Which plastids are found in the guard cells

Cell Mol. Mochizuki, A. Genetic studies on the number of plastid in stomata I. Effects of autopolyploidy in sugar beets. Cytologia 20, — Mullet, J. Chloroplast development and gene expression. Plant Mol. Nadeau, J. Stomatal development in Arabidopsis. The Arabidopsis Book 1, e Verma, D. Springer, Berlin, Heidelberg , — Redistribution of Golgi stacks and other organelles during mitosis and cytokinesis in plant cells.

Springer-Verlag , — Okazaki, K. Plant Cell 21, — Osteryoung, K. Division and dynamic morphology of plastids. Possingham, J. Controls to plastid division. Changes in chloroplast number per cell during leaf development in spinach. Planta 86, — Pyke, K. Plastid division and development. The control of chloroplast number in wheat mesophyll cells.

Rapid image analysis screening procedure for identifying chloroplast number mutants in mesophyll cells of Arabidopsis thaliana L. A genetic analysis of chloroplast division and expansion in Arabidopsis thaliana. Qin, X. Chloroplast number in guard cells as ploidy indicator of in vitro -grown androgenic pepper plantlets.

Plant Cell Tissue Organ Cult. Robertson, E. Sachs, J. Textbook of Botany. Oxford: Clarendon Press. Sakisaka, M. On the number of chloroplasts in the guard cells of seed plants. Mitochondrial reticulation in shoot apical meristem cell provides a mechanism for homogeneization of mtDNA prior to gamete formation. Sheahan, M. Rose, R.

Organelle inheritance in plant cell division: the actin cytoskeleton is required for unbiased inheritance of chloroplasts, mitochondria and endoplasmic reticulum in dividing protoplasts. Singsit, C. Chloroplast density in guard cells of leaves of anther-derived potato plants grown in vitro and in vivo.

HortScience 26, — Stokes, K. Chloroplast division and morphology are differentially affected by overexpression of FtsZ1 and FtsZ2 genes in Arabidopsis. Taiz, L. Plant Physiology and Development, Sixth Ed. Thomas, M. Plastid number and plastid structural changes associated with tobacco mesophyll protoplast culture and plant regeneration. Tirlapur, U. Femtosecond near-infrared lasers as a novel tool for non-invasive real-time high-resolution time-lapse imaging of chloroplast division in living bundle sheath cells of Arabidopsis.

Planta , 1— Vitha, S. FtsZ ring formation at the chloroplast division site in plants. Cell Biol. ARC6 is a J-domain plastid division protein and an evolutionary descendant of the cyanobacterial cell division protein Ftn2.

Plant Cell 15, — Wada, M. Chloroplast and nuclear photorelocation movements. B Phys. Wild, A. The effect of different light intensities on the frequency and size of stomata, the size of cells, the number, size and chlorophyll content of chloroplasts in the mesophyll and the guard cells during the ontogeny of primary leaves of Sinapis alba.

Yoder, D. Effects of mutations in Arabidopsis FtsZ1 on plastid division, FtsZ ring formation and positioning, and FtsZ filament morphology in vivo. Yu, Z. A large number of tetraploid Arabidopsis thaliana lines, generated by a rapid strategy, reveal high stability of neo-tetraploids during consecutive generations.

Zhao, L. Ultrastructure of stomatal development in Arabidopsis Brassicaceae leaves. Plastid-derived protrusions lacking chlorophyll in guard cells have also been reported in su Forth and Pyke Our results indicate that similar plastid-derived protrusions lacking chlorophyll were also generated from enlarged chloroplasts in crl and arc6 guard cells Fig.

Thus, enlarged chloroplasts in crl and arc6 probably undergo a similar process of plastid proliferation, not only in guard cells, but also in mesophyll cells and in cells of the cotyledon primordia of embryos. Moreover, the FtsZ ring does not form a ring structure in arc6 chloroplasts Vitha et al. This mode of plastid division was not observed in wild-type cells Fig. Such plastids were also found in arc6 embryos, but not in wild-type embryos data not shown.

Thus plastid DNA might not always be transmitted to the daughter plastids in the budding and fragmentation mode of plastid proliferation.

For growth on plates, the plants were grown on MS growth medium containing 0. The mtDsRed-expressing plants were gene-rated by a genetic cross with a mtDsRed-expressing line, which was a gift from S. A total of transgenic Arabidopsis seedlings were selected on bialaphos-containing MS plates, and one transgenic line, FL, which showed high and stable expression of the plastid-targeted YFP designated as TP FtsZ :YFP , was further characterized for microscopic observations.

Samples for plastid analysis were prepared by the method described by Pyke and Leech with slight modifications. Chloroplast and plastid numbers were determined in pavement cells, mesophyll cells and cells from the cotyledons of mature embryos in plants expressing TP ftsZ :YFP. Cotyledons of mature embryos were dissected under a stereomicroscope before analysis. Chloroplasts and plastids in guard cells in the cotyledons of plants expressing TP ftsZ :YFP were counted without fixation.

Cells having condensed chromosomes were scanned in three dimensions with an FV confocal microscope. The ratio was obtained by dividing the area of the plastids in each cell by the area of the cell itself. Whole-mount immunolocalization of Tic was performed as described by Friml et al. We thank S. Google Scholar. Oxford University Press is a department of the University of Oxford.

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Close mobile search navigation Article Navigation. Volume Article Contents Abstract. Materials and Methods. Yuling Chen , Yuling Chen. Oxford Academic. Tomoya Asano. Makoto T. Shigeo Yoshida. Yasunori Machida. Select Format Select format. Permissions Icon Permissions. Abstract Plastids are maintained in cells by proliferating prior to cell division and being partitioned to each daughter cell during cell division. Open in new tab Download slide. Table 1 Average numbers of plastids per cell.

Plant phenotype. Cotyledon in embryos a. Stomatal guard cell b. Mesophyll cell c. Pavement cell d. Wild type Open in new tab. The EMB gene encodes a novel ankyrin repeat containing protein that is essential for the normal development of Arabidopsis embryos.

Google Scholar Crossref. Search ADS. The Arabidopsis immutans mutation affects plastid differentiation and the morphogenesis of white and green sectors in variegated plants. A defect in atToc of Arabidopsis thaliana causes severe defects in leaf development. Google Scholar PubMed. DAG , a gene required for chloroplast differentiation and palisade development in Antirrhinum majus.

Floral dip: a simplified method for Agrobacterium -mediated transformation of Arabidopsis thaliana. A homologue of the bacterial cell division site-determining factor MinD mediates placement of the chloroplast division apparatus. Partial complementation of embryo defective mutations: a general strategy to elucidate gene function. Overexpression of the Arabidopsis thaliana MinD1 gene alters chloroplast size and number in transgenic tobacco plants. The suffulta mutation in tomato reveals a novel method of plastid replication during fruit ripening.

Therefore, despite the known phenotype of the arc5 mesophyll chloroplasts, a large portion of arc5 PC plastids are capable of completing their division process. The ARC5 gene product, DRP5B, assembles into a constriction ring at the central division site on the cytosolic surface of the chloroplast, and its Cyanidioschyzon homolog generates the force needed for constriction via the chloroplast division machinery [ 8 ] for a review, see [ 52 ]. Our results raise new possibilities for the role of DRP5B in plastid division.

The first possibility is that DRP5B is only required for the fission of well-expanded or expanding plastids, such as those observed in mesophyll cells, but it is dispensable for poorly sized plastids, such as those observed in PCs. The second possibility which is not exclusive of the first is that PCs contain a plastid division machinery that is distinct from that of mesophyll chloroplasts and does not involve DRP5B.

The possibility that DRP5B is dispensable for plastid division in certain types or developmental stages of cells is supported by an earlier report showing that the expression of DRP5B was too low to detect plastidial DRP5B rings in shoot apical meristems and young emerging leaves of Arabidopsis [ 53 ]. The possibility that dumbbell-shaped PC plastids of arc5 contain a normal FtsZ ring at the mid-plastid constriction should be addressed in a future study. This issue is intriguing because the division-arrested mesophyll chloroplasts of arc5 possess multiple, uncondensed FtsZ rings or spirals at or near the constriction site [ 13 , 54 ].

In the mesophyll cells of both arc6 and atminE1 and another allele, arc12 , chloroplasts are unable to initiate the division process but instead continue to expand, resulting in the uniform occurrence of mesophyll cells containing one or a few, extremely large chloroplasts [ 7 , 27 , 30 , 46 ], indicating that ARC6 and MinE are critical initiation factors in the conventional chloroplast division system. On the other hand, PC plastids in arc6 and atminE1 assume various morphological features in addition to the formation of giant plastids: size heterogeneity, extensive stromule formation, and the formation of putative division constrictions, although only in small plastids Fig 2.

The distinct roles of ARC6 and MinE in chloroplast division machinery suggest that the aberrant morphologies of epidermal plastids in arc6 and atminE1 cannot be ascribed to a defect in the specific functions of ARC6 or MinE, but rather, they might be due to a common failure to complete an upstream event in the plastid division process for instance, assembly of the contractile FtsZ ring. The finding that grape-like plastid clusters occur in arc6 Fig 3 and atminE1 [ 28 ] provides important insights into the defective control of plastid division in PCs.

This finding, and the absence of node-like structures connecting bulges at the center of the grape-like cluster in arc6 Fig 3B and atminE1 [ 28 ], support the hypothesis that the cluster is derived from one or a few plastids containing many bulges, at least some of which undergo fission to produce discrete plastidic entities but are incapable of detaching themselves from the site of membrane fission, and which may generally be induced by serious inhibition of PC plastid division.

The process by which plastid bulges are formed, maintained, and possibly separated in arc6 and atminE1 PCs should be investigated in more detail to expand our knowledge of the non-mesophyll mode of plastid division.

Stomatal GCs lacking chloroplasts, but containing chlorophyll-deficient plastids, have been observed in the tomato suffulta mutant [ 21 ] and Arabidopsis arc6 and crl mutants [ 19 ], leading to a model in which such non-green plastids in GCs arise by budding and fragmentation of giant chloroplasts model A. This model was originally proposed for plastid replication in the pericarp cells of ripening tomato fruits [ 3 , 21 , 30 , 55 ]. Conceivably, the morphological patterns of the leaf epidermal plastids in arc5 , arc6 , and atminE1 observed in the present study might result from a combination of these three models.

As represented by the class IV pattern, plastid biogenesis in GCs is a unique, markedly different process from that of the surrounding PCs, even though both GCs and PCs are present in the same epidermal tissue. Thus, in the future, it would be interesting to examine plastid biogenesis in GMCs to explore how the switching of the plastid biogenesis mode occurs. The current results indicate that size heterogeneity in plastids can occur in PCs and GCs of arc5 and arc6 Figs 2 and 4 , both with responsible loci that encode the key components of the division ring complex in mesophyll chloroplasts.

Furthermore, plastid size heterogeneity was also observed in PCs and GCs of atminE1 Figs 2 and 4 , in which mesophyll chloroplasts fail to initiate their division [ 27 ]. These phenotypes were exactly the same as those observed in arc6 [ 7 ]. Nevertheless, a more plausible explanation is that correct division site positioning in plastids is more vulnerable to malfunctioning of the division ring machinery in non-mesophyll tissues than in the mesophyll.

Perhaps such increased vulnerability is associated with the higher fluidity or lower rigidity of the plastid envelope membranes, as indicated by the more frequent formation of stromules compared with that of mesophyll chloroplasts. Under this scenario, the timing or extent of bulge and stromule development may contribute to the plastid heterogeneity detected in PCs. Our current and recent observations [ 28 , 63 ] strengthen the notion that leaf epidermal plastids tend to display high morphological variability on a cell-by-cell or even plastid-by-plastid basis, as induced by mutations in chloroplast division genes.

Concerning the developmental regulation of plastid morphology in the division mutants, we previously proposed that development-associated chloroplast expansion prevents the constriction events of the plastid envelope in leaf mesophylls of an AtMinE1 overexpressor and arc11 , a chloroplast division site positioning mutant [ 56 , 64 ]. Based on this, thylakoid development may be a critical index for distinguishing the plastid phenotypes of leaf epidermis and mesophylls. The thylakoid system itself has mechanical strength, which enables it to retain well-preserved forms following isolation from chloroplasts.

Upon defects in the plastid division apparatus, epidermal plastids that are poor in thylakoid membranes might undergo division via a rudimentary FtsZ1 ring [ 28 ], whereas mesophyll chloroplasts tend to continuously grow because of restrictions caused by the presence or growth of their thylakoids. In support of this idea, a majority of FtsZ1 rings in epidermal plastids of arc11 achieved plastid fission, unlike mesophyll chloroplasts [ 65 ]. Furthermore, it has been recently shown that thylakoid division in Arabidopsis mesophyll chloroplasts requires the chloroplast division machinery [ 66 ].

The relationship between thylakoid organization and plastid division is almost unknown and will require substantial investigation in the future [ 54 ]. The differences in phenotypes reported for epidermal plastids in arc5 , arc6 or atminE1 are an important consideration.

In particular, in arc6 , despite well-conserved GC structures, there have been some differences in the literature concerning the size, shape, and number of GC plastids chloroplasts [ 5 , 7 , 18 , 19 ]. As a whole, the GC plastids observed in the current study were more highly developed, and more chloroplasts were produced in the plant samples examined than in other studies. Differences in the study materials, such as the use of rosette leaves [ 5 , 7 , 18 ] and this study or cotyledons [ 19 ], the adaxial this study or abaxial [ 5 , 7 , 18 ] side of leaves, and soil [ 5 , 7 , 18 ] or solid medium this study for plant cultivation, may explain the variations in GC phenotypes among studies.

As recently reported for the hypocotyl and root epidermal cells [ 67 ], differences in the distance from key proliferative zones for leaf development, such as the shoot apical meristem and the leaf blade—petiole junction [ 68 , 69 ], could also affect GC phenotypes.

Furthermore, differences in organelle states could affect the physiological functions or activities of the GCs. The plastid morphology reported in this study should be of help in future studies investigating the roles of GCs in the control of stomatal opening and closing [ 70 , 71 ].

This would also be the case for studies designed to examine the roles of PCs. A CLSM image of maximal intensity projection is shown. The arrowhead indicates two stromules that appear to align or wind together. See also Fig 2. Serial optical sections of Fig 4D are shown. Serial optical sections of Fig 4I are shown. The GCs were randomly shuffled to create GC pairs, and the number of each GC-pair type was counted in each simulation trial.

The average counts from 1, trials for each mutant are shown gray boxes , along with standard deviation error bars. The actual counts of each GC-pair type in GC pairs Fig 5F are also shown for each mutant black boxes for comparison. The simulation program was written in Python 3. A three-dimensionally reconstructed image of the GC plastids in Fig 4J is shown with rotation around two axes. A three-dimensionally reconstructed image of the GC plastids in Fig 4K is shown with rotation around two axes.

Kevin Pyke and Dr. Rachel Leech UK for their donation of arc mutant seeds. The authors also thank Y. Kazama and H. Ishikawa for helpful discussions and T. Motoyama, I.

Yamaguchi, H. Ichikawa, N. Suzuki, S. Shimada, A. Yamagami, S. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Chloroplasts, or photosynthetic plastids, multiply by binary fission, forming a homogeneous population in plant cells.

Confocal laser scanning microscopy and epifluorescence microscopy The basal parts of the leaf including the petiole were excised from seedlings with tweezers. Results Plastid morphology in leaf PCs of arc5 and arc6 To investigate the morphology of leaf epidermal plastids in the Arabidopsis arc5 and arc6 mutants, we introduced expression cassettes encoding stroma-targeted YFP or GFP under the control of the CaMV35S promoter into both mutants via crossing for details, see the Materials and Methods.

Download: PPT. Fig 1. Plastid phenotypes in leaf mesophyll cells of Arabidopsis WT, arc5 , arc6 , and atminE1. Giant plastid formation. Fig 2. Stromule development. Grape-like plastid clusters. Fig 3. Grape-like plastid clusters in leaf PCs of arc6. Plastid morphology in leaf stomatal GCs of arc5 and arc6 Extending the analysis of epidermal plastids in leaf PCs, we examined those in stomatal GCs.

Fig 4. Fig 5. Quantitative evaluation of plastid morphology, pigmentation, and partitioning in leaf stomatal GCs in arc5 and arc6 We then observed chlorophyll autofluorescence and stromal YFP fluorescence simultaneously in GCs, as exemplified in atminE1 Fig 5A—5D.

Fig 6. Table 1. Discussion Exploring differences between PC plastid division and mesophyll chloroplast division In this study, we investigated the plastid phenotypes in leaf epidermal cells of the arc5 and arc6 mutants, which showed distinct morphological differences compared with the known chloroplast phenotypes reported for mesophyll cells of the corresponding mutants [ 5 — 7 ]. F Chlorophyll autofluorescence Chl was used as a chloroplast marker. G Extended model 2, representing the involvement of equal and unequal chloroplast partitioning following GMC division and subsequent division of GC chloroplasts with equal blue line or selective red line division competency, which would result in four types of chloroplast number determination Fates 1—4 during late stomatal development of Arabidopsis leaves.

A , C — F Epifluorescence microscopy was performed with an Olympus IX71 inverted microscope using plant materials as previously described Fujiwara et al.

Indications in panels are as follows: arrowhead, the FtsZ1 ring; arrow, enlarged GC chloroplast; dashed line, cell shape. Leaf mesophyll cells have long been employed as a primary model for the analysis of chloroplast number. While they have advantages for the study of the effects of environmental conditions on chloroplast division e. Firstly, leaf mesophyll cells vary in size and shape and are distributed deep within the leaf, which makes it difficult to manipulate intact tissues.

Secondly, the susceptibility of leaf mesophyll chloroplast proliferation to environmental stress and plant growth conditions can prevent reliable comparisons between studies. The leaf mesophyll chloroplast number per cell in Arabidopsis Columbia Col ecotype has been reported variously as 76 Kinsman and Pyke, , 80— Stokes et al. Furthermore, it is almost impossible to assess the contribution of chloroplast partitioning to final chloroplast number per mesophyll cell during leaf development, although this is thought to be determined by the balance between the rate of cell division and rate of chloroplast division.

To uncover the mechanism of chloroplast number control in vegetative leaf cells, a model system that overcomes the above issues is required. Stomatal GCs see Figure 1A exhibit the characteristics of a model system for understanding the mechanism of chloroplast number control. GCs are highly uniform in size and shape within a tissue, and their scattered but dense distribution in the outermost layer of shoots facilitates their detection by light and fluorescence microscopy.

GCs are also derived from protodermal cells in the shoot apical meristem or from embryonic epidermal cells, and their developmental sequence through meristemoids a stomatal precursor with meristematic activity and guard mother cells GMCs; a precursor of GC pairs is established in detail Zhao and Sack, ; Nadeau and Sack, ; Kalve et al. Late stomatal development involves a single round of symmetric GMC division, which enables the assessment of chloroplast distribution and partitioning before and after cytokinesis.

From the perspective of practical experiments, leaf GCs are suitable for microscopy. It was previously shown that chloroplast number per cell in leaf GCs of Sinapis alba was less affected by different light conditions than that in leaf mesophyll cells Wild and Wolf, Additionally, the difference in GC chloroplast number in leaf petioles is relatively minor among the three Arabidopsis ecotypes Col, L er , and Ws Fujiwara et al.

Furthermore, endoreduplication, which impacts the development of leaf mesophyll, pavement, and trichome cells, has not been detected in Arabidopsis leaf GCs Melaragno et al.

Together, these reports suggest that leaf GCs are potentially an excellent model for the systematic analysis of chloroplast number dynamics in a particular cell lineage.

In the history of GC chloroplast research, chloroplast counting at the stomatal GC pair level has served an equally important role in determining the chloroplast number as counting at the individual GC level. Both methods produce the same mean chloroplast number Butterfass, When the variation in chloroplast distribution in paired GCs and its underlying mechanism is a subject of focus, detailed information of chloroplasts at the individual cell level, i.

Chloroplast plastid proliferation during the GMC—GC differentiation was previously investigated in several plant species Butterfass, , Butterfass These studies proposed two models for determining the terminal chloroplast number in GCs in different plant species Figure 1B : one model 1; sugar beet involves only chloroplast partitioning at GMC division, and the other [model 2; alsike clover Trifolium hybridum ] involves not only chloroplast partitioning but also chloroplast proliferation during GC development.

In the era of molecular genetics, genomics, cell imaging, and other interdisciplinary analyses, there are many possibilities for the further characterization of the chloroplast partitioning mechanism. Arabidopsis leaf GCs may be one of the best model systems for this purpose. Several studies have examined the GC chloroplast number in the leaves or cotyledons of Arabidopsis Hoffmann, ; Pyke and Leech, ; Pyke et al. These GCs exhibit a modest number of chloroplasts, ranging from 3. To date, no studies have examined the alterations in chloroplast plastid number during stomatal development.

However, microscopic evidence from stomatal development analyses e. Expanding leaf petioles fifth leaves of 4-week-old seedlings were employed. As a result, GCs with symmetrically constricting chloroplasts were detected Figure 1C. These chloroplasts formed the FtsZ1 ring, a chloroplast division ring on the stromal surface of the inner envelope membrane in leaf mesophyll and pavement cells Vitha et al.

Consistent with the stomatal patterning in Arabidopsis leaf development Donnelly et al. Thus, model 2 is most likely the best fit for Arabidopsis leaf GCs. When epidermal peels of fully expanded leaves third—fourth leaf blades of 4-week-old seedlings from a TP-fused yellow fluorescent protein YFP line were microscopically characterized FL line; Fujiwara et al.

Within the GC pair of a stoma, the size of chloroplasts in the GC containing smaller numbers of chloroplasts was larger than in the other GCs in the pair containing larger numbers of chloroplasts Figures 1D, E. In this way, GCs probably maintain the total chloroplast volume per cell at a constant level during cell growth. Enlarged chloroplasts represented the terminal phenotype and could no longer divide in expanded leaves.

These results were confirmed in several independent experiments, irrespective of the expression of a TP-fused fluorescent protein for stroma labeling Figure 1F. This GC chloroplast phenotype is interpreted as a compensation mechanism for chloroplast expansion, which was well documented in leaf mesophyll cells defective in the control of chloroplast division Pyke and Leech, ; Pyke et al.

To date, only one study Ellis and Leech, has reported a negative correlation between chloroplast number and chloroplast size in leaf mesophyll cells of wheat, whereas many studies have reported a positive correlation between cell volume and chloroplast number in normal leaf mesophyll cells Leech and Pyke, ; Pyke, Whereas imbalances in GC chloroplast number occur at low frequency Fujiwara et al.

The chloroplast compensation effect in GCs may be less strict than in leaf mesophyll cells. GCs might be able to withstand scarcity or complete loss of total chloroplast volume per cell in severely impaired chloroplast division mutants, such as in Arabidopsis arc6 and atminE1 and tomato suffulta , whereas many mutant GCs showed reduced chloroplast number and enlarged chloroplast size similarly to the leaf mesophyll cells Robertson et al.

In a late chloroplast division mutant, arc5 , the reduction in GC chloroplast number was not associated with a significant increase in chloroplast size, unlike in leaf mesophyll cells Pyke and Leech, A lower degree of chloroplast expansion in GCs than in mesophyll cells Pyke and Leech, ; Barton et al.

Furthermore, the timing of chloroplast division during GMC—GC differentiation might significantly affect the terminal GC chloroplast phenotype. Although further detailed characterization is required to address this issue, it seems plausible that Arabidopsis leaf GCs represent a system to investigate the unexploited aspects of chloroplast number control in plant cells.

On the basis of the above, we propose a working model an extended model 2 for the analysis of chloroplast number in GCs Figure 1G. During GMC division, chloroplasts may undergo either equal or unequal partitioning. During chloroplast proliferation, GC chloroplasts will proliferate with either equal blue line or selective magenta line division competency. For example, if equally partitioned chloroplasts possess equivalent division competency, equal chloroplast numbers will occur in the GC pair Fate 1.

If unequally partitioned chloroplasts possess equivalent division competency, chloroplasts will increase at the same rate within the GC pair Fate 3. The model raises two issues: i Are GC chloroplasts properly partitioned into daughter cells and how do they partition? And ii is division competency of GC chloroplasts coordinately regulated? Intriguingly, in Arabidopsis arc6 , leaf or cotyledon GCs have zero to three chloroplasts, and in chloroplast-deficient GCs, non-photosynthetic plastids still exist in vesicular to elongated forms Robertson et al.

No GCs devoid of plastids per se have been found in arc6 , and no explanation for this has been forthcoming, despite the disruption of the chloroplast division apparatus Vitha et al.