Comparative ultrastructure of fruit plastids in three genetically diverse genotypes of apple (Malus × domestica Borkh.) during development

Abstract

Comparative ultrastructural developmental time-course analysis has identified discrete stages at which the fruit plastids undergo structural and consequently functional transitions to facilitate subsequent development-guided understanding of the complex plastid biology. Plastids are the defining organelle for a plant cell and are critical for myriad metabolic functions. The role of leaf plastid, chloroplast, is extensively documented; however, fruit plastids-chromoplasts-are poorly understood, especially in the context of the diverse metabolic processes operating in these diverse plant organs. Recently, in a comparative study of the predicted plastid-targeted proteomes across seven plant species, we reported that each plant species is predicted to harbor a unique set of plastid-targeted proteins. However, the temporal and developmental context of these processes remains unknown. In this study, an ultrastructural analysis approach was used to characterize fruit plastids in the epidermal and collenchymal cell layers at 11 developmental timepoints in three genotypes of apple (Malus × domestica Borkh.): chlorophyll-predominant 'Granny Smith', carotenoid-predominant 'Golden Delicious', and anthocyanin-predominant 'Top Red Delicious'. Plastids transitioned from a proplastid-like plastid to a chromoplast-like plastid in epidermis cells, while in the collenchyma cells, they transitioned from a chloroplast-like plastid to a chloro-chromo-amyloplast plastid. Plastids in the collenchyma cells of the three genotypes demonstrated a diverse array of structures and features. This study enabled the identification of discrete developmental stages during which specific functions are most likely being performed by the plastids as indicated by accumulation of plastoglobuli, starch granules, and other sub-organeller structures. Information regarding the metabolically active developmental stages is expected to facilitate biologically relevant omics studies to unravel the complex biochemistry of plastids in perennial non-model systems.

Keywords: Chloroplast; Chromoplast; Malus × domestica Borkh.; Plastid transition; Rosaceae.

Fig. 1

Phenotypic changes in the fruit of three cultivars of Malus × domestica Borkh. ‘Golden Delicious’, ‘Granny Smith’, and ‘Top Red Delicious’. Fruit was collected throughout the 2010 growing season. Images are shown for samples collected at 8, 39, 64, 93, 121, and 148 days after anthesis (DAA) for each cultivar which represents the developmental continuum to maturity

Fig. 2

Changes in the epidermal and outer collenchyma cellular structures of three cultivars of Malus × domestica Borkh. Thick sections were made from tissue derived from fruit of ‘Golden Delicious’, ‘Granny Smith’, and ‘Top Red Delicious’ at 8, 39, 64, 93, 121, and 148 days after anthesis (DAA). The outermost layer with elongated cells is the epidermal layer and the rounded cells visible 2–3 layers deep represent the collenchymal layer. Images were recorded at ×400 magnification

Fig. 3

Ultrastructural changes in the epidermal cell plastids of ‘Golden Delicious’, ‘Granny Smith’, and ‘Top Red Delicious’ apple fruit during development. Transmission electron microscopy of plastids of the three investigated cultivars was performed throughout fruit development. Micrographs display plastid ultrastructure from early (8 DAA—a, d, g), intermediate (64 DAA—b, e, h) and mature (148 DAA—c, f, i) developmental stages. Structures including plastoglobuli (Pg), osmiophilic bodies (OB), phytoferritin (Pf), starch granules (SG), osmiophilic stacked membranes (SM), vesiculate lamellae (VL), and neighboring mitochondria (Mt) are labeled. Phytoferritins are visible in b and c during the intermediate and mature stage in ‘Golden Delicious’ cultivar only

Fig. 4

Representative plastid from epidermal tissue of ‘Golden Delicious’ apple fruit at 78 DAA highlighting the area of membrane coalescence (AOC). Early plastids contain small globulous structures and osmiophilic stacking membranes, while later plastids largely contain a single large osmiophilic body (OB). It appears that these two structures are related and perhaps the membranes coalesce and contribute to the formation of the large osmiophilic body. Other structures visible in the micrograph include plastoglobuli (Pg) and osmiophilic stacked membranes (SM)

Fig. 5

Ultrastructural changes in plastids of the outer collenchyma cell layer of ‘Golden Delicious’, ‘Granny Smith’, and ‘Top Red Delicious’ fruit during development. Transmission electron microscopy of plastids of the three investigated cultivars was performed throughout fruit development. Representative micrographs display early (8 DAA—a, d, g), intermediate (64 DAA—b, e, h) and mature (148 DAA—c, f, i) plastid morphologies. ‘Golden Delicious’ distinctively demonstrates the presence of osmiophilic stacked membranes (SM), while ‘Granny Smith’ retains thylakoid membranes (Th) compared to the other two cultivars. Other structures visible in the micrograph include plastoglobuli (Pg), starch granules (SG), vesiculate lamellae (VL), and neighboring mitochondria (Mt)

Fig. 6

Changes in area of plastids in epidermal (a) and collenchymal (b) cells of ‘Golden Delicious’, ‘Granny Smith’, and ‘Top Red Delicious’ fruit during development. Plastid area was measured at each timepoint for six plastids in each cultivar using ImageJ. Average plastid area with standard error was plotted against sampling date. Standard error bars (n = 6) are provided for each timepoint

Fig. 7

Measurements of average number of plastoglobuli per plastid, average area, average area per plastid and plastoglobule area as a percent of total plastid area in epidermal (a, c, e, g) and collenchyma (b, d, f, h) cells. Plastoglobuli measurements were made using ImageJ in six plastids representing each sample. Average plastoglobule size, area, and percent area of total plastid area were plotted against stage of growth. Standard error bars (n = 6) are provided for each timepoint

Fig. 8

Measurements of starch granule average area, total area, and as a percent of total plastid area in epidermal (a, c, e) and collenchyma (b, d, f) cells. Starch granules were measured using ImageJ in six plastids representing each sample. Average starch granule size, area, and percent area of total plastid area were plotted against stage of growth. Standard error bars (n = 6) are provided for each timepoint

Fig. 9

Comparative depiction of sub-organeller transitions in the epidermal and collenchymal plastids in the context of the developmental continuum of the fruit