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.     

THE STRUCTURE OF GRANA IN FLOWERING PLANTS

A model of granum structure, consisting of multiple helical frets wound around each cylindrical granum, is proposed as a representation of granum structure for flowering plants in general. This model was originally formulated for grana in the mesophyll plastids of Zea mays and was subsequently extended to grana of Phaseolus vulgaris by additional studies. The model is now shown to be applicable also to grana in the plastids of Cannabis sativa, Elodea canadensis, Nicotiana rustica, Pisum sativum, and Spinacia oleracea. This sample of seven angiosperms includes genera commonly studied by other workers in their ultrastructural investigations of plastid structure.     

The chloroplast nucleoids of the bundle sheath and mesophyll cells of Zea mays

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

Flash-induced 515 nm absorbance change in chloroplasts with various granum contents

The 515 nm absorbance change was studied in mesophyll and bundle sheath chloroplasts of maize, which contain different amounts of grana. The amplitude of the 515 nm signal (induced by 3 μs flashes repeated at 4 s intervals) has shown a correlation with the granum content of the samples. However, upon addition of N-methylphenazonium methosulphate the 515 nm signal became independent of the amount of grana: in agranal thylakoids a large pool of silent Photosystem I was activated and, as a result, the amplitude of the 515 nm signal of agranal chloroplasts increased to the level exhibited by granal chloroplasts.These data show that the 515 nm absorbance change is not limited to small closed vesicles like grana, but in the presence of suitable electron donors single lamellae of bundle sheath chloroplasts can also be active.     

Flash-induced 515 nm absorbance change in chloroplasts with various granum contents.

The 515 nm absorbance change was studied in mesophyll and bundle sheath chloroplasts of maize, which contain different amounts of grana. The amplitude of the 515 nm signal (induced by 3 micro seconds flashes repeated at 4 s intervals) has shown a correlation with the granum content of the samples. However, upon addition of N-methylphenazonium methosulphate the 515 nm signal became independent of the amount of grana: in agranal thylakoids a large pool of silent Photosystem I was activated and, as a result, the amplitude of the 515 nm signal of agranal chloroplasts increased to the level exhibited by granal chloroplasts. These data show that the 515 nm absorbance change is not limited to small closed vesicles like grana, but in the presence of suitable electron donors single lamellae of bundle sheath chloroplasts can also be active.     

THE ULTRASTRUCTURE OF GRANA IN MESOPHYLL PLASTIDS OF ZEA MAYS

The compartments of the grana of mesophyll plastids of Zea mays are extensively interconnected. In median sections of grana, 16.5% of compartment margins were involved in a Y-shaped conjunction of the loculi of two consecutive compartments with the loculus of an attached fret. Various configurations accounted for the remainder of the compartment margins seen in median sections of grana. Tangential views of grana and serial sections revealed an oblique relationship between the frets and the grana. Analysis of serial sections allows the conclusion that the components of the fretwork are helically wound around a granum.     

PLASTID ORGANIZATION IN PHENOTYPICALLY GREEN LEAF TISSUE OF A GENETIC ALBINO STRAIN OF NICOTIANA (SOLANACEAE)

Plastid organization within phenotypically green leaf tissue that forms in albino plants of a genetic albino strain of Nicotiana has been examined with the transmission electron microscope. Studies revealed the presence of plastids with and without thylakoids. When present, thylakoids were loosely and irregularly scattered in the stroma or organized either into several large spindle-shaped grana or into a single compound granum with deeply indented margins. Plastids without thylakoids were vesiculated and resembled the typical genetic albino type. Plastid types were not segregrated into individual cells and no plastid type appeared to be typical for the mutant tissue. Orientation of grana and thylakoid membrane associations were noted as well as the presence of osmiophilic globules, starch grains and DNA-like fibrillar areas.     

Localization of photosynthetic electron transport components in mesophyll and bundle sheath chloroplasts of Zea mays

The organization of the electron transport components in mesophyll and bundle sheath chloroplasts of Zea mays was investigated. Grana-containing mesophyll chloroplasts (chlorophyll a to chlorophyll b ratio of about 3.0) possessed the full complement of the various electron transport components, comparable to chloroplasts from C₃ plants. Agranal bundle sheath chloroplasts ($\text{Chl}\text{a}\text{Chl}\text{b}\text{> 5.0}$) contained the full complement of photosystem (PS) I and of cytochrome (cyt) f but lacked a major portion of PS II and its associated Chl $\text{a}\text{b}$ light-harvesting complex (LHC), and most of the cyt b₅₅₉. The kinetic analysis of system I photoactivity revealed that the functional photosynthetic unit size of PS I was unchanged and identical in mesophyll and bundle sheath chloroplasts. The results suggest that PS I is contained in stroma-exposed thylakoids and that it does not receive excitation energy from the Chl $\text{a}\text{b}$ LHC present in the grana. A stoichiometric parity between PS I and cyt f in mesophyll and bundle sheath chloroplasts indicates that biosynthetic and functional properties of cyt f and P₇₀₀ are closely coordinated. Thus, it is likely that both cyt f and P₇₀₀ are located in the membrane of the intergrana thylakoids only. The kinetic analysis of PS II photoactivity revealed the absence of PS II_αfrom the bundle sheath chloroplasts and helped identify the small complement of system II in bundle sheath chloroplasts as PS II_β. The distribution of the main electron transport components in grana and stroma thylakoids is presented in a model of the higher plant chloroplast membrane system.     

Comparative Studies of the Thylakoid Proteins of Mesophyll and Bundle Sheath Plastids of Zea mays

The proteins from both grana and stroma lamellae of maize (Zea mays) mesophyll plastids and from maize bundle sheath plastid membranes have been compared by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels using a discontinuous buffer system. Peptide differences between grana and stroma lamellae were essentially quantitative and not qualitative. Bundle sheath plastid membrane peptides more closely resembled those of the ultrastructurally similar stroma lamellae. However, bundle sheath membranes contained several peptides not apparent in the stroma lamellae.     

Comparative ultrastructural properties of pallisade tissue and spongy tissue chloroplasts from normal and mutant leaves of Pisum sativum L.*

Abstract. The ultrastructure of chloroplasts from palisade and spongy tissue was studied in order to analyse the adaptation of chloroplasts to the light gradient within the bifacial leaves of pea. Chloroplasts of two nuclear gene mutants of Pisum sativum (chlorotica-29 and chlorophyll b-less 130A), grown under normal light conditions, were compared with the wild type (WT) garden-pea cv. ‘Dippes Gelbe Viktoria’. The differentiation of the thylakoid membrane system of plastids from normal pea leaves exhibited nearly the same degree of grana formation in palisade and in spongy tissue. Using morphometrical measurements, only a slight increase in grana stacking capacity was found in chloroplasts of spongy tissue. In contrast, chloroplasts of mutant leaves differed in grana development in palisade and spongy tissue, respectively. Their thylakoid systems appeared to be disorganized and not developed as much as in chloroplasts from normal pea leaves. Grana contained fewer lamellae per granum, the number of grana per chloroplast section was reduced and the length of appressed thylakoid regions was decreased. Nevertheless, chloroplasts of the mutants were always differentiated into grana and stroma thylakoids. The structural changes observed and the reduction of the total chlorophyll content correlated with alterations in the polypeptide composition of thylakoid membrane preparations from mutant chloroplasts. In sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), polypeptide bands with a relative molecular mass of 27 and 26 kilodalton (kD) were markedly reduced in mutant chloroplasts. These two polypeptides represented the major apoproteins of the light harvesting chlorophyll a/b complex from photosystem II (LHC-II) as inferred from a comparison with the electrophoretic mobility of polypeptides isolated from the LHC-II.     

ULTRASTRUCTURE OF THE LAMELLAE AND GRANA IN THE CHLOROPLASTS OF ZEA MAYS L

The fine structure of the chloroplasts of maize (Zea mays L.) has been investigated by electron microscopic examination of ultrathin sections of leaves fixed in buffered osmium tetroxide solutions. Both the parenchyma sheath and mesophyll chloroplasts contain a system of densely staining lamellae about 125 A thick immersed in a finely granular matrix material (the stroma), and are bounded by a thin limiting membrane which often appears as a double structure. In the parenchyma sheath chloroplasts, the lamellae usually extend the full width of the disc-shaped plastids, and grana are absent. The mesophyll chloroplasts, however, contain numerous grana of a fairly regular cylindrical form. These consist of highly ordered stacks of dense lamellae, the interlamellar spacing being ca. 125 A. The grana are interlinked by a system of lamellae (intergrana lamellae) which are on the average about one-half as numerous as the lamellae within the grana. In general, this appears to be due to a bifurcation of the lamellae at the periphery of the granum, but more complex interrelationships have been observed. The lamellae of the parenchyma sheath chloroplasts and those of both the grana and intergrana regions of the mesophyll chloroplasts exhibit a compound structure when oriented normally to the plane of the section. A central exceptionally dense line (ca. 35 A thick) designated the P zone is interposed between two less dense layers (the L zones, ca. 45 A thick), the outer borders of which are defined by thin dense lines (the C zones). Within the grana, the C zones, by virtue of their close apposition, give rise to thin dense intermediate lines (I zones) situated midway between adjacent P zones. A model of the lamellar structure is proposed in which mixed lipide layers (L zones) are linked to a protein layer (P zone) by non-polar interaction. Chlorophyll is distributed over the entire lamellar surface and held in the structure by van der Waals interaction of the phytol "tail" with the hydrocarbon moieties of the mixed lipide layers. The evidence in favour of the model is briefly discussed.     

DEVELOPMENTAL ANATOMY OF EPIDERMAL AND MESOPHYLL CHLOROPLASTS IN OPUNTIA BASILARIS LEAVES

Developmental studies of Opuntia basilaris Engelm. & Bigel. leaves revealed the presence of three morphologically distinct types of plastids. All epidermal cells examined contained chloroplasts. After 13 days of growth in the dark the plastids of epidermal and mesophyll cells were characterized by the presence of a prolamellar body and fibril inclusions. Epidermal plastids which developed under light conditions contained large stromacentres and a limited grana-fret membrane system. Guard cell plastids developed under similar conditions had a much poorer developed grana system with fibril inclusions apparent only during the developmental stages. At maturity these plastids appeared swollen or dilated. Mesophyll plastids had fibril inclusions during all stages of development and at maturity contained a very extensive grana-fret membrane system. Microbodies were found in association with the mesophyll plastids. Starch accumulation was common in subsidiary cell and guard cell plastids.     

Accumulation of delta-Aminolevulinic Acid and Its Relation to Chlorophyll Synthesis and Development of Plastid Structure in Greening Leaves

Levulinic acid inhibited the greening of etiolated maize (Zea mays) and bean (Phaseolus vulgaris) leaves and caused accumulation of δ-aminolevulinic acid (ALA). ALA accumulation in maize was equivalent to the decrease in chlorophyll, over a wide range of experimental conditions. It was saturated at low light intensities and was not limited by the supply of substrates during the early hours of greening. During 20 hours in light, levulinic acid had little effect on the structural development of thylakoids in bundle sheath chloroplasts but significantly reduced the number and size of thylakoids in grana of mesophyll chloroplasts. Recrystallization of prolamellar bodies and their reformation was inhibited. Mitochondria appeared not to be affected.     

Differences in chlorophyll-protein complexes and composition of polypeptides between thylakoids from bundle sheaths and mesophyll cells in maize

Thylakoids from enzymatically separated bundle sheath and mesophyll tissue chloroplasts were examined for their chlorophyll-proteins by tube sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). Differences were found in distribution of chlorophyll among peaks. The chlorophyll-protein a peak (CPa), considered to be the photosystem II (PSII) reaction centre by many authors, was seen to be absent in bundle sheath thylakoid samples. The slab SDS-PAGE revealed the absence of the polypeptides present in PSII preparations of chloroplast subfractions having only PSII activity. This finding confirms Anderson's hypothesis of the structure of grana and stroma thylakoids.     

Photosystem II Activity in Agranal Bundle Sheath Chloroplasts from Zea mays

The photochemical activities of chloroplasts isolated from bundle sheath and mesophyll cells of maize (Zea mays var. DS606A) have been measured. Bundle sheath chloroplasts are almost devoid of grana, except in very young leaves, while mesophyll chloroplasts contain grana at all stages of leaf development.     

Structure and function of developing barley plastids

Five different regions of the first foliage leaf of etiolated barley seedlings were studied with respect to leaf growth, plastid growth and replication, differentiation of etioplasts, and conversion of etioplasts into chloroplasts upon illumination. Ultrastructural changes of the plastids were correlated with chlorophyll synthesis and development of photosynthetic activity as measured by (14)CO(2) incorporation and O(2) evolution. The first foliage leaf has greater linear growth over a longer period of time in the dark than in the light. Only the bottom two regions (4 and 5) are still growing in the 5-day etiolated leaf. Region 4 grows by cell elongation, and region 5 grows by both cell division and elongation. Plastids in all five regions of the leaf are capable of enlarging when exposed to light. This is true both for the intact plant and for excised sections. Plastid replication occurs predominantly in the younger regions of the leaf (regions 3, 4, and 5). The amount of chlorophyll synthesized by different regions in the intact plant is significantly higher (3-40 times) than that made by excised sections. Ultrastructural changes occurring in each region when excised sections are illuminated were classified into five stages involving increased membrane synthesis and appression into grana, and these changes were correlated with the first appearance of photosynthetic activity. The earliest detectable photosynthetic activity occurs in region 1 after 2 hours of illumination when chloroplasts show only a few overlaps in the thylakoids. Plastids in younger regions of the leaf require up to 24 hours of light to form grana and develop photosynthetic activity. Plastids in each region of the leaf are in different stages of development when photosynthesis is initiated, indicating that development of photosynthetic activity is not strictly correlated with a certain stage of plastid development. Membrane appression is not indicative of photosynthetic activity since overlaps are formed in the dark, but it was always present when photosynthetic activity was detectable. Likewise, there does not appear to be any strict correlation between the presence of chlorophyll and membrane appression. These results show that the particular structural and functional correlations that can be made depend to a large degree on age of the tissue.     

Investigations of the role of the main light-harvesting chlorophyll- protein complex in thylakoid membranes. Reconstitution of depleted membranes from intermittent-light-grown plants with the isolated complex

The functions of the light-harvesting complex of photosystem II (LHC- II) have been studied using thylakoids from intermittent-light-grown (IML) plants, which are deficient in this complex. These chloroplasts have no grana stacks and only limited lamellar appression in situ. In vitro the thylakoids showed limited but significant Mg2+-induced membrane appression and a clear segregation of membrane particles into such regions. This observation, together with the immunological detection of small quantities of LHC-II apoproteins, suggests that the molecular mechanism of appression may be similar to the more extensive thylakoid stacking seen in normal chloroplasts and involve LHC-II polypeptides directly. To study LHC-II function directly, a sonication- freeze-thaw procedure was developed for controlled insertion of purified LHC-II into IML membranes. Incorporation was demonstrated by density gradient centrifugation, antibody agglutination tests, and freeze-fracture electron microscopy. The reconstituted membranes, unlike the parent IML membranes, exhibited both extensive membrane appression and increased room temperature fluorescence in the presence of cations, and a decreased photosystem I activity at low light intensity. These membranes thus mimic normal chloroplasts in this regard, suggesting that the incorporated LHC-II interacts with photosystem II centers in IML membranes and exerts a direct role in the regulation of excitation energy distribution between the two photosystems.     

Benzyladenine effects on the development of the photosynthetic apparatus in Zea mays: Studies on photosynthetic activity, enzymes and (etio)chloroplast ultrastructure

Primary leaf segments from 8-day-old dark-grown, and from 4- and 8-day-old light-grown seedlings of Zea mays L. cv. Fronica, were treated with 10^-bM benzyladenine (BA) in the dark for 14 h. The segments were then studied after an exposure to light for 14 h. Photosynthetic activity (O₂ evolution and CO₂ fixation) and chlorophyll accumulation were stimulated by BA in dark-grown leaf segments with etioplastids in the earliest stage of development. In these segments BA stimulated the activities of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39), phosphoenolpyruvate carboxylase (EC 4.1.1.31), NADP⁺-malic enzyme (EC 1.1.1.40) and pyruvate, orthophosphate dikinase (EC 2.7.9.1). In segments taken from 4- and 8-day light-grown seedlings, BA did not enhance the photosynthetic activity nor the chlorophyll accumulation. The activity of the enzymes mentioned above, was significantly enhanced by the BA-treatment. BA mainly affected grana stacking in mesophyll cell chloroplasts in primary leaf segments taken from 3- to 5-day light-grown seedlings. Stroma thylakoid development was stimulated only in leaf segments from 3-day-old plants. At the same time BA accelerated grana loss in chloroplasts of bundle sheath cells, a typical phenomenon of development in such chloroplasts. Stroma thylakoid length in these chloroplasts increased by a BA treatment in segments from 3- and 4-day light-grown plants. A significantly higher number of chloroplasts was only observed with segments taken from 8-day light-grown seedlings and treated with BA. The etiochloroplast number in segments taken from 8-day etiolated plants was significantly higher in BA-treated segments after 26 h illumination. In etiochloroplasts from both mesophyll and bundle sheath cells, BA enhanced grana stacking after illumination for 4 h or more, whereas stroma membrane length was significantly higher only after 26 h light. It is concluded that the effects of BA depend on the developmental stage. BA accelerates the development of mesophyll and bundle sheath cell (etio)chloroplasts, but does not affect the ultrastructure of mature chloroplasts.     

Tissue localization of polyphenol oxidase inSorghum

Summary Plastidic polyphenol oxidase (PPO) was localized in various plastid types ofSorghum bicolor (L.) Moench using cytochemical and biochemical franctionation techniques. PPO was found to be present in the mesophyll plastids yet absent from the bundle sheath and guard cell plastids. Mechanical fractionation of mesophyll and bundle sheath plastids, with subsequent electrophoretic or spectrophotometric assay of the preparations, also indicated that PPO was absent from the bundle sheath but present in the mesophyll fraction. A developmental study revealed that, although all leaf plastids near the basal meristem were ultrastructurally similar, the mesophyll and bundle sheath plastids were already differentiated with respect to PPO activity.     

Transgenic maize lines with cell‐type specific expression of fluorescent proteins in plastids

Plastid number and morphology vary dramatically between cell types and at different developmental stages. Furthermore, in C4 plants such as maize, chloroplast ultrastructure and biochemical functions are specialized in mesophyll and bundle sheath cells, which differentiate acropetally from the proplastid form in the leaf base. To develop visible markers for maize plastids, we have created a series of stable transgenics expressing fluorescent proteins fused to either the maize ubiquitin promoter, the mesophyll‐specific phosphoenolpyruvate carboxylase (PepC) promoter, or the bundle sheath‐specific Rubisco small subunit 1 (RbcS) promoter. Multiple independent events were examined and revealed that maize codon‐optimized versions of YFP and GFP were particularly well expressed, and that expression was stably inherited. Plants carrying PepC promoter constructs exhibit YFP expression in mesophyll plastids and the RbcS promoter mediated expression in bundle sheath plastids. The PepC and RbcS promoter fusions also proved useful for identifying plastids in organs such as epidermis, silks, roots and trichomes. These tools will inform future plastid‐related studies of wild‐type and mutant maize plants and provide material from which different plastid types may be isolated.