Transcriptome and Metabolome Analyses Reveal a Complex Stigma Microenvironment for Pollen Tube Growth in Tobacco

Abstract

In flowering plants, the success of fertilization depends on the rapid polar extension of a pollen tube, which delivers sperm cells to the female gametophyte for fertilization. Numerous studies have shown that the microenvironment in planta is more conducive to the growth and development of pollen tubes than that in vitro. However, how stigma factors coordinate to regulate pollen tube growth is still poorly understood. Here, we demonstrate that in tobacco, mature stigma extract, but not immature stigma extract, facilitates pollen tube growth. Comparative transcriptomic and qRT-PCR analyses showed that the differentially expressed genes during stigma maturation were mainly enriched in the metabolism pathway. Through metabolome analyses, about 500 metabolites were identified to be differently accumulated; the significantly increased metabolites in the mature stigmas mainly belonged to alkaloids, flavonoids, and terpenoids, while the downregulated differential metabolites were related to lipids, amino acids, and their derivatives. Among the different kinds of plant hormones, the cis-form contents of zeatin were significantly increased, and more importantly, cis-zeatin riboside promoted pollen tube growth in vitro. Thus, our results reveal an overall landscape of gene expression and a detailed nutritional microenvironment established for pollen tube growth during the process of stigma maturation, which provides valuable clues for optimizing in vitro pollen growth and investigating the pollen-stigma interaction.

Keywords: cis-zeatin riboside; metabolome; microenvironment; pollen tube growth; stigma; tobacco; transcriptome.

Figure 1

Mature stigma extract promotes pollen tube growth in tobacco. (A) Representative images of tobacco flower buds at 25–30 mm and at the anther dehiscence stage. (B) Representative images of pistils from tobacco flower buds (25–30 mm) and opening flowers. S_immature, stigma at flower buds (25–30 mm); S_mature, stigma at opening flowers. Bars = 10 mm. (C) Images of pollen germinated with S_immature or S_mature extracts at 2 h after germination (HAG). Without stigma extracts as the control (CK). Bar = 100 μm. (D) Statistical data of wild-type pollen tube length at 1 or 2 HAG, with and without stigma extracts. The data for pollen tube length are presented in the box-and-whisker plots. The centerline in the plot represents the 50th percentile. The bottom and top of each box indicate the 25th and 75th percentiles, respectively, and the whiskers represent the minimum and maximum values. For 1 HAG, the number of pollen tubes in CK, S_immature, and S_mature are 245, 255, and 254, respectively. For 2 HAG, the number of pollen tubes in CK, S_immature, and S_mature are 252, 231, and 236, respectively. For statistical comparisons of the multiple groups in (D), different letters denote that these groups differ significantly (p < 0.0001) in one-way ANOVA and Tukey’s range test.

Figure 2

Comparative transcriptomic analysis of immature and mature stigmas. (A) Heat map showing Pearson’s correlation across RNA-seq datasets. Three biological replicates for each sample. (B) Volcano plot depicts differentially expressed genes between S_immature and S_mature. Red dots indicate upregulated genes, while blue dots indicate downregulated genes. FC, fold change; FDR, false discovery rate; NS, not significant. (C) Hierarchical clustering of differentially expressed genes (DEGs). DEGs between S_immature and S_mature could be categorized into seven distinct clusters. (D) KEGG pathway analysis of DEGs. The number of enriched genes is shown above the column. (E) High correlation between quantitative real-time PCR (qRT-PCR) and RNA sequencing results (75 genes). (F) Verification of DEGs in flavonoid biosynthesis, terpenoid biosynthesis, starch and sucrose metabolism, pentose and glucuronate interconversions, carotenoid biosynthesis, and fatty acid degradation and biosynthesis pathways using qRT-PCR. The expression level of each gene was estimated using log₂ (FC).

Figure 3

Determination of significantly changed metabolites between immature and mature stigmas. (A) Pearson’s correlation of all six stigma samples. (B) A volcano map of differentially regulated metabolites between S_immature and S_mature. Red and green dots represent significantly up- or downregulated metabolites, respectively. The dot size indicates the VIP (variable importance in projection) values. FC, fold change. (C,D) Heat map clustering and KEGG enrichment of differentially regulated metabolites between S_immature and S_mature.

Figure 4

cZR promotes tobacco pollen tube growth in vitro. (A) The quantification of different kinds of plant hormones in S_immature and S_mature. The data are presented as the mean ± SD from three independent replicates. The up-regulated hormones were indicated in red and the down-regulated shown in blue. Two-tailed Student’s t-test was used for statistical analysis (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). IAA, 3-indolylacetic acid; oxIAA, 2-oxo-3-indolineacetic acid; IBA, 4-(3-indolyl)butyric acid; ABA, abscisic acid; JA, jasmonic acid; JA-Ile, jasmonoyl-L-isoleucine; OPDA, 12-oxo-phytodienoic acid; SA, salicylic acid; iP, N6-isopentenyladenine; iPR, N6-isopentenyladenosine; tZ, trans-zeatin; tZR, trans-zeatin riboside; cZ, cis-zeatin; cZR, cis-zeatin riboside; DHZ, dihydrozeatin; DHZR, dihydrozeatin riboside; CS, castasterone; 6-deoxoCS, 6-deoxocastasterone. (B) Images of pollen germinated with supplement of tZR or cZR at 2 h after germination (HAG). Without the addition of tZR or cZR as a control (CK). Bar = 100 μm. (C) Statistical data of wild-type pollen tube length at 1 or 2 HAG. The data for the pollen tube length are presented in the box-and-whisker plots. The centerline in the plot represents the 50th percentile. The bottom and top of each box indicate the 25th and 75th percentiles, respectively, and the whiskers represent the minimum and maximum values. For 1 HAG, the number of pollen tubes in CK, tZR, and cZR is 173, 191, and 197, respectively. For 2 HAG, the number of pollen tubes in CK, tZR, and cZR is 194, 216, and 173, respectively. Ordinary one-way ANOVA with Tukey’s multiple comparisons test was used for statistical difference analysis among different groups. Different letters above the bars indicate significant differences (p < 0.05) among the groups.