Epidermal Pavement Cells of Arabidopsis Have Chloroplasts

Plastids are multifunctional, pleomorphic organelles of purported endosymbiotic origin that in plants and green algae display a characteristic double membrane envelope (Wise, 2007). All plastids originate fromcolorless proplastids, and a simple pigmentation-based classification distinguishes chloroplasts from other plastids by the presence of chlorophyll, chromoplasts by the predominance of other pigments, and leucoplasts by the absence of all pigmentation (Schimper, 1883, 1885). Plastids are able to interconvert according to tissue and developmental requirements (Schimper, 1883, 1885). In higher plants themajority of chloroplasts are found in the leaf mesophyll tissue. The presence of chloroplasts in the epidermis of some higher plant species, including tobacco (Nicotiana tabacum), is also generally accepted (Shaw and MacLachlan, 1954; Dupree et al., 1991; Brunkard et al., 2015). However, several modern textbooks and primary publications categorically state that the epidermis of higher plants contains chloroplasts only in the guard cells, while pavement and trichome cells have leucoplasts (MacDonald, 2003; Smith, 2005; Bowes andMauseth, 2008; Solomon et al., 2010; Vaughan, 2013). In themodel plantArabidopsis (Arabidopsis thaliana), observations of leucoplasts in the unicellular trichomes are consistent, but there is considerable ambiguity regarding the presence or absence of chloroplasts in pavement cells (Table I). Several publications clearly show chloroplasts in the pavement cells ofArabidopsis, and aprecise, observationbased statement that contradicts the common textbook knowledge has beenmade by Pyke (2009): “In a leaf, the chloroplasts in the epidermal cells covering the leaf surface are significantly smaller and poorly developed compared with mesophyll chloroplasts, but do contain low levels of chlorophyll and should be considered as chloroplasts” (p. 15). Nevertheless, a degree of uncertainty has remained since other investigators who have observed chlorophyll fluorescence in pavement cells have either dismissed it as artifactual or have described such chloroplasts as not being fully developed (Haseloff et al., 1997; Chiang et al., 2012; Higa et al., 2014). Still others report an absence of chlorophyll fluorescence in the pavement cells (Table I). The significance of this issue is highlighted by a recent publication that uses the purported absence of chloroplasts in the pavement cells to explain differences in plastid behavior between cotyledon pavement and guard cells in response to chemically induced redox stress (Brunkard et al., 2015). The plastid type identified in a tissue creates an association with specific attributes. The name influences our comprehension of its internal biochemistry, its response and susceptibility to environmental stimuli such as redox imbalances, and its overall behavior and interactions with other cytoplasmic components and compartments. For example, photosynthesis in chloroplasts suggests a primary source of sugars, whereas leucoplasts are recognized as sink plastids that receive already synthesized sugar molecules. For models that rely on identifying a plastid type to explain plastid behavior, a changed label can suggest a different but perhaps experimentally unsubstantiated interpretation. After recognizing the present ambiguity on the subject, we investigated the presence of chloroplasts in the pavement cells of Arabidopsis. Representative images and observations obtained independently in several different labs are presented (Fig. 1). Chlorophyll autofluorescence (emission peak 485 nm) is routinely detected using epifluorescent microscopy (B-3A long-pass filter set) as well as confocal laser scanning microscopy (excitation 488 nm; emission collected 650–750 nm) in pavement cell plastids. The observations remain consistent for plants in different stages of development, grown on soil or on Suc-containing medium under varying light conditions (Fig. 1, A and B). In accordance with an earlier report by Pyke and Leech (1994), the number of chloroplasts in a pavement cell is one-tenth (106 3) of that observed inmesophyll cells (1106 10). In comparison to the clustered chloroplasts in mesophyll cells, pavement cell chloroplasts appear very dispersed, often located near the edges of the jigsawpuzzle-shaped cells. The average size of pavement cell chloroplasts is approximately one-half the size of a mesophyll chloroplasts but slightly larger than guard cell chloroplasts. The average chlorophyll a fluorescence values of pavement cell chloroplasts lies between that of guard cell and mesophyll cell chloroplasts. Observations of low chlorophyll signal are matched by ultrastructural details that show a small number of clearly defined grana (Fig. 1, C–E). Moreover, under actinic illumination pavement cell chloroplasts exhibit a fluorescence transient comparable to that shown by mesophyll chloroplasts, suggesting that they do have an active photosystem II and can utilize light energy for carbon fixation. Whereas each observation presented here supports earlier publications (referenced in Table I) and the presence of chloroplasts in Arabidopsis pavement cells, there is some basis for their perceived absence too. One reason that they may be overlooked lies in their low number and sparse distribution in pavement cells. Further, like chloroplasts in the mesophyll, pavement cell chloroplasts exhibit light avoidance responses (Higa et al., 2014) and relocate to the lower lateral regions of the cells in tissue exposed to light. This location *Address correspondence to [email protected]. www.plantphysiol.org/cgi/doi/10.1104/pp.16.00608