Transcriptional profiling of Arabidopsis tissues reveals the unique characteristics of the pollen transcriptome

Abstract

Pollen tubes are a good model for the study of cell growth and morphogenesis because of their extreme elongation without cell division. Yet, knowledge about the genetic basis of pollen germination and tube growth is still lagging behind advances in pollen physiology and biochemistry. In an effort to reduce this gap, we have developed a new method to obtain highly purified, hydrated pollen grains of Arabidopsis through flowcytometric sorting, and we used GeneChips (Affymetrix, Santa Clara, CA; representing approximately 8,200 genes) to compare the transcriptional profile of sorted pollen with those of four vegetative tissues (seedlings, leaves, roots, and siliques). We present a new graphical tool allowing genomic scale visualization of the unique transcriptional profile of pollen. The 1,584 genes expressed in pollen showed a 90% overlap with genes expressed in these vegetative tissues, whereas one-third of the genes constitutively expressed in the vegetative tissues were not expressed in pollen. Among the 469 genes enriched in pollen, 162 were selectively expressed, and most of these had not been associated previously with pollen. Their functional classification reveals several new candidate genes, mainly in the categories of signal transduction and cell wall biosynthesis and regulation. Thus, the results presented improve our knowledge of the molecular mechanisms underlying pollen germination and tube growth and provide new directions for deciphering their genetic basis. Because pollen expresses about one-third of the number of genes expressed on average in other organs, it may constitute an ideal system to study fundamental mechanisms of cell biology and, by omission, of cell division.

Figure 1.

Purification steps and viability of Arabidopsis pollen. Pollen and impurities before sorting (A) were separated into non-hydrated (B) and hydrated pollen (C) by flowcytometric sorting. D, A viability stain of the sorted, hydrated pollen grains.

Figure 2.

Flowcytometric sorting of Arabidopsis pollen. Arabidopsis pollen was identified through its size (A) and autofluorescence properties (B). A, Hydrated pollen was located in region R1 of the pulse width versus Forward Scatter (FSC) display, whereas non-hydrated pollen was mostly contained in R2. B, For sorting, a logical gate combination of regions R1 and R3 was used.

Figure 3.

“Snail view” representation of tissue-dependent gene expression patterns. The expression for 5,999 genes is represented in angular coordinates, in which angle encodes gene rank (clockwise from top) and radius encodes the logarithm of gene expression (values <1 were set to 1 for better visualization). Genes were ranked according to increasing mean expression in seedlings. For each tissue, the mean pattern in seedlings (black line) is coplotted with the mean pattern of specific tissue (gray dots), except for seedling where one replicate is coplotted. The values are the correlation coefficient between the gene expression patterns in the two tissues (for seedlings between the replicates).

Figure 4.

Analysis of genes enriched or selectively expressed in pollen. A, Hierarchical cluster analysis of the 1,584 genes called Present in pollen. Default parameters in dChip were used (standardization and clustering methods follow Golub et al. [1999] and Eisen et al. [1998]). Each column represents a single gene, and each row a single tissue type. Genes mainly enriched in pollen are found in the right cluster, whereas those expressed in pollen but with higher expression values in at least one of the vegetative tissues are found in the left cluster. P, Pollen; Se, seedling; L, leaf; Si; silique; R, root. B, Venn diagram depicting total numbers of genes expressed in vegetative tissues and in pollen. Genes were scored as Present in the vegetative tissues, if they received at least one Present call (MAS 5) in the seedling, leaf, root, or silique sample (5,775 genes). In pollen, 1,422 genes of these were also called Present. A set of 162 genes was exclusively called Present in pollen. Genes recognized by more than one probe set in the Affymetrix array were only counted once. C, Overview of the functional classification of 150 pollen selectively expressed genes listed in Table I. Total number of genes in each category is given in brackets. Functional classification is based on established as well as putative functions.