Sequential Deposition and Remodeling of Cell Wall Polymers During Tomato Pollen Development

Abstract

The cell wall of a mature pollen grain is a highly specialized, multilayered structure. The outer, sporopollenin-based exine provides protection and support to the pollen grain, while the inner intine, composed primarily of cellulose, is important for pollen germination. The formation of the mature pollen grain wall takes place within the anther with contributions of cell wall material from both the developing pollen grain as well as the surrounding cells of the tapetum. The process of wall development is complex; multiple cell wall polymers are deposited, some transiently, in a controlled sequence of events. Tomato (Solanum lycopersicum) is an important agricultural crop, which requires successful fertilization for fruit production as do many other members of the Solanaceae family. Despite the importance of pollen development for tomato, little is known about the detailed pollen gain wall developmental process. Here, we describe the structure of the tomato pollen wall and establish a developmental timeline of its formation. Mature tomato pollen is released from the anther in a dehydrated state and is tricolpate, with three long apertures without overlaying exine from which the pollen tube may emerge. Using histology and immunostaining, we determined the order in which key cell wall polymers were deposited with respect to overall pollen and anther development. Pollen development began in young flower buds when the premeiotic microspore mother cells (MMCs) began losing their cellulose primary cell wall. Following meiosis, the still conjoined microspores progressed to the tetrad stage characterized by a temporary, thick callose wall. Breakdown of the callose wall released the individual early microspores. Exine deposition began with the secretion of the sporopollenin foot layer. At the late microspore stage, exine deposition was completed and the tapetum degenerated. The pollen underwent mitosis to produce bicellular pollen; at which point, intine formation began, continuing through to pollen maturation. The entire cell wall development process was also punctuated by dynamic changes in pectin composition, particularly changes in methyl-esterified and de-methyl-esterified homogalacturonan.

Keywords: 3-glucan); callose (β-1; cell wall; cellulose; exine development; intine; pectin; pollen; tomato.

Figure 1

Structure of mature tomato pollen grain. (A) Scanning electron micrograph of a mature, released tomato pollen grain. Arrows mark the apertures (Ap). (B) Transmission electron micrograph of a mature pollen grain cross section. Nu, nucleus; ER, endoplasmic reticulum; Ap, aperture. (C) TEM of mature pollen, showing the layers of the pollen wall. Cyt, cytoplasm; In, intine; Ne, nexine; Ba, bacula; Te, tectum. (D) TEM close-up of pollen aperture. Arrow marks the aperture. Cyt, cytoplasm; In, intine; Zw, zwischenkorper. Scale bars 1 μm in (A), 2 μm in (B), 200 nm in (C), and 600 nm in (D).

Figure 2

Tomato pollen development staging. (A) photographic series of floral development in tomato, with bud length as a marker for each stage in pollen development inside the anther. Bud lengths, 2 mm (microspore mother cell stage), 4 mm (tetrad stage), 5 mm (uninucleate microspore stage), 6 mm (bicellular pollen stage), and 8 mm (mature pollen stage). (B) Micrographs of paraffin-embedded 8-μm-thick anther cross sections, stained with toluidine blue. (C) Micrographs of LR White embedded 500-nm-thick anther cross sections, also stained with toluidine blue. (D) Developing pollen grain at higher magnification, close-up of individual cells. MMC, microspore mother cell; Te, tetrad; MS, microspore; Tap, tapetum; Po, pollen; MP, mature pollen. Scale bars 1 mm in (A), 100 μm in (B), 20 μm in (C), and 10 μm in (D).

Figure 3

Progression of exine deposition. Micrographs of LR White embedded 500-nm thick anther cross sections, stained with toluidine blue. (A) The uninucleate microspore stage at 5-mm bud length. Cross section of anther locule (Left), The arrow marks the tapetum. Close-up of microspores (right), black arrow marks aperture; the blue arrow marks the exine. (B) The bicellular pollen stage at 6-mm bud length. Cross section of anther locule (left); the arrow marks the tapetum. Close-up of microspores (right), black arrow marks aperture; the blue arrow marks the exine. (C) The mature pollen stage at 8-mm bud length. Cross section of anther locule (left); the arrow marks the tapetum. Close-up of microspores (right); the black arrow marks aperture; the blue arrow marks the exine. Ex, exine; Tap, tapetum; Po, pollen; Ap, aperture. All scale bars are 10 μm.

Figure 4

Calcofluor white staining of pollen development. Fluorescent micrographs of anther cross sections (500 nm) in LR white, stained with calcofluor white. (A) The microspore mother cell stage in the 2-mm bud anther. (B) The tetrad stage in the 4-mm bud anther. (C) The uninucleate microspore stage in the 5-mm bud anther. The arrow marks the microspore wall. (D) Bicellular pollen in 6-mm bud anther. The arrow marks the pollen wall. (E) Mature pollen in 8-mm bud anther. The arrow marks pollen wall. Te, tetrad. Scale bars. (A) 50 μm (left), 25 μm (center and right). (B) 75 μm (left), 50 μm (center and right). (C) 50 μm (left), 25 μm (center and right). (D) 75 μm (left), 50 μm (center and right). (E) 75 μm (left), 50 μm (center and right).

Figure 5

Aniline blue staining of the tetrad callose wall. Fluorescent micrographs of anther cross sections (500 nm) in LR white, of the 4-mm bud anther, stained with calcofluor white and aniline blue fluorochrome. Calcofluor white staining (cyan) (left). Aniline blue staining (magenta) (center). Merged staining (right). Scale bars 50 μm.

Figure 6

LM20 and LM19 immunostaining of the 2-mm and 4-mm bud anthers. Fluorescent micrographs of anther cross sections (500 nm) in LR white, of the 2-mm and 4-mm bud anther, stained with Calcofluor white and FITC conjugated anti-rat secondary for LM20 or LM19. Calcofluor white staining (Cyan) (left). Aniline blue staining (magenta) (center). Merged staining (right). (A) LM20 staining of the 2-mm anther. The right panel inset, showing zoomed-in view of the MMCs wall with the merged staining. (B) LM19 staining of the 2-mm anther. The right panel inset, showing zoomed-in view of the MMCs with the merged staining. (C) LM20 staining of the 4-mm anther. The right panel inset, showing zoomed-in view of the tetrad cells with the merged staining. (D) LM19 staining of the 4-mm anther. The right panel inset, showing zoomed-in view of the tetrad cells with the merged staining. Scale bars 50 μm.

Figure 7

LM20 and LM19 immunostaining of the 5-mm bud anthers. Fluorescent micrographs of anther cross sections (500 nm) in LR white, of the 5-mm bud anther, stained with Calcofluor white and FITC conjugated secondary for LM20 or LM19. Calcofluor white staining (Cyan) (left). Aniline blue staining (magenta) (center). Merged staining (right). (A) LM20 staining of the 5-mm anther. The right panel inset, showing zoomed-in view of the microspore wall with the merged staining. (B) LM19 staining of the 5-mm anther. The right panel inset, showing zoomed-in view of the microspore with the merged staining. Scale bars 50 μm.

Figure 8

LM20 and LM19 immunostaining of the 6-mm and 8-mm bud anthers. Fluorescent micrographs of anther cross sections (500 nm) in LR white, of the 6-mm and 8-mm bud anther, stained with Calcofluor white and FITC conjugated anti-rat secondary for LM20 or LM19. Calcofluor white staining (cyan) (left). Aniline blue staining (magenta) (center). Merged staining (right). (A) LM20 staining of the 6-mm anther.The right panel inset, showing zoomed-in view of the pollen wall with the merged staining. (B) LM19 staining of the 6-mm anther. The right panel inset, showing zoomed-in view of the pollen with the merged staining. (C) LM20 staining of the 8-mm anther. The right panel inset, showing zoomed-in view of the pollen wall with the merged staining. (D) LM19 staining of the 8-mm anther. The right panel inset, showing zoomed-in view of the pollen wall with the merged staining. Scale bars 50 μm.

Figure 9

Summary of tomato pollen wall development process. (A) A graphic summary of the major hallmarks of the tomato pollen development timeline. The pollen development stage is given above, and the bud length corresponding to each stage is given below, along with the major cell wall identities. (B) Cell wall polymer changes during development. The cellulose of the primary cell wall of the MMC breaks down at the 2-mm bud stage. Following meiosis, the tetrad cells are surrounded by a thick callose and pectin wall, which is broken down to separate the tetrad cells into free uninucleate microspores. Exine deposition by the tapetal cells begins at the 5-mm bud stage and continues through pollen maturation. The deposition of the cellulose and pectin, which make up the intine, initiates at the 6-mm bud stage and also continues through maturation. meHG deposition at the bicellular pollen stage is initially limited to the aperture regions, later spreading to the whole intine.