Epidermal Chloroplasts Research Articles

Morphological, photosynthetic, and CAM physiological responses of the submerged macrophyte Ottelia alismoides to light quality

Light quality strongly affect a great many physiological processes in terrestrial plants. Whatever, few studies have investigated how the submerged macrophytes response to different light quality. The present research aimed to understand how light quality affect morphogenesis, photosynthesis, as well as crassulacean acid metabolism (CAM) operation during the growth of Ottelia alismoides , a freshwater facultative CAM macrophyte. The plants were grown under three light qualities of white (W), red (R) and blue (B) lights with same light intensity of 100 μmol m −2 s −1 . Leaf development, leaf anatomic characteristics, photosynthetic traits, as well as CAM metabolism were determined on the newly produced leaves during light treatment. Monochromatic R stimulated leaf blade and petiole elongation, leaf area and dry weight, but reduced leaf thickness, negatively influenced chlorophyll content and Fv/Fm. On the contrary, B- grown plants were dwarf and with compact size as well as lower leaf dry weight. The content of photosynthetic product starch under B was significantly lower as compared with R, moreover, combined with the analysis of ultrastructural characteristics of epidermal and mesophyll chloroplasts, it showed that B inhibited starch to be translocated out of epidermal chloroplasts. Furthermore, both R- and B- grown plants exhibited obvious diurnal acidity fluctuation comparable to W- grown plants, and had high activity of enzymes including ribulose-bisphosphate carboxylase/oxygenase (Rubisco), phosphoenolpyruvate carboxylase (PEPC) and pyruvate phosphate dikinase (PPDK) that required for CAM cycle, indicating that CAM was present in both R- and B- grown plants and monochromatic R and B did not affect CAM activity. In conclusion, monochromatic red and blue light differentially played their special roles in regulating leaf growth and morphogenesis, as well as photosynthetic metabolism in O. alismoides . • O. alismoides displayed strong morphological plasticity to light quality. • Leaf elongation was promoted by red light but inhibited by blue light. • Red light favoured O. alismoides leaf development and photosynthesis. • Blue light inhibited translocation of starch out of epidermal chloroplasts. • O. alismoides exhibited comparable CAM activity under white, red and blue light.

Emerging Roles of Motile Epidermal Chloroplasts in Plant Immunity.

Plant epidermis contains atypical small chloroplasts. However, the physiological role of this organelle is unclear compared to that of large mesophyll chloroplasts, the well-known function of which is photosynthesis. Although knowledge of the involvement of chloroplasts in the plant immunity has been expanded to date, the differences between the epidermal and mesophyll chloroplasts are beyond the scope of this study. Given the role of the plant epidermis as a barrier to environmental stresses, including pathogen attacks, and the immune-related function of chloroplasts, plant defense research on epidermal chloroplasts is an emerging field. Recent studies have revealed the dynamic movements of epidermal chloroplasts in response to fungal and oomycete pathogens. Furthermore, epidermal chloroplast-associated proteins and cellular events that are tightly linked to epidermal resistance against pathogens have been reported. In this review, I have focused on the recent progress in epidermal chloroplast-mediated plant immunity.

Epidermal chloroplasts are defense-related motile organelles equipped with plant immune components

In addition to conspicuous large mesophyll chloroplasts, where most photosynthesis occurs, small epidermal chloroplasts have also been observed in plant leaves. However, the functional significance of this small organelle remains unclear. Here, we present evidence that Arabidopsis epidermal chloroplasts control the entry of fungal pathogens. In entry trials, specialized fungal cells called appressoria triggered dynamic movement of epidermal chloroplasts. This movement is controlled by common regulators of mesophyll chloroplast photorelocation movement, designated as the epidermal chloroplast response (ECR). The ECR occurs when the PEN2 myrosinase-related higher-layer antifungal system becomes ineffective, and blockage of the distinct steps of the ECR commonly decreases preinvasive nonhost resistance against fungi. Furthermore, immune components were preferentially localized to epidermal chloroplasts, contributing to antifungal nonhost resistance in the pen2 background. Our findings reveal that atypical small chloroplasts act as defense-related motile organelles by specifically positioning immune components in the plant epidermis, which is the first site of contact between the plant and pathogens. Thus, this work deepens our understanding of the functions of epidermal chloroplasts.

Responses of Leaf Anatomy and CO2 Concentrating Mechanisms of the Aquatic Plant Ottelia cordata to Variable CO2.

Acclimation to variable CO2 was studied in floating leaves of the freshwater monocot Ottelia cordata grown in either low or high CO2. The most striking anatomical variations responding to high CO2 included the enlarged upper epidermal cells and the decreased area of epidermal chloroplasts. Stomata that distributed on the upper surface, and the stomatic chamber area, showed no significant response to high CO2. pH-drift experiments indicated that floating leaves of O. cordata were able to use bicarbonate regardless of CO2 concentrations. Photosynthetic enzyme activities and patterns of organic acids fluctuation confirmed that floating leaves of O. cordata can operate CAM only at low CO2, and perform C4-like metabolism at both high and low CO2. Overall, the present results imply that the floating leaves of O. cordata does not just rely on the atmospheric CO2 for its inorganic carbon, but is also dependent on CO2 and bicarbonate in the water. By showing these effects of CO2 variation, we highlight the need for further experimental studies on the regulatory mechanisms in O. cordata floating leaves, that prevent futile cycling among the three CO2 concentrating mechanisms (bicarbonate use, C4, and CAM metabolism) and the strategy for exploiting atmospheric CO2, as well as studies on the detailed biochemical pathway for C4 and CAM metabolism in this species.

Structural basis for C4 photosynthesis without Kranz anatomy in leaves of the submerged freshwater plant Ottelia alismoides.

Ottelia alismoides (Hydrocharitaceae) is a freshwater macrophyte that, unusually, possesses three different CO2-concentrating mechanisms. Here we describe its leaf anatomy and chloroplast ultrastructure, how these are altered by CO2 concentration and how they may underlie C4 photosynthesis. Light and transmission electron microscopy were used to study the anatomy of mature leaves of O. alismoides grown at high and low CO2 concentrations. Diel acid change and the activity of phosphoenolpyruvate carboxylase were measured to confirm that CAM activity and C4 photosynthesis were present. When O. alismoides was grown at low CO2, the leaves performed both C4 and CAM photosynthesis whereas at high CO2 leaves used C4 photosynthesis. The leaf comprised an upper and lower layer of epidermal cells separated by a large air space occupying about 22 % of the leaf transverse-section area, and by mesophyll cells connecting the two epidermal layers. Kranz anatomy was absent. At low CO2, chloroplasts in the mesophyll cells were filled with starch even at the start of the photoperiod, while epidermal chloroplasts contained small starch grains. The number of chloroplasts in the epidermis was greater than in the mesophyll cells. At high CO2, the structure was unchanged but the thicknesses of the two epidermal layers, the air space, mesophyll and the transverse-section area of cells and air space were greater. Leaves of O. alismoides have epidermal and mesophyll cells that contain chloroplasts and large air spaces but lack Kranz anatomy. The high starch content of mesophyll cells suggests they may benefit from an internal source of CO2, for example via C4 metabolism, and are also sites of starch storage. The air spaces may help in the recycling of decarboxylated or respired CO2. The structural similarity of leaves at low and high CO2 is consistent with the constitutive nature of bicarbonate and C4 photosynthesis. There is sufficient structural diversity within the leaf of O. alismoides to support dual-cell C4 photosynthesis even though Kranz anatomy is absent.

The Arabidopsis minD mutation causes aberrant FtsZ1 ring placement and moderate heterogeneity of chloroplasts in the leaf epidermis

ABSTRACTSymmetric division of leaf mesophyll chloroplasts requires MinD and MinE, which work together to suppress division other than at the mid-chloroplast. arc11 is a MinD loss-of-function mutant of Arabidopsis thaliana. In arc11 plants, asymmetric chloroplast division, as well as its delay or arrest, results in extreme size polymorphism of chloroplasts in mature mesophyll cells. The current study examined chloroplast phenotypes in the epidermis of arc11 leaves. Fluorescence microscopy analysis revealed that epidermal chloroplasts in mature leaves exhibited moderate heterogeneity in size. This probably resulted from completion of many of the previous non-equatorial or multiple division events in expanding leaves. Additionally, analyses of plastids found that epidermal chloroplasts in arc11 mutants showed several phenotypes that have not previously been described.

Visualizing Stromule Frequency with Fluorescence Microscopy.

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

The influence of salinity on growth, morphology, leaf ultrastructure, and cell viability of the seagrass Halodule wrightii Ascherson.

Halodule wrightii is an ecologically important seagrass; however, little is known about the adaptation of this species in the context of environmental change, particularly changes arising from alterations in salinity of coastal ecosystems. This study aimed to determine the effects of different salinities on growth, morphology, leaf ultrastructure, and cell viability of H. wrightii. To accomplish this, plants were cultivated for 21days in salinities of 25, 35, and 45. More hydropotens were observed in samples exposed to salinity of 45 with increased invagination of the plasma membrane and cell wall. These invaginations were also observed in other epidermal cells of the leaf blade. In particular, a significant retraction of plasma membrane was seen in samples exposed to salinity of 45, with possible deposition of compounds between the membrane and cell wall. Osmotic stress in samples exposed to salinity of 45 affected the chloroplasts through an increase in plastoglobules and thylakoids by granum in the epidermal chloroplasts of the leaf and decrease in the number of chloroplasts. Overall, this study showed that H. wrightii can survive within salinities that range between 25 and 45 without changing growth rate. However, the plant did have higher cell viability at salinity of 35. Salt stress in mesocosms, at both salinity of 25 and 45, decreased cell viability in this species. H . wrightii had greater changes in salinity of 45; this showed that the species is more tolerant of salinities below this value.

Photonic multilayer structure of Begonia chloroplasts enhances photosynthetic efficiency.

Enhanced light harvesting is an area of interest for optimizing both natural photosynthesis and artificial solar energy capture1,2. Iridescence has been shown to exist widely and in diverse forms in plants and other photosynthetic organisms and symbioses3,4, but there has yet to be any direct link demonstrated between iridescence and photosynthesis. Here we show that epidermal chloroplasts, also known as iridoplasts, in shade-dwelling species of Begonia5, notable for their brilliant blue iridescence, have a photonic crystal structure formed from a periodic arrangement of the light-absorbing thylakoid tissue itself. This structure enhances photosynthesis in two ways: by increasing light capture at the predominantly green wavelengths available in shade conditions, and by directly enhancing quantum yield by 5-10% under low-light conditions. These findings together imply that the iridoplast is a highly modified chloroplast structure adapted to make best use of the extremely low-light conditions in the tropical forest understorey in which it is found5,6. A phylogenetically diverse range of shade-dwelling plant species has been found to produce similarly structured chloroplasts7-9, suggesting that the ability to produce chloroplasts whose membranes are organized as a multilayer with photonic properties may be widespread. In fact, given the well-established diversity and plasticity of chloroplasts10,11, our results imply that photonic effects may be important even in plants that do not show any obvious signs of iridescence to the naked eye but where a highly ordered chloroplast structure may present a clear blue reflectance at the microscale. Chloroplasts are generally thought of as purely photochemical; we suggest that one should also think of them as a photonic structure with a complex interplay between control of light propagation, light capture and photochemistry.

Chloroplast dimorphism in leaves of Cabomba caroliniana (Cabombaceae)

Cabomba Aublet is useful as a genetic model of angiosperm evolution. Previous anatomical studies and preliminary light microscopic observations on C. caroliniana A Gray revealed the presence of dimorphic chloroplasts in both types of leaves, floating and submerged leaves. Leaf anatomy and chloroplasts ultrastructure were analyzed and compared. The chloroplast ultrastructure of the mesophyll and epidermal cells is different. Mesophyll chloroplasts have several plastoglobuli, large starch grains and grana formed by an average of 29 thylakoids. Epidermal chloroplasts are smaller, have grana formed by an average of 9 thylakoids and starch grains are occasional and smaller. A statistical analysis (t-test) was made to determine if differences between chloroplasts are significant. The ultrastructure of the epidermis chloroplast is similar to the one of sun leaves chloroplasts and the ultrastructure of the mesophyll chloroplast is similar to the one of shade chloroplast observed in other species.

3D Confocal Laser Scanning Microscopy for the Analysis of Chlorophyll Fluorescence Parameters of Chloroplasts in Intact Leaf Tissues

We analyzed the chlorophyll fluorescence parameters in a 3D cellular arrangement in vivo by using a modified Nipkow disk-type confocal laser scanning microscope (CLSM). We first defined the 3D values of Phi(PSII) (photochemical yield of PSII) and NPQ (non-photochemical quenching) in mesophyll, epidermal and guard cell chloroplasts from the leaf surface to several tens of microns in depth. We also used this CLSM method to analyze the relationships between actinic light intensity and the chlorophyll fluorescence parameters for Boston fern and broad bean leaf specimens. As the actinic light intensity increased, the mean Phi(PSII) values decreased and the NPQ values increased in all chloroplasts of Boston fern and broad bean leaf. These values differed with cell type and species. The Boston fern chloroplasts had lower Phi(PSII) values than the broad bean chloroplasts, and vice versa for the NPQ values. The Phi(PSII) values of Boston fern chloroplasts decreased in the order mesophyll, epidermal and guard cell chloroplasts. The NPQ values decreased in the order guard cell, mesophyll and epidermal chloroplasts, except at 12 micromol m(-2) s(-1) actinic light, when the mesophyll value was slightly lower than that of the epidermis. The trend in the Phi(PSII) and NPQ values of broad bean mesophyll and guard cell chloroplasts was opposite to that of Boston fern chloroplasts. As 3D CLSM can provide the Phi(PSII) and NPQ values of each chloroplast in a 3D cellular arrangement, this method has potential for investigating differences in the functions of chloroplasts in vivo.

Photosynthetic energy storage in aquatic leaves measured by photothermal deflection

In a study of photosynthetic energy storage efficiency (ES), the adaxial surface of the leaves of Vallisneria americana exhibited the highest ES values (22%) of the four aquatic plants examined. V. americana leaves have a dispersed structure and it was possible to measure the energy storage properties of the epidermal cells independently of the rest of the leaf. The abaxial epidermis had a higher value of ES at zero light fluence than the adaxial epidermis but ES in the abaxial epidermis declined much more rapidly with light fluence. Thus the abaxial epidermis is more suited to lower light fluences than the adaxial epidermis. ES declined as the pH rose from 4.0 to 8.0 at a constant dissolved inorganic carbon concentration. This paralleled the change from carbon dioxide to bicarbonate and suggests that these leaves utilise CO2 more efficiently than bicarbonate. ES increased by about 50% at pH 8.0 as leaf sections further from the leaf tip were examined which demonstrates that the older epidermal cells are less well able to use bicarbonate. Exposure to 30 min of a saturating light fluence caused the epidermal chloroplasts to move from the periclinal walls to the anticlinal walls. This decreased the photothermal signal by increasing the thermal diffusion distance and lowering the light fluence due to greater chloroplast shading. The latter effect increased ES. It appears that chloroplast movement could assist the epidermis to survive harmful light fluences.

On Megaceros aenigmaticus Schust.

The description ofMegaceros aenigmaticus Schust., briefly treated Schuster (1992), is enhanced. Two phenotypes (both lacking visible gametangia) were collected 1952: a) a weakly to moderately frilled-margined, closely pinnatifid to pinnate phase and b) an entire-margined phase, often remotely 2-3-pinnate, with very narrow ultimate pinnae, mimicking a large Riccardia mul- tifida or R. chamedryfolia. Both phases had only solitary chloroplasts of dorsal epidermal cells. It was tentatively concluded that a distinct species of Anthoceros was at hand, the single chloroplasts seeming to exclude Megaceros (to which phase a bore some resemblances). Lack ofgametangia and sporophytes precluded resolution of both generic provenance and of whether one or two intermingled taxa were at hand; the plant has remained in limbo for forty years. A preliminary account by Renzaglia and Hicks (1984) of a third phenotype appeared: c) a 9 phase with frilled thallus margins, bearing 1-4 epidermal chloroplasts per cell. The question arose: are these phases members of one species, and to what genus do they belong? In October 1987, mass collections were made order to find 6 plants and sporophytes and to try to resolve whether all these enigmatic plants belonged to one or more species. Among thousands ofplants collectedfrom several stations, afourth phenotype was found: d) vigorous ! plants with strikingly lobulate-laciniate and crispulate thallus margins, with dorsal epidermal cells with either paired or single dumbbell-shaped chloroplasts. This vari- ability-from Anthoceros-like to Megaceros-like epidermal cells-and gross variability four other criteria: a) development, or lack thereof of endogenous slime cells; b) presence or absence ofNostoc symbionts; c) number of cells delimiting the mouth of the archegonium; and d) number of cells peripheral to the ventral gametophytic stomata, are subject to in-depth investigation. It is concluded that a single, strikingly malleable species of Megaceros is at hand, differing from all well-known taxa of that primarily antipodal genus being unisexual and relying on fragmentation and re- generant-formation for its dispersal. The assumption is documented that reduction to sterile and purely 9 clonal populations, isolated limited environments western North Carolina to South Carolina, along small rills, is probably a recent (surely post-Oligocene, perhaps post-Pliocene) phenomenon. Extinction of the 6 sex is shown to be matched a number of other cases the Appalachian system.

Dimorphic epidermal cell chloroplasts in the mesophyll‐less leaves of an extreme–shade tropical fern, Teratophyllum rotundifoliatum (R. Bonap.) Holtt.: a light and electron microscope study

summaryThis light and electron microscope study of the iridescent blue leaves of the fern Teratophyllum rotundifoliatum, reveals several novel anatomical and cytological features. In this extreme shade species [0.4—‐(3)–4 W m−2] of lowland tropical rainforest in peninsular Malaya, the greater part of the lamina between the simple collateral vascular bundles, consists only of epidermal cells. From 4–(6.9)–12 large chloroplasts 9.4–(17.9)–27 μm in diameter are located adjacent to the inner walls of the lens‐shaped upper epidermal cells. Those in the tessellated lower epidermal cells are smaller [5.0–(7.6)–19.5 μm], much more numerous [21–(53)–83] and regularly distributed around the periphery. They are identical in size and ultrastructure to the chloroplasts in the guard cells of the stomata. Whereas the upper epidermal chloroplasts and those in the stelar tissues contain very little starch, numerous starch grains occupy up to Sl‐9% of the volume of the lower epidermal chloroplasts. As in other extreme‐shade plants the epidermal chloroplasts in T. rotundifoliatum contain numerous large grana, with no preferred orientation, and integranal lamellae completely filling the stroma. The numbers of thylakoids per granum in the lower epidermal cells [9–(29.3)–84] are significantly smaller than those in the upper [22–(86)–28O]. The latter appear to be the largest yet recorded in land plants and this probably accounts for the remarkably low chlorophyll a: b ratio (1.8) in T. rotundifoliatum. Record amounts of chlorophyll per unit leaf fresh weight and per unit leaf area in T. rotundifoliatum reflect the high proportion of leaf cells containing chloroplasts. The unusual features of leaf anatomy and cytology in T. rotundifoliatum appear to be associated with maximizing the efficiency of absorption of very low diffuse radiation.

Microspectrofluorometric Measurement of Chloroplast DNA in Dividing and Expanding Leaf Cells of Spinacia oleracea

Absolute DNA amounts of individual chloroplasts from mesophyll and epidermal cells of developing spinach leaves were measured by microspectrofluorometry using the DNA-specific stain, 4,6-diamidino-2-phenyl indole, and the bacterium, Pediococcus damnosus, as an internal standard. Values obtained by this method showed that DNA amounts of individual chloroplasts from mesophyll cells fell within a normal distribution curve, although mean DNA amounts changed during leaf development and also differed from the levels in epidermal chloroplasts. There was no evidence in the data of plastids containing either the high or low levels of DNA which would be indicative of discontinuous polyploidy of plastids, or of division occurring in only a small subpopulation of chloroplasts. By contrast, the distribution of nuclear DNA amounts in the same leaf tissues in which cell division was known to be occurring showed a clear bimodal distribution. We consider that the distribution of chloroplast DNA in the plastid population shows that there is no S-phase of chloroplast DNA synthesis, all chloroplasts in the population in young leaf cells synthesize DNA, and all chloroplasts divide.

Chloroplasts in the epidermis of Sarracenia (the American pitcher plant) and their possible role in carnivory ‐ An immunocytochemical approach

A photosynthetic apparatus is present in the epidermis of the bottom zone of the pitcher of Sarracenia purpurea L. ssp. purpurea. This has been demonstrated using conventional light and electron microscopy, as well as fluorescent and immunohistochemical techniques. Red intrinsic fluorescence by these chloroplasts indicates photochemical activity. Antibodies against the coupling factor of chloroplast ATPase and against the subunits of ribulose‐bis‐phosphate‐carboxylase were bound to the epidermal chloroplasts. This has been visualized using a ferritin‐isothiocyanate labeled second antibody. These results unequivocally prove the existence of the two main proteins which are associated with the photophosphorylation (membrane protein) and carbon dioxide fixation (stromal protein). The possible implication of this system to interrelationships between the carnivorous plant and aquatic insects inhabiting its leaves is discussed.

Light-controlled sugar-starch interconversion in epidermal chloroplasts of light-grown cucumber hypocotyl sections and its relationship to cell elongation

Sugar-starch interconversion in epidermal chloroplasts of light-grown cucumber hypocotyl sections as a regulatory mechanism of the osmotic potential of the cell was studied in relation to cell elongation. The presence of chloroplasts in epidermal cells was confirmed by electron microscopy, and also the chloroplasts were shown to act as the site of sucrose-starch interconversion. Chloroplast starch formation was induced by light, which was more distinct in the presence of sucrose (50 mM). The starch formation was microscopically detectable even at 1 hr incubation in the light with sucrose. On the other hand, no starch formation was observed in the dark both in the presence and absence of sucrose. Red light was effective, but not blue light. A photosynthetic inhibitor, 3-(4-chlorophenyl)-1:1-dimethylurea, also inhibited starch formation. Thus, epidermal chloroplast starch formation was induced under conditions where cell elongation is small and the osmotic potential (Ψ 0) of the epidermal cell is high. The sugar quantity (free sugar and reducing sugar) as the osmotica of the cell was larger in the dark than in the light, whereas the quantity of starch was greater in the light than in the dark. It is assumed from these results that one of the regulatory mechanisms of the osmotic potential of the epidermal cells in sugar-starch interconversion which occurs in epidermal chloroplasts.

A membrane-bound plastid inclusion in the epidermis of leaves of Taraxacum officinale

Epidermal chloroplasts of Taraxacum officinale agg. contain large electron-dense inclusion bodies enclosed by a single membrane. These inclusion bodies were not observed in mesophyll chloroplasts. The origin and functional role of these structures is discussed.

Photosynthetic Carbon Metabolism of a Marine Grass

The delta(13)C value of a tropical marine grass Thalassia testudinum is -9.04 per thousand. This value is similar to the delta(13)C value of terrestrial tropical grasses. The delta(13)C values of the organic acid fraction, the amino acid fraction, the sugar fraction, malic acid, and glucose are: -11.2 per thousand, -13.1 per thousand, -10.1 per thousand, -11.1 per thousand, and -11.5 per thousand, respectively. The delta(13)C values of malic acid and glucose of Thalassia are similar to the delta(13)C values of these intermediates in sorghum leaves and attest to the presence of the photosynthetic C(4)-dicarboxylic acid pathway in this marine grass. The inorganic HCO(3) (-) for the growth of the grass fluctuates between -6.7 to -2.7 per thousand during the day. If CO(2) fixation in Thalassia is catalyzed by phosphoenolpyruvate carboxylase (which would result in a -3 per thousand fractionation between HCO(3) (-) and malic acid), the predicted delta(13)C value for Thalassia would be -9.7 to -5.7 per thousand. This range is close to the observed range of -12.6 to -7.8 per thousand for Thalassia and agree with the operation of the C(4)-dicarboxylic acid pathway in this plant. The early products of the fixation of HCO(3) (-) in the leaf sections are malic acid and aspartic acid which are similar to the early products of CO(2) fixation in C(4) terrestrial plants.Electron microscopy of the leaves of Thalassia reveal thick walled epidermal cells exceedingly rich in mitochondria and C(3)-type chloroplasts. The mesophyll cells have many different shapes and surround air lacunae which contain O(2) and CO(2). The mesophyll cells are highly vacuolated and the parietal cytoplasm contains an occasional chloroplast. This chloroplast contains grana but the lamellar system is not as developed as the system in epidermal chloroplasts. Extensive phloem tissue is present but the xylem elements are reduced in this aquatic grass. The vascular tissue is not surrounded by bundle sheath cells.This work does not establish the exact relation between structure and function in Thalassia, but it does show the C(4)-type photosynthetic carbon metabolism in this grass involves epidermal and mesophyll cells and internally produced O(2) and CO(2) in the air lacunae.

Unusual structures associated with peripheral reticulum in chloroplasts of Myriophyllum spicatum L.

A peripheral reticulum has been observed in epidermal chloroplasts of leaves of Myriophyllum spicatum seedlings. Associated with this membrane system are packets containing 5–80 unbranched tubular or rodlike structures which are individually about 250 A wide by 1500 A long. They are surrounded by the peripheral reticulum and are closely associated with the inner chloroplast membrane.