Planta (Berl.) 86, 295~-298 (1969)
Rod-Shaped Accumulations in Cisternae of the Endoplasmic Reticulum in Root Cells of Lepidium sativum Seedlings TOI~-HEN~ING IvE~sE~ a n d PE~ t~. FLOOD Botanical Laboratory and Institute of Anatomy, University of Bergen, Bergen, Norway Received March 13, 1969
Summary. Ultrathin sections through the median plane of norm al, 48-hrs-old seedling roots of cress, showed the presence of accumulations of a finely fibrillar material. These inclusions were confined to the cisternae of the granular endoplasmic reticulum of surface cell layers of the root tip and the root-hair zone. The rod-shaped structure and random orientation of these inclusions were clearly seen in 3-dimensional reconstructions of serial ultrathin sections. I n connection with u l t r a s t r u c t u r a ] studies of geotropism in cress seedlings (Iv~asE~, unpublished), u n c o m m o n inclusions were f o u n d i n cisternae of the g r a n u l a r endoplasmic r e t i c u l u m (ER) of certain root-tip cells. Similar structures were described b y B O ~ E T T a n d NEWCOMB (1965) i n cells of the root-hair zone of radish seedlings. The p r e s e n t c o m m u n i c a t i o n reports on the frequency a n d d i s t r i b u t i o n of such inclusions in cress seedling roots. Seeds of cress (Lepidium sativum, L.) from two sources, were sterilized with 3% hypoeMorite and grown for 48 hr at 21 =~ 0.5~ in the dark on filter paper moistened with distilled water. The radicles were then fixed in potassium permanganate, in gluteraldehyde followed by osmium tetroxide, or in osmium tetroxide alone; details will be described later. Dehydration was carried out in ice-cooled ethanol of increasing concentrations, and infiltration and embedding were performed in Epon 812 according to Jv~rIP~,g (1962). Ultrathin sections from near the median plane of the root tips and the root-hair zones were cut with glass or diamond knives and stained with uranyl acetate and lead citrate. Irrespective of the fixative used, large a c c u m u l a t i o n s of finely g r a n u l a r or fibrillar m a t e r i a l were f o u n d in cisternae of the EI~ i n some root cells of cress seedlings. Their presence in all the p l a n t s e x a m i n e d (more t h a n 20), grown from seeds of two distinct sources, indicates t h a t t h e y are n o r m a l c o n s t i t u e n t s of certain cress root cells. W h e n fixed i n K M n O 4 (Fig. la), the a c c u m u l a t i o n s measured u p to 0.7 ~ across a n d consisted of a finely granular, electron-dense material. T h e y were e v i d e n t l y limited b y a smooth m e m b r a n e continous with the m e m b r a n e s of the endoplasmic reticulum. Ribosomes were a b s e n t from these preparations (cp. LV~T, 1956).
Fig. 1. Sections through cress-root cells showing the rod-shaped bodies, a. Section through a cytoplasma-rich portion of a cortical cell fixed in K M n 0 t . A membraneous elongation (arrows), indistinguishable from sacs of the endoplasmic reticulum (E), is projecting from one of the rodshaped bodies (B). Mitochondria (M), vacuole (V), Golgi apparatus (G) a n d cell wall (W) are also indicated. • 12,000 - - b. Section through two rod-shaped bodies in a root cap cell fixed in OsO 4. Note the faint, longitudinal striation in the bodies and their limiting membrane. Ribosomes and endoplasmic reticulum are also present, x 30,000 - - e. Section through a eytoplasmic trabeculum of an epidermal cell fixed in glutaraldehyde and OsO 4. Two rod-shaped bodies are tangentially sectioned (eontrolled b y serial sectioning) and exhibit several polyribosome chains a t their surface membranes (arrows). Mitoehondrion (M) and Golgi apparatus (G) are also present. • 25,000
T.-H. IVERSENand P. R. FLOOD: Accumulations in ER of Lepidium
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7( Fig. 2. Semi-schematic map showing the mean number of rod-shaped bodies per eell in median sections through the root tip and the root hair zone. (See text for further explanation) W h e n fixed in Os04, or gluteraldehyde followed b y osmium, (Fig. l b and e), the bodies were about 0.4 ~ across and seemed to be identical with the cisternal accumulations of the granular EI~ of cells in the root hair zone in radish as described b y B O ~ E T T and NEWCOMB (1965). The n u m b e r of ribosomes in each polyribosome chain, attached to the limiting m e m b r a n e of these eisternal accumulations, seems to be somew h a t lower in cress t h a n in radish (10--15 against 20--.95 particles). Although yet unable to prove or disprove the proteinaceous nature of the eisternae accumulations, as advocated b y BONNETT and NEWCOMS (1965), we have gathered some more information on their frequency and distribution in an a t t e m p t to clarify their significance. The n u m b e r of eisternal accumulations in individual cells of the root cap and root hair zone was counted in median sections t h r o u g h 5 different roots (Fig. 2). I t is evident t h a t the profiles are not present in the meristem, phloem, and xylem, but are present in the cells of the root cap, the outermost cortical layers, and the epidermis. I n the latter, their n u m b e r is quite high, up to 20 isolated profiles in one section of a single cell. I t is assumed t h a t similar structures are also present in cortical and epidermal cells in other regions of the root (el. BO~NETT and N:EWCOMB,1965, p. 424). As is indicated in Fig. 2, the cell walls are frequently thicker between the cells containing eisterna] accumulations t h a n between those lacking them. The relation of the accumulations to the cell walls is, however, not a direct one as a continuity between the two structures was not seen. The accumulations do not seem to be mechanically anchored to the cell walls as t h e y move easily in centrifugal fields (IVERSES, unpublished results). I n reconstructions based on series of ultra-thin sections, the threedimensional structure and the interrelationships of the eisternal accumulations are easily depicted (Fig. 3). T h e y are found in the cytoplasmic
T.-H. IV~RSE~ and P. 1~. FLOOD: Accumulations in ER of Lepidium
Fig. 3. Diagram of a vacuolated part of a cortical cell, reconstructed from 34 serial sections, each about 1000 A thick. The course of the cell border, and the outline of the cytoplasmic regions of the uppermost section are indicated
l a y e r a d j o i n i n g t h e cell wall a n d in t h e c y t o p l a s m i c t r a b e e n l a e of h i g h l y v a c u o l a t e d cells. The m a j o r i t y of t h e a c c u m u l a t i o n s are elongated, slender rods. M a n y of t h e m are a l m o s t straight, b u t some are n o t i c e a b l y bent. T h e y do n o t seem to be o r i e n t e d in a n y special w a y with reference to t h e axes of t h e cells or t h e root. I t is concluded t h a t t h e cisternal a c c u m u l a t i o n s show t o p o g r a p h i c a l r e l a t i o n to t h e superficial cell layers of the root, i. e. to older, more v a c u o l a t e d a n d d i f f e r e n t i a t e d cells t h a t are s e p a r a t e d from each o t h e r b y t h i c k e r cell walls t h a n t h e r e m a i n d e r of t h e cells of the y o u n g root. T h e y m a y r e p r e s e n t p r o t e i n precursors for the f o r m a t i o n of these t h i c k cell walls. Cisternal a c c u m u l a t i o n s of the endoplasmic r e t i c u l u m m a y be u n i q u e for the Cruci[erae to which f a m i l y b o t h r a d i s h a n d cress belong. BONZev~TT a n d NEWCO~B (1965) r e p o r t e d t h a t t h e y are a b s e n t in Melilotus albus a n d Phaseolus vulgaris. W e m a d e a similar o b s e r v a t i o n in Helianthus annuus a n d Zea mays.
References Bo~sETT, H.T., and E.H. N~.wco~s: Polyribosomes and cisternal accumulations in root cells of radish. J. Cell Biol. 27, 423--432 (1965). JUNIPER, B.E. : Embedding large slices of plant tissue for electron microscopy. Nature (Lond.) 194, 1296--1297 (1962). LusT, J. H. : Permanganate. - - A new fixative for electron microscopy. J. biophys. biochem. Cytol. 2, 799 802 (1956). Cand. real. ToR-HENNING IVERSEN Botanisk laboratorium Universitetet i Bergen All6gt. 7O Bergen, Norway