Regulation of Brassica rapa chloroplast proliferation in vivo and in cultured leaf disks

Summary. To understand the regulatory mechanisms of chloroplast proliferation, chloroplast replication was studied in cultured leaf disks cut from plants of 25 species. In leaf disks from Brassica rapa var. perviridis, the number of chloroplasts per cell increased remarkably in culture. We examined chloroplast replication in this plant in vivo and in culture media with and without benzyladenine, a cytokinin. In whole plants, leaf cells undergo two phases from leaf emergence to full expansion: an early proliferative stage, in which mitosis occurs, and a differentiational stage after mitosis has diminished. During the proliferative stage, chloroplast replication keeps pace with cell division. In the differentiational phase, cell division ceases but chloroplast replication continues for two or three more cycles, with the number of chloroplasts per cell reaching about 60. In the leaf disks, the number of chloroplasts per cell increased from about 18 to 300 without benzyladenine, and to over 600 with benzyladenine, indicating that this cytokinin enhances chloroplast replication in cultured tissue. We also studied changes in ploidy and cell volume between in vivo cells and cells grown in culture with and without benzyladenine. Ploidy and cell volume increased in a manner very similar to that of the number of chloroplasts, suggesting a relationship between these phenomena.Correspondence and reprints: Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo 113-0033, Japan.     

Combined effects of light and water stress on chloroplast volume regulation.

A nuclear magnetic resonance technique was used to measure changes in the water content of Acer platanoides chloroplasts in leaf discs that had reached osmotic equilibrium with external solutions either in the dark or under exposure to light. Results showed that chloroplast volume regulation (CVR) maintained constant water content in the chloroplasts over a range of water potentials in the dark, but CVR failed when the water potential fell below a critical value. The critical potential was lower in the dark in sun leaves than in shade leaves. Upon exposure to intense light, CVR remained effective in sun leaves over the same range as in the dark, but it failed in shade leaves at all water potentials. Osmolytes are necessary for CVR, but KCl is relatively ineffective; increased concentrations of intracellular KCl did not fully support an increase in the range of CVR. The results indicate that leaves need reserve supplies of cytosolic osmolytes to maintain CVR at low water potentials, and a larger reserve supply is needed in leaves that are exposed to intense light.     

Stimulation by Light of Rapid pH Regulation in the Chloroplast Stroma in Vivo as Indicated by CO2 Solubilization in Leaves

Leaves of Brassica oleracea, Helianthus annuus, and Nicotiana rustica were exposed for 20 s to high concentrations of CO2. CO2 uptake by the leaf, which was very fast, was measured as a transient increase in the concentration of oxygen. Rapid solubilization of CO2 in excess of that which is physically dissolved in aqueous phases is proposed to be caused by bicarbonate formation in the stroma of chloroplasts, which contain carbonic anhydrase. On this basis, pH values and bicarbonate accumulation in the chloroplast stroma were calculated. Buffer capacities were far higher than expected on the basis of known concentrations in the chloroplast stroma. Moreover, apparent buffer capacities increased with the time of exposure to high CO2, and they were higher when the measurements were performed in the light than in the dark. During prolonged exposure of leaves to 16% CO2, calculated bicarbonate concentrations in the chloroplast stroma exceeded 90 mM in the dark and 120 mM in the light. The observations are interpreted as indicating that under acid stress protons are rapidly exported from the chloroplasts in exchange for cations, which are imported. The data are discussed in terms of effective metabolic pH control by ion transport, first across the chloroplast envelope and, then, across the tonoplast of leaf mesophyll cells. The direct involvement of the vacuole in the regulation of the chloroplast pH in leaf cells is suggested.     

Photosynthesis under osmotic stress

The reversibility of the inhibition of photosynthetic reactions by water stress was examined with four systems of increasing complexity—stromal enzymes, intact chloroplasts, mesophyll protoplasts, and leaf slices. The inhibition of soluble chloroplast enzymes by high solute concentrations was instantly relieved when solutes were properly diluted. In contrast, photosynthesis was not restored but actually more inhibited when isolated chloroplasts exposed to hypertonic stress were transferred to conditions optimal for photosynthesis of unstressed chloroplasts. Upon transfer, chloroplast volumes increased beyond the volumes of unstressed chloroplasts, and partial envelope rupture occurred. In protoplasts and leaf slices, considerable and rapid, but incomplete restoration of photosynthesis was observed during transfer from hypertonic to isotonic conditions. Chloroplast envelopes did not rupture in situ during water uptake. It is concluded that inhibition of photosynthesis by severe water stress is at the biochemical level brought about in part by reversible inhibition of chloroplast enzymes and in part by membrane damage which requires repair mechanisms for reversibility. Both soluble enzymes and membranes appear to be affected by the increased concentration of internal solutes, which is caused by dehydration.     

Changes in the Number and Size of Chloroplasts during Senescence of Primary Leaves of Wheat Grown under Different Conditions

Changes in the number and size of chloroplasts in mesophyllcells were investigated in primary leaves of wheat from fullexpansion to yellowing under different growth conditions. Thenumber of chloroplasts per cell decreased slowly, although thedecrease was steady and statistically significant, until thelast stage of leaf senescence, when rapid degradation of chloroplaststook place. Rates of leaf senescence, or the decline in thenumber of chloroplasts, varied greatly among plants grown atdifferent seasons of the year, but about 20% of chloroplastsalways disappeared during the phase when steady loss of chloroplastsoccurred. The area of chloroplast disks also decreased graduallybut significantly, with a rapid decrease late in senescence.Thus, the total quantity of chloroplasts per mesophyll celldecreased substantially during leaf senescence. Yellowed leavescontained numerous structures that resemble oil drops but nochloroplasts. Decreases in rates of photosynthesis that occurduring senescence may, therefore, be largely due to decreasesin the quantity of chloroplasts. However, a better correlationwas found between the decrease in the maximum capacity for photosynthesisand the degradation of RuBP carboxylase. When plants had beengrown with a sufficient supply of nutrients, the number of chloroplastsdecreased steadily but at a reduced rate and the reduction inthe area of chloroplast disks was strongly suppressed. Thus,the quantitative decrease in chloroplasts in senescing leavesappears to be regulated by the requirements for nutrients (nitrogen)of other part of the plant. ³Present address: Department of Biology, Faculty of Science,Toho University, Miyama, Funabashi, Chiba, 274 Japan     

Chloroplast osmotic adjustment and water stress effects on photosynthesis

Previous studies have suggested that chloroplast stromal volume reduction may mediate the inhibition of photosynthesis under water stress. In this study, the effects of spinach (Spinacia oleracea, var `Winter Bloomsdale') plant water deficits on chloroplast photosynthetic capacity, solute concentrations in chloroplasts, and chloroplast volume were studied. In situ (gas exchange) and in vitro measurements indicated that chloroplast photosynthetic capacity was maintained during initial leaf water potential (Ψ_w) and relative water content (RWC) decline. During the latter part of the stress period, photosynthesis dropped precipitously. Chloroplast stromal volume apparently remained constant during the initial period of decline in RWC, but as leaf Ψ_w reached −1.2 megapascals, stromal volume began to decline. The apparent maintenance of stromal volume over the initial RWC decline during a stress cycle suggested that chloroplasts are capable of osmotic adjustment in response to leaf water deficits. This hypothesis was confirmed by measuring chloroplast solute levels, which increased during stress. The results of these experiments suggest that stromal volume reduction in situ may be associated with loss of photosynthetic capacity and that one mechanism of photosynthetic acclimation to low Ψ_w may involve stromal volume maintenance.     

The leaf anatomy of a broad-leaved evergreen allows an increase in leaf nitrogen content in winter

In temperate regions, evergreen species are exposed to large seasonal changes in air temperature and irradiance. They change photosynthetic characteristics of leaves responding to such environmental changes. Recent studies have suggested that photosynthetic acclimation is strongly constrained by leaf anatomy such as leaf thickness, mesophyll and chloroplast surface facing the intercellular space, and the chloroplast volume. We studied how these parameters of leaf anatomy are related with photosynthetic seasonal acclimation. We evaluated differential effects of winter and summer irradiance on leaf anatomy and photosynthesis. Using a broad-leaved evergreen Aucuba japonica , we performed a transfer experiment in which irradiance regimes were changed at the beginning of autumn and of spring. We found that a vacant space on mesophyll surface in summer enabled chloroplast volume to increase in winter. The leaf nitrogen and Rubisco content were higher in winter than in summer. They were correlated significantly with chloroplast volume and with chloroplast surface area facing the intercellular space. Thus, summer leaves were thicker than needed to accommodate mesophyll surface chloroplasts at this time of year but this allowed for increases in mesophyll surface chloroplasts in the winter. It appears that summer leaf anatomical characteristics help facilitate photosynthetic acclimation to winter conditions. Photosynthetic capacity and photosynthetic nitrogen use efficiency were lower in winter than in summer but it appears that these reductions were partially compensated by higher Rubisco contents and mesophyll surface chloroplast area in winter foliage.     

Chloroplast Response to Low Leaf Water Potentials: III. Differing Inhibition of Electron Transport and Photophosphorylation

Cyclic and noncyclic photophosphorylation and electron transport by photosystem 1, photosystem 2, and from water to methyl viologen (“whole chain”) were studied in chloroplasts isolated from sunflower (Helianthus annus L. var Russian Mammoth) leaves that had been desiccated to varying degrees. Electron transport showed considerable inhibition at leaf water potentials of −9 bars when the chloroplasts were exposed to an uncoupler in vitro, and it continued to decline in activity as leaf water potentials decreased. Electron transport by photosystem 2 and coupled electron transport by photosystem 1 and the whole chain were unaffected at leaf water potentials of −10 to −11 bars but became progressively inhibited between leaf water potentials of −11 and −17 bars. A low, stable activity remained at leaf water potentials below −17 bars. In contrast, both types of photophosphorylation were unaffected by leaf water potentials of −10 to −11 bars, but then ultimately became zero at leaf water potentials of −17 bars. Although the chloroplasts isolated from the desiccated leaves were coupled at leaf water potentials of −11 to −12 bars, they became progressively uncoupled as leaf water potentials decreased to −17 bars. Abscisic acid and ribonuclease had no effect on chloroplast photophosphorylation. The results are generally consistent with the idea that chloroplast activity begins to decrease at the same leaf water potentials that cause stomatal closure in sunflower leaves and that chloroplast electron transport begins to limit photosynthesis at leaf water potentials below about −11 bars. However, it suggests that, during severe desiccation, the limitation may shift from electron transport to photophosphorylation.     

Abscisic acid immunolocalization and ultrastructural changes in water-stressed lavender (Lavandula stoechas L.) plants

The relationship between the bulk abscisic acid (ABA) content, ABA compartmental redistribution, and chloroplast ultrastructural changes was studied in leaves of lavender ( Lavandula stoechas L.) plants subjected to water stress. ABA was uniformly distributed in the cytosol, nucleus, chloroplasts, and cell walls of mesophyll cells in well-watered plants. In plants subjected to water stress (−2.6 MPa water potential) the bulk leaf ABA increased from 900 to 3 600 pmol g⁻¹ fresh weight. At the ultrastructural level, the first indication of this rise in ABA was a 4-fold increase in ABA immunolabeling in the cell wall in which the highest labeling values were recorded. This increase in apoplastic ABA in lavender was not attributable to ABA release from the chloroplast, because a simultaneous increase in ABA labeling was observed in both the chloroplast and nucleus (2- and 3-fold, respectively). Water stress induced a progressive increase in bulk leaf ABA concentration to 13 600 pmol g⁻¹ fresh weight coincident, with the highest immunolabeling of ABA in the nucleus and chloroplast. Under severe water stress, the chloroplast membrane broke down, resulting in leakage of ABA from the chloroplast. The stress-induced increase of ABA in chloroplasts and nuclei may serve a function other than affecting stomatal movement.     

The localization of (3H) thymidine incorporation in the DNA of replicating spinach chloroplasts by electron-microscope autoradiography.

Electron-microscope autoradiography has been used to obtain information on the localization of DNA labelled with [3H]thymidine in chloroplasts known to be replicating and concomitantly synthesizing and segregating DNA, in cultured leaf disks. The studies were made using both Microdol-X developer and a 'compact' developer which gave a smaller grain size. About 80% of the grains were associated with the granal membranes and with presumptive DNA regions (3-nm fibril material in clear areas). Few grains occurred in association with the chloroplast envelope. We suggest that the DNA of chloroplasts is associated with the grana lamellae and extends into the stroma. Some light-microscope autoradiographs of whole chloroplasts show spiral or helical-like labelling patterns. We interpret these patterns as demonstration of the possibility that DNA occurs along the length of a continuous lamellar membrane system. Chloroplast fractionation experiments provided data consistent with the electron-microscope autoradiographic studies as most of the label was associated with chlorophyll-containing lamellae. We consider an association of chloroplast DNA molecules along the length of a continuous lamellar system would ensure an orderly segregation of DNA to daughter chloroplasts, during the binary fission of spinach chloroplasts by constriction division.     

Control of starch granule numbers in Arabidopsis chloroplasts

The aim of this work was to investigate starch granule numbers in Arabidopsis (Arabidopsis thaliana) leaves. Lack of quantitative information on the extent of genetic, temporal, developmental, and environmental variation in granule numbers is an important limitation in understanding control of starch degradation and the mechanism of granule initiation. Two methods were developed for reliable estimation of numbers of granules per chloroplast. First, direct measurements were made on large series of consecutive sections of mesophyll tissue obtained by focused ion beam-scanning electron microscopy. Second, average numbers were calculated from the starch contents of leaves and chloroplasts and estimates of granule mass based on granule dimensions. Examination of wild-type plants and accumulation and regulation of chloroplast (arc) mutants with few, large chloroplasts provided the following new insights. There is wide variation in chloroplast volumes in cells of wild-type leaves. Granule numbers per chloroplast are correlated with chloroplast volume, i.e. large chloroplasts have more granules than small chloroplasts. Mature leaves of wild-type plants and arc mutants have approximately the same number of granules per unit volume of stroma, regardless of the size and number of chloroplasts per cell. Granule numbers per unit volume of stroma are also relatively constant in immature leaves but are greater than in mature leaves. Granule initiation occurs as chloroplasts divide in immature leaves, but relatively little initiation occurs in mature leaves. Changes in leaf starch content over the diurnal cycle are largely brought about by changes in the volume of a fixed number of granules.     

A plant-specific protein essential for blue-light-induced chloroplast movements

In Arabidopsis (Arabidopsis thaliana), light-dependent chloroplast movements are induced by blue light. When exposed to low fluence rates of light, chloroplasts accumulate in periclinal layers perpendicular to the direction of light, presumably to optimize light absorption by exposing more chloroplast area to the light. Under high light conditions, chloroplasts become positioned parallel to the incoming light in a response that can reduce exposure to light intensities that may damage the photosynthetic machinery. To identify components of the pathway downstream of the photoreceptors that mediate chloroplast movements (i.e. phototropins), we conducted a mutant screen that has led to the isolation of several Arabidopsis mutants displaying altered chloroplast movements. The plastid movement impaired1 (pmi1) mutant exhibits severely attenuated chloroplast movements under all tested fluence rates of light, suggesting that it is a necessary component for both the low- and high-light-dependant chloroplast movement responses. Analysis of pmi1 leaf cross sections revealed that regardless of the light condition, chloroplasts are more evenly distributed in leaf mesophyll cells than in the wild type. The pmi1-1 mutant was found to contain a single nonsense mutation within the open reading frame of At1g42550. This gene encodes a plant-specific protein of unknown function that appears to be conserved among angiosperms. Sequence analysis of the protein suggests that it may be involved in calcium-mediated signal transduction, possibly through protein-protein interactions.     

Osmotic adjustment by intact isolated chloroplasts in response to osmotic stress and its effect on photosynthesis and chloroplast volume

Spinach leaf chloroplasts isolated in isotonic media (330 millimolar sorbitol, −1.0 megapascals osmotic potential) had optimum rates of photosynthesis when assayed at −1.0 megapascals. When chloroplasts were isolated in hypertonic media (720 millimolar sorbitol, −2.0 megapascals osmotic potential) the optimum osmotic potential for photosynthesis was shifted to −1.8 megapascals and the chloroplasts had higher rates of CO₂-dependent O₂ evolution than chloroplasts isolated in 330 millimolar sorbitol when both were assayed at high solute concentrations.

Transfer of chloroplasts isolated in 330 millimolar sorbitol to 720 millimolar sorbitol resulted in decreased chloroplast volume but this shrinkage was only transient and the chloroplasts subsequently swelled so that within 2 to 3 minutes at 20°C the chloroplast volume had returned to near the original value. Thus, actual steady state chloroplast volume was not decreased in hypertonic media. In isotonic media, there was a slow but significant uptake of sorbitol by chloroplasts (10 to 20 micromoles per milligram chlorophyll per hour at 20°C). Transfer of chloroplasts from 330 millimolar sorbitol to 720 millimolar sorbitol resulted in rapid uptake of sorbitol (up to 280 micromoles per milligram chlorophyll per hour at 20°C) and after 5 minutes the concentration of sorbitol inside the chloroplasts exceeded 500 millimolar. This uptake of sorbitol resulted in a significant underestimation of chloroplast volume unless [¹⁴C]sorbitol was added just prior to centrifuging the chloroplasts through silicone oil. Sudden exposure to osmotic stress apparently induced a transient change in the permeability of the chloroplast envelope since addition of [¹⁴C]sorbitol 3 minutes after transfer to hypertonic media (when chloroplast volume had returned to normal) did not result in rapid uptake of labeled sorbitol.

It is concluded that chloroplasts can osmotically adjust in vitro by uptake of solutes which do not normally penetrate the chloroplast envelope, resulting in a restoration of normal chloroplast volume and partially preventing the inhibition of photosynthesis by high solute concentrations. The results indicate the importance of matching the osmotic potential of isolation media to that of the tissue, particularly in studies of stress physiology.

     

Two Isoforms of Dihydroxyacetone Phosphate Reductase from the Chloroplasts of Dunaliella tertiolecta

Three isoforms of dihydroxyacetone phosphate reductase in extracts from Dunaliella tertiolecta have been separated by a diethylaminoethyl cellulose column chromatography with a shallow NaCl gradient. The chloroplasts contained the two major isoforms, and the third, minor form was in the cytosol. The isoforms are unstable in the absence of glycerol and they are cold labile, but they may be partially reactivated at 35[deg]C. The first chloroplast form to elute from the DEAE cellulose column was the major form when the cells were grown on high NaCl and it has been referred to as the form for glycerol production for osmoregulation or "osmoregulator form." The second form increased in specific activity when inorganic phosphate was increased in the growth media to stimulate growth, and it has been given the designation for the form for glyceride synthesis, "glyceride form." The osmoregulator form was stimulated by NaCl added to the enzyme assay, but not by reduced Escherichia coli thioredoxin. The glyceride form had properties similar to the enzyme in leaf chloroplast, such as inhibition by NaCl and by fatty acyl-coenzyme A derivatives and some stimulation by dithiothreitol, uridine diphosphate galactose, cyti-dine diphosphate dipalmatoyl diglyceride, and reduced E. coli thioredoxin. Thus, Dunaliella chloroplasts have a salt-stimulated osmoregulatory form of dihydroxyacetone phosphate reductase, which seems to have a role in glycerol production, and an isoform, which may be involved in glyceride synthesis and which has properties similar to the enzyme in chloroplasts of higher plants.     

干旱胁迫对不同耐旱性大麦品种叶片超微结构的影响

选用耐旱性不同的3个大麦(Hordeum sativum)品种作为研究对象, 分析干旱胁迫对其叶肉细胞叶绿体、线粒体和细胞核超微结构的影响。结果表明, 3个大麦品种在非胁迫条件下其超微结构无明显差异。遭受干旱胁迫后, 不耐旱大麦品种Moroc9-75叶片细胞核中染色质的凝聚程度高, 叶绿体变形, 外被膜出现较大程度的波浪状和膨胀, 同时基粒出现弯曲、膨胀、排列混乱的现象; 线粒体外形及膜受到破坏、内部嵴部分消失等。耐旱大麦品种HS41-1叶片细胞中染色质虽出现凝聚, 但凝聚程度低; 其叶绿体及线粒体与非胁迫条件下基本相似, 多数未见明显损伤。耐旱中等的大麦品种Martin叶片超微结构的变化则介于二者之间。因此, 干旱胁迫下叶绿体外形、基粒和基质类囊体膜结构的完整性与基粒的排列次序、染色质的凝聚度和线粒体膜及嵴的完整性与大麦的耐旱性相关, 这些特性可作为评价大麦耐旱性强弱的形态结构指标。     

Clumping and dispersal of chloroplasts in succulent plants

Plants have evolved various photoprotective mechanisms to mitigate photodamage. Here we report the diurnal movement of chloroplasts in the leaves of succulent crassulacean acid metabolism (CAM) plants under combined light and water stress. In leaves of water-stressed plants, the chloroplasts became densely clumped in one or sometimes two areas in the cytoplasm under light and dispersed during darkness. The chloroplast clumping resulted in leaf optical changes, with a decrease in absorptance and an increase in transmittance. The plant stress hormone abscisic acid induced chloroplast clumping in the leaf cells under light. We suggest that the marked chloroplast movement in these CAM plants is a photoprotective strategy used by the plants subjected to severe water stress.Abbreviations ABA Abscisic acid - CAM Crassulacean acid metabolism     

Nuclear magnetic resonance study of spin relaxation and magnetic field gradients in maple leaves.

1H Nuclear magnetic resonance techniques were used to measure the distributions of spin-spin relaxation times, T2, and of magnetic field gradients in both the chloroplast and nonchloroplast water compartments of maple leaves (Acer platanoides). Results showed that encounters between water molecules and membranes inside chloroplasts provide an inefficient relaxation mechanism; i.e., chloroplast membranes interact weakly with water molecules. Gradient measurements indirectly measured the sizes of chloroplasts by showing that water in the chloroplasts is confined to small compartments a few microns in diameter. A comparison between measured gradients and gradients calculated for a model leaf indicated that chloroplasts are somewhat more likely to occupy positions along cell walls adjacent to air spaces, but also they may be found in the interiors of cells.     

Isolation of mutant lines with decreased numbers of chloroplasts per cell from a tagged mutant library of the moss Physcomitrella patens

Eleven mutant lines exhibiting decreased numbers of chloroplasts per cell were isolated from 8 800 tagged mutant lines of Physcomitrella patens by microscopic observations. Chloronema subapical cells in wild-type plants had a mean of 48 chloroplasts, whereas chloroplast numbers in subapical cells in mutant lines 215 and 222 decreased to 75 % of that in the wild type. Seven mutant lines - 473, 122, 221, 129, 492, 207, and 138 - had about half as many chloroplasts as the wild type. Mutant line 11 had a few remarkably enlarged chloroplasts, and mutant line 347 had chloroplasts of various sizes. Whereas the cell volume was the same as in the wild type in mutant lines 222, 473, 221, 129, 492, and 207, the cell volume of the other mutants increased. The chloroplast number of leaf cells was the same as that of chloronema cells in each mutant line when gametophores could be formed. Treatment with ampicillin decreased the number of chloroplasts in all mutant lines. Southern hybridization using DNA in tags as probes showed that only one insertion occurred in mutant lines 473 and 221. To determine whether the tagged DNA inserted into the known genes for plastid division, we isolated the PpMinD1, PpMinD2, and PpMinE1 genes. Genomic polymerase chain reaction analysis showed that the PpFtsZ and PpMinD/E genes were not disrupted by the insertion of the tags in mutant lines 11 and 347, respectively.     

Physiological and ultrastructural responses of sour orange (Citrus aurantium L.) clones to water stress

Water stress is a major abiotic constraint leading to serious crop losses. Recently, in the Mediterranean region, water stress has become markedly sensed, especially in Citrus orchards. This study investigated the physiological responses of local sour orange (Citrus aurantium L.) clones to severe water stress. Water stress was applied by withholding irrigation during weeks, followed by a rewatering phase during three months. Under water stress, sour orange clones decreased their stomatal conductance, net photosynthetic rate, and transpiration rate. On the contrary, biomass was stable, especially in the Kliaa clone. In addition, reduced leaf water potentials (-3 MPa) and water contents were measured in most of the clones, except Kliaa which kept the highest water potential (-2.5 MPa). After rewatering, all clones recovered except of the Ghars Mrad (GM) clone. Ultrastructural observations of leaf sections by transmission electron microscopy did not reveal marked alterations in the mesophyll cells and chloroplast structure of Kliaa in comparison to the sensitive clone GM, in which palisade parenchyma cells and chloroplasts were disorganized. This contrasting behavior was mainly attributed to genetic differences as attested by molecular analysis. This study highlighted GM as the drought-sensitive clone and Kliaa as the tolerant clone able to develop an avoidance strategy based on an efficient stomatal regulation. Although a high percentage of polyembryony characterizes C. aurantium and justifies its multiplication by seeds, heterogeneous water-stress responses could be observed within sour orange plants in young orchards.     

Immunogold Localization and Quantification of Cellular and Subcellular Abscisic Acid, Prior to and During Drought Stress

An Immunogold labeling procedure and experimental data are presented, which demonstrate that antibodies produced against a bovine serum albumin-abscisic acid conjugate can be used both to characterize the cellular and subcellular localization of abscislc acid (ABA), and to permit quantitative comparisons of this hormone in the subcellular compartments prior to and at times of drought stress. At the control leaf water potential (approximately -0.45 MPa), a quantitatively similar positive labeling pattern was observed in the chloroplasts and apoplast. A twofold drought stress-induced increase in the apoplastic ABA concentration was observed in the drought stressed leaf tissue (i.e., at a leaf water potential of approximately -1.55 MPa), while the ABA concentration in the chloroplasts did not differ from that of the controls. Three histochemical controls and the physiological observations validated the specificity of the procedure. Based on the labeling patterns we observed and literature cited, the validity of the hypothesis that drought stress induces a release of chloroplastic ABA is questioned. We interpreted our results as providing indirect evidence for a drought stress-induced root source origin for the increased apoplastic ABA concentrations.