Differential expression of members of the annexin multigene family in Arabidopsis

Abstract

Although in most plant species no more than two annexin genes have been reported to date, seven annexin homologs have been identified in Arabidopsis, Annexin Arabidopsis 1-7 (AnnAt1--AnnAt7). This establishes that annexins can be a diverse, multigene protein family in a single plant species. Here we compare and analyze these seven annexin gene sequences and present the in situ RNA localization patterns of two of these genes, AnnAt1 and AnnAt2, during different stages of Arabidopsis development. Sequence analysis of AnnAt1--AnnAt7 reveals that they contain the characteristic four structural repeats including the more highly conserved 17-amino acid endonexin fold region found in vertebrate annexins. Alignment comparisons show that there are differences within the repeat regions that may have functional importance. To assess the relative level of expression in various tissues, reverse transcription-PCR was carried out using gene-specific primers for each of the Arabidopsis annexin genes. In addition, northern blot analysis using gene-specific probes indicates differences in AnnAt1 and AnnAt2 expression levels in different tissues. AnnAt1 is expressed in all tissues examined and is most abundant in stems, whereas AnnAt2 is expressed mainly in root tissue and to a lesser extent in stems and flowers. In situ RNA localization demonstrates that these two annexin genes display developmentally regulated tissue-specific and cell-specific expression patterns. These patterns are both distinct and overlapping. The developmental expression patterns for both annexins provide further support for the hypothesis that annexins are involved in the Golgi-mediated secretion of polysaccharides.

Figure 1

An alignment of the deduced amino acid sequences of seven Arabidopsis annexin genes obtained using clustal on Macvector. Identical amino acids are indicated in dark gray, conserved amino acids in light gray. A potential nuclear localization domain in AnnAt3 is boxed. The potential heme-binding domain is underlined and an arrow marks the critical His residue in that domain. Various amino acid motifs discussed in the text are aligned above the annexin sequences, and the numbers found below the corresponding amino acid residue are based on the AnnAt1 protein sequence. GenBank accession nos. are as follows: AnnAt1 (AF083913), AnnAt2 (AF083914), AnnAt3 (AF1888362), AnnAt4 (AF188363), AnnAt5 (AY014797), AnnAt6 (AY014798), and AnnAt7 (AY014799).

Figure 2

Northern-blot analysis of Arabidopsis tissue-specific RNA using gene-specific 3′ UTR probes for AnnAt1 and AnnAt2. R, Roots; S, stems; F, flowers; Y, young leaves; O, old leaves.

Figure 3

RT-PCR analysis of the expression profiles of seven Arabidopsis annexins. Total RNA (2 μg) from leaf (L), stem (S), flower (F), and root (R) tissue was used to synthesize cDNA. A fraction (1/30) of the synthesized cDNA was used to amplify and quantitate AnnAt1–7 (accession nos. AF083913, AF083914, AF1888362, AF188363, AY014797, AY014798, and AY014799, respectively) gene transcripts. The sizes of the annexin PCR products are 954; 954; 966; 960; 951; 1,003; and 1,005 bp, respectively. The RT-PCR product of the APT1 gene (accession no. Y07681; 478 bp) was used as an internal control. A, Ethidium bromide-stained RT-PCR products separated in 1% (w/v) agarose gel. B, A chart depicting the relative amount of each annexin gene transcript. Optical densitometry was performed for each PCR product and normalized against the optical density obtained for the APT1 gene transcript for each tissue type. For each gene transcript, the tissue with the highest normalized optical density was designated 100% and the normalized optical densities for the remaining three tissues were expressed as a percentage.

Figure 4

AnnAt1 expression. A, Early expression in the epidermis in the zone of elongation in a root (r) tip, and in the root cap (arrow) of the germinating seedling; cotyledon (c), hypocotyl (h), and seedcoat (sc). B, Later expression includes internal cells of the root, and hypocotyl (h) epidermal cells. C, Expression in the seedling hypocotyl (h) occurs in the epidermis and vascular tissue. A small arrow shows the hypocotyl-root junction, and the large arrow indicates the root tip of another seedling. D, Cross section of a hypocotyl showing staining in the epidermis and vascular tissue. E, Root from a germinating seedling showing expression throughout the root base, including the hair cells, and excluded from the center of the root tip (arrow). F, Close-up of a root tip showing expression restricted to the epidermis of the root tip and outer rootcap cells. G, Longitudinal section through d 7 seedling showing expression in the trichomes (arrows), the hypocotyl vasculature (hv), and cotyledon (c) vasculature; lp, leaf primordia. H, Longitudinal section through d 14 plant showing expression in trichomes (arrow), pith meristem (p), leaf bases (lb), hypocotyl vasculature (hv), and leaf vascular tissue (vt). I, Close-up of d 14 plant showing vascular expression occurs in the phloem (ph) and not the xylem (x); floral meristem (f), shoot meristem (s), and pith meristem (p). J, Close-up of a cross section through a leaf base showing expression in the phloem (ph) and not the xylem (x). Scale bar lower left: A, 60 μm; B, 75 μm; C and E, 500 μm; D, 140 μm; F, 130 μm; G and H, 400 μm; I, 180 μm; and J, 50 μm.

Figure 5

AnnAt2 expression. A, Expression in the germinating embryo begins in epidermal cells of the root (r); cotyledon (c) and seed coat (sc). B, Seedling showing epidermal expression in the epidermis of the hypocotyl (h) and cotyledons (c). C, Oblique longitudinal section through the hypocotyl (h)-root (r) junction showing expression in the hypocotyl epidermis and the endodermis (e) of the root. D, Cross section through a seedling hypocotyl showing epidermal expression. E, Cross section through seedling root showing expression in the endodermis (e). F, Oblique longitudinal section through a root showing expression in two lateral root primordia. G, Oblique section through a root tip showing enhanced expression in the epidermis. H, Longitudinal section through d 14 plant showing epidermal expression in the creases (arrows) between floral primordia (f) and the shoot meristem (sm). An arrowhead shows patchy epidermal expression in a flower bud; leaf base (lb). Scale bar lower left: A and C, 60 μm; B, 70 μm; D and E, 130 μm; F and G, 100 μm; and H, 200 μm.