Al-hyperaccumulator Vochysiaceae from the Brazilian Cerrado store aluminum in their chloroplasts without apparent damage

We investigated the pattern of aluminum (Al) accumulation in leaf tissues of native hyperaccumulator Vochysiaceae species Qualea grandiflora,Callisthene major, and Vochysia pyramidalis, from the Brazilian Cerrado. Non-accumulator Sclerolobium paniculatum was used as a control species. We expected a strong compartmentalization of Al in non-active leaf cell compartments such as cell walls and vacuoles in Al-accumulating species and the absence of Al in critical metabolic sites such as the chloroplasts. Plant leaves were harvested in the field and cut in small segments for histological analysis; hematoxylin dye was used for Al localization in tissues. Results of soil analysis of the three sites and the concentration of Al in leaves indicated that there is no direct relationship between Al availability in soils and Al hyperaccumulation among the Vochysiaceae species evaluated. The cross-sections of leaf tissues showed hematoxylin color in the palisade and spongy parenchyma cells (chloroplast) of Q. grandiflora and C. major. The vascular system of Q. grandiflora was not colored, but some cells from the xylem region of C. major were stained. In contrast, the adaxial and abaxial epidermal cells of V. pyramidalis were colored by hematoxylin, as were some cells from the vascular bundle, but color formation was not observed in the cells of palisade parenchyma. Al was not detected in leaves of S. paniculatum. We concluded that, although hyperaccumulation of Al is a common trait in the Vochysiaceae family, the processes of storage and detoxification in leaf tissues differ among the species. Two of the three hyperaccumulator species use chloroplasts as a sink for Al, with no apparent signs of toxicity. Therefore, the physiological role of Al in plant tissues remains to be elucidated.     

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.     

SPAD chlorophyll meter reading can be pronouncedly affected by chloroplast movement

Non-destructive assessment of chlorophyll content has recently been widely done by chlorophyll meters based on measurement of leaf transmittance (e.g. the SPAD-502 chlorophyll meter measures the leaf transmittance at 650 and 940 nm). However, the leaf transmittance depends not only on the content of chlorophylls but also on their distribution in leaves. The chlorophyll distribution within leaves is co-determined by chloroplast arrangement in cells that depends on light conditions. When tobacco leaves were exposed to a strong blue light (about 340 μmol of photons m⁻² s⁻¹), a very pronounced increase in the leaf transmittance was observed as chloroplasts migrated from face position (along cell walls perpendicular to the incident light) to side position (along cell walls parallel to the incoming light) and the SPAD reading decreased markedly. This effect was more pronounced in the leaves of young tobacco plants compared with old ones; the difference between SPAD values in face and side position reached even about 35%. It is shown how the chloroplast movement changes a relationship between the SPAD readings and real chlorophyll content. For an elimination of the chloroplast movement effect, it can be recommended to measure the SPAD values in leaves with a defined chloroplasts arrangement.     

DEVELOPMENT OF THE DIMORPHIC CHLOROPLASTS OF SUGAR CANE

The vascular bundle sheath cells of sugar cane contain starch-storing chloroplasts lacking grana, whereas the adjacent mesophyll cells contain chloroplasts which store very little starch and possess abundant grana. This study was undertaken to determine the ontogeny of these dimorphic chloroplasts. Proplastids in the two cell types in the meristematic region of light-grown leaves cannot be distinguished morphologically. Bundle sheath cell chloroplasts in tissue with 50% of its future chlorophyll possess grana consisting of 2-8 thylakoids/granum. Mesophyll cell chloroplasts of the same age have better developed grana and large, well structured prolamellar bodies. A few grana are still present in bundle sheath cell chloroplasts when the leaf tissue has 75% of its eventual chlorophyll, and prolamellar bodies are also found in mesophyll cell chloroplasts at this stage. The two cell layers in mature dark-grown leaves contain morphologically distinct etio-plasts. The response of these two plastids to light treatment also differs. Plastids in tissue treated with light for short periods exhibit protrusions resembling mitochondria. Plastids in bundle sheath cells of dark-grown leaves do not go through a grana-forming stage. It is concluded that the structure of the specialized chloroplasts in bundle sheath cells of sugar cane is a result of reduction, and that the development of chloroplast dimorphism is related in some way to leaf cell differentiation.     

A Genetic Analysis of Chloroplast Division and Expansion in Arabidopsis thaliana

A nuclear recessive mutant of Arabidopsis thaliana, arc5, has been isolated in which there is no significant increase in chloroplast number during leaf mesophyll cell expansion and in which there are only 13 chloroplasts per mesophyll cell compared with 121 in wild-type cells. Mature arc5 chloroplasts in fully expanded mesophyll cells are 6-fold larger than in wild-type cells. A large proportion of arc5 chloroplasts also show some degree of central constriction, suggesting that the mutation has prevented the completion of the chloroplast division process. To examine the interaction of arc loci, a double mutant was constructed between arc1, a mutant possessing many small chloroplasts, and arc5. A second double mutant was also constructed between arc3, a previously discovered mutant also possessing few large chloroplasts per cell, and arc1. Analysis of these double mutants shows that chloroplast number per mesophyll cell is greater when arc5 and arc3 mutations are expressed in the arc1 background than when expressed alone. The cell-specific nature of arc mutants was also analyzed. The phenotypic traits characteristic of arc3 and arc5 are a reduction in chloroplast number and an increase in chloroplast size in mesophyll cells: these changes are also observed in reduced form in the epidermal and guard cell chloroplasts of arc3 and arc5 plants. Analysis of parenchyma sheath cell chloroplasts suggests that in leaves of arc1 plants the normal developmental distinction between mesophyll and parenchyma sheath chloroplasts is perturbed. The relevance of these findings to the analysis of the control of chloroplast division in mesophyll cells is discussed.     

The Heterogeneity of Structural and Functional Photosynthetic Characteristics of Mesophyll Chloroplasts in Various Parts of Mature or Senescing Leaf Blade of Two Maize (Zea Mays L.) Genotypes

Differences in ultrastructural parameters of mesophyll cell (MC) chloroplasts, contents of photosynthetic pigments, and photochemical activities of isolated MC chloroplasts were studied in the basal, middle, and apical part of mature or senescing leaf blade of two maize genotypes. A distinct heterogeneity of leaf blade was observed both for structural and functional characteristics of chloroplasts. In both mature and senescing leaves the shape of MC chloroplasts changed from flat one in basal part of leaf to nearly spherical one in leaf apex. The volume density of granal thylakoids decreased from leaf base to apex in both types of leaves examined, while the amount of intergranal thylakoids increased in mature leaves but decreased in senescing leaves. The most striking heterogeneity was found for the quantity of plastoglobuli, which strongly increased with the increasing distance from leaf base. The differences in chloroplast ultrastructure were accompanied by differences in other photosynthetic characteristics. The Hill reaction activity and activity of photosystem 1 of isolated MC chloroplasts decreased from leaf base to apex in mature leaves. Apical part of senescing leaf blade was characterised by low contents of chlorophyll (Chl) a and Chl b, whereas in mature leaves, the content of Chls as well as the content of total carotenoids (Car) slightly increased from basal to apical leaf part. This was reflected also in the ratio Chl (a+b)/total Car; the ratio of Chl a/b did not significantly differ between individual parts of leaf blade. Both genotypes examined differed in the character of developmental gradient observed along whole length of leaf blade.     

Ultrastructural aspects of kranz anatomy inDigitaria sanguinalis andSetaria viridis (poaceae)

Structural differentiation of Kranz anatomy has been investigated in leaf cross sections of two C-4 Poaceae:Digitaria sanguinalis andSetaria viridis. The study mainly focused on cellular and interfacial features of bundle sheath (BS) and mesophyll (MS) cells of the C-4 structure. Prominent BS, spaced by only two MS cells apart, were surrounded concentrically by a layer of MS cells. BS cells ofS. viridis had centrifugally arranged relatively large chloroplasts containing much starch, but the chloroplasts had agrana to rudimentary grana. Structural and size dimorphisms, when starch was present, were detected between BS and MS chloroplasts. Loosely arranged MS cells had peripherally displaced smaller chloroplasts containing little to none starch. BS chloroplasts ofD. sanguinalis were similar to those ofS. viridis, but had very little starch and well-developed long agranal stroma lamella. Features of MS cells were similar in both species, but well-defined peripheral reticulum (PR) was easily recognized in MS chloroplasts ofS. viridis. Virtually no PR was developed in BS chloroplasts examined. BS cells contained more mitochondria and microbodies, but no structural dimorphism was noticed. The electron-dense suberized lamella were often observed between BS and MS cells, especially in the primary wall of BS cells. It was most frequently found at the BS and MS cell interfaces and terminated in radial walls of the adjacent BS cells. Prominent pits with plasmodesmata (pd) were seen in the walls of both cells. There also were numerous pd in outer tangential walls of the BS cells. The number of pd ranged from 20 to 60. The pd trasversed a segment of cell wall much thinner than the adjacent wall. The current cellular data have been compared to the ultrastructural features known in leaves of other C-4 plants, especially NADP-ME species.     

Blue light-dependent nuclear positioning in Arabidopsis thaliana leaf cells

The plant nucleus changes its intracellular position not only upon cell division and cell growth but also in response to environmental stimuli such as light. We found that the nucleus takes different intracellular positions depending on blue light in Arabidopsis thaliana leaf cells. Under dark conditions, nuclei in mesophyll cells were positioned at the center of the bottom of cells (dark position). Under blue light at 100 mumol m(-2) s(-1), in contrast, nuclei were located along the anticlinal walls (light position). The nuclear positioning from the dark position to the light position was fully induced within a few hours of blue light illumination, and it was a reversible response. The response was also observed in epidermal cells, which have no chloroplasts, suggesting that the nucleus has the potential actively to change its position without chloroplasts. Light-dependent nuclear positioning was induced specifically by blue light at >50 mumol m(-2) s(-1). Furthermore, the response to blue light was induced in phot1 but not in phot2 and phot1phot2 mutants. Unexpectedly, we also found that nuclei as well as chloroplasts in phot2 and phot1phot2 mutants took unusual intracellular positions under both dark and light conditions. The lack of the response and the unusual positioning of nuclei and chloroplasts in the phot2 mutant were recovered by externally introducing the PHOT2 gene into the mutant. These results indicate that phot2 mediates the blue light-dependent nuclear positioning and the proper positioning of nuclei and chloroplasts. This is the first characterization of light-dependent nuclear positioning in spermatophytes.     

ANATOMY OF SOME LEAF GALLS OF ROSA WOODSII (ROSACEAE)

Infection of Rosa woodsii by some members of the order Hymenoptera results in neoplasmic outgrowths on the leaves. One type of outgrowth produces a spherical swelling (leaf gall) while the other has extensive hair-like proliferations (hairy gall). The anatomy and ultrastructure of these galls were examined by light microscopy and transmission electron microscopy. The leaf gall cells were considerably larger than normal cells, lacked well-developed chloroplasts and were loosely arranged with prominent intercellular spaces. Vascular bundles were scattered throughout the gall tissue. The upper three cell layers of the leaf gall tissue resembles a periderm, having many suberin lamellae. The suberin lamellae were often traversed by pores which may represent incomplete plasmodesmata. Phenolic compounds were commonly seen both in the normal and gall cells. A layer of internal cells of the hairy galls have remarkably thickened cell walls, presumably due to the deposition of cellulosic substances. Unlike leaf galls, the epidermal cells of the hairy galls were not heavily cuticularized and no periderm was found. The hair-like outgrowths present on the outer surface of these galls had a central vascular bundle. The epidermis of the outgrowths also had thickened cell walls, and trichomes occurred on the outer surface. The structural modifications brought about by the insect invasion in these two galls are compared and their roles in gall formation are discussed.     

Changes in chloroplast number per cell during leaf development in spinach

Summary The amounts of chlorophyll and nitrogen and the numbers of cells per unit area change as the green leaves of spinach plants grow and increase in size in the light. The changes in the numbers of chloroplasts per cell were measured by a new method. A 5-fold increase in the numbers of chloroplasts per cell took place in both palisade and mesophyll cells over a growing period of 10 days during which time the area of the leaves increased from 1 to 50 cm². Proplastids were not present in the young green leaves but electron-microscope and phase-contrast observations showed the presence of grana-containing chloroplasts, many of which appeared to be undergoing division by constriction. It is suggested that the large increase in chloroplast numbers as leaf cells grow and expand in the light is from the division of differentiated chloroplasts containing grana.     

Cadmium distribution and microlocalization in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis>) after long-term growth on cadmium-contaminated soil

Brassica napus (L.) was grown from seeds on a reconstituted soil contaminated with 100 mg Cd kg(-1). Compared with roots and stems, leaves accumulated high amounts of Cd. Although the Cd concentration in the leaves remained high throughout plant growth and no appreciable change was noticed in the total, extractable or soluble Cd in the soil adhering to the roots, the symptoms of Cd toxicity (leaf chlorosis, growth retardation) decreased with time. Cd induced a noticeable accumulation of phytochelatins in young plants (aged 22 days), which decreased in parallel to the disappearance of the symptoms of Cd intoxication. The subcellular distribution of Cd in leaves of Cd-acclimated plants was determined using biochemical, microscopic and metal-imaging techniques. Leaf fractionation by differential centrifugations showed that Cd was present predominantly in the 'soluble' fraction corresponding to the vacuoles and the cytoplasm. Transmission electron microscopic analyses revealed that those cell compartments contained electron-dense granules associated with needle-like structures. Cd, and also high amounts of sulfur, was detected in those structures by electron-spectroscopic imaging. This technique also showed Cd binding to cell walls by a mechanism that does not involve sulfur atoms. In contrast, very little Cd was found in chloroplasts, and this is consistent with the preservation of photosynthesis in plants grown on Cd-polluted soil. The microanalytical results presented here confirm that long-term growth of B. napus on Cd-contaminated soil is accompanied by preferential storage of Cd in the vacuoles and the cell walls. This phenomenon diverted Cd ions from metabolically active compartments (cytosol, chloroplasts, mitochondria), resulting in a reduction of Cd toxicity in the leaves.     

Photoacoustic analysis indicates that chloroplast movement does not alter liquid-phase CO2 diffusion in leaves of Alocasia brisbanensis

Light-mediated chloroplast movements are common in plants. When leaves of Alocasia brisbanensis (F.M. Bailey) Domin are exposed to dim light, mesophyll chloroplasts spread along the periclinal walls normal to the light, maximizing absorbance. Under high light, the chloroplasts move to anticlinal walls. It has been proposed that movement to the high-light position shortens the diffusion path for CO(2) from the intercellular air spaces to the chloroplasts, thus reducing CO(2) limitation of photosynthesis. To test this hypothesis, we used pulsed photoacoustics to measure oxygen diffusion times as a proxy for CO(2) diffusion in leaf cells. We found no evidence that chloroplast movement to the high-light position enhanced gas diffusion. Times for oxygen diffusion were not shorter in leaves pretreated with white light, which induced chloroplast movement to the high-light position, compared with leaves pretreated with 500 to 700 nm light, which did not induce movement. From the oxygen diffusion time and the diffusion distance from chloroplasts to the intercellular gas space, we calculated an oxygen permeability of 2.25 x 10(-)(6) cm(2) s(-)(1) for leaf cells at 20 degrees C. When leaf temperature was varied from 5 degrees C to 40 degrees C, the permeability for oxygen increased between 5 degrees C and 20 degrees C but changed little between 20 degrees C and 40 degrees C, indicating changes in viscosity or other physical parameters of leaf cells above 20 degrees C. Resistance for CO(2) estimated from oxygen permeability was in good agreement with published values, validating photoacoustics as another way of assessing internal resistances to CO(2) diffusion.     

Osmotic Adaptation in Panicum repens. Differences Between Organ, Cellular and Subcellular Levels

The process of osmotic adaptation was studied in leaves of Panicum repens. Two phases were observed: the first phase, which continued for 2–4 days, was mainly characterized by dehydration of leaves, a fast synthesis of organic acids and penetration of sodium into leaf cells. Chloride becomes dominant in the leaves only from the third day of exposure and onwards. The second phase of adaptation lasted for 4–6 days. During this phase, a decrease in organic acid content and an increase in leaf-chloride content was observed. In spite of the fact that the osmotic potential of the leaves reached lower values than that of the external medium already after 2 days, the rate of growth of the plants was hampered. Such inhibition of growth disappeared 6–8 days after exposure to salinity. Ion content of the cell walls, chloroplasts and vacuoles of Panicum leaf cells was investigated during the various stages of osmotic adaptation. An increase in sodium and chloride content of the cell walls during the early period of adaptation probably prevented the full osmotic adaptation of the protoplasts. It is suggested that a locally unbalanced distribution of ions may be one of the reasons for the decrease in growth rate during the process of osmotic adaptation and frequently after that.     

Heterophylly in Ranunculus flabellaris: The Effect of Abscisic Acid on Leaf Ultrastructure

Ranunculus flabellaris Rafin., an aquatic buttercup, exhibitsheterophylly at the level of cellular ultrastructure. Comparedto terrestrial leaves, underwater leaves have thinner epidermalcell walls and more numerous paramural bodies per epidermaland mesophyll cell cross-section. The number of chloroplastsand mitochondria in cell cross-sections also contrasts betweenthe two leaf types. Despite within-and between-leaf variations,different patterns of organelle distribution for the two leafforms were found using principal coordinates analysis. In addition,underwater leaf chloroplasts are smaller, have fewer grana,a greater number of thylakoids/granum, and less starch comparedto chloroplasts from terrestrial leaves. At the ultrastructurallevel, submergence in ABA solution does not produce a leaf withas many characteristics of the terrestrial environment, as shownin previous studies of leaf morphology and anatomy. While numberand distribution of organelles in ABA-treated leaves are similarto terrestrial leaves, some features of chloroplast internalstructure and paramural body number and distribution resembleunderwater leaves. It is postulated that ABA acts as a morphogeninvolved in guiding the irreversible processes of leaf development,but certain subcellular characteristics may be determined directlyby the physical environment. Difficulties encountered in quantitativeanalyses of cellular ultrastructure are discussed. Ranunculus flabellaris, ABA, heterophylly, leaf ultrastructure, principal coordinates analysis     

Chloride localization in Phaseolus vulgaris leaves exposed to HCl gas

An investigation of chloride accumulation and distribution in unifoliate leaves of Phaseolus vulgaris following supra-acute exposures to gaseous hydrogen chloride (HCl) was conducted. Plants which were 8-22 days old (post-seeding) were exposed for 20 min to HCl gas in concentrations ranging from 6.0-54.2 mg m-3 in different combinations of age and concentration. Aqueous extracts of treated unifoliates contained more chloride than was present in control leaves. In both 8 and 12-day-old leaves, the amount of chloride accumulated was proportional to the exposure concentration of HCl, but 12-day-old plants accumulated less chloride than 8-day-old plants. The number of leaves with macroscopic injury symptoms (epidermal glazing and interveinal necrosis) was also related to chloride content. Precipitation of chloride ions by silver salts was employed to examine the ultrastructural distribution of chloride. Deposits of AgCl were present in cell walls, ground plasm, vacuoles, and chloroplasts of both control and HCl-treated leaf tissues. In leaves sampled immediately after treatment, chloroplastic AgCl precipitates were less than in controls, but as sampling was delayed from the HCl treatment, AgCl deposits in chloroplasts regained control levels. The frequency and distribution of AgCl deposits indicated that chloride passed through the leaf cuticle and migrated through the apopolast. Chloride also entered the cytoplasm of cells and appeared to be sequestered in vacuoles of treated cells.     

Cellular Structure and Light Absorbtion in Leaves of Frithia pulchra (Mesembryanthemaceae)

In order to quantify the structural differences between celltypes of leaves from a ‘ window’ plant, an ultrastructuralmorphometric analysis was made of the epidermal, window andchlorenchyma tissues of Frithia pulchra. Epidermal cells arethe largest cells found in Frithia leaves and are characterizedby the presence of a thick outer tangential cell wall and numerousvacuolar inclusions. Epidermal tissue has an optical densityof 0.30. The transparent window tissue (i.e. optical density= 0.08) has a uniform ultrastructure throughout the length ofthe leaf. The vacuome comprises aproximately 97 per cent ofthe protoplasmic volume of window cells. Chlorenchyma cellspossess thin cell walls and are surrounded by numerous intercellularspaces. Cells of the apical chlorenchyma tissue possess approximately30 plastids per cell. These chloroplasts have an average individualvolume of 220 µm². Cells of the basal chlorenchyma tissuecontain chloroplasts that are five to six times smaller andmore numerous than those in cells of the apical chlorenchyma.The increased volume of chloroplasts in the apical comparedwith basal chlorenchyma cells (i.e. 31.4 and 20.2 per cent ofthe protoplasm, respectively) is positively correlated withtheir optical densities of 1.46 and 0.97, respectively. Frithia pulchra, stereology, leaf, light absorption, window plant     

Variation in Mesophyll Cell Number and Size in Wheat Leaves

The numbers of mesophyll cells in wheat leaves were determinedin a variety of wheat species differing in ploidy level andin leaves from different positions on the wheat plant. Leafsize and mesophyll cell number are linearly related in bothcases but differences were observed in mesophyll cell numberper unit leaf area with changing leaf size. Where changes incell size are caused either by nuclear ploidy or leaf position,differences in mesophyll cell number per unit leaf are negativelycorrelated with mesophyll cell plan area. The decrease in cellsize with increasing leaf position also results in a greaternumber of chloroplasts per unit leaf area. These results arediscussed in relation to anatomical variation of the wheat leaf. Mesophyll cell, cell numbers, leaf size, Triticum     

Chloroplast avoidance movement is not functional in plants grown under strong sunlight

Chloroplast movement in nine climbing plant species was investigated. It is thought that chloroplasts generally escape from strong light to avoid photodamage but accumulate towards weak light to perform photosynthesis effectively. Unexpectedly, however, the leaves of climbing plants grown under strong sunlight showed very low or no chloroplast photorelocation responses to either weak or strong blue light when detected by red light transmittance through leaves. Direct observations of Cayratia japonica leaves, for example, revealed that the average number of chloroplasts in upper periclinal walls of palisade tissue cells was only 1.2 after weak blue‐light irradiation and almost all of the chloroplasts remained at the anticlinal wall, the state of chloroplast avoidance response. The leaves grown under strong light have thin and columnar palisade tissue cells comparing with the leaves grown under low light. Depending on our analyses and our schematic model, the thinner cells in a unit leaf area have a wider total plasma membrane area, such that more chloroplasts can exist on the plasma membrane in the thinner cells than in the thicker cells in a unit leaf‐area basis. The same strategy might be used in other plant leaves grown under direct sunlight.     

Internal Detoxification Mechanism of Al in Hydrangea (Identification of Al Form in the Leaves)

An internal detoxification mechanism for Al was investigated in an Al-accumulating plant, hydrangea (Hydrangea macrophylla), focusing on Al forms present in the cells. The leaves of hydrangea contained as much as 15.7 mmol Al kg-1 fresh weight, and more than two-thirds of the Al was found in the cell sap. Using 27Al- nuclear magnetic resonance, the dominant peak of Al was observed at a chemical shift of 11 to 12 parts per million in both intact leaves and the extracted cell sap, which is in good accordance with the chemical shift for the 1:1 Al-citrate complex. Purification of cell sap by molecular sieve chromatography (Sephadex G-10) combined with ion-exclusion chromatography indicated that Al in fractions with the same retention time as citric acid contributed to the observed 27Al peak in the intact leaves. The molar ratio of Al to citric acid in the crude and purified cell sap approximated 1. The structure of the ligand chelated with Al was identified to be citric acid. Bioassay experiments showed that the purified Al complex from the cell sap did not inhibit root elongation of corn (Zea mays L.) and the viability of cells on the root tip surface was also not affected. These observations indicate that Al is bound to citric acid in the cells of hydrangea leaves.