Variations in morphology and PSII photosynthetic capabilities during the early development of tetraspores of Gracilaria vermiculophylla (Ohmi) Papenfuss (Gracilariales, Rhodophyta)

Abstract

Background: Red algae are primitive photosynthetic eukaryotes, whose spores are ideal subjects for studies of photosynthesis and development. Although the development of red alga spores has received considerable research attention, few studies have focused on the detailed morphological and photosynthetic changes that occur during the early development of tetraspores of Gracilaria vermiculophylla (Ohmi) Papenfuss (Gracilariales, Rhodophyta). Herein, we documented these changes in this species of red algae.

Results: In the tetraspores, we observed two types of division, cruciate and zonate, and both could develop into multicellular bodies (disks). During the first 84 hours, tetraspores divided several times, but the diameter of the disks changed very little; thereafter, the diameter increased significantly. Scanning electron microscopy observations and analysis of histological sections revealed that the natural shape of the disk remains tapered over time, and the erect frond grows from the central protrusion of the disk. Cultivation of tissue from excised disks demonstrated that the central protrusion of the disk is essential for initiation of the erect frond. Photosynthetic (i.e., PSII) activities were measured using chlorophyll fluorescence analysis. The results indicated that freshly released tetraspores retained limited PSII photosynthetic capabilities; when the tetraspores attached to a substrate, those capabilities increased significantly. In the disk, the PSII activity of both marginal and central cells was similar, although some degree of morphological polarity was present; the PSII photosynthetic capabilities in young germling exhibited an apico-basal gradient.

Conclusions: Attachment of tetraspores to a substrate significantly enhanced their PSII photosynthetic capabilities, and triggered further development. The central protrusion of the disk is the growth point, may have transfer of nutritive material with the marginal cells. Within the young germling, the hetero-distribution of PSII photosynthetic capabilities might be due to the differences in cell functions.

Figure 1

Cruciate development of a G. vermiculophylla tetraspore. This is the main type of division (98.4% ± 0.0045), and it is characterized by the crossing of the first and second cleavage furrow. A, Freshly released tetraspores; B-H, Various developmental phases at 12 hour intervals; I-L, Various developmental phases at 24 hours intervals. Scale bar = 30 μm.

Figure 2

Zonate development of a G. vermiculophylla tetraspore. This type of division is rarely observed (1.6% ± 0.0045). The spore elongates before the first division, and the second furrow is always parallel to the first one. A, Freshly released tetraspores; B-H, Various developmental phases at 12 hour intervals; I-L, Various development phases at 24 hour intervals. Scale bar = 30 μm.

Figure 3

Variations in tetraspore/disk diameter during cruciate development. The time point at which the spores were freshly released was set as 0 hours. Statistical results suggest that there is no significant difference in diameter of spores or disks before 84 hours. After 84 hours, morphological changes occurred in marginal cells of the disk, the diameter began to increase (refer to Figure1-A-H). A one-way ANOVA and Students-Newman-Keuls Post Hoc comparison were used to test for statistical significance.

Figure 4

Changes in Y (II) and Fv/Fm during early development of G. vermiculophylla. A, In freshly released tetraspores, both Fv/Fm and Y (II) were very low; B, The PSII photosynthetic capabilities in attached tetraspores before the first division, compared with A, were enhanced significantly; C, Attached tetraspores after the first division; D, Attached tetraspores after the second division. There was no significant difference in the photosynthetic capabilities of PSII among E-M; E-M, multicellular bodies (disks) at different developmental stages. Statistical significance was tested by one-way ANOVA and Student-Newman-Keuls post hoc comparison. The arrow shows the start of tissue cultivation of excised disk. Y (II) was determined under constant illumination of 156 μmol photon m^-2.s^-1.

Figure 5

SEM micrographs of disks. The disks were cultured for 13 days after the attachment of tetraspores and then were detached from the substrate for the SEM observations. A and B were observed from different angles.

Figure 6

Photos of serial paraffin slices of disks at various developmental stages. Disks cultured 14, 15, 16, and 20 days after the attachment of tetraspores were detached from the substrate for preparation of serial paraffin slices. A, After cultivation for 14 days; B, After cultivation for 15 days; C, After cultivation for 16 days; D, After cultivation for 21 days. Scale bar = 100 μm.

Figure 7

Comparison of basal diameter and vertical height of disks at various developmental stages. The basal diameter (solid black square) and vertical height (solid black circle) were measured in photos of serial paraffin slices, and the ratio was calculated (hollow triangle).

Figure 8

Germlings of G. vermiculophylla cultured for 30 days showing the erect axis. A, A young germling with an erect frond detached from the substrate; B, A close-up view of A; C, a young germling with two erect fronds. Scale bars = 100 μm.

Figure 9

The 3D profile of the maximal PSII quantum yield (Fv/Fm) of a disk. Using the transect tool in the Imaging Win v2.21d software, the distribution of Fv/Fm within a disk, which was cultured for 13 days after the attachment of the tetraspore, was analyzed. It is clear that PSII photosynthetic capabilities of different areas within the disk are very similar. The Z-coordinate represents the value of Fv/Fm.

Figure 10

The repair process of excised disks that contained the protrusion. Disks cultured for 13 days were excised in situ, and then the remaining part with the central protrusion was cultivated continuously. A-J, At 24 hours intervals after cutting. K, 2 weeks after cutting. Scale bars = 100 μm.

Figure 11

The repair process of excised disks that did not contain the protrusion. Disks cultured for 13 days were excised in situ, and then the remaining part without the central protrusion was cultivated continuously. They formed a spindle-like disk (A-H) first and then initiated the erect frond at the center of the new disk (I, i). A-L, At 24 hours intervals; I and i, J and j, K and k, and L and l, were captured at the same time but with different focus, respectively. Scale bars = 50 μm.

Figure 12

Changes in photosynthetic capabilities of excised disks that contained the protrusion. The photosynthetic capabilities of PSII in excised disks were determined every day to compare them with the abilities of normal disks. Y (II) was determined under constant illumination of 156 μmol photon m^-2.s^-1.

Figure 13

Changes in photosynthetic capabilities of excised disks that did not contain the protrusion. Y (II) was determined under constant illumination of 156 μmol photon m^-2.s^-1.

Figure 14

Heterogeneous distribution of Fv/Fm and Y (II) along the length of a young germling. The germling was cultured for 30 days after the attachment of the tetraspore. In some parts, the value of Fv/Fm overlaps with that of Y (II). A, PSII Photosynthetic capabilities of different positions within the germling. The values of Fv/Fm and Y (II) represent the mean of the pixel value included in the black line across the young germling (see B); B, False color image of Fv/Fm of the young germling.