Differential expression of endo-β-1,4-xylanase isoenzymes X-I and X-II at various stages throughout barley development

Barley endo-β-1,4-xylanases (EC 3.2.1.8) hydrolyse arabinoxylan, which is an abundant cell wall constituent in grass species. In addition to isoenzyme X-I, the major endo-β-1,4-xylanase released from the aleurone layer of germinating barley, another isoenzyme, named X-II, is expressed during germination. Using PCR on barley genomic DNA, we isolated the complete gene encoding sequence of this less-characterized isoenzyme. The gene, shown to be located on chromosome arm 5HL, has a coding sequence of 1665 bp interrupted by two introns. The corresponding protein sequence shows a high overall homology to X-I (88%) and reveals a modular structure consisting of a carbohydrate binding module and a glycosyl hydrolase family 10 domain but no signal peptide. Isoenzyme-specific sequence differences were exploited to develop X-I- and X-II-specific reverse transcriptase-PCR assays. These showed that X-I and X-II portray distinct expression profiles throughout plant development. Both isoenzymes are transcribed during germination but, in contrast to X-I, X-II transcripts accumulate in the developing shoot and root of the seedling embryo and not in the aleurone layer surrounding the endosperm. During subsequent stages of development, up to anthesis, X-II remains expressed in various organs. In developing grains, X-II is transcribed in the early stages of grain filling whereas X-I transcription is switched on during the later stages. The possible implications of these findings are discussed in regard to the distinct physiological role of the two isoenzymes.