Cell Wall Invertase and Sugar Transporters Are Differentially Activated in Tomato Styles and Ovaries During Pollination and Fertilization

Abstract

Flowering plants depend on pollination and fertilization to activate the transition from ovule to seed and ovary to fruit, namely seed and fruit set, which are key for completing the plant life cycle and realizing crop yield potential. These processes are highly energy consuming and rely on the efficient use of sucrose as the major nutrient and energy source. However, it remains elusive as how sucrose imported into and utilizated within the female reproductive organ is regulated in response to pollination and fertilization. Here, we explored this issue in tomato by focusing on genes encoding cell wall invertase (CWIN) and sugar transporters, which are major players in sucrose phloem unloading, and sink development. The transcript level of a major CWIN gene, LIN5, and CWIN activity were significantly increased in style at 4 h after pollination (HAP) in comparison with that in the non-pollination control, and this was sustained at 2 days after pollination (DAP). In the ovaries, however, CWIN activity and LIN5 expression did not increase until 2 DAP when fertilization occurred. Interestingly, a CWIN inhibitor gene INVINH1 was repressed in the pollinated style at 2 DAP. In response to pollination, the style exhibited increased expressions of genes encoding hexose transporters, SlHT1, 2, SlSWEET5b, and sucrose transporters SlSUT1, 2, and 4 from 4 HAP to 2 DAP. Upon fertilization, SlSUT1 and SlHT1 and 2, but not SlSWEETs, were also stimulated in fruitlets at 2 DAP. Together, the findings reveal that styles respond promptly and more broadly to pollination for activation of CWIN and sugar transporters to fuel pollen tube elongation, whereas the ovaries do not exhibit activation for some of these genes until fertilization occurs.

Highlights: Expression of genes encoding cell wall invertases and sugar transporters was stimulated in pollinated style and fertilized ovaries in tomato.

Keywords: cell wall invertase; fertilization; fruit set; pollen tube elongation; pollination; sugar transporter; sugar utilization; tomato.

FIGURE 1

The phenotype of style and ovary during flowering stage in tomato and the illustration of time of manual pollination and sampling. (A) The phenotypes of tomato flower, ovary, and style from flower bud to fruitlets. The elongation of the style stopped at ∼2 days before anthesis. Fruitlet enlargement was not apparent until 3 DAP. (B) A schematic diagram about the timing of emasculation, pollination, and sampling. The ovary and style were harvested at 4 HAP and 2 DAP, representing the time points before and after fertilization, respectively. Emasculated flower with no pollination served as a control. (C) The phenotypes of manually pollinated ovary and style from 4 HAP to 8 DAP. The pollinated ovary increased its size at 2 DAP onward (red arrows). In contrast, the non-pollinated control ovaries were stunted and aborted at 6∼8 DAP. The findings validated the success of emasculation and pollination treatments. HAP, hours after pollination; DAP, days after pollination.

FIGURE 2

Visualization of pollen tube elongation within tomato pistils. (A,B) Longitudinal sections of ovary and fruitlet at 24 and 48 h after pollination (HAP), respectively. Pollen tubes, exhibiting fluorescence from the callose, entered the ovary through the junction of ovary and style (white arrowheads) at 24 HAP (A) and arrived at ovules at 48 HAP (B), as indicated by white arrows. (C) Negative control I, fruitlet derived from pollinated flower at 48 HAP without staining with aniline blue. No fluorescence was detected in the fruit. (D) Negative control II, fruitlet at 48 HAP, derived from non-pollinated flower, but stained with aniline blue. Fluorescence was only found at the bottom of fruit, but not in ovules. Red arrows at the bottom of ovaries in (A,B,D) indicate fluorescence from the pre-existing callose of ovary tissues, mainly the vascular bundles. White arrowheads indicate the connection between ovary and style in (A–D). Each image is one representative out of four biological replicates. c, columella; pl, placenta; o, ovule; Scale bars represent 0.25 mm in (A,B), 0.5 mm in (C,D). (E) An illustration showing that pollen tube elongates along the style. (F) A schematic diagram of a tomato ovary. The pollen tube enters through the top of the ovary and reaches ovules to finish the fertilization process.

FIGURE 3

Relative expression of CWIN-related genes LIN5, LIN7, and INVINH1, in style and fruitlet in response to pollination at 4 HAP and fertilization at 2 DAP. Asterisks indicate significant differences (Student’s t-test, ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001, n = 4) between non-pollination and pollination treatments. DAP, days after pollination; HAP, hours after pollination.

FIGURE 4

The activities of CWIN, VIN, and CIN in style and fruitlet in response to pollination at 4 HAP and fertilization at 2 DAP. Asterisks indicate significant differences (Student’s t-test, ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001, n = 4) between non-pollination and pollination treatments. DAP, days after pollination; HAP, hours after pollination.

FIGURE 5

Relative gene expression of SlHT1, SlHT2, and SlHT3 in style and fruitlet in response to pollination at 4 HAP and fertilization at 2 DAP. Asterisks indicate significant differences (Student’s t-test, ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001, n = 4) between non-pollination and pollination treatments. DAP, days after pollination; HAP, hours after pollination.

FIGURE 6

Relative expression of SlSWEET transporter genes in response to pollination at 4 HAP and fertilization at 2 DAP. Asterisks indicate significant differences (Student’s t-test, ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001, n = 4) between non-pollination and pollination treatments. DAP, days after pollination; HAP, hours after pollination.

FIGURE 7

Relative expression of SlSUT transporter genes in response to pollination at 4 HAP and fertilization at 2 DAP. Asterisks indicate significant differences (Student’s t-test, ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001, n = 4) between non-pollination and pollination treatments. DAP, days after pollination; HAP, hours after pollination.

FIGURE 8

A model of how sugars transported into and metabolism within tomato style and ovary are regulated during pollination and fertilization. The occurrence of pollination stimulates and repressed cell wall invertase and its inhibitor gene (INVINH1), respectively, in the style, leading to high CWIN activity to hydrolyze sucrose into hexoses in the style apoplasm. Meanwhile, the expressions of sucrose transporter genes SlSUT1 and 2 as well as SlHT1 and 2 and SWEET5b for hexoses are also stimulated. The increased SlSWEET5b on the pollen tube might function in the uptake of apoplasmic hexose into pollen tubes or inversely exported intercellular hexose out of the pollen tube. The coexistences of SlSWEET and SlHT may contribute to the maintenance of cytosolic sugar homeostasis, which is crucial for metabolism and cellular function. These pollination-elicited changes enhance carbon input into and uses within pollen tubes to fuel their rapid elongation. For the ovaries or fruitlets, instead of pollination effect, the accomplishment of fertilization selectively stimulates the expression of genes for CWIN (LIN5), SlHT (SlHT1 and 2), and SlSUT (SlSUT1), but not those for SlSWEETs (SlSWEET10b and 12a), to support the growth of the newly formed fruitlets.