Transcriptome analyses show changes in gene expression to accompany pollen germination and tube growth in Arabidopsis

Abstract

Pollen germination, along with pollen tube growth, is an essential process for the reproduction of flowering plants. The germinating pollen with tip-growth characteristics provides an ideal model system for the study of cell growth and morphogenesis. As an essential step toward a detailed understanding of this important process, the objective of this study was to comprehensively analyze the transcriptome changes during pollen germination and pollen tube growth. Using Affymetrix Arabidopsis (Arabidopsis thaliana) ATH1 Genome Arrays, this study is, to our knowledge, the first to show the changes in the transcriptome from desiccated mature pollen grains to hydrated pollen grains and then to pollen tubes of Arabidopsis. The number of expressed genes, either for total expressed genes or for specifically expressed genes, increased significantly from desiccated mature pollen to hydrated pollen and again to growing pollen tubes, which is consistent with the finding that pollen germination and tube growth were significantly inhibited in vitro by a transcriptional inhibitor. The results of Gene Ontology analyses showed that expression of genes related to cell rescue, transcription, signal transduction, and cellular transport was significantly changed, especially for up-regulation, during pollen germination and tube growth. In particular, genes of the calmodulin/calmodulin-like protein, cation/hydrogen exchanger, and heat shock protein families showed the most significant changes during pollen germination and tube growth. These results demonstrate that the overall transcription of genes, both in the number of expressed genes and in the levels of transcription, was increased. Furthermore, the appearance of many novel transcripts during pollen germination as well as tube growth indicates that these newly expressed genes may function in this complex process.

Figure 1.

Effects of Act D on Arabidopsis PG and PTG. The experiments were repeated three times, and each treatment in one experiment had four replicates. Each data point is presented as mean ± se (n = 4). ^#, Data point for the control; *, significantly different from the control at P < 0.05 by Student's t test; **, significantly different from the control at P < 0.01 by Student's t test.

Figure 2.

Large-scale in vitro PG in liquid medium and pollen viability assay. A to C show the thin liquid layer method for PG and PTG. A, A 35-mm dish with a steel-wire net (80 μm) in it. The pollen grain suspension with 30 μL of basic medium was spread on the steel-wire net. (B) The pollen tubes under the steel-wire net after 4 h of incubation. (C) The collected pollen tubes after filtering through a 50-μm nylon mesh. D to F show FDA staining of the hydrated pollen grains after incubation in liquid medium for 45 min. D, FDA fluorescence image. E, Bright-field image. F, Merged image of D and E.

Figure 3.

Confirmation of transcriptional profiles of the selected genes by real-time PCR. HP-MP and PT-MP ratios in microarray data of 24 tested genes were compared with the real-time PCR results. The sequences of the gene-specific primers used for PCR are listed in Supplemental Table S8.

Figure 4.

Functional category distribution of transcriptionally changed genes in PG and PTG. The FunCat Scheme version 2.0 Web service at the Munich Information Center for Protein Sequences was used to analyze the GO categories of transcriptionally changed genes in PG (A) and PTG (B). The numbers of changed genes in 14 main biological categories are represented.