How this change occurs at the molecular level is uncertain, although comparative transcriptomics implicate decreased expression of genes whose products comprise the plastid division rings in reducing M chloroplast numbers and increasing their size in the C 4 Flaveria species. The presence of reduced chloroplast numbers and coverage in Flaveria and all other C 4 lineages examined to date is strong evidence that the C 3 pattern of chloroplast distribution is maladapted in the C 4 context.
As such, reduction in M chloroplast numbers, and possibly increases in size, should be included in efforts to engineer the C 4 pathway into major C 3 crops of warm regions such as rice, soybean and cotton von Caemmerer at al. Based on the results here, this would require independent changes to the genetic control over M and BS chloroplast properties.
In the case of the BS cells, these gene changes may control chloroplast size, but the number of changes may reflect a distinct mechanism associated with GC1 and CLMP. Identifying the regulatory elements that determine the C 4 transcriptome pattern with respect to chloroplast division stands out as the next major step in understanding how the C 4 pattern of chloroplast investment evolves. The 18 Flaveria species used in this study are F. Their original collection locations if known and photosynthetic classification are listed in Supplementary Table S1.
Plants were grown either from seeds or from cuttings in 10 or 20 liter buckets filled with a sandy-loam soil in the spring and summer of and Plants sampled were generally 2—3 months in age, although some of the perennial species were over a year old. All individuals sampled were healthy, with deep-green leaves and, where measured, they exhibited photosynthesis rates close to peak values reported in the literature not shown.
Sampling and preparation for light microscopy single-cell isolates and TEM, and imaging of cellular features was conducted as previously described Sage and Williams , Sage et al. As previously noted, these procedures were designed to ensure rapid fixation of tissue and cellular components and to prevent post-sampling movement of chloroplasts Stata et al. The cellular features that were quantified are illustrated in Supplementary Fig.
S1 and include: i the number of chloroplasts per M cell area in planar sections; ii the area of chloroplasts in planar sections; iii the fractional area of an M cell that was covered by chloroplasts in a planar section; iv the proportion of the entire M cell perimeter covered by chloroplasts; v the proportion of the M cell perimeter facing the intercellular spaces that was covered by chloroplasts; and vi the fraction of the outer chloroplast edge that was in contact with the M cell periphery Supplementary Fig.
To minimize the possibility of artifacts associated with plane of section, we also measured parameters i — iv in paradermal sections of leaves, and examined chloroplast number and size in single-cell isolates of adaxial palisade parenchyma and abaxial spongy parenchyma M cells, from two species, i.
Using TEM, we also assessed chloroplast number per planar area of BS cells in transverse sections, BS chloroplast size as estimated by planer area of chloroplasts in transverse sections, and total chloroplast area per BS cell area in planar sections.
Transcriptome assemblies and expression profiles of chloroplast division genes were obtained from the 1KP database www. Plants were grown in a greenhouse at the University of Toronto and sampled in June and July of Within a species, one immature leaf from each of three plants was pooled to form a species sample.
Fifteen genes implicated in plastid division and development were selected from table 1 of Osteryoung and Pyke for copy number and expression level analysis see Tables 3 and 4 for the list of transcripts examined. For each, we identified orthologous sequences in the Flaveria transcriptomes using a phylogenetic approach. Using these alignments and trees, we distinguished between genuine duplication events and multiple sequence matches due to partial transcript assemblies.
In cases where partially assembled transcripts were present, the longest for each gene copy was used for expression level analysis. Phylogenetic reconstruction was conducted using Mesquite Maddison and Maddison , with squared parsimony reconstruction.
Tree topologies are based on the phylogenies presented in both Lyu et al. The topology in Lyu et al. For each species, one leaf from each of three plants was sampled. For each leaf, 10 sections were obtained and one M and BS cell per section analyzed; the resulting values were pooled to provide one value per plant per parameter.
Measured M cells were from the upper adaxial half of the leaf, unless otherwise indicated. M cells were selected at random from the subpopulation of M cells where the plane of section passed through the central portion of the cell. Plant means were then averaged to obtain species means, which were compared using one-way analysis of variance ANOVA and a Student—Neuman—Kuels multiple range test to assess differences between species Sigma-plot v Species values within a photosynthetic category were pooled, and differences between the photosynthetic category means were tested using one-way ANOVA and a Student—Neuman—Kuels post-hoc test.
To evaluate parameters as a function of C 4 cycle strength, we obtained values for the percentage of initial 14 C fixation into the C 4 acids malate and aspartate, compiled by Vogan and Sage for 15 of the 18 species in the study, using data from published sources Rumpho et al.
Percentage 14 C fixation into malate and aspartate is the single best indicator of relative C 4 cycle strength as it directly reflects the fractional incorporation of CO 2 into organic acids via PEP carboxylation vs. Rubisco activity Edwards and Ku Other indexes such as O 2 sensitivity of carbon fixation and the CO 2 compensation point of carbon fixation are affected by photorespiratory glycine shuttling, and thus can vary for reasons other than C 4 cycle activity Monson and Rawsthorne We assumed that the values for the percentage 14 C fixation into malate and aspartate for the species lacking these data F.
Using least-squares regression routines in Sigmaplot v Responses of M parameters vs. C 4 cycle strength were fit to second-order polynomials as these gave better fits than linear regressions. All other responses all responses of BS parameters and M responses to transcriptome abundance were analyzed using linear regressions as these best fit the relationships. Altschul S. Madden T. Zhang J. Zhang Z. Miller W. Nucleic Acids Res. Google Scholar. Weber A. Ediited by Raghavendra A.
Sage R. Springer , Dordrecht, The Netherlands. Google Preview. Brown R. Hattersley P. Plant Physiol. Brown N. Parsley K. Hibberd J. Trends Plant Sci. Busch F. Sage T. Cousins A. Plant Cell Environ. Bioinformatics 25 : — Christin P. Besnard G. Samaritani E. Duvall M. Hodkinson T. Savolainen V. Osborne C. Arakaki M. Edwards E. Covshoff S. Burgess S. Dai Z.
Edwards G. Planta : — Dengler N. Nelson T. In C 4 Plant Biology. Edited by Sage R. Monson R. Academic Press , San Diego.
In The Biochemistry of Plants , Vol. Edited by Hatch M. Academic Press , London. Voznesenskya E. Epstein E. Bloom A. Sinauer Associates , Sunderland, MA. Evans J. Loreto F. In Photosynthesis: Physiology and Metabolism. Edited by Leegood R. Sharkey T.
Kluwer Academic , Dordrecht, The Netherlands. Frederick S. Newcomb E. Planta 96 : — Glynn J. Yang Y. Vitha S. Schmitz A. Hemmes M. Miyagishima S. Plant J. Cell Mol. Gowik U. Westhoff P. Holaday A. Lee K. Chollet R. Planta : 25 — Johnson M. Carpenter E. Tian Z. Bruskiewich R. Burris J. Carrigan C. PLoS One 7 : e Kanai R. A loosely packed layer of irregularly-shaped cells.
Air spaces that surround this cell layer allow gas exchange to take place. Small pores holes located on leaves. They are usually present on the underside of leaves but can also be found on the upper side as well.
A membrane bound structure within the chloroplast. Thylakoids consist of a thylakoid membrane surrounding a thylakoid space or lumen. Thylakoids contain chlorophyll and are where photosynthesis takes place. The process of water movement through plants and eventual evaporation from small pores, or stomata, in leaves. Strands of vascular tissues connecting all of plant parts in order to transport nutrients and water through phloem and xylem.
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Above Image: Diagram showing the special types of cells present in leaves Without leaves, there would not be life on Earth. Leaf Structure Leaves are complex organs consisting of many different cell types see Figure 1 including the epidermis, palisade mesophyll layer, spongy mesophyll layer, and vascular bundles. Leaves have an upper epidermis that is located on the upper part of the leaf. A cuticle can also sometimes be present on the outside of the epidermis.
This waxy layer helps prevent water loss, especially in dry regions. The palisade mesophyll layer is made up of closely-packed, elongated cells located just below the upper epidermis. They contain chloroplasts and carry out most of the photosynthesis. Vascular bundles are made up of xylem and phloem cells. These are the cells that carry water and nutrients throughout the plant and are visible as the veins in leaves. Unlike palisade cells, however, spongy cells are located deeper in the leaf below the upper epidermis and the palisade tissue.
With regards to photosynthesis, this is a disadvantage given that light does not penetrate to this region easily. As a result, spongy cells do not receive enough sunlight required for photosynthesis to occur ideally.
Although spongy cells are not well suited for photosynthesis processes, their arrangement are ideal for gaseous exchange. As previously mentioned, spongy cells are loosely packed above the lower epidermis.
This creates large spaces between the cells which is ideal for gaseous exchange. Small openings located on the epidermis allow such gases as carbon dioxide to enter the leaf and reach the mesophyll cells. On the other hand, photosynthetic processes in the mesophyll result in the production of oxygen. The loosely packed cells spongy cells in this region of the cell allow these gases to be exchanged where oxygen is released while carbon dioxide is used for photosynthesis.
Using an electron microscope , it's possible to not only clearly observe mesophyll cells, but also the architecture of the thylakoid membrane. However, for the purposes of observing mesophyll cells, a light microscope is sufficient. With various samples, a vibratome is used for cutting in order to obtain thin sections that can be viewed under the microscope. However, with some samples, such as very thin leaves, alternative approaches may be used to cut in order to obtain the thinnest needed.
Here, a young, thin cassava stem is first cleaned and dried under the sun or in the oven. A small leaf sample 1cm sq is then cut out and inserted between the sliced cassava cork so that the sample is held between the sliced cork - Here, it's important to ensure that the cassava cork can fit in the hole of the mini microtome.
When viewed under the microscope, well prepared slices will display preserved mesophyll cells. Here, the epidermis will appear thin and darker while spongy cells will appear scattered below well organized palisade cells. Return to Plant Biology overview. Return to Leaf Structure under the Microscope. See also info on Meristem cells of plants and Transgenic Plants.
Return to learning about Guard Cells. Return to Organelles - Animal and Plant. Return from Mesophyll Cells page to MicroscopeMaster home. David S. Shatelet, et al. Chicago Journals.
An alternative simple method for preparing and preserving cross-section of leaves and roots in herbaceous plants: Case study in Orchidaceae. Leaves are thin — ensures all cells receive light. Leaves have a large surface area. Adaptations to maximise gas exchange: Spongy mesophyll — have very few chloroplasts and a large surface area to increase the diffusion of carbon dioxide and oxygen. Intercellular air spaces within the spongy mesophyll layer — they allow the diffusion of carbon dioxide and oxygen.
Stomata small pores usually found on the lower surface of the leaf — allow carbon dioxide and oxygen to enter and leave the leaf.
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