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Anatomy and Morphology |
Department of Biological Science Education, College of Education, Chosun University, Gwangju 501-759, Republic of Korea
Received for publication August 22, 2006. Accepted for publication April 8, 2007.
ABSTRACT
The anatomical and ultrastructural development of the haustorium of the Cuscuta japonica, a holoparasitic angiosperm, growing on the host plant Impatiens balsamina was studied. After the shoot tips of light-grown parasite seedlings contacted the host, the upper haustorium (external to the host organ) developed through three main successive stages of the haustorial initials, the meristem, and the endophyte primoridium (EP) within the middle layer of the cortex of the parasite stem. The haustorial initial cells were characterized by abundant starch-bearing amyloplasts and mitochondria with an expanded intermembrane space. The meristem cells had numerous large chloroplasts with well-developed thylakoids, reflecting the capability for photosynthesis. Commonly, all three stages of haustorial cells contained conspicuous, large nuclei with enlarged nucleoli and dense cytoplasm including many other organelles, indicating a very active metabolism. In the final stage of upper haustorium development, the meristem cells differentiated into the EP, a host-penetrating tissue. The primordium had smaller file cells at the proximal end and elongate digitate cells at the distal end. The file cells divided actively, while the digitate cells contained abundant chloroplasts, dictyosomes, rough endoplasmic reticulum, and other organelles, suggesting that the EP was cytohistologically well organized for penetration into the host tissue.
Key Words: anatomy • Cuscuta japonica • endophyte primordium • parasitic angiosperm • ultrastructure • upper haustorium
The embryos of the parasitic angiosperm genus Cuscuta (dodder) lack cotyledons; young seedlings have a radicle that withers with age, and their shoots have tiny, scale-like leaves. Because the Cuscuta must obtain organic and inorganic nutrients and water from their host plants (Parker and Riches, 1993
; Pate, 1995
; Press, 1995
), the parasite is generally thought to be holoparasitic. Chlorophyll, thylakoids, and activity of enzyme Rubisco have not been detected in the post-parasitic C. europaea (Machado and Zetsche, 1990
) or C. grandiflora and C. odorata (van der Kooij et al., 2000
). Deletion of photosynthetically related genes has been decribed for the post-parasitic C. europaea (Freyer et al., 1995
). On the other hand, several Cuscuta species do have chlorophyll, especially in the tips of seedlings (Panda and Choudhury, 1992
; Dawson et al., 1994
). Photosynthetic pigments in C. campestris (Dinelli et al., 1993
) and various photosynthetic proteins in C. pentagona (Sherman et al., 1999
) have been detected in the pre-parasitic seedlings and parasitic stems. Thus, the Cuscuta cannot be considered a strict holoparasite.
To take water and nutrients from their host plants, the Cuscuta develop a specialized absorptive organ called the haustorium, which is divided into the upper haustorium external to the host and the endophyte inside the host (Kuijt, 1977
). Although most studies on haustorial structure of Cuscuta plants have focused on the endophyte (Kuijt, 1977
), a few focused on the upper haustorium (Heide-Jørgensen, 1987
; Lee and Lee, 1986
, 1989
). Heide-Jørgensen (1987)
revealed that, in the early stage of haustorial development of C. gronovii and C. reflexa, the epidermal cells of the stem develop into secretory trichomes, which upon attachment to the host, produce finger-like projections at the apical end. Lee and Lee (1986)
reported that the elongate digitate cells in the upper haustorium of C. australis are metabolically very active. Lee and Lee (1989)
also detailed the anatomy and ultrastructure of the upper haustorium of C. australis in relation to subsequent developmental stages. Contact stimuli between the C. australis stem and its host initiate the development of the upper haustorium from the cortical cells of the parasite stem: in the mature haustorium, the endophyte primordium consists of three cell types, i.e., file, digitate, and compressed, that differentiate from the meristem, which develops from the initials (Lee and Lee, 1989
). However, the cytological features of the haustorial cells early in development have not been studied in detail. Here I describe the development of the upper haustorium in Cuscuta japonica. This report is the third part of a comprehensive study on the development of the embryos, seedlings, and haustoria of C. japonica.
MATERIALS AND METHODS
Plant materials
Mature, dry seeds of the parasite Cuscuta japonica Choisy (Convolvulaceae) were scarified with concentrated sulfuric acid for 45 min and then rinsed in tap water followed by distilled water. They were placed on moist filter paper in petri dishes, germinated in the dark at 30°C, and grown to the seedling stage. The roots of 3-d-old, dark-grown seedlings were wrapped with wet cotton, placed in 500-mL covered beakers, and exposed to sunlight near windows for 3 d. Shoot tips of these 6-d-old seedlings, when placed in contact with the stem of the host plant Impatiens balsamina L., entwined the host stem and produced upper haustoria on the side of the dodder stem touching the host. Shoot tips of free-living and 6-d-old dodder seedlings and the tissues of the developing upper haustorium were examined with light and electron microscopy.
Transmission electron microscopy
Seedling tissue was sliced into approximately 1-mm3 segments and prefixed in a mixture of 2.5% glutaraldehyde–2% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 6.8, for 2–3 h at room temperature. Segments were then exposed twice to microwave radiation for 10 and 20 s at 70% of the maximum in an 800-W Pelco Model 3450 Laboratory Microwave Processor (Ted Pella, Inc., Redding, California, USA) equipped with a thermistor copper temperature probe and an auxiliary Pelco 3420 Microwave Load Cooler (Ted Pella, Inc.). Tissues were postfixed in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer at pH 6.8, and microwaved three times for 40 s. Pre- and postfixation temperature in the microwave oven was 45°C. Segments were washed in buffer and dehydrated in a graded series of acetone (30, 50, 70, 90, 100, 100, and 100%) with 40 s at each step; dehydration was done in the microwave oven at 37°C. Tissue segments were then infiltrated and embedded in Spurr's resin (Spurr, 1969
). Thick sections (1 to 2 µm) cut with an LKB-V ultramicrotome were stained with 0.05% toluidine blue and examined with an Olympus BH2 light microscope. Thin sections cut with a RCM MT-7000 ultramicrotome were mounted on grids, stained with uranyl acetate and lead citrate, and then examined and photographed with a Hitachi H-7600 transmission electron microscope, at 80 kV.
RESULTS
Haustorial initiation and development
In transverse section, the stem of 6-d-old C. japonica seedlings that had not contacted the host had a one-cell-layer epidermis, a cortex, and a central stele. The cortex was typically 6–7 cells thick. The epidermal cells were vacuolated, and the cortex consisted of vacuolated parenchyma cells. After the hooked and greenish tip regions of the Cuscuta seedlings were placed lightly on the host stem, the vacuolated cortical cells began to have more darkly stained cytoplasm containing prominent nuclei and abundant, starch-bearing amyloplasts (Fig. 1). The cortical cells were recognized as the initials that gave rise to the upper haustorium. These cells contained large nuclei with enlarged nucleoli and starch-containing amyloplasts (Fig. 2). The initial cells had also other abundant organelles including dictyosomes, rough endoplasmic reticulum (r-ER), and several small vacuoles; a large number of mitochondria had well-developed cristae and an electron-dense matrix (Fig. 3). In these mitochondria, the intermembrane space between the inner and outer membranes was expanded. Proplastids contained densely stained stroma, numerous small vesicles, and plastoglobuli (Fig. 4). At the earliest stage, the initial cells were at the interphase of cell cycle (Figs. 1, 2). Subsequently, they began to divide anticlinally and periclinally (Fig. 5). At this time, the epidermal cells at the contact side of the parasite stem were round in transverse section and had dense cytoplasm and more evident nuclei than did epidermal cells before contact. Some dividing cells contained plastids with thylakoids and dense particles in small vacuoles. The haustorial initials developed into a group of meristem cells (Figs. 6, 7). Concurrently, the epidermal cells at the contact side began to elongate (Fig. 6) and to branch at their tips (Fig. 7). During these events, the cortical cells between the meristem and the stele began to expand toward the contact side (Fig. 6). The meristem cells contained large nuclei with enlarged nucleoli and many other organelles, especially large chloroplasts with thylakoids (Figs. 8–10). The meristem cells sometimes contained extremely elongate chloroplasts (Fig. 9) and vacuoles, and the vacuoles contained electron-dense particles, some of which were alveolated (Figs. 8, 10, 11).
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). For example, in Fig. 15, six cells forming the file marked by asterisks appeared to originate from only one cell that expanded before division; they probably arose from four periclinal divisions, and the lowest two cells probably arose from one anticlinal division. Collectively, the smaller file cells were arranged regularly in many radial rows. The digitate cells at the distal region possessed large nuclei with a couple of enlarged nucleoli and very densely stained cytoplasm containing numerous organelles such as dictyosomes, mitochondria, plastids, and r-ER (Figs. 16, 17). Several small vacuoles with or without electron-dense particles were near dictyosomes and r-ER (Fig. 16). The digitate cells contained some type of plastid with membrane-bound cryatalline inclusions (Figs. 18, 19), starch grains (Fig. 19), and thylakoids (Figs. 16, 19, 20). In contrast, those cortical cells of the parasite stem not involved with haustorial cells had starch-containing amyloplasts.
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Induction of the upper haustorium
The first event of haustorial development in C. japonica occurred in the vacuolated cortical cells in the middle layers of the parasite stem at the contact side. Once the parasite stem contacted the host, the cortical cells produced denser cytoplasm, larger nuclei with enlarged nucleoli, and many amyloplasts. Subsequently, they divided. These cells were referred to as haustorial initials and were very similar to those in C. australis (Lee and Lee, 1989
). This cellular response and subsequent division may be triggered by tactile stimuli when the Cuscuta stem contacts the host. When the haustorium in C. australis is not in contact with the host, the endophyte primordium (EP), a host-penetrating tissue, and thus a functional, mature upper haustorium, does not develop (Lee and Lee, 1989
). Tada et al. (1996)
also described that contact stimuli induced development of the haustorium in C. japonica.
Cytological features of haustorial initials, meristem, and EP
Maturation of the upper haustorium of C. japonica progressed through three main stages: the successive development of the haustorial initials, meristem, and EP. The EP was composed of two cell types, digitate and file, that differentiated in the fully matured upper haustorium. The initial cells had numerous starch-containing amyloplasts and mitochondria with well-developed cristae and a dense matrix. Notably, in these mitochondria, the intermembrane space between the inner and outer membranes was swollen. In contrast, the intermembrane space was less swollen in the mitochondria of the meristem cells (Figs. 8, 10) and the digitate cells (Figs. 16, 17) in the EP. Generally, the intermembrane compartment may be considerably expanded under conditions of active respiration (Karp, 2002
). Thus, the initial cells indicate a high level of cellular respiration.
It is also notable that the meristem cells contained many large chloroplasts with well-developed thylakoids, which were usually more developed than those of the digitate cells in the EP. These chloroplasts are believed to be responsible for the photosynthetic capability of Cuscuta before it parasitizes the host plant. The presence of chloroplasts is consistent with reports that free-living Cuscuta seedlings have higher photosynthetic rates (Pattee et al., 1965
) and more chlorophyll (Dinelli et al., 1993
) and photosynthetic proteins (Sherman et al., 1999
) than do the parasitic seedlings. Plastids of the cortical cells in the stems of C. pentagona (Sherman et al., 1999
) and C. reflexa (van der Kooij et al., 2000
) have thylakoids organized into grana. Thus far, however, chloroplasts with grana have not been reported in the haustorial meristem of the other Cuscuta species: ours is the first report of such chloroplasts in C. japonica (Figs. 9, 10). This evidence suggests that C. japonica, at least in the seedling stage, is not strictly holoparasitic (Dinelli et al., 1993
).
The prominent, large nuclei with enlarged nucleoli, as well as the very dense cytoplasm with numerous organelles probably reflect increased protein synthesis during haustorial development, as has been reported for seedlings of the root parasite Striga (Timko et al., 1989
; Wolf and Timko, 1992
; Riopel and Timko, 1995
). Haustoria-specific proteins are more abundant in the early stage of haustorial induction in Striga seedlings; concurrently, densely-stained haustorial cells appear. According to Stranger et al. (1995)
, addition of host-derived quinone induces new protein synthesis and haustoria development in Striga seedlings.
In the cells of C. japonica embryos and seedlings, numerous protein bodies with electron-dense, globoid crystals are degraded and transformed into vacuoles with or without crystals (Lee, 2006
). Thus, the dense particles in vacuoles of the initial and meristem cells (as in Figs. 8, 10, 11) are probably these globoid crystals. The crystals possess P, K, Mg, and phytate, which is an iron storage compound detected in protein bodies of seeds (Maldonado and Lott, 1991
; Pergo et al., 1998
; Maroder et al., 2003
). Therefore, the electron-dense particles may be used as a source of nutrients for the growth of those haustorial cells. On the other hand, the dense particles detected in the vacuoles of the digitate cells (Fig. 16) are likely to be different from those in the initial and meristem cells, considering that the particles in the digitate cells were irregular in shape and more abundant than in the other two types of cells. Because the small vacuoles or particle-containing vacuoles were visible near the numerous dictyosomes and ER, they may be involved in the secretion of the dense particles as the haustoria penetrates into the host tissue.
Characteristics of endophyte primordium (EP)
While the C. japonica upper haustorium matured, the EP developed from the meristem, which was derived from the haustorial initials. This pattern of haustorial development is very similar to that of C. australis (Lee and Lee, 1989
). Moreover, the anatomical structure and histological organization of the cells into the initials, meristem, and EP are also nearly the same in both species. The two species differ, however, in some respects in the composition of the cell types in the EP and the ultrastructure of the digitate cells of the EP. In the EP of C. australis, three cell types (digitate, file, and compressed) are clearly distinguished (Lee and Lee, 1989
), while the EP of C. japonica consisted of only two cell types (file and digitate). When the digitate cells elongate, however, they will compress the cells below them. An EP was not described for C. americana (Peirce, 1893) or C. reflexa (Thomson, 1925
; Forstreuter and Weber, 1984
). Even though a structure similar to the EP is visible in C. epilinum (Koch, 1874
) and C. pentagona (Tripodi, 1970
), no detailed structures were described.
On the other hand, the elongate digitate cells in an EP are thought to be remarkably specialized because of their very dense cytoplasm, large nuclei with enlarged nucleoli, and other abundant organelles. The morphology of the digitate cells in C. japonica is very similar to that in C. australis (Lee and Lee, 1989
); the two species differ, however, in the presence of some organelles in the digitate cells. Namely, in the C. australis–Trifolium repens system, the digitate cells have cytosegresomes, microfilament bundles, multilamellar structures, and proplastids (Lee and Lee, 1986
), while in the C. japonica–Impatiens balsamina system, the digitate cells have several small vacuoles containing irregular-shaped dense particles, plastids with thylakoids, crystals, and starch grains. The composition of cell organelles in the digitate cells is thus likely to differ in the two host–parasite systems.
Based on the cellular structure of the EP, we can reasonably speculate that physical pressure and enzymatic degradation drive haustorial penetration into the host tissue as they do for C. australis (Lee and Lee, 1989
). The cellular proliferation and enlargement after division of the file cells in the proximal zone of the EP may generate mechanical forces that enable the digitate cells to advance and penetrate the host. In addition, the digitate cells possessed a great abundance of dictyosomes and r-ER. Considering the function of these organelles, the digitate cells may actively synthesize and secrete enzymes to degrade the host tissue, enzymes like acid phosphatase in C. pentagona (Tripodi, 1970
) and wall-lytic enzymes in C. reflexa (Nagar et al., 1984
). Therefore, in the EP of the mature upper hasustorium, the file cells and the digitate cells may produce mechanical pressure and enzymes, respectively, for penetration. With respect to attachment of the haustorium to the host, modifications in the epidermal cells of the stem of C. japonica will be the subject of another paper.
In summary, the upper haustorium in C. japonica matures via three major developmental stages: initials, meristem, and endophyte primordium (EP) appear successively in the middle layers of the cortex in the parasite stem. The initial cells had numerous amyloplasts and mitochondria with an expanded intermembrane space between the outer and inner membranes, suggesting highly activated cellular respiration. The meristem cells are notable in having large chloroplasts with well-organized thylakoids, reflecting photosynthetic capability. In the haustorial cells of initials, meristem, and EP, large nuclei with enlarged nucleoli and dense cytoplasm with other abundant organelles indicate that these cells support the protein synthesis necessary for haustorial development. The file cells of the EP in the mature upper haustorium divide actively, whereas the digitate cells of the EP have abundant chloroplasts, dictyosomes, and r-ER. These cytological features and the histological arrangement of both types of cells in the EP contribute to a structure that is well suited for haustorial invasion into the host tissue.
FOOTNOTES
1 The author thanks two anonymous reviewers for helpful comments on the manuscript. This study was supported by a research fund from Chosun University in 2004. 
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