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Changing Plastid Dynamics within Early Root and Shoot Apical Meristem-Derived Tissue of A. thaliana

Changing Plastid Dynamics within Early Root and Shoot Apical Meristem-Derived Tissue of A. thaliana BioscienceHorizons Volume 10 2017 10.1093/biohorizons/hzx001 .............................................. .................................................. .................................................. ............... Research article Changing Plastid Dynamics within Early Root and Shoot Apical Meristem-Derived Tissue of A. thaliana Lawrence Bramham and Kevin Pyke School of Life Sciences, University of Warwick, Wellesbourne Campus, Warwick, CV35 9EF *Corresponding author: School of Life Sciences, University of Warwick, Wellesbourne Campus, Warwick, CV35 9EF. Email: lawrencebram@hotmail.co.uk Supervisor: Dr Kevin Pyke, School of Biosciences, University of Nottingham, Sutton Bonington LE12 5RD, UK. .............................................. .................................................. .................................................. ............... Whilst plastids are fundamental to many aspects of plant biology and the production of enhanced crop cultivars, research into the dynamics of non-green plastids has remained somewhat disregarded by the scientific community compared to chloroplasts. They are equally pivotal to normal plant development however, and are now increasingly becoming the focus of research made possible by genetic manipulation and reporter gene constructs. The total plastid content of all plant cells originates from small, undifferentiated plastids termed proplastids found within the meristematic regions of both root and shoot tissue. The cellular regulatory mechanisms controlling the development of plastids in young tissues are poorly understood, especially in the case of non-green plastids in roots. This investigation con- sequently aimed to elucidate the differences in plastid content, morphology and subcellular localization within epidermal cells derived from the root and shoot apical meristems (RAM and SAM respectively) of Arabidopsis thaliana. Quantification of non-green plastids was facilitated via the use of confocal laser scanning microscopy in conjunction with the expression of plastid-targeted green fluorescent protein driven by a constitutive promoter. Characterization of early seedling development and tissue diversification was also achieved by assessing epidermal cell size relative to devel- opmental progression, ultimately facilitating comparative analyses of plastid dynamics on both a temporal and tissue- varietal basis. The number of plastids in epidermal cells within RAM-derived tissue was shown to increase across regions of cell division before being regulated throughout subsequent zones of elongation and maturing root tissue. In contrast, epidermal cells of the hypocotyl exhibit a more generalized increase in plastid number and less strict maintenance of cell plan area coverage during tissue expansion. The findings presented here suggest the functioning of distinct mechanisms regulating plastid division and growth in relation to cell size within shoot and root apical meristem-derived tissues. Key words: Plastids, GFP, Epidermal Cells, Root, Hypocotyl, Arabidopsis thaliana Submitted on 18 May 2016; editorial decision on 6 January 2017 .............................................. .................................................. .................................................. ............... This research was carried out as part of my undergraduate dissertation, which attributed towards the degree of BSc (Hons) in Biotechnology at The University of Nottingham, Sutton Bonington Campus, UK. ............................................................................................... .................................................................. © The Author 2017. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Research article Bioscience Horizons � Volume 10 2017 ............................................................................................... .................................................................. pollen and seed dispersal by attracting pollinators or herbi- Introduction vores at specific developmental stages (Galpaz et al., 2006). Due to their fundamental roles in plant biology, the ability to Plastids are organelles that ubiquitously reside in the cells of accumulate novel compounds and other unique advantages, higher plants, adopting different forms in order to facilitate a plastids present significant targets for agronomic enhance- diverse array of intracellular functions. They are believed to ment and new biotechnological applications. They have have originated through a primordial endosymbiotic event already been widely used within the field of genetic manipula- with a free-living cyanobacterium-like prokaryote and remain tion (Medina-Bolíva and Cramer, 2004; Daniell, 2006; fundamentally distinct from other eukaryotic organelles Clarke and Daniell, 2011) but a large potential for plastid (Gould, Waller and McFadden, 2008; Pyke, 2009). The fore- genetic engineering still exists, perhaps emerging from most role of plastids, or more specifically green-pigmented increased research into non-green plastids and their develop- chloroplasts, is the fixation of atmospheric carbon dioxide mental biology. into a plethora of organic molecules that facilitate growth and development (Robertson and Laetsch, 1974; Pyke, 2009). Higher plant embryogenesis transforms a fertilized ovum Unsurprisingly, due to the pivotal nature of photosynthesis into a juvenile form of the plant lacking most species-specific amidst aims to improve its efficiency, chloroplasts within features (Fig. 1). Post-embryonic development subsequently foliar tissue have been the focus of significantly higher levels occurs from meristematic tissue in a progressive and highly of research compared to plastids of other tissues. This is also organized wave-like manner, generating new tissue through partly due to methodological difficulties in quantifying non- successive cell replication and expansion (Malamy and green plastids before the advent of genetic manipulation, the Benfey, 1997; Cary, Che and Howell, 2002). The following use of reporter gene constructs and confocal laser scanning increase in architectural complexity is thus heavily influenced microscopy (CLSM). These non-green plastids, however, are by changing factors within the early stages of germination no less intrinsic to plant development. (Jürgens, 2001). Leucoplasts are one such plastid subgroup crucial to lipid The total plastid content of all plant cells originates from biosynthesis, amino acid metabolism and as storage sites for small, undifferentiated plastids termed proplastids found starch grains (amyloplasts), lipids (elaioplasts) and proteins within dividing cells of the root and shoot apical meristems (aleuroplasts) (Ohlrogge and Browse, 1995; Finkemeier and (RAM and SAM, respectively) (Pyke, 2009; Finkemeier and Leister, 2010). Of these, amyloplasts are deemed particularly Leister, 2010). They replicate via the prokaryotically-derived critical to correct development as sources of long-term energy mechanism of binary fission to form daughter plastids which, reserves essential for growth and the formation of viable seeds having segregated into individual cells during mitosis, differ- and desirable grain (MacCleery and Kiss, 1999; Pyke, 2009). entiate between plastid varieties depending on intracellular They are also fundamental to plants’ gravitropism where suf- conditions and gene expression (Pyke, 2009; Osteryoung and ficiently dense amyloplasts termed statoliths are translocated Pyke, 2014). The renowned example of this is during chloro- by gravity and promote the redistribution of auxin through plast development where, in the absence of light, proplastids membrane-based influx/efflux proteins, enabling directional develop into etioplasts containing semi-crystalline prolamellar growth under conditions of otherwise limited stimuli (Swarup bodies which, when later exposed to light, transform into the et al., 2005). characteristic, chlorophyll-containing thylakoid membranes of chloroplasts (Finkemeier and Leister, 2010). Whilst classi- In addition to key intracellular roles, plastids are also fied into different plastid sub-types according to their function important for determining interactions between plants and their immediate environment. Chromoplasts, for example, and internal structure, differentiation between plastid types is synthesize and accumulate pigments including yellow xantho- both highly prevalent and fundamental to their success as phylls and red lycopene in tomatoes, which can encourage dynamic elements of plant biology. Figure 1. Seed germination in A. thaliana.(A) Diagram of internalized embryo. (B) Germination observed from 0 to 72 h. Radicle emergence and testa shedding were seen to occur at approximately 36 and 52 h after sowing, respectively. ............................................................................................... .................................................................. 2 Bioscience Horizons � Volume 10 2017 Research article ............................................................................................... .................................................................. Green fluorescent protein (GFP) has been used extensively then added to 1.5 g bacto-agar and sterilized at 121°C for 1 h in modified forms as a reporter for gene expression (Chalfie using an autoclave. et al., 1994). Unlike other methods of analysing expression, Seventy-five millilitre agar was poured into standard 120 × its introduction within target gene constructs facilitates the 120 mm square petri dishes onto which two rows of 10 ster- direct visualization of expression in vivo by naturally fluores- ile seeds were evenly distributed. cing green when exposed to light in the blue to ultraviolet range (Chiu et al., 1996). Targeting GFP to plastids when Seed sterilization and growth used in partnership with CLSM can therefore be used to Within a laminar flow hood, seeds were suspended in a solu- quantify their size, structure and subcellular localization. tion of 0.75 ml 50% bleach/water and 0.75 ml 0.1% Triton/ Whilst the mechanism of chloroplast division has been water. After 6 min, any superfluous solution was discarded studied in depth (Maple and Møller, 2007; Osteryoung and from sedimented seeds before 1 ml 70% ethanol solution was Pyke, 2014), relatively little is known about how other plastid used to resuspend the seeds. Seed suspensions were then varieties divide, especially proplastids and other root-based inverted over 20 s before excess solution was removed and plastids. A wide array of plastid-derived structures including seeds distributed on cooled agar. stromules and vesicles have been observed in cultured root tis- Seeds were cooled to 4°C for 12 h in order to ensure even sue however, suggesting that division mechanisms additional germination before being placed in a growth room main- to those of chloroplasts are present and required to facilitate tained at 20–25°C on a 24 h light cycle. A total of twenty the regulation of a population of increasingly heterogeneous plates containing 400 seeds were monitored over 156 h in plastid forms (Pyke, 2013). 12-h increments for comparative growth between root and Considering the importance of plastids to plant biology and hypocotyl tissue whilst, where appropriate, seedlings were their potential in enhancing cultivars of high socioeconomic excised for further analysis. importance, it is somewhat surprising that relatively limited research has been undertaken into their development during Confocal microscopy and image analysis embryogenesis and germination; particularly into the differ- Ten seedlings were harvested every 12 h from 60 h after plat- ences seen between the plastid dynamics of root and shoot ing (until 156 h) and were imaged using a Leica SP2 confocal meristem-derived tissues. As individual systems of gene expres- laser scanning microscope (Leica Microsystems, Heidelberg, sion and development, these early differences could have pro- Germany). A single plane of peak GFP and chlorophyll fluor- found impacts on general plant biology, crop improvement escence within the epidermal layer was measured in each sam- and biotechnology. Before further research can be performed, ple from the root tip, across the root–hypocotyl tissue however, a detailed characterization of a suitable model system boundary and into hypocotyl tissue, retaining accurate plastid is fundamental. quantification throughout all tissues under investigation. This investigation consequently aimed to elucidate some of As an indication of whether the observed prevalence of the differences seen between meristem-derived tissues by visu- plastids was representative of each cell’s actual content, Z alizing plastid number and size across Arabidopsis thaliana stacks were performed and assessed on samples of 20 similar seedling transects during the first 7 days after imbibition. sized epidermal cells in each major tissue variety and a range of seedlings (4 × 96 h, 120 h, 144 h and 156 h). GFP imaging was accomplished by directing a 488 nm laser for excitation Materials and methods onto samples while detecting fluorescence at an emission wavelength of 509 nm. Chlorophyll autofluorescence was Seeds of A. thaliana (var. Columbia) were donated by George imaged at a peak emission wavelength of 673 nm whilst Bassell and originated from the study of Cutler et al. (2000). bright field images of tissue were also collected. Through Agrobacterium-mediated transformation, a trans- gene containing plastid-targeted GFP under the control of a Cell walls were labelled with propidium iodide (PI); seed- constitutive promoter was introduced, facilitating the intra- ling tissue samples were excised using a scalpel and soaked cellular visualization of all plastid forms by GFP fluorescence for 5 min in 3 ml 0.5 μm PI solution before being carefully using CLSM. rinsed several times with water and imaged (excitation wave- length = 535 nm, emission wavelength = 617 nm). Growth media All recorded images consisted of an average of eight com- Bacto-agar enriched with Murashige and Skoog (MS) salts plete scans that were later compiled into single, layered was used as an optimal growth medium that enabled unobtru- images of each channel across the entire length of individual sive observation and excision of seedlings (Murashige and seedlings (Figs 3, 4B, 5D and 7B). These images were then Skoog, 1962). All growth media was produced in the ratio of analysed using ImageJ software (http://imagej.nih.gov/ij/ 150 ml purified water, 1.5 g sucrose and 0.645 g MS basal download.html, downloaded on 20/10/2014), ensuring at salts mixed to a pH of 5.8–6.0. The resulting solution was least 80% of visible epidermal cells were assessed for plan cell ............................................................................................... .................................................................. 3 Research article Bioscience Horizons � Volume 10 2017 ............................................................................................... .................................................................. area, plastid number and percentage coverage (percentage more to total seedling length than hypocotyl growth (Fig. 2). plan cell area occupied by plastids). Statistical analyses and Total seedling, root and hypocotyl tissue length were all also graphs were performed and produced respectively using seen to increase by a near-consistent 3% per hour after root Microsoft Excel 2013 (Figs 2, 4A, 6, 7A and 8). emergence, suggesting a conserved and relatively stable rate of growth within the two types of tissue. Tissue under investigation can be classified into regions Results based on gross tissue morphology, cell replication and size. Moving across root tissue from the root tip to the root– Seedling growth characterization hypocotyl tissue boundary, successive zones of high (but Measuring the average length of the hypocotyl and root tissue decreasing) cell division, elongation and root hair tissue exist. during germination showed that root growth contributed Many of these tissues were seen to overlap however, restrict- ing the ability to conclusively investigate discrete tissue var- ieties. Within the hypocotyl, less visually recognizable changes in morphology were observed during the early stages of development except for a notable increase in chlorophyll content revealed by autofluorescence (Fig. 3). Regions of higher rates of cell division, elongation and mature hypocotyl tissue were also present and can be qualified by changes in epidermal cell size (Fig. 4A). An increase in epidermal cell size with distance from root tip was seen to occur consistently in different seedlings at near-constant distances (Fig. 4A). In root tissue, the zone of cell division extended approximately 1100 μm from the root tip, within which cells of the root cap were shown to uni- formly increase in plan cell area from an average of 85.7 to 351.2 μm . Subsequently, a major increase in cell size was Figure 2. Length of total A. thaliana seedling ( ), root ( ) and observed beyond 1100 μm where average epidermal cell size hypocotyl ( ) tissue during germination. Error bars represent standard increased by roughly 70 μm per 100 μm until a distance of error. Number of seedlings assessed = 340. Figure 4. (A) Exemplified trend within 96 h A. thaliana of changing epidermal plan cell area against distance from root tip. Root epidermal Figure 3. Superimposed confocal image of chlorophyll fluorescence cells ( ), hypocotyl epidermal cells ( ). (B) Annotated compilation of (red) and bright field image of A. thaliana seedling after 52 h. Red confocal bright field channel images of 96 h A. thaliana seedling across chlorophyll fluorescence can be seen in the hypocotyl and cotyledons the root–hypocotyl transect. Labels indicate the approximate regions but not within root tissue after the root–hypocotyl tissue boundary. of distinct tissue morphology. ............................................................................................... .................................................................. 4 Bioscience Horizons � Volume 10 2017 Research article ............................................................................................... .................................................................. 4500 μm from the root tip. After 4500 μm until the root– hypocotyl tissue boundary, rate of cell expansion in all sam- ples declined and epidermal cells averaged a plan area of 3497 μm . Epidermal cells within the hypocotyl followed a similar trend of progressive expansion as distance from the SAM increased but exhibited higher levels of variation (Fig. 4A). Within the zone of elongation, cells were seen to consistently increase at an average rate of 142 μm per 100 μm; double that seen in comparable root tissue. Hypocotyl epidermal cell size was also seen to stabilize at comparatively lower levels than root epidermal cells on the root–hypocotyl tissue boundary; on average approximately 2100 and 3600 μm , respectively. These changes to epidermal cell size were seen to be dependent on the morphology and developmental status of hypocotyl and root tissues from 60 to 156 h in all samples. Epidermal cell size was consequently used as a way to qualify developmental progression against the distinct regions of plastid prevalence seen across the root–hypocotyl boundary (Fig. 5A), tissue within the zone of elongation/mature root (Fig. 5B) and in the root tip (Fig. 5C). Plastid number with epidermal cell size The number of plastids within root epidermal cells exhibited two principal stages when plotted against cell plan area (Fig. 6A). Within cells of an average size of 85.7 μm upwards Figure 5. Superimposed confocal GFP and bright field images of 96 h to 500 μm , an increase in plastid number was seen from 3 to A. thaliana seedling showing the changing plastid dynamics within 16. Past the zone of cell replication characterized by plan cell 2 epidermal cells of: (A) the root–hypocotyl tissue boundary; (B) mature areas higher than 500 μm , a relatively consistent number of root tissue and (C) root tip. (D) Compilation of superimposed confocal plastids were subsequently observed averaging slightly higher GFP and bright field channel images of whole 96 h A. thaliana seedling than 17 (17.44) but with a range of 9–26 (SD = 4.18). In across the root–hypocotyl tissue transect. Labels indicate the hypocotyl tissue, the number of plastids per epidermal cell approximate regions of distinct tissue morphology and root–hypocotyl remained within the range of 3–10 across cell sizes below tissue boundary. 500 μm (average of 5.84, SD = 1.95) (Fig. 6B). Plastid num- ber subsequently increased to approximately 26 (26.44) within epidermal cells larger than 1700 μm . Variation in In root tissue, percentage plastid coverage was observed to plastid number was seen however, consistently ranging from be higher within epidermal cells of a plan area lower than 20 to 36 plastids per cell (SD = 4.62). 500 μm (Fig. 8A). Due to their size however, relatively small changes caused large differences leading to variation in plastid coverage from 25% to 55% (average = 37.7%, SD = 8.8%). Plastid coverage against transect distance In cells indicative of the zone of elongation and mature root and epidermal cell size tissue (cell sizes of 500–4500 μm ), percentage coverage was Across all samples, epidermal plan cell area plastid coverage shown to average 20.2% (SD = 5.1%) (Fig. 8A). in root tissue was seen to decrease from an average of 39.7% By comparison, hypocotyl tissue exhibited a less fixed within the root tip and across the zone of cell division (up to trend of reducing plastid coverage against increasing epider- 1100 μm from the root tip) to 20.8% throughout the zones of malcellsize(Fig. 8B). Higher levels of variation from 20% cell elongation and mature root tissue (Fig. 7A). In hypocotyl to 75% were seen across all cells lower than 2000 μm epidermal cells, plastid coverage followed a similar trend. As (average = 41.8%, SD = 13.6%). In epidermal cells larger distance from the SAM increased, percentage plastid coverage than 2000 μm , coverage was seen to decrease until an aver- was seen to drop from an average of 52.1% within the zone age of 28.3% (SD = 8.5%). of cell division (up to 900 μm from SAM) to 24.9% through- out the remaining tissue. In all equivalent hypocotyl and root In order to qualify if the recorded number of plastids and epidermal cells, plastid coverage was seen to be generally percentage plan cell area coverage correlated to the actual higher in hypocotyl tissue. content of epidermal cells, Z stacks of individual cells were ............................................................................................... .................................................................. 5 Research article Bioscience Horizons � Volume 10 2017 ............................................................................................... .................................................................. Figure 6. Aggregated 96 h, 120 h and 144 h A. thaliana seedling data Figure 8. Aggregated 96 h, 120 h and 144 h A. thaliana seedling data of changing number of plastids per cell against epidermal plan cell of changing percentage epidermal cell plastid coverage against area. (A) Root epidermal cells ( ). (B) Hypocotyl epidermal cells ( ). epidermal plan cell area. (A) Root epidermal cells ( ). (B) Hypocotyl epidermal cells ( ). performed. Due to difficulties in determining intercellular boundaries on a Z-plane and reduced fluorescence however, no conclusive scale factor between observable plastid number and total plastid content could be established across the range of tissues investigated. A statistically significant correlation between increases in observed plastid content and that seen within Z stacks was found though, suggesting that data col- lected can be used for comparative analyses. Intracellular plastid numbers measured also corresponded generally to pre- vious studies; 10–20 proplastids within meristematic cells and up to 100 chloroplasts depending on cell size and function (Pyke, 1999; Segui-Simarro and Staehelin, 2009). Plastid morphology and subcellular localization Post-root tissue containing statoliths but within the zone of cell replication, no significant patterning of intracellular local- ization was observed in any samples. Plastids were seen to be distributed between cell peripheries, cytoplasm and in close proximity to nuclei (Fig. 9A). Thin, stromule-like projections and ‘dumbbell’-shaped plastids were also seen within root tip Figure 7. (A) Exemplified trend within 96 h A. thaliana of changing tissue and regions of cell elongation (Fig. 9B) but not as preva- percentage epidermal cell plastid coverage against distance from root lently in zones of cell division and hypocotyl cells (data not tip. Root epidermal cells ( ), hypocotyl epidermal cells ( ). (B) shown). Within cells of the zone of elongation and mature Annotated compilation of superimposed confocal GFP and bright field root tissue, plastid morphology was seen to stabilize into channel images of 96 h A. thaliana seedling across the root–hypocotyl tissue transect. Labels indicate the approximate regions of distinct mostly spherical and elliptical forms distributed along inter- tissue morphology. cellular boundaries (Fig. 9C). ............................................................................................... .................................................................. 6 Bioscience Horizons � Volume 10 2017 Research article ............................................................................................... .................................................................. tissue, whilst a relatively stable area of plastid cell coverage was maintained (Figs 6A and 8A, respectively). By compari- son, the number of plastids within equivalent tissues of the hypocotyl remained within the lower spectrum of those seen in root tissue whilst a higher discrepancy for area of plastid cell coverage was observed (Figs 6B and 8B, respectively). This suggests that within actively dividing tissue, plastids are also replicating whilst a system for regulating plastid num- ber and size is in effect. It also implies that within hypocotyl regions of cell replication, somewhat less rigorous or func- tionally different regulatory pathways exist. Within maturing root tissue, percentage plastid coverage was seen to be approximately half that of replicating regions (Fig. 8A) whilst plastid number was also relatively tightly main- tained (Fig. 6A). This suggests the presence of strong regulatory systems assessing both intracellular plastid number and size in root epidermal cells. Conversely, epidermal cells of the hypo- cotyl exhibited an increase in plastid number throughout the zone of elongation and mature tissue alongside a reduction in plastid coverage (Figs 6Band 8B, respectively). This indicates that plastid number rather than size is increasing whilst allud- ing to additional differences in regulatory systems between the Figure 9. Superimposed confocal GFP and bright field channel two tissues. The disparities in plastid replication rates and images showing subcellular plastid localization within 96 h (A and B) morphology may have a number of evolutionary advantages, and 120 h (C and D) A. thaliana epidermal cells. (A) Irregular patterning within replicating root cells. (B) Stromules and dividing (dumbbell- potentially including the preservation of resources by inhibiting shaped) plastids within elongating root cells highlighted by broad and the formation of functionally superfluous plastids. thin arrowheads respectively. (C) Peripheral patterning within Within the hypocotyl and subsequent foliar tissue, higher elongating/mature root cells. Dashed lines indicate approximate location of plant cell walls (D) Peripheral patterning within mature numbers of plastids (specifically chloroplasts) are beneficial hypocotyl tissue. due to their increased presence enabling more efficient light absorbance and photosynthesis (Finkemeier and Leister, 2010). Increasingly higher levels of peripheral localization Throughout the zones of cell replication and elongation of and chlorophyll autofluorescence were seen across transects the hypocotyl, larger plastids were observed compared to of the hypocotyl, signifying a definitive increase in chloroplast within the equivalent root epidermal layer and, whilst differentiation and functional relocation (Figs 3 and 9D) dumbbell-shaped plastids and stromules were seen towards (Trojan and Gabrys, 1996). It is unclear, however, what fac- the SAM, their prevalence was significantly lower compared tors induce these changes and limit potential plastid differenti- to RAM-derived tissue. In all instances, plastids of the hypo- ation. All seedlings were grown laterally across agar in a 24-h cotyl were also predominantly localized around cell peripher- light cycle, suggesting that any abiotic factors present equally ies (Fig. 9D). affected the two tissues, potentially implying that some degree of plastid predefinition according to function may exist. Discussion The presence of stromules (stroma-filled tubules) projecting from plastids (Fig. 9B) have been shown to facilitate the traffick- Interpretation of results ing of molecules between plastids (Hanson and Sattarzadeh, Plastid dynamics within hypocotyl and RAM-derived epider- 2011), however, their role in the exchange of molecules remains mal cells of A. thaliana have been shown to deviate in terms of a controversial subject. Their presence could still be potentially plastid number, plan cell area coverage and morphology at important within the zone of elongation and mature root tissue near-fixed stages of early plant development. Characterization where root hairs are present and nutrient uptake occurs of A. thaliana growth and epidermal cell plan area assisted in (Benning, Xu and Awai, 2006). Plastids have also been shown qualifying these changes whilst additionally providing a meth- to play a major role in nitrate assimilation by reducing it into od of assessing plastid dynamics across a range of sample seed- nitrite (formed within the cytosol from nitrate by nitrate reduc- lings and tissue varieties (Fig. 4). tase) and further into ammonia using nitrite reductase (Finkemeier and Leister, 2010). In root meristematic tissue and the zone of cell division, the observable number of plastids per epidermal cell was seen to Increased plastid surface area and molecular trafficking increase with cell size, and thus distance from meristematic facilitated by stromules may contribute to more efficient ............................................................................................... .................................................................. 7 Research article Bioscience Horizons � Volume 10 2017 ............................................................................................... .................................................................. nutrient absorption, and is perhaps why the presence of plas- shoot and root tissue formation in early embryogenesis tids within RAM-derived tissues is so highly regulated; espe- (Okazaki et al.,2010; Holtsmark et al.,2013). The phase of cially during early development where the use of nutrients individual cells’ replicative cycles, tightly coupled nuclear- must be optimized for survival. It is also interesting to note based gene expression and tissue variety have the potential to the discrepancy seen in equivalent hypocotyl tissue where directly mediate plastid division (Gillard et al.,2008). stromules were seen less frequently, both here and in previous Homeostatic mechanisms also control the levels of intra- investigations (Pyke and Howells, 2002). cellular PDVs and thus rates of plastid division. One such mechanism is the ubiquitin-proteasome system formed of a Plastid replication and regulatory number of protein complexes capable of degrading unneeded mechanisms or damaged target proteins by proteolysis (proteasomes) and a regulatory protein that facilitates the post-translational ‘tag- Extensive research has been undertaken into plastid division ging’ of target proteins for degradation (ubiquitin) (Haas and regulation by characterizing Arabidopsis accumulation et al., 1982; Kimura and Tanaka, 2010). Through targeted and replication of chloroplasts (arc) mutants. In particular, ubiquitin-activating enzymes, PDVs can thus be degraded and arc6, a mutant known to interfere with proplastid division in plastid division regulated in accordance to the expression of root and shoot meristematic tissues has helped define distinct specific genes including MinC; an inhibitor of FtsZ’s polymer- gene regions fundamental to controlling intracellular plastid ization into the contractile ring’s precursor (Margolin, 2005). number (Robertson, Pyke and Leech, 1995; Vitha et al., 2003). Coupling of plastid and cellular division Plastid division is facilitated through the formation of a Whilst many of these regulatory mechanisms are shown to double ringed protein complex encircling dividing plastids at coincide with discrete phases of the cell cycle, they cannot be their centre, perpendicular to their longitudinal axis. One ring considered to be ‘coupled’ with the division of plastids, as each forms on the stromal side of plastids’ inner envelope whilst process can be altered independently without impairing the the other forms on the cytosolic side of their outer envelope other (Osteryoung and Nunnari, 2003). In arc mutants and (Pyke, 1999). Using a dynamin-related protein that produces plants overexpressing PDVs (a factor that counterintuitively cytokinetic force (DRP5B/ARC5), this ring constricts until inhibits plastid division), no defects in development or cell div- envelope membranes deliquesce and reform to produce two ision have been seen (Pyke and Leech, 1992; Osteryoung et al., plastids (Maple and Møller, 2007; Yang et al., 2008). 1998). Reciprocally, stopping cell division via inhibition of Increased numbers of dumbbell-shaped plastids are there- CDKs does not, whilst seriously hindering normal plant devel- fore representative of higher levels of plastid replication, as opment, necessarily affect plastid division as seen in plants seen within cells of both RAM (Fig. 9B) and SAM prior to the overexpressing NtKIS1a; a highly characterized CDK inhibitor zone of division and in later, maturing tissue (data not (Jasinski et al.,2002, 2003). shown). Whilst morphology of plastids can indicate levels of replication, the disparities seen in the regulation of plastid Future research and potentials of defined content between meristematic and mature root tissues are per- plastid dynamics haps derived from a combination of plastids’ primordial ori- gins and homoeostatic mechanisms. Further research into the regulatory factors differing between early hypocotyl and root tissue could be undertaken by profil- During the simultaneous evolution of the ancestral precur- ing gene expression whilst assessing the plastid dynamics of sors to plastids and their eukaryotic hosts, genes once present respective tissues (Bustin, 2002; Trevino, Falciani and in the endosymbiont’s genome were either lost, retained or Barrera-Saldaña, 2007). The extension of this study into the translocated to the host’s nucleus and levels of synchrony cell layers beyond the epidermis will also be a necessary focus between them were established (Gould et al.,2008). Of the pro- for defining plastid dynamics against plant development teins encoded by such genes, some are essential to plastid repli- because, whilst epidermal cells are considerably easier to cation and have been shown to accumulate during discrete assess and provide comparative analysis across both temporal phases of the cell cycle in accordance with cyclin-dependent and surface tissue-based development, they do not form cer- kinases (CDKs). These include ARC3, MinD, MinE (specify tain key tissues of mature plants. Plastid prevalence could also the site of plastid division), FtsZ proteins (tubulin-like proteins be compared between wild type A. thaliana, meristematic that form the early ring structure), ARC6 (recruits other pro- mutants such as those investigated by Aida, Ishida and teins to the ring/site of division) and DRP5B/ARC5 (generates Tasaka (1999) and important crop cultivars in order to qual- the cytokinetic force necessary for division) (Okazaki, Kabeya ify the impacts of early plastid variation upon later growth. and Miyagishima, 2010). Other essential proteins, such as the nuclear-encoded plastid division proteins (PDVs), have been Ultimately, the data collected here highlights the paucity of shown to regulate DRP5B/ARC5 and consequently plastid div- knowledge relating to the early discrepancies of plastid ision according to cell cycle progression and/or differentiation dynamics within early plant tissues and, whether due to determined by cytokinin, a phytohormone fundamental to changes in gene expression, cellular identity and/or abiotic ............................................................................................... .................................................................. 8 Bioscience Horizons � Volume 10 2017 Research article ............................................................................................... .................................................................. Clarke, J. L. and Daniell, H. (2011) Plastid biotechnology for crop pro- conditions, these observations could correlate to plastid duction: present status and future perspectives, Plant Molecular prevalence within mature plant tissue. Defining the causes of Biology,76(3–5), 211–220. early plastid dynamics could be potentially exploited for future research alongside the development of biofactories of Cutler, S. R., Ehrhardt, D. W., Griffitts, J. S. et al. (2000) Random GFP: important molecules and enhanced cultivars of high socio- cDNA fusions enable visualization of subcellular structures in cells economic importance. of Arabidopsis at a high frequency, Proceedings of the National Academy of Sciences of the USA, 97 (7), 3718–3723. Author biography Daniell, H. (2006) Production of biopharmaceuticals and vaccines in plants via the chloroplast genome, Biotechnology Journal, 1 (10), Molecular biology appeals to his avid interest in science, both 1071–1079. in terms of how elements intrinsic to life function and how they Finkemeier, I. and Leister, D. (2010) Plant chloroplasts and other plas- can be adapted. He aims to forge a career in science under- tids, in Encyclopedia of Life Sciences (ELS), John Wiley and Sons, Ltd, pinned by this fascination of how minute intracellular changes Chichester, United Kingdom. induce huge phenotypic impacts, specifically researching molecular plant pathology and crop cultivar improvement Galpaz, N., Ronen, G., Khalfa, Z. et al. (2006) A chromoplast- through genetic engineering. In 2015, he graduated from The specific carotenoid biosynthesis pathway is revealed by clon- University of Nottingham with a first-class honours degree in ing of the tomato white-flower locus, The Plant Cell,18 (8), Biotechnology, and is about to embark on a PhD at The 1947–1960. University of Warwick where he will research resistance to Turnip mosaic virus in members of the Brassica genus. Gillard, J., Devos, V., Huysman, M. J. J. et al. (2008) Physiological and transcriptomic evidence for a close coupling between chloroplast ontogeny and cell cycle progression in the pennate diatom Acknowledgements Seminavis robusta, Plant Physiology, 148 (3), 1394–1411. Gould, S. B., Waller, R. F. and McFadden, G. I. (2008) Plastid evolution, I would like to especially thank Dr Kevin Pyke for his signifi- Annual Review of Plant Biology, 59, 491–517. cant guidance throughout this study and all the fellow under- graduate students, postgraduate students and staff of UoN Haas, A. L., Warms, J. V., Hershko, A. et al. (1982) Ubiquitin-activating Sutton Bonington campus’ plant science laboratories. Addi- enzyme: mechanism and role in protein-ubiquitin conjugation, The tional thanks to George Bassell for donating the transgenic A. Journal of Biological Chemistry, 25 (5), 2543–2548. thaliana seeds used throughout this experiment and the grant- aided support that research at The University of Nottingham Hanson, M. R. and Sattarzadeh, A. (2011) Stromules: recent insights into receives from the Biotechnological and Biological Sciences a long neglected feature of plastid morphology and function, Plant Research Council UK (BBSRC). Physiology, 155, 1486–1492. Holtsmark, I., Lee, S., Lunde, K. A. et al. (2013) Plastid division control: the PDV proteins regulate DRP5B dynamin activity, Plant Molecular References Biology, 82 (3), 255–266. Aida, M., Ishida, T. and Tasaka, M. (1999) Shoot apical meristem and Jasinski, S., Leite, C. S., Domenichini, S. et al. (2003) NtKIS2, a novel cotyledon formation during Arabidopsis embryogenesis: interaction tobacco cyclin-dependent kinase inhibitor is differentially among the CUP-SHAPED COTYLEDON and SHOOT MERISTEMLESS expressed during the cell-cycle and plant development, Plant genes, Development, 126 (8), 1563–1570. Physiology and Biochemistry, 41, 667–676. Benning, C., Xu, C. and Awai, K. (2006) Non-vesicular and vesicular lipid Jasinski, S., Riou-Khamlichi, C., Roche, O. et al. (2002) The CDK inhibitor trafficking involving plastids, Current Opinion in Biotechnology,9, NtKIS1a is involved in plant development, endoreduplication and 241–247. restores normal development of cyclin D3; 1-overexpressing plants, Bustin, S. A. (2002) Quantification of mRNA using real-time reverse tran- Journal of Cell Sciences, 115 (5), 973–982. scription PCR (RT-PCR): trends and problems, Journal of Molecular Jürgens, G. 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Physiology, 120, 183–192. ............................................................................................... .................................................................. 9 Research article Bioscience Horizons � Volume 10 2017 ............................................................................................... .................................................................. Malamy, J. E. and Benfey, P. N. (1997) Organization and cell differentiation Pyke, K. A. and Howells, C. A. (2002) Plastid and stromule morphogen- in lateral roots of Arabidopsis thaliana, Development, 124, 33–44. esis in tomato, Annals of Botany, 90, 559–566. Maple, J. and Møller, S. G. (2007) Plastid division: evolution mechanism Pyke, K. A. and Leech, R. (1992) Chloroplast division and expansion is and complexity, Annals of Botany, 99, 565–579. radically altered by nuclear mutations in Arabidopsis thaliana, Plant Physiology, 99, 1005–1008. Margolin, W. (2005) FtsZ and the division of prokaryotic cells and orga- nelles, Nature Reviews Molecular Cell Biology, 6, 862–871. Robertson, D. and Laetsch, W. M. (1974) Structure and function of developing barley plastids, Plant Physiology, 54 (2), 148–159. Medina-Bolíva, F. and Cramer, C. (2004) Production of recombinant proteins by hairy roots cultured in plastic sleeve bioreactors, Robertson, E. J., Pyke, K. A. and Leech, R. M. (1995) arc6, an extreme Methods in Molecular Biology, 267, 351–363. chloroplast division mutant of Arabidopsis also alters proplastid proliferation and morphology in shoot and root apices, Journal of Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth Cell Science, 108, 2937–2944. and bioassays with tobacco tissue cultures, Physiologica Plantarum, 15 (3), 473–497. Segui-Simarro, J. M. and Staehelin, L. A. 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(2014) Division and dynamic morph- Microarrays: a powerful genomic tool for biomedical and clinical ology of plastids, Annual Review of Plant Biology, 65, 443–472. research, Molecular Medicine,13 (9–10), 527–541. Osteryoung, K. W., Stokes, K. D., Rutherford, S. M. et al. (1998) Trojan, A. and Gabrys, H. (1996) Chloroplast distribution in Arabidopsis Chloroplast division in higher plants requires members of two func- thaliana (L.) depends on light conditions during growth, Plant tionally divergent gene families with homology to bacterial ftsZ, Physiology, 111, 419–425. The Plant Cell, 10, 1991–2004. Vitha, S., Froehlich, J. E., Koksharova, O. et al. (2003) ARC6 is a J-domain Pyke, K. A. (1999) Plastid division and development, The Plant Cell,11 plastid division protein and an evolutionary descendent of the (4), 549–556. cyanobacterial cell division process, Journal of Bacteriology, 15, Pyke, K. A. (2009) Plastid Biology, Cambridge University Press, United 1918–1933. Kingdom. Yang, Y., Glynn, J. M., Bradley, J. S. C. et al. (2008) Plastid division: across Pyke, K. A. (2013) Divide and shape: an edosymbiont in action, Planta, time and space, Current Opinion in Plant Biology, 11, 577–584. 237, 381–387. ............................................................................................... .................................................................. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioscience Horizons Oxford University Press

Changing Plastid Dynamics within Early Root and Shoot Apical Meristem-Derived Tissue of A. thaliana

Bioscience Horizons , Volume 10 – Feb 18, 2017

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Abstract

BioscienceHorizons Volume 10 2017 10.1093/biohorizons/hzx001 .............................................. .................................................. .................................................. ............... Research article Changing Plastid Dynamics within Early Root and Shoot Apical Meristem-Derived Tissue of A. thaliana Lawrence Bramham and Kevin Pyke School of Life Sciences, University of Warwick, Wellesbourne Campus, Warwick, CV35 9EF *Corresponding author: School of Life Sciences, University of Warwick, Wellesbourne Campus, Warwick, CV35 9EF. Email: lawrencebram@hotmail.co.uk Supervisor: Dr Kevin Pyke, School of Biosciences, University of Nottingham, Sutton Bonington LE12 5RD, UK. .............................................. .................................................. .................................................. ............... Whilst plastids are fundamental to many aspects of plant biology and the production of enhanced crop cultivars, research into the dynamics of non-green plastids has remained somewhat disregarded by the scientific community compared to chloroplasts. They are equally pivotal to normal plant development however, and are now increasingly becoming the focus of research made possible by genetic manipulation and reporter gene constructs. The total plastid content of all plant cells originates from small, undifferentiated plastids termed proplastids found within the meristematic regions of both root and shoot tissue. The cellular regulatory mechanisms controlling the development of plastids in young tissues are poorly understood, especially in the case of non-green plastids in roots. This investigation con- sequently aimed to elucidate the differences in plastid content, morphology and subcellular localization within epidermal cells derived from the root and shoot apical meristems (RAM and SAM respectively) of Arabidopsis thaliana. Quantification of non-green plastids was facilitated via the use of confocal laser scanning microscopy in conjunction with the expression of plastid-targeted green fluorescent protein driven by a constitutive promoter. Characterization of early seedling development and tissue diversification was also achieved by assessing epidermal cell size relative to devel- opmental progression, ultimately facilitating comparative analyses of plastid dynamics on both a temporal and tissue- varietal basis. The number of plastids in epidermal cells within RAM-derived tissue was shown to increase across regions of cell division before being regulated throughout subsequent zones of elongation and maturing root tissue. In contrast, epidermal cells of the hypocotyl exhibit a more generalized increase in plastid number and less strict maintenance of cell plan area coverage during tissue expansion. The findings presented here suggest the functioning of distinct mechanisms regulating plastid division and growth in relation to cell size within shoot and root apical meristem-derived tissues. Key words: Plastids, GFP, Epidermal Cells, Root, Hypocotyl, Arabidopsis thaliana Submitted on 18 May 2016; editorial decision on 6 January 2017 .............................................. .................................................. .................................................. ............... This research was carried out as part of my undergraduate dissertation, which attributed towards the degree of BSc (Hons) in Biotechnology at The University of Nottingham, Sutton Bonington Campus, UK. ............................................................................................... .................................................................. © The Author 2017. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Research article Bioscience Horizons � Volume 10 2017 ............................................................................................... .................................................................. pollen and seed dispersal by attracting pollinators or herbi- Introduction vores at specific developmental stages (Galpaz et al., 2006). Due to their fundamental roles in plant biology, the ability to Plastids are organelles that ubiquitously reside in the cells of accumulate novel compounds and other unique advantages, higher plants, adopting different forms in order to facilitate a plastids present significant targets for agronomic enhance- diverse array of intracellular functions. They are believed to ment and new biotechnological applications. They have have originated through a primordial endosymbiotic event already been widely used within the field of genetic manipula- with a free-living cyanobacterium-like prokaryote and remain tion (Medina-Bolíva and Cramer, 2004; Daniell, 2006; fundamentally distinct from other eukaryotic organelles Clarke and Daniell, 2011) but a large potential for plastid (Gould, Waller and McFadden, 2008; Pyke, 2009). The fore- genetic engineering still exists, perhaps emerging from most role of plastids, or more specifically green-pigmented increased research into non-green plastids and their develop- chloroplasts, is the fixation of atmospheric carbon dioxide mental biology. into a plethora of organic molecules that facilitate growth and development (Robertson and Laetsch, 1974; Pyke, 2009). Higher plant embryogenesis transforms a fertilized ovum Unsurprisingly, due to the pivotal nature of photosynthesis into a juvenile form of the plant lacking most species-specific amidst aims to improve its efficiency, chloroplasts within features (Fig. 1). Post-embryonic development subsequently foliar tissue have been the focus of significantly higher levels occurs from meristematic tissue in a progressive and highly of research compared to plastids of other tissues. This is also organized wave-like manner, generating new tissue through partly due to methodological difficulties in quantifying non- successive cell replication and expansion (Malamy and green plastids before the advent of genetic manipulation, the Benfey, 1997; Cary, Che and Howell, 2002). The following use of reporter gene constructs and confocal laser scanning increase in architectural complexity is thus heavily influenced microscopy (CLSM). These non-green plastids, however, are by changing factors within the early stages of germination no less intrinsic to plant development. (Jürgens, 2001). Leucoplasts are one such plastid subgroup crucial to lipid The total plastid content of all plant cells originates from biosynthesis, amino acid metabolism and as storage sites for small, undifferentiated plastids termed proplastids found starch grains (amyloplasts), lipids (elaioplasts) and proteins within dividing cells of the root and shoot apical meristems (aleuroplasts) (Ohlrogge and Browse, 1995; Finkemeier and (RAM and SAM, respectively) (Pyke, 2009; Finkemeier and Leister, 2010). Of these, amyloplasts are deemed particularly Leister, 2010). They replicate via the prokaryotically-derived critical to correct development as sources of long-term energy mechanism of binary fission to form daughter plastids which, reserves essential for growth and the formation of viable seeds having segregated into individual cells during mitosis, differ- and desirable grain (MacCleery and Kiss, 1999; Pyke, 2009). entiate between plastid varieties depending on intracellular They are also fundamental to plants’ gravitropism where suf- conditions and gene expression (Pyke, 2009; Osteryoung and ficiently dense amyloplasts termed statoliths are translocated Pyke, 2014). The renowned example of this is during chloro- by gravity and promote the redistribution of auxin through plast development where, in the absence of light, proplastids membrane-based influx/efflux proteins, enabling directional develop into etioplasts containing semi-crystalline prolamellar growth under conditions of otherwise limited stimuli (Swarup bodies which, when later exposed to light, transform into the et al., 2005). characteristic, chlorophyll-containing thylakoid membranes of chloroplasts (Finkemeier and Leister, 2010). Whilst classi- In addition to key intracellular roles, plastids are also fied into different plastid sub-types according to their function important for determining interactions between plants and their immediate environment. Chromoplasts, for example, and internal structure, differentiation between plastid types is synthesize and accumulate pigments including yellow xantho- both highly prevalent and fundamental to their success as phylls and red lycopene in tomatoes, which can encourage dynamic elements of plant biology. Figure 1. Seed germination in A. thaliana.(A) Diagram of internalized embryo. (B) Germination observed from 0 to 72 h. Radicle emergence and testa shedding were seen to occur at approximately 36 and 52 h after sowing, respectively. ............................................................................................... .................................................................. 2 Bioscience Horizons � Volume 10 2017 Research article ............................................................................................... .................................................................. Green fluorescent protein (GFP) has been used extensively then added to 1.5 g bacto-agar and sterilized at 121°C for 1 h in modified forms as a reporter for gene expression (Chalfie using an autoclave. et al., 1994). Unlike other methods of analysing expression, Seventy-five millilitre agar was poured into standard 120 × its introduction within target gene constructs facilitates the 120 mm square petri dishes onto which two rows of 10 ster- direct visualization of expression in vivo by naturally fluores- ile seeds were evenly distributed. cing green when exposed to light in the blue to ultraviolet range (Chiu et al., 1996). Targeting GFP to plastids when Seed sterilization and growth used in partnership with CLSM can therefore be used to Within a laminar flow hood, seeds were suspended in a solu- quantify their size, structure and subcellular localization. tion of 0.75 ml 50% bleach/water and 0.75 ml 0.1% Triton/ Whilst the mechanism of chloroplast division has been water. After 6 min, any superfluous solution was discarded studied in depth (Maple and Møller, 2007; Osteryoung and from sedimented seeds before 1 ml 70% ethanol solution was Pyke, 2014), relatively little is known about how other plastid used to resuspend the seeds. Seed suspensions were then varieties divide, especially proplastids and other root-based inverted over 20 s before excess solution was removed and plastids. A wide array of plastid-derived structures including seeds distributed on cooled agar. stromules and vesicles have been observed in cultured root tis- Seeds were cooled to 4°C for 12 h in order to ensure even sue however, suggesting that division mechanisms additional germination before being placed in a growth room main- to those of chloroplasts are present and required to facilitate tained at 20–25°C on a 24 h light cycle. A total of twenty the regulation of a population of increasingly heterogeneous plates containing 400 seeds were monitored over 156 h in plastid forms (Pyke, 2013). 12-h increments for comparative growth between root and Considering the importance of plastids to plant biology and hypocotyl tissue whilst, where appropriate, seedlings were their potential in enhancing cultivars of high socioeconomic excised for further analysis. importance, it is somewhat surprising that relatively limited research has been undertaken into their development during Confocal microscopy and image analysis embryogenesis and germination; particularly into the differ- Ten seedlings were harvested every 12 h from 60 h after plat- ences seen between the plastid dynamics of root and shoot ing (until 156 h) and were imaged using a Leica SP2 confocal meristem-derived tissues. As individual systems of gene expres- laser scanning microscope (Leica Microsystems, Heidelberg, sion and development, these early differences could have pro- Germany). A single plane of peak GFP and chlorophyll fluor- found impacts on general plant biology, crop improvement escence within the epidermal layer was measured in each sam- and biotechnology. Before further research can be performed, ple from the root tip, across the root–hypocotyl tissue however, a detailed characterization of a suitable model system boundary and into hypocotyl tissue, retaining accurate plastid is fundamental. quantification throughout all tissues under investigation. This investigation consequently aimed to elucidate some of As an indication of whether the observed prevalence of the differences seen between meristem-derived tissues by visu- plastids was representative of each cell’s actual content, Z alizing plastid number and size across Arabidopsis thaliana stacks were performed and assessed on samples of 20 similar seedling transects during the first 7 days after imbibition. sized epidermal cells in each major tissue variety and a range of seedlings (4 × 96 h, 120 h, 144 h and 156 h). GFP imaging was accomplished by directing a 488 nm laser for excitation Materials and methods onto samples while detecting fluorescence at an emission wavelength of 509 nm. Chlorophyll autofluorescence was Seeds of A. thaliana (var. Columbia) were donated by George imaged at a peak emission wavelength of 673 nm whilst Bassell and originated from the study of Cutler et al. (2000). bright field images of tissue were also collected. Through Agrobacterium-mediated transformation, a trans- gene containing plastid-targeted GFP under the control of a Cell walls were labelled with propidium iodide (PI); seed- constitutive promoter was introduced, facilitating the intra- ling tissue samples were excised using a scalpel and soaked cellular visualization of all plastid forms by GFP fluorescence for 5 min in 3 ml 0.5 μm PI solution before being carefully using CLSM. rinsed several times with water and imaged (excitation wave- length = 535 nm, emission wavelength = 617 nm). Growth media All recorded images consisted of an average of eight com- Bacto-agar enriched with Murashige and Skoog (MS) salts plete scans that were later compiled into single, layered was used as an optimal growth medium that enabled unobtru- images of each channel across the entire length of individual sive observation and excision of seedlings (Murashige and seedlings (Figs 3, 4B, 5D and 7B). These images were then Skoog, 1962). All growth media was produced in the ratio of analysed using ImageJ software (http://imagej.nih.gov/ij/ 150 ml purified water, 1.5 g sucrose and 0.645 g MS basal download.html, downloaded on 20/10/2014), ensuring at salts mixed to a pH of 5.8–6.0. The resulting solution was least 80% of visible epidermal cells were assessed for plan cell ............................................................................................... .................................................................. 3 Research article Bioscience Horizons � Volume 10 2017 ............................................................................................... .................................................................. area, plastid number and percentage coverage (percentage more to total seedling length than hypocotyl growth (Fig. 2). plan cell area occupied by plastids). Statistical analyses and Total seedling, root and hypocotyl tissue length were all also graphs were performed and produced respectively using seen to increase by a near-consistent 3% per hour after root Microsoft Excel 2013 (Figs 2, 4A, 6, 7A and 8). emergence, suggesting a conserved and relatively stable rate of growth within the two types of tissue. Tissue under investigation can be classified into regions Results based on gross tissue morphology, cell replication and size. Moving across root tissue from the root tip to the root– Seedling growth characterization hypocotyl tissue boundary, successive zones of high (but Measuring the average length of the hypocotyl and root tissue decreasing) cell division, elongation and root hair tissue exist. during germination showed that root growth contributed Many of these tissues were seen to overlap however, restrict- ing the ability to conclusively investigate discrete tissue var- ieties. Within the hypocotyl, less visually recognizable changes in morphology were observed during the early stages of development except for a notable increase in chlorophyll content revealed by autofluorescence (Fig. 3). Regions of higher rates of cell division, elongation and mature hypocotyl tissue were also present and can be qualified by changes in epidermal cell size (Fig. 4A). An increase in epidermal cell size with distance from root tip was seen to occur consistently in different seedlings at near-constant distances (Fig. 4A). In root tissue, the zone of cell division extended approximately 1100 μm from the root tip, within which cells of the root cap were shown to uni- formly increase in plan cell area from an average of 85.7 to 351.2 μm . Subsequently, a major increase in cell size was Figure 2. Length of total A. thaliana seedling ( ), root ( ) and observed beyond 1100 μm where average epidermal cell size hypocotyl ( ) tissue during germination. Error bars represent standard increased by roughly 70 μm per 100 μm until a distance of error. Number of seedlings assessed = 340. Figure 4. (A) Exemplified trend within 96 h A. thaliana of changing epidermal plan cell area against distance from root tip. Root epidermal Figure 3. Superimposed confocal image of chlorophyll fluorescence cells ( ), hypocotyl epidermal cells ( ). (B) Annotated compilation of (red) and bright field image of A. thaliana seedling after 52 h. Red confocal bright field channel images of 96 h A. thaliana seedling across chlorophyll fluorescence can be seen in the hypocotyl and cotyledons the root–hypocotyl transect. Labels indicate the approximate regions but not within root tissue after the root–hypocotyl tissue boundary. of distinct tissue morphology. ............................................................................................... .................................................................. 4 Bioscience Horizons � Volume 10 2017 Research article ............................................................................................... .................................................................. 4500 μm from the root tip. After 4500 μm until the root– hypocotyl tissue boundary, rate of cell expansion in all sam- ples declined and epidermal cells averaged a plan area of 3497 μm . Epidermal cells within the hypocotyl followed a similar trend of progressive expansion as distance from the SAM increased but exhibited higher levels of variation (Fig. 4A). Within the zone of elongation, cells were seen to consistently increase at an average rate of 142 μm per 100 μm; double that seen in comparable root tissue. Hypocotyl epidermal cell size was also seen to stabilize at comparatively lower levels than root epidermal cells on the root–hypocotyl tissue boundary; on average approximately 2100 and 3600 μm , respectively. These changes to epidermal cell size were seen to be dependent on the morphology and developmental status of hypocotyl and root tissues from 60 to 156 h in all samples. Epidermal cell size was consequently used as a way to qualify developmental progression against the distinct regions of plastid prevalence seen across the root–hypocotyl boundary (Fig. 5A), tissue within the zone of elongation/mature root (Fig. 5B) and in the root tip (Fig. 5C). Plastid number with epidermal cell size The number of plastids within root epidermal cells exhibited two principal stages when plotted against cell plan area (Fig. 6A). Within cells of an average size of 85.7 μm upwards Figure 5. Superimposed confocal GFP and bright field images of 96 h to 500 μm , an increase in plastid number was seen from 3 to A. thaliana seedling showing the changing plastid dynamics within 16. Past the zone of cell replication characterized by plan cell 2 epidermal cells of: (A) the root–hypocotyl tissue boundary; (B) mature areas higher than 500 μm , a relatively consistent number of root tissue and (C) root tip. (D) Compilation of superimposed confocal plastids were subsequently observed averaging slightly higher GFP and bright field channel images of whole 96 h A. thaliana seedling than 17 (17.44) but with a range of 9–26 (SD = 4.18). In across the root–hypocotyl tissue transect. Labels indicate the hypocotyl tissue, the number of plastids per epidermal cell approximate regions of distinct tissue morphology and root–hypocotyl remained within the range of 3–10 across cell sizes below tissue boundary. 500 μm (average of 5.84, SD = 1.95) (Fig. 6B). Plastid num- ber subsequently increased to approximately 26 (26.44) within epidermal cells larger than 1700 μm . Variation in In root tissue, percentage plastid coverage was observed to plastid number was seen however, consistently ranging from be higher within epidermal cells of a plan area lower than 20 to 36 plastids per cell (SD = 4.62). 500 μm (Fig. 8A). Due to their size however, relatively small changes caused large differences leading to variation in plastid coverage from 25% to 55% (average = 37.7%, SD = 8.8%). Plastid coverage against transect distance In cells indicative of the zone of elongation and mature root and epidermal cell size tissue (cell sizes of 500–4500 μm ), percentage coverage was Across all samples, epidermal plan cell area plastid coverage shown to average 20.2% (SD = 5.1%) (Fig. 8A). in root tissue was seen to decrease from an average of 39.7% By comparison, hypocotyl tissue exhibited a less fixed within the root tip and across the zone of cell division (up to trend of reducing plastid coverage against increasing epider- 1100 μm from the root tip) to 20.8% throughout the zones of malcellsize(Fig. 8B). Higher levels of variation from 20% cell elongation and mature root tissue (Fig. 7A). In hypocotyl to 75% were seen across all cells lower than 2000 μm epidermal cells, plastid coverage followed a similar trend. As (average = 41.8%, SD = 13.6%). In epidermal cells larger distance from the SAM increased, percentage plastid coverage than 2000 μm , coverage was seen to decrease until an aver- was seen to drop from an average of 52.1% within the zone age of 28.3% (SD = 8.5%). of cell division (up to 900 μm from SAM) to 24.9% through- out the remaining tissue. In all equivalent hypocotyl and root In order to qualify if the recorded number of plastids and epidermal cells, plastid coverage was seen to be generally percentage plan cell area coverage correlated to the actual higher in hypocotyl tissue. content of epidermal cells, Z stacks of individual cells were ............................................................................................... .................................................................. 5 Research article Bioscience Horizons � Volume 10 2017 ............................................................................................... .................................................................. Figure 6. Aggregated 96 h, 120 h and 144 h A. thaliana seedling data Figure 8. Aggregated 96 h, 120 h and 144 h A. thaliana seedling data of changing number of plastids per cell against epidermal plan cell of changing percentage epidermal cell plastid coverage against area. (A) Root epidermal cells ( ). (B) Hypocotyl epidermal cells ( ). epidermal plan cell area. (A) Root epidermal cells ( ). (B) Hypocotyl epidermal cells ( ). performed. Due to difficulties in determining intercellular boundaries on a Z-plane and reduced fluorescence however, no conclusive scale factor between observable plastid number and total plastid content could be established across the range of tissues investigated. A statistically significant correlation between increases in observed plastid content and that seen within Z stacks was found though, suggesting that data col- lected can be used for comparative analyses. Intracellular plastid numbers measured also corresponded generally to pre- vious studies; 10–20 proplastids within meristematic cells and up to 100 chloroplasts depending on cell size and function (Pyke, 1999; Segui-Simarro and Staehelin, 2009). Plastid morphology and subcellular localization Post-root tissue containing statoliths but within the zone of cell replication, no significant patterning of intracellular local- ization was observed in any samples. Plastids were seen to be distributed between cell peripheries, cytoplasm and in close proximity to nuclei (Fig. 9A). Thin, stromule-like projections and ‘dumbbell’-shaped plastids were also seen within root tip Figure 7. (A) Exemplified trend within 96 h A. thaliana of changing tissue and regions of cell elongation (Fig. 9B) but not as preva- percentage epidermal cell plastid coverage against distance from root lently in zones of cell division and hypocotyl cells (data not tip. Root epidermal cells ( ), hypocotyl epidermal cells ( ). (B) shown). Within cells of the zone of elongation and mature Annotated compilation of superimposed confocal GFP and bright field root tissue, plastid morphology was seen to stabilize into channel images of 96 h A. thaliana seedling across the root–hypocotyl tissue transect. Labels indicate the approximate regions of distinct mostly spherical and elliptical forms distributed along inter- tissue morphology. cellular boundaries (Fig. 9C). ............................................................................................... .................................................................. 6 Bioscience Horizons � Volume 10 2017 Research article ............................................................................................... .................................................................. tissue, whilst a relatively stable area of plastid cell coverage was maintained (Figs 6A and 8A, respectively). By compari- son, the number of plastids within equivalent tissues of the hypocotyl remained within the lower spectrum of those seen in root tissue whilst a higher discrepancy for area of plastid cell coverage was observed (Figs 6B and 8B, respectively). This suggests that within actively dividing tissue, plastids are also replicating whilst a system for regulating plastid num- ber and size is in effect. It also implies that within hypocotyl regions of cell replication, somewhat less rigorous or func- tionally different regulatory pathways exist. Within maturing root tissue, percentage plastid coverage was seen to be approximately half that of replicating regions (Fig. 8A) whilst plastid number was also relatively tightly main- tained (Fig. 6A). This suggests the presence of strong regulatory systems assessing both intracellular plastid number and size in root epidermal cells. Conversely, epidermal cells of the hypo- cotyl exhibited an increase in plastid number throughout the zone of elongation and mature tissue alongside a reduction in plastid coverage (Figs 6Band 8B, respectively). This indicates that plastid number rather than size is increasing whilst allud- ing to additional differences in regulatory systems between the Figure 9. Superimposed confocal GFP and bright field channel two tissues. The disparities in plastid replication rates and images showing subcellular plastid localization within 96 h (A and B) morphology may have a number of evolutionary advantages, and 120 h (C and D) A. thaliana epidermal cells. (A) Irregular patterning within replicating root cells. (B) Stromules and dividing (dumbbell- potentially including the preservation of resources by inhibiting shaped) plastids within elongating root cells highlighted by broad and the formation of functionally superfluous plastids. thin arrowheads respectively. (C) Peripheral patterning within Within the hypocotyl and subsequent foliar tissue, higher elongating/mature root cells. Dashed lines indicate approximate location of plant cell walls (D) Peripheral patterning within mature numbers of plastids (specifically chloroplasts) are beneficial hypocotyl tissue. due to their increased presence enabling more efficient light absorbance and photosynthesis (Finkemeier and Leister, 2010). Increasingly higher levels of peripheral localization Throughout the zones of cell replication and elongation of and chlorophyll autofluorescence were seen across transects the hypocotyl, larger plastids were observed compared to of the hypocotyl, signifying a definitive increase in chloroplast within the equivalent root epidermal layer and, whilst differentiation and functional relocation (Figs 3 and 9D) dumbbell-shaped plastids and stromules were seen towards (Trojan and Gabrys, 1996). It is unclear, however, what fac- the SAM, their prevalence was significantly lower compared tors induce these changes and limit potential plastid differenti- to RAM-derived tissue. In all instances, plastids of the hypo- ation. All seedlings were grown laterally across agar in a 24-h cotyl were also predominantly localized around cell peripher- light cycle, suggesting that any abiotic factors present equally ies (Fig. 9D). affected the two tissues, potentially implying that some degree of plastid predefinition according to function may exist. Discussion The presence of stromules (stroma-filled tubules) projecting from plastids (Fig. 9B) have been shown to facilitate the traffick- Interpretation of results ing of molecules between plastids (Hanson and Sattarzadeh, Plastid dynamics within hypocotyl and RAM-derived epider- 2011), however, their role in the exchange of molecules remains mal cells of A. thaliana have been shown to deviate in terms of a controversial subject. Their presence could still be potentially plastid number, plan cell area coverage and morphology at important within the zone of elongation and mature root tissue near-fixed stages of early plant development. Characterization where root hairs are present and nutrient uptake occurs of A. thaliana growth and epidermal cell plan area assisted in (Benning, Xu and Awai, 2006). Plastids have also been shown qualifying these changes whilst additionally providing a meth- to play a major role in nitrate assimilation by reducing it into od of assessing plastid dynamics across a range of sample seed- nitrite (formed within the cytosol from nitrate by nitrate reduc- lings and tissue varieties (Fig. 4). tase) and further into ammonia using nitrite reductase (Finkemeier and Leister, 2010). In root meristematic tissue and the zone of cell division, the observable number of plastids per epidermal cell was seen to Increased plastid surface area and molecular trafficking increase with cell size, and thus distance from meristematic facilitated by stromules may contribute to more efficient ............................................................................................... .................................................................. 7 Research article Bioscience Horizons � Volume 10 2017 ............................................................................................... .................................................................. nutrient absorption, and is perhaps why the presence of plas- shoot and root tissue formation in early embryogenesis tids within RAM-derived tissues is so highly regulated; espe- (Okazaki et al.,2010; Holtsmark et al.,2013). The phase of cially during early development where the use of nutrients individual cells’ replicative cycles, tightly coupled nuclear- must be optimized for survival. It is also interesting to note based gene expression and tissue variety have the potential to the discrepancy seen in equivalent hypocotyl tissue where directly mediate plastid division (Gillard et al.,2008). stromules were seen less frequently, both here and in previous Homeostatic mechanisms also control the levels of intra- investigations (Pyke and Howells, 2002). cellular PDVs and thus rates of plastid division. One such mechanism is the ubiquitin-proteasome system formed of a Plastid replication and regulatory number of protein complexes capable of degrading unneeded mechanisms or damaged target proteins by proteolysis (proteasomes) and a regulatory protein that facilitates the post-translational ‘tag- Extensive research has been undertaken into plastid division ging’ of target proteins for degradation (ubiquitin) (Haas and regulation by characterizing Arabidopsis accumulation et al., 1982; Kimura and Tanaka, 2010). Through targeted and replication of chloroplasts (arc) mutants. In particular, ubiquitin-activating enzymes, PDVs can thus be degraded and arc6, a mutant known to interfere with proplastid division in plastid division regulated in accordance to the expression of root and shoot meristematic tissues has helped define distinct specific genes including MinC; an inhibitor of FtsZ’s polymer- gene regions fundamental to controlling intracellular plastid ization into the contractile ring’s precursor (Margolin, 2005). number (Robertson, Pyke and Leech, 1995; Vitha et al., 2003). Coupling of plastid and cellular division Plastid division is facilitated through the formation of a Whilst many of these regulatory mechanisms are shown to double ringed protein complex encircling dividing plastids at coincide with discrete phases of the cell cycle, they cannot be their centre, perpendicular to their longitudinal axis. One ring considered to be ‘coupled’ with the division of plastids, as each forms on the stromal side of plastids’ inner envelope whilst process can be altered independently without impairing the the other forms on the cytosolic side of their outer envelope other (Osteryoung and Nunnari, 2003). In arc mutants and (Pyke, 1999). Using a dynamin-related protein that produces plants overexpressing PDVs (a factor that counterintuitively cytokinetic force (DRP5B/ARC5), this ring constricts until inhibits plastid division), no defects in development or cell div- envelope membranes deliquesce and reform to produce two ision have been seen (Pyke and Leech, 1992; Osteryoung et al., plastids (Maple and Møller, 2007; Yang et al., 2008). 1998). Reciprocally, stopping cell division via inhibition of Increased numbers of dumbbell-shaped plastids are there- CDKs does not, whilst seriously hindering normal plant devel- fore representative of higher levels of plastid replication, as opment, necessarily affect plastid division as seen in plants seen within cells of both RAM (Fig. 9B) and SAM prior to the overexpressing NtKIS1a; a highly characterized CDK inhibitor zone of division and in later, maturing tissue (data not (Jasinski et al.,2002, 2003). shown). Whilst morphology of plastids can indicate levels of replication, the disparities seen in the regulation of plastid Future research and potentials of defined content between meristematic and mature root tissues are per- plastid dynamics haps derived from a combination of plastids’ primordial ori- gins and homoeostatic mechanisms. Further research into the regulatory factors differing between early hypocotyl and root tissue could be undertaken by profil- During the simultaneous evolution of the ancestral precur- ing gene expression whilst assessing the plastid dynamics of sors to plastids and their eukaryotic hosts, genes once present respective tissues (Bustin, 2002; Trevino, Falciani and in the endosymbiont’s genome were either lost, retained or Barrera-Saldaña, 2007). The extension of this study into the translocated to the host’s nucleus and levels of synchrony cell layers beyond the epidermis will also be a necessary focus between them were established (Gould et al.,2008). Of the pro- for defining plastid dynamics against plant development teins encoded by such genes, some are essential to plastid repli- because, whilst epidermal cells are considerably easier to cation and have been shown to accumulate during discrete assess and provide comparative analysis across both temporal phases of the cell cycle in accordance with cyclin-dependent and surface tissue-based development, they do not form cer- kinases (CDKs). These include ARC3, MinD, MinE (specify tain key tissues of mature plants. Plastid prevalence could also the site of plastid division), FtsZ proteins (tubulin-like proteins be compared between wild type A. thaliana, meristematic that form the early ring structure), ARC6 (recruits other pro- mutants such as those investigated by Aida, Ishida and teins to the ring/site of division) and DRP5B/ARC5 (generates Tasaka (1999) and important crop cultivars in order to qual- the cytokinetic force necessary for division) (Okazaki, Kabeya ify the impacts of early plastid variation upon later growth. and Miyagishima, 2010). Other essential proteins, such as the nuclear-encoded plastid division proteins (PDVs), have been Ultimately, the data collected here highlights the paucity of shown to regulate DRP5B/ARC5 and consequently plastid div- knowledge relating to the early discrepancies of plastid ision according to cell cycle progression and/or differentiation dynamics within early plant tissues and, whether due to determined by cytokinin, a phytohormone fundamental to changes in gene expression, cellular identity and/or abiotic ............................................................................................... .................................................................. 8 Bioscience Horizons � Volume 10 2017 Research article ............................................................................................... .................................................................. Clarke, J. L. and Daniell, H. (2011) Plastid biotechnology for crop pro- conditions, these observations could correlate to plastid duction: present status and future perspectives, Plant Molecular prevalence within mature plant tissue. Defining the causes of Biology,76(3–5), 211–220. early plastid dynamics could be potentially exploited for future research alongside the development of biofactories of Cutler, S. R., Ehrhardt, D. W., Griffitts, J. S. et al. (2000) Random GFP: important molecules and enhanced cultivars of high socio- cDNA fusions enable visualization of subcellular structures in cells economic importance. of Arabidopsis at a high frequency, Proceedings of the National Academy of Sciences of the USA, 97 (7), 3718–3723. Author biography Daniell, H. (2006) Production of biopharmaceuticals and vaccines in plants via the chloroplast genome, Biotechnology Journal, 1 (10), Molecular biology appeals to his avid interest in science, both 1071–1079. in terms of how elements intrinsic to life function and how they Finkemeier, I. and Leister, D. (2010) Plant chloroplasts and other plas- can be adapted. He aims to forge a career in science under- tids, in Encyclopedia of Life Sciences (ELS), John Wiley and Sons, Ltd, pinned by this fascination of how minute intracellular changes Chichester, United Kingdom. induce huge phenotypic impacts, specifically researching molecular plant pathology and crop cultivar improvement Galpaz, N., Ronen, G., Khalfa, Z. et al. (2006) A chromoplast- through genetic engineering. In 2015, he graduated from The specific carotenoid biosynthesis pathway is revealed by clon- University of Nottingham with a first-class honours degree in ing of the tomato white-flower locus, The Plant Cell,18 (8), Biotechnology, and is about to embark on a PhD at The 1947–1960. University of Warwick where he will research resistance to Turnip mosaic virus in members of the Brassica genus. Gillard, J., Devos, V., Huysman, M. J. J. et al. (2008) Physiological and transcriptomic evidence for a close coupling between chloroplast ontogeny and cell cycle progression in the pennate diatom Acknowledgements Seminavis robusta, Plant Physiology, 148 (3), 1394–1411. Gould, S. B., Waller, R. F. and McFadden, G. I. (2008) Plastid evolution, I would like to especially thank Dr Kevin Pyke for his signifi- Annual Review of Plant Biology, 59, 491–517. cant guidance throughout this study and all the fellow under- graduate students, postgraduate students and staff of UoN Haas, A. L., Warms, J. 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