Expansins are conserved in conifers and expressed in hypocotyls in response to exogenous auxin

Abstract

Differential display reverse transcription-polymerase chain reaction was used to detect the induction of gene expression during adventitious root formation in loblolly pine (Pinus taeda) after treatment with the exogenous auxin indole-3-butyric acid. A BLAST search of the GenBank database using one of the clones obtained revealed very strong similarity to the alpha-expansin gene family in angiosperms. A near-full-length loblolly pine alpha-expansin sequence was obtained using 5'- and 3'-rapid amplification of cDNA end cloning, and the deduced amino acid sequence was highly conserved relative to those of angiosperm expansins. Northern analysis indicates that alpha-expansin mRNA expression increases 50- to 100-fold in the base of hypocotyl stem cuttings from loblolly pine seedlings in response to indole-3-butyric acid, with peak expression occurring 24 to 48 h after induction.

Figure 1

The DNA and deduced amino acid sequence for a loblolly pine expansin. The expansin cDNA sequence was submitted to the GenBank/EMBL DNA databases (accession no. AF085330). The putative translation initiation codon is underlined. The translation termination codon is indicated by an asterisk. Nucleotide positions are given by numbers to the left of the DNA sequence. Amino acid positions are indicated by numbers to the right of the amino acid sequence. The sequence includes the 5′ primer used for 3′-RACE cloning but does not include additional 5′ residues found in a 5′-RACE clone (accession no. U64895) from which the 5′ PCR primer was derived.

Figure 2

Alignment of loblolly pine amino acid sequence for expansin with angiosperm expansin sequences. Deduced amino acid sequences for expansins from loblolly pine (accession no. AF085330), Arabidopsis (accession no. U30481), cucumber (accession no. U30382), and rice (accession no. U85246) were aligned using the Pileup program from the Wisconsin Package DNA sequence. Output was produced using the program Boxshade (www.isrec.isb-sib.ch/software/BOX_form.html). Amino acids printed in inverse represent positions where at least 50% of the residues are identical. Amino acids that are similar to the consensus are shaded. The location of the putative N terminus of the mature peptide from loblolly pine as predicted by SignalP (Nielsen et al., 1997) is indicated by an arrow (↓).

Figure 3

Northern blot of hypocotyl RNA from loblolly pine seedling cuttings. A, RNA was extracted from the base of hypocotyl cuttings and placed in either distilled water (−) or distilled water plus 10 mm IBA (+) for the times indicated. One microgram of total RNA was electrophoresed in a 1% agarose gel, northern blotted, and hybridized with the clone pDD21.4.1. After exposure to x-ray film, the filter was erased and rehybridized with an 18S rRNA probe from eastern larch and with a loblolly pine partial cDNA clone for actin (accession no. AF085331), as described by Hutchison et al. (1990). B, RNA extracted from hypocotyl cuttings at the times indicated in an independent rooting experiment. Four micrograms of RNA was used per sample, and the filter was hybridized with the larch 18S rRNA probe and the expansin probe.