Photosystem II Activity, Plastoquinone A Levels, and Fluorescence Characterization of a Virescens Mutant of Barley

Chloroplasts isolated from seedlings of a virescens mutant of barley (Hordeum vulgare L cv Gateway) grown for 6 days under continuous illumination had lower levels of photosystem II activities on a chlorophyll basis than wild-type seedlings. After 8 days, however, the photosystem II rates of the mutant and wild-type were approximately equal. Lower levels of the photosystem II activities in the mutant were correlated with a smaller functional plastoquinone pool size as determined by room temperature fluorescence induction. Higher levels of extractable plastoquinone A on a chlorophyll basis, however, were obtained from mutant chloroplasts. An increased room temperature fluorescence yield in the mutant was shown to be due to a higher level of initial fluorescence. A decreased sigmoidicity in the room temperature fluorescence induction transient in the presence of diuron and an increased 77 K fluorescence emission at 680 nanometers lead us to believe that a certain population of the light harvesting chlorophyll protein complex in the mutant membranes is unconnected to photo-system II reaction centers. Although photochemical activities of the mutant approach wild-type values as the mutant develops, the population of dissociated light harvesting complexes does not appear to change.     

The Effect of Antibiotics on the Ultrastructure and Photochemical Activity of a Developing Chloroplast

The effects of the antibiotics chloramphenicol and cycloheximideon the ultrastructure of the chloroplast in greening cells ofEuglena gracilis strain Z have been investigated. The rate ofchloroplaat development in the presence of either antibioticwas closely related to that of chlorophyll production. Chloramphenicol,which at 10 mg/ml inhibits chlorophyll synthesis but not celldivision, caused a marked inhibition of the development of chloroplaststructure. The chloroplasts were smaller than those of untreatedcells and contained a smaller number of internal lamellae. Mostof these lamellae were not appressed and the results supportthe suggestion that chloramphenicol inhibits the synthesis ofa protein responsible for the fusion of individual lamellaein the chloroplast. Measurement of the photochemical activityof chloroplasts isolated from chloramphenicol-treated cellsshowed that the photoreduction of NADP from water (photosystemI+II), photosystem II activity, and photosystem I activity weregreatly inhibited when compared with chloroplasts from untreatedcells. In contrast, cycloheximide at 2.5 to 5.0 µg/mlinhibited cell division but allowed the chloroplasts to developafter a lag phase, the length of which was related to the concentrationof antibiotic employed. The number of lamellae per chloroplastand the degree of appression of the lamellae in chloroplastsof cycloheximide-treated cells and untreated cells were comparable.After 48-h illumination the photochemical activities of chloroplastsisolated from cycloheximide-treated cells were about 50 percent of those of the untreated cells.     

Biogenesis of water splitting by photosystem II during de‐etiolation of barley (Hordeum vulgare L.)

Etioplasts lack thylakoid membranes and photosystem complexes. Light triggers differentiation of etioplasts into mature chloroplasts, and photosystem complexes assemble in parallel with thylakoid membrane development. Plastids isolated at various time points of de‐etiolation are ideal to study the kinetic biogenesis of photosystem complexes during chloroplast development. Here, we investigated the chronology of photosystem II (PSII) biogenesis by monitoring assembly status of chlorophyll‐binding protein complexes and development of water splitting via O₂ production in plastids (etiochloroplasts) isolated during de‐etiolation of barley (Hordeum vulgare L.). Assembly of PSII monomers, dimers and complexes binding outer light‐harvesting antenna [PSII‐light‐harvesting complex II (LHCII) supercomplexes] was identified after 1, 2 and 4 h of de‐etiolation, respectively. Water splitting was detected in parallel with assembly of PSII monomers, and its development correlated with an increase of bound Mn in the samples. After 4 h of de‐etiolation, etiochloroplasts revealed the same water‐splitting efficiency as mature chloroplasts. We conclude that the capability of PSII to split water during de‐etiolation precedes assembly of the PSII‐LHCII supercomplexes. Taken together, data show a rapid establishment of water‐splitting activity during etioplast‐to‐chloroplast transition and emphasize that assembly of the functional water‐splitting site of PSII is not the rate‐limiting step in the formation of photoactive thylakoid membranes.     

Changes in the structure of DNA molecules and the amount of DNA per plastid during chloroplast development in maize

We examined the DNA from chloroplasts obtained from different tissues of juvenile maize seedlings (from eight to 16 days old) and adult plants (50-58 days old). During plastid development, we found a striking progression from complex multigenomic DNA molecules to simple subgenomic molecules. The decrease in molecular size and complexity of the DNA paralleled a progressive decrease in DNA content per plastid. Most surprising, we were unable to detect DNA of any size in most chloroplasts from mature leaves, long before the onset of leaf senescence. Thus, the DNA content per plastid is not constant but varies during development from hundreds of genome copies in the proplastid to undetectable levels in the mature chloroplast. This loss of DNA from isolated, mature chloroplasts was monitored by three independent methods: staining intact chloroplasts with 4',6-diamidino-2-phenylindole (DAPI); staining at the single-molecule level with ethidium bromide after exhaustive deproteinization of lysed chloroplasts; and blot-hybridization after standard DNA isolation procedures. We propose a mechanism for the production of multigenomic chloroplast chromosomes that begins at paired DNA replication origins on linear molecules to generate a head-to-tail linear concatemer, followed by recombination-dependent replication.     

ARC6 is a J-domain plastid division protein and an evolutionary descendant of the cyanobacterial cell division protein Ftn2

Replication of chloroplasts is essential for achieving and maintaining optimal plastid numbers in plant cells. The plastid division machinery contains components of both endosymbiotic and host cell origin, but little is known about the regulation and molecular mechanisms that govern the division process. The Arabidopsis mutant arc6 is defective in plastid division, and its leaf mesophyll cells contain only one or two grossly enlarged chloroplasts. We show here that arc6 chloroplasts also exhibit abnormal localization of the key plastid division proteins FtsZ1 and FtsZ2. Whereas in wild-type plants, the FtsZ proteins assemble into a ring at the plastid division site, chloroplasts in the arc6 mutant contain numerous short, disorganized FtsZ filament fragments. We identified the mutation in arc6 and show that the ARC6 gene encodes a chloroplast-targeted DnaJ-like protein localized to the plastid envelope membrane. An ARC6-green fluorescent protein fusion protein was localized to a ring at the center of the chloroplasts and rescued the chloroplast division defect in the arc6 mutant. The ARC6 gene product is related closely to Ftn2, a prokaryotic cell division protein unique to cyanobacteria. Based on the FtsZ filament morphology observed in the arc6 mutant and in plants that overexpress ARC6, we hypothesize that ARC6 functions in the assembly and/or stabilization of the plastid-dividing FtsZ ring. We also analyzed FtsZ localization patterns in transgenic plants in which plastid division was blocked by altered expression of the division site-determining factor AtMinD. Our results indicate that MinD and ARC6 act in opposite directions: ARC6 promotes and MinD inhibits FtsZ filament formation in the chloroplast.     

Developmental changes in energy dissipation in etiolated wheat seedlings during the greening process

We studied the developmental changes in photosynthetic and respiration rates and thermal dissipation processes connected with chloroplasts and mitochondria activity in etiolated wheat (Triticum aestivum L., var. Irgina) seedlings during the greening process. Etioplasts gradually developed into mature chloroplasts under continuous light [190 μmol(photon) m^?2 s^?1] for 48 h in 5-day-dark-grown seedlings. The net photosynthetic rate of irradiated leaves became positive after 6 h of illumination and increased further. The first two hours of de-etiolation were characterized by low values of maximum (F_v/F_m) and actual photochemical efficiency of photosystem II (PSII) and by a coefficient of photochemical quenching in leaves. F_v/F_m reached 0.8 by the end of 24 h-light period. During greening, energy-dependent component of nonphotochemical quenching of chlorophyll fluorescence, violaxanthin cycle (VXC) operation, and lipoperoxidation activity changed in a similar way. Values of these parameters were the highest at the later phase of de-etiolation (4–12 h of illumination). The respiration rate increased significantly after 2 h of greening and it was the highest after 4–6 h of illumination. It was caused by an increase in alternative respiration (AP) capacity. The strong, positive linear correlation was revealed between AP capacity and heat production in greening tissues. These results indicated that VXC in chloroplasts and AP in mitochondria were intensified as energy-dissipating systems at the later stage of greening (after 4 h), when most of prolamellar bodies converted into thylakoids, and they showed the greatest activity until the photosynthetic machinery was almost completely developed.     

The DCL gene of tomato is required for chloroplast development and palisade cell morphogenesis in leaves.

The defective chloroplasts and leaves-mutable (dcl-m) mutation of tomato was identified in a Ds mutagenesis screen. This unstable mutation affects both chloroplast development and palisade cell morphogenesis in leaves. Mutant plants are clonally variegated as a result of somatic excision of Ds and have albino leaves with green sectors. Leaf midribs and stems are light green with sectors of dark green tissue but fruit and petals are wild-type in appearance. Within dark green sectors of dcl-m leaves, palisade cells are normal, whereas in albino areas of dcl-m leaves, palisade cells do not expand to become their characteristic columnar shape. The development of chloroplasts from proplastids in albino areas is apparently blocked at an early stage. DCL was cloned using Ds as a tag and encodes a novel protein of approximately 25 kDa, containing a chloroplast transit peptide and an acidic alpha-helical region. DCL protein was imported into chloroplasts in vitro and processed to a mature form. Because of the ubiquitous expression of DCL and the proplastid-like appearance of dcl-affected plastids, the DCL protein may regulate a basic and universal function of the plastid. The novel dcl-m phenotype suggests that chloroplast development is required for correct palisade cell morphogenesis during leaf development.     

Characterization of the Snowy Cotyledon 1 Mutant of Arabidopsis Thaliana: The Impact of Chloroplast Elongation Factor G on Chloroplast Development and Plant Vitality

During seedling development chloroplast formation marks the transition from heterotrophic to autotrophic growth. The development and activity of chloroplasts may differ in cotyledons that initially serve as a storage organ and true leaves whose primary function is photosynthesis. A genetic screen was used for the identification of genes that affect selectively chloroplast function in cotyledons of Arabidopsis thaliana. Several mutants exhibiting pale cotyledons and green true leaves were isolated and dubbed snowy cotyledon (sco).One of the mutants, sco1, was characterized in more detail. The mutated gene was identified using map-based cloning. The mutant contains a point mutation in a gene encoding the chloroplast elongation factor G, leading to an amino acid exchange within the predicted 70S ribosome-binding domain. The mutation results in a delay in the onset of germination. At this early developmental stage embryos still contain undifferentiated proplastids, whose proper function seems necessary for seed germination. In light-grown sco1 seedlings the greening of cotyledons is severely impaired, whereas the following true leaves develop normally as in wild-type plants. Despite this apparent similarity of chloroplast development in true leaves of mutant and wild-type plants various aspects of mature plant development are also affected by the sco1 mutation such as the onset of flowering, the growth rate, and seed production. The onset of senescence in the mutant and the wild-type plants occurs, however, at the same time, suggesting that in the mutant this particular developmental step does not seem to suffer from reduced protein translation efficiency in chloroplasts.     

Independent effects of leaf growth and light on the development of the plastid and its DNA content in Zea species

In maize (Zea mays L.), chloroplast development progresses from the basal meristem to the mature leaf tip, and light is required for maturation to photosynthetic competence. During chloroplast greening, it was found that chloroplast DNA (cpDNA) is extensively degraded, falling to undetectable levels in many individual chloroplasts for three maize cultivars, as well as Zea mexicana (the ancestor of cultivated maize) and the perennial species Zea diploperennis. In dark-grown maize seedlings, the proplastid-to-etioplast transition is characterized by plastid enlargement, cpDNA replication, and the retention of high levels of cpDNA. When dark-grown seedlings are transferred to white light, the DNA content per plastid increases slightly during the first 4 h of illumination and then declines rapidly to a minimum at 24 h during the etioplast-to-chloroplast transition. Plastid autofluorescence (from chlorophyll) continues to increase as cpDNA declines, whereas plastid size remains constant. It is concluded that the increase in cpDNA that accompanies plastid enlargement is a consequence of cell and leaf growth, rather than illumination, whereas light stimulates photosynthetic capacity and cpDNA instability. When cpDNA from total tissue was monitored by blot hybridization and real-time quantitative PCR, no decline following transfer from dark to light was observed. The lack of agreement between DNA per plastid and cpDNA per cell may be attributed to nupts (nuclear sequences of plastid origin).     

FtsHi1/ARC1 is an essential gene in Arabidopsis that links chloroplast biogenesis and division

The Arabidopsis arc1 (accumulation and replication of chloroplasts 1) mutant has pale seedlings and smaller, more numerous chloroplasts than the wild type. Previous work has suggested that arc1 affects the timing of chloroplast division but does not function directly in the division process. We isolated ARC1 by map‐based cloning and discovered it encodes FtsHi1 (At4g23940), one of several FtsHi proteins in Arabidopsis. These poorly studied proteins resemble FtsH metalloproteases important for organelle biogenesis and protein quality control but are presumed to be proteolytically inactive. FtsHi1 bears a predicted chloroplast transit peptide and localizes to the chloroplast envelope membrane. Phenotypic studies showed that arc1 (hereafter ftsHi1‐1), which bears a missense mutation, is a weak allele of FtsHi1 that disrupts thylakoid development and reduces de‐etiolation efficiency in seedlings, suggesting that FtsHi1 is important for chloroplast biogenesis. Consistent with this finding, transgenic plants suppressed for accumulation of an FtsHi1 fusion protein were often variegated. A strong T‐DNA insertion allele, ftsHi1‐2, caused embryo‐lethality, indicating that FtsHi1 is an essential gene product. A wild‐type FtsHi1 transgene rescued both the chloroplast division and pale phenotypes of ftsHi1‐1 and the embryo‐lethal phenotype of ftsHi1‐2. FtsHi1 overexpression produced a subtle increase in chloroplast size and decrease in chloroplast number in wild‐type plants while suppression led to increased numbers of small chloroplasts, providing new evidence that FtsHi1 negatively influences chloroplast division. Taken together, our analyses reveal that FtsHi1 functions in an essential, envelope‐associated process that may couple plastid development with division.     

Development of Photosystem I and Photosystem II Activities in Leaves of Light-grown Maize (Zea mays)

To compare chloroplast development in a normally grown plant with etiochloroplast development, green maize plants (Zea mays), grown under a diurnal light regime (16-hour day) were harvested 7 days after sowing and chloroplast biogenesis within the leaf tissue was examined. Determination of total chlorophyll content, ratio of chlorophyll a to chlorophyll b, and O₂-evolving capacity were made for intact leaf tissue. Plastids at different stages of development were isolated and the electron-transporting capacities of photosystem I and photosystem II measured. Light saturation curves were produced for O₂-evolving capacity of intact leaf tissue and for photosystem I and photosystem II activities of isolated plastids. Structural studies were also made on the developing plastids. The results indicate that the light-harvesting apparatus becomes increasingly efficient during plastid development due to an increase in the photosynthetic unit size. Photosystem I development is completed before that of photosystem II. Increases in O₂-evolving capacity during plastid development can be correlated with increased thylakoid fusion. The pattern of photosynthetic membrane development in the light-grown maize plastids is similar to that found in greening etiochloroplasts.