Characterization and expression of four proline-rich cell wall protein genes in Arabidopsis encoding two distinct subsets of multiple domain proteins

Abstract

We have characterized the molecular organization and expression of four proline-rich protein genes from Arabidopsis (AtPRPs). These genes predict two classes of cell wall proteins based on DNA sequence identity, repetitive motifs, and domain organization. AtPRP1 and AtPRP3 encode proteins containing an N-terminal PRP-like domain followed by a C-terminal domain that is biased toward P, T, Y, and K. AtPRP2 and AtPRP4 represent a second, novel group of PRP genes that encode two-domain proteins containing a non-repetitive N-terminal domain followed by a PRP-like region rich in P, V, K, and C. Northern hybridization analysis indicated that AtPRP1 and AtPRP3 are exclusively expressed in roots, while transcripts encoding AtPRP2 and AtPRP4 were most abundant in aerial organs of the plant. Histochemical analyses of promoter/beta-glucuronidase fusions localized AtPRP3 expression to regions of the root containing root hairs. AtPRP2 and AtPRP4 expression was detected in expanding leaves, stems, flowers, and siliques. In addition, AtPRP4 expression was detected in stipules and during the early stages of lateral root formation. These studies support a model for involvement of PRPs in specifying cell-type-specific wall structures, and provide the basis for a genetic approach to dissect the function of PRPs during growth and development.

Figure 1

Genomic Southern analysis of AtPRP sequences. One microgram of Arabidopsis genomic DNA was digested with EcoRI and analyzed by Southern hybridization using coding region probes for AtPRP1 and AtPRP2. The relative positions of the molecular mass markers are indicated. From top to bottom, the four restriction fragments correspond to AtPRP3, AtPRP1, AtPRP4, and AtPRP2.

Figure 2

DNA and predicted amino acid sequence of AtPRP1. The ORF for AtPRP1 and the predicted amino acid sequence are presented in uppercase, while upstream, downstream, and intron genomic sequences are presented in lowercase. The predicted cleavage site for the signal peptide is indicated with an arrowhead. A potential TATA box and polyadenylation signal are underlined.

Figure 3

DNA and predicted amino acid sequence of AtPRP3. The ORF for AtPRP3 and the predicted amino acid sequence is presented in uppercase, while upstream, downstream, and intron genomic sequences are presented in lowercase. The predicted cleavage site for the signal peptide is indicated with an arrowhead. A potential TATA box and polyadenylation signal are underlined.

Figure 4

DNA and predicted amino acid sequence of AtPRP2. The ORF for AtPRP2 and the predicted amino acid sequence are presented in uppercase, while upstream, downstream, and intron genomic sequences are presented in lowercase. The predicted cleavage site for the signal peptide is indicated with an arrowhead. A potential TATA box and polyadenylation signal are underlined.

Figure 5

DNA and predicted amino acid sequence of AtPRP4. The ORF for AtPRP4 and the predicted amino acid sequence is presented in uppercase while upstream, downstream, and intron genomic sequences are presented in lowercase. The predicted cleavage site for the signal peptide is indicated with an arrowhead. A potential TATA box and polyadenylation signal are underlined.

Figure 6

AtPRP expression analyzed by northern hybridization. A, Poly(A⁺) RNA (1.5 μg) isolated from the following organs of soil-grown plants was loaded onto each lane: 1, expanding rosette leaves; 2, mature rosette leaves; 3, roots (obtained from plants grown in liquid culture); 4, stems; 5, cauline leaves; and 6, flowers. Equal loading was confirmed by ethidium bromide staining. B, The specificity of the probes was analyzed using northern hybridization. The left two panels illustrate the difference in transcript size that was observed using the AtPRP2 and AtPRP4 probes to analyze northern blots of mRNA from expanding rosette leaves. In the right two panels, parallel northern blots containing in vitro-synthesized sense AtPRP1 and AtPRP3 transcripts (which are of the same predicted size) were hybridized with gene-specific probes for either AtPRP1 or AtPRP3.

Figure 7

Histochemical localization of AtPRP expression using AtPRP promoter/GUS analysis. a and b, AtPRP3/GUS. a, 2-d-old seedling; b, 8-d-old seedling. c to l, AtPRP4/GUS. c, 1-d-old seedling; d, 2-d-old seedling; e, 23-d-old seedling; f, detail stipules; g, detail roots; h, immature inflorescence; i, flower cluster; j, young silique; k, detail nectaries; l, maturing silique. m to o, AtPRP2/GUS. m, 23-d-old seedling; n, immature inflorescence; o, flower cluster.