-2-phenylindole to observe DNA. Fluorimetry was performed using a video-intensified microscope photon-counting system. The DNA content of individual mitochondria was more than 1 Mbp in the shoot apical meristem and the young leaf primordium, and decreased to approximately 170 kbp in the mature foliage leaf. In contrast, the DNA content of individual plastids was low in the shoot apical meristem and increased until day 7 after sowing. Application of 5-bromo-2-deoxyuridine, an analogue of thymidine, was usesd to investigate DNA synthesis in situ. The activities of DNA synthesis in the mitochondria and plastids changed according to the stage of development. Mitochondrial DNA was actively synthesized in the shoot apical meristem and young leaf primordia. This strongly suggests that the amount of mitochondrial DNA per mitochondrion, which has been synthesized in the shoot apical meristem and young leaf primordium, is gradually reduced due to continual divisions of the mitochondria during low levels of mitochondrial DNA synthesis. Synthesis of DNA in the plastid became active in the leaf primordia following DNA synthesis in the mitochondria, and the small plastids were filled with large plastid nucleotids. This enlargement of the plastid nucleoids occurred before the synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase and the development of thylakoids.Abbreviations BrdU 5-bromo-2-deoxyuridine - DAPI 4-6-diamidino-2-phenylindole - DiOC₆a 3,3-dihexyloxacarbocyanine - mtDNA mitochondrial DNA - mt-nucleoid mitochondrial nucleoid - ptDNA plastid DNA - pt-nucleoid plastid nucleoid - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase This work was supported by grant No. 2553 to M.F. and Nos. 04454019, 03304005 and 06262204 to T.K. from the Ministry of Education, Science and Culture of Japan, and by a grant for a pioneering research project in biotechnology from the Ministry of Agriculture, Forestry and Fisheries of Japan.     

Cellular basis of developmental plasticity observed in heterophyllous leaf formation of Ludwigia arcuata (Onagraceae)

When heterophyllous plants of Ludwigia arcuata Walt. (Onagraceae) were transferred from aerial condition to submergence, young developing leaves were matured into leaves with intermediate shape between aerial-type and submerged-type, showing spatulate shape (spoon-shaped). This change was also induced by the exposure of plants to ethylene. On the other hand, when the plants were transferred from submergence to aerial conditions, young developing leaves were matured into intermediate-type leaves with elliptic shape (spearhead shape). Anatomical analysis revealed that the formation of spatulate leaf was caused by the reduction of the number of epidermal cells aligned in the leaf transverse direction in the basal region of the leaf while the tip regions remained as before and did not respond to this treatment. During development, the ethylene-induced spatulate leaves showed that three types of alterations in epidermal cell division were involved in this process. Changes in the distribution of cell divisions in leaf lamina were detected by the first day of ethylene exposure, and changes in the orientation of cell division planes were detected by the second day. However, changes in the number of cells aligned in the leaf transverse direction were not detected by this time. Three days after ethylene exposure, frequency of cell divisions changed, and by the time changes of cell numbers aligned in the leaf transverse direction were observed. Thus, the formation of intermediate-type leaves in L. arcuata was ascribed to the alterations of cell division patterns which was induced by ethylene.     

14C fixation,metabolic labeling patterns,and translocation profiles during leaf development in Populus deltoides

The incorporation of photosynthetically fixed ¹⁴CO₂ and the distribution of ¹⁴C among the main chemical constituents of laminae and petioles were examined in cottonwood (Populus deltoides Bartr. ex Marsh.) leaves ranging in age from Leaf Plastochron Index (LPI) 3 (about one-quarter to one-third expanded) to LPI 30 (beginning of senescence). In addition, carbon flow among chemical fractions and translocation from leaves of LPI 7 and 14 were examined periodically up to 24 h after labeling. Specific activity of ¹⁴C (on dry-weight basis) increased in developing laminae to full leaf expansion, decreased in the mature leaves to LPI 16, then remained constant to LPI 30. In developing leaves (LPI 3-5), after 2 h, most of the ¹⁴C was found in protein, pigments, lipids, and other structural and metabolic components necessary for cell development; only 28% was in the sugar fraction of the lamina. In fully expanded leaves (LPI 6-8), after 2 h, the sugar fraction contained 50–60% and about 90% of fixed ¹⁴C in the lamina and the petiole, respectively. In a pulsechase kinetic series with recently mature leaves, 60% of the ¹⁴C was found in the sugar fraction after 15 min of ¹⁴CO₂ fixation. Over the 24-h translocation period, ¹⁴C decreased in sugars to 23% and increased in the combined residue fraction (protein, starch, and structural carbohydrates) to about 60% of the total activity left in the lamina. Within 24 h after labeling, the turnover of ¹⁴C-organic acids,-sugar, and-amino acids (either metabolzed or translocated from the leaf) was 30, 70 and 80%, respectively, of that initially incorporated into these fractions by a leaf at LPI 7 (turnover was 55% of ¹⁴C-organic acids, 80% of ¹⁴C-sugar, and 95% of ¹⁴C-amino acids at LPI 14). Anatomical maturity in cottonwood leaves is closely correlated with physiological maturity and with production of translocatable sugar.Abbreviations LPI leaf plastochron index - PI plastochron index Research Plant Physiologist and Chief Plant Physiologist, respectively     

Veratridine induces a Ca2+ influx,cyclic GMP formation,and backward swimming inParamecium tetraurelia wildtype cells and Ca2+ current-deficient pawn mutant cells

Summary Veratridine opens voltage-dependent Na⁺ channels in many metazoans. InParamecium, which has voltage-dependent Ca²⁺ channels and a Ca/K action potential, no such Na⁺ channels are known. A Ca-inward current is correlated to an intracellular increase in cGMP. The addition of veratridine toParamecium wildtype and to pawn mutant cells, which lack the Ca-inward current, transiently increased intracellular levels of cGMP about sevenfold to 40 pmol/mg protein. A half-maximal effect was obtained with 250 m veratridine. The increase in cGMP was maximal about 15 sec after the addition of veratridine and declined rapidly afterwards. Intracellular cAMP levels were not affected. The effect of veratridine on cGMP was dependent on the presence of extracellular Ca²⁺. The time dependence and extent of stimulation closely resembled the effects observed after stimulation by Ba²⁺, which causes the repetitive firing of action potentials, Ca-dependent ciliary reversal, and cGMP formation. The effects of Ba²⁺ and veratridine were not additive. Wildtype cells and, surprisingly, also pawn mutant cells showed avoiding reactions upon addition of veratridine indicating that it induced a Ca²⁺ influx into the cilia, which causes ciliary reversal. The potency of veratridine to stimulate cGMP formation was little affected by Na⁺ in wildtype cells, three pawn mutant strains, and in the cell line fast-2, which is defective in a Ca-dependent Na-inward current. Divalent cations (Ca²⁺, Mg²⁺, and Ba²⁺) inhibited the effects the veratridine similar to metazoan cells. The results indicate that veratridine can open the voltage-operated Ca²⁺ channels inParamecium wildtype and, most interestingly, in pawn mutant cells. The pawn mutation is suggested to represent a defect in the activation of the Ca²⁺ channel. This explains the lack of differences in ciliary proteins between wildtype and pawn cells reported earlier.     

Alterations in Ca2+-channels during the development of diabetic cardiomyopathy

In order to examine the status of Ca²⁺ channels in heart sarcolemma during the development of diabetes, rats were injected intravenously with 65 mg/kg streptozotocin and hearts were removed 1, 3 and 8 weeks later. Crude membranes from the ventricular muscle were prepared and the specific binding of ³H-nitrendipine was studied by employing different concentrations of this Ca ²⁺-antagonist. A significant decrease in both dissociation constant and maximal number of 3H-nitrendipine binding was observed in 3 and 8 weeks diabetic preparations. No such alterations were evident in diabetic brain membranes. Treatment of diabetic animals with insulin prevented the occurrence of these changes in the myocardium. The altered ³H-nitrendipine binding characteristics in diabetic heart membranes may not be due to the high levels of circulating catecholamines in this experimental model because no such changes were seen upon injecting a high dose (40 mg/kg) of isoproterenol in rats for 24 hr. The reduced number of ³H-nitrendipine binding sites may decrease Ca²⁺-influx through voltage sensitive Ca²⁺ channels and partly explain the depressed cardiac contractile force development in chronic diabetes whereas the increased affinity of Ca²⁺ channels may partly explain the increased sensitivity of diabetic heart to Ca ²⁺.     

Analysis of the Ca2+ domain in the Arabidopsis H+/Ca2+ antiporters CAX1 and CAX3

Ca²⁺ levels in plants are controlled in part by H⁺/Ca²⁺ exchangers. Structure/function analysis of the Arabidopsis H⁺/cation exchanger, CAX1, revealed that a nine amino acid region (87–95) is involved in CAX1-mediated Ca²⁺ specificity. CAX3 is 77% identical (93% similar) to CAX1, and when expressed in yeast, localizes to the vacuole but does not suppress yeast mutants defective in vacuolar Ca²⁺ transport. Transgenic tobacco plants expressing CAX3 containing the 9 amino acid Ca²⁺ domain (Cad) from CAX1 (CAX3-9) displayed altered stress sensitivities similar to CAX1-expressing plants, whereas CAX3-9-expressing plants did not have any altered stress sensitivities. A single leucine-to-isoleucine change at position 87 (CAX3-I) within the Cad of CAX3 allows this protein to weakly transport Ca²⁺ in yeast (less than 10% of CAX1). Site-directed mutagenesis of the leucine in the CAX3 Cad demonstrated that no amino acid change tested could confer more activity than CAX3-I. Transport studies in yeast demonstrated that the first three amino acids of the CAX1 Cad could confer twice the Ca²⁺ transport capability compared to CAX3-I. The entire Cad of CAX3 (87–95) inserted into CAX1 abolishes CAX1-mediated Ca²⁺ transport. However, single, double, or triple amino acid replacements within the native CAX1 Cad did not block CAX1 mediated Ca²⁺ transport. Together these findings suggest that other domains within CAX1 and CAX3 influence Ca²⁺ transport. This study has implications for the ability to engineer CAX-mediated transport in plants by manipulating Cad residues.     

Distinct leaf developmental and gene expression responses to light quantity depend on blue-photoreceptor or plastid-derived signals,and can occur in the absence of phototropins

Leaf palisade cell development and the composition of chloroplasts respond to the fluence rate of light to maximise photosynthetic light capture while minimising photodamage. The underlying light sensory mechanisms are probably multiple and remain only partially understood. Phototropins (PHOT1 and PHOT2) are blue light receptors regulating responses which are light quantity-dependent and which include the control of leaf expansion. Here we show that genes for proteins in the reaction centres show long-term responses in wild type plants, and single blue photoreceptor mutants, to light fluence rate consistent with regulation by photosynthetic redox signals. Using contrasting intensities of white or broad-band red or blue light, we observe that increased fluence rate results in thicker leaves and greater number of palisade cells, but the anticlinal elongation of those cells is specifically responsive to the fluence rate of blue light. This palisade cell elongation response is still quantitatively normal in fully light-exposed regions of phot1 phot2 double mutants under increased fluence rate of white light. Plants grown at high light display elevated expression of RBCS (for the Rubisco small subunit) which, together with expected down-regulation of LHCB1 (for the photosynthetic antenna primarily of photosystem II), is also observed in phot double mutants. We conclude that an unknown blue light photoreceptor, or combination thereof, controls the development of a typical palisade cell morphology, but phototropins are not essential for either this response or acclimation-related gene expression changes. Together with previous evidence, our data further demonstrate that photosynthetic (chloroplast-derived) signals play a central role in the majority of acclimation responses.     

Changes in photosynthetic characteristics during leaf development in apple

A comprehensive developmental survey of leaf area, chlorophyll, photosynthetic rate, leaf resistance, transpiration ratio, CO₂ compensation point and photorespiration was conducted in apple. The largest changes in each of the photosynthetic characteristics studied took place during the earliest stages of leaf development, coinciding with the period of greatest leaf expansion and chlorophyll synthesis. During early development, photosynthesis increased 5-fold, reaching a maximum rate of 40 mg CO₂ dm^-2 hr^-1 at a leaf plastochron index (LPI) of 10. During this same period, leaf resistance, transpiration ratio, CO₂ compensation point and mesophyll resistance decreased, while carboxylation efficiency increased. Two especially interesting aspects of the data discussed are simultaneous changes that occur at a LPI of 10 and 12 in all of the photosynthetic characteristics examined and an apparent decrease in photorespiration as leaves age. From our results it is clear that stage of leaf development is an important factor affecting the rate of photosynthesis and photorespiration.Scientific Paper No. 5687, College of Agriculture, Washington State University, Pullman. This work is supported by the National Science Foundation Grant 80-10958 and the Columbia River Orchards Foundation.     

Non-random distribution patterns of leaf miners on oak trees

Summary Leaf-mining Stilbosis quadricustatella larvae are distributed non-randomly within leaves of their host plants, sand live oak (Quercus geminata) and water oak (Q. nigra), in north Florida. Fewer mines are found together on the same side of the mid-vein than separated, on opposite sides of the mid-vein. Larvae do not normally cross the mid-vein but create small blotch-like mines along subsidiary veins. Investigations of the usual mortality factors acting on these leaf-miner populations, including competition, parasitism, and predation, revealed no significant differences in these factors between mines separated by the mid-vein and those on the same side of the leaf. However, early leaf abscission, which kills the larvae present in the leaf, occurs significantly more frequently in cases where larvae are clustered on one leaf side. The reasons for this differential leaf abscission are not yet clear.     

Expression patterns of dachshund during head development of Gryllus bimaculatus (cricket)

We report that Gryllus bimaculatus dachshund (Gbdac), a cricket homologue of Drosophila dachshund (Dmdac), is expressed in the developing eye and brain. During brain development, Gbdac was first expressed in the medial head region, corresponding to a part of developing protocephalic region, and expressed in the primordial and adult Kenyon cells. During eye development, Gbdac was first expressed in the lateral head region, becoming to the eye primordium and a part of the deutocerebrum. Then, Gbdac was expressed in the posterior region of the eye primordium, prior to the formation of compound eyes. The expression domain shifted to the anterior domain concomitantly with the movement of morphogenetic furrows. Gbdac was also expressed in the developing optic lobes during differentiation of the retina. These expression patterns were compared with those of Dmdac. We found that although developmental processes of the Gryllus eye and brain differ from those of the Drosophila ones, the expression patterns of Gbdac are essentially similar to those of the Dmdac.     

Photosynthetic carbon metabolism during leaf ontogeny in Zea mays L.: Enzyme studies

The activities of several enzymes, including ribulose-1,5-diphosphate (RuDP) carboxylase (EC 4.1.1.39) and phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) were measured as a function of leaf age in Z. mays. Mature leaf tissue had a RuDP-carboxylase activity of 296.7 mol CO₂ g^-1 fresh weight h^-1 and a PEP-carboxylase activity of 660.6 mol CO₂ g^-1 fresh weight h^-1. In young corn leaves the activity of the two enzymes was 11 and 29%, respectively, of the mature leaves. In senescent leaf tissue, RuDP carboxylase activity declined more rapidly than that of any of the other enzymes assayed. On a relative basis the activities of NADP malic enzyme (EC 1.1.1.40), aspartate (EC 2.6.1.1) and alanine aminotransferase (EC 2.6.1.2), and NAD malate dehydrogenase (EC 1.1.1.37) exceeded those of both PEP and RuDP carboxylase in young and senescent leaf tissue. Pulse-chase labeling experiments with mature and senescent leaf tissue show that the predominant C₄ acid differs between the two leaf ages. Labeling of alanine in senescent tissue never exceeded 4% of the total ¹⁴C remaining during the chase period, while in mature leaf tissue alanine accounted for 20% of the total after 60 s in ¹²CO₂. The activity of RuDP carboxylase during leaf ontogeny in Z. mays parallels the development of the activity of this enzyme in C₃ plants.Abbreviations RuDP ribulose-1,5-diphosphate - PEP phosphoenol pyruvate - PGA 3-phosphoglycerate     

Calcium and oxalate content of the leaves of Phaseolus vulgaris at different calcium supply in relation to calcium oxalate crystal formation

Soluble and insoluble oxalate and insoluble calcium were measured in the leaves of Phaseolus vulgaris. The plants were grown in nutrient solutions with two different concentrations of calcium. Two developmental stages of the leaves were studied. Although the content of insoluble calcium differs widely according to leaf age and growth conditions, the percentage bound in crystals is nearly the same in all cases. In the growing leaves, concentrations of total oxalate are independent of calcium supply, thus, showing that the known rise in numbers of crystals, and of cells containing them, is not induced via oxalate biosynthesis. Fully expanded leaves contain more oxalate when grown in a nutrient solution with higher calcium concentration. Amounts of oxalate in percent of dry weight are similar to those given in the literature for other legume leaves.     

Regulation of photosynthesis during Arabidopsis leaf development in continuous light

Previous investigations in our laboratory have shown that leaf developmental programming in tobacco is regulated by source strength. One hypothesis to explain how source strength is perceived is that hexokinase acts as a sensor of carbohydrate flux to regulate the expression of photosynthetic genes, possibly as a result of sucrose cycling through acid invertase and hexokinase. We have turned to Arabidopsis as a model system to study leaf development and have examined various photosynthetic parameters during the ontogeny of a single leaf on the Arabidopsis rosette grown in continuous light. We found that photosynthetic rates, photosynthetic gene expression, pigment contents and total protein amounts attain peak levels early in the expansion phase of development, then decline progressively as development proceeds. In contrast, the flux of ¹⁴CO₂ into hexoses increases modestly until full expansion is attained, then falls in the fully expanded leaf. Partitioning of carbon into hexoses versus sucrose increases until full expansion is attained, then falls. The in vitro activities of hexokinase, vacuolar acid invertase, and cell wall acid invertase do not change until the late stages of senescence, when they increase markedly. At this time there are also dramatic increases in hexose pool sizes and in senescence-associated gene (SAG) expression. Taken together, our results suggest that invertase and hexokinase activities do not control the partitioning of label into hexoses during development. We conclude that our data are not readily compatible with a simple model of leaf development, whereby alterations in photosynthetic rates are mediated directly by hexose flux or by hexose pool sizes. Yet, these factors might contribute to the control of gene expression. This revised version was published online in June 2006 with corrections to the Cover Date.