Genes with homologies to known α‐galactosidases are expressed during senescence of barley leaves

Abstract

α‐Galactosidase activity increases during dark induced senescence as well as during natural senescence under field conditions. Two cDNA clones named pHvSF11 and pHvSF23 were isolated from a cDNA library of senescent barley flag leaves by screening with a coffee bean α‐galactosidase cDNA as probe. Sequences of the two barley cDNAs show high homology to those of known α‐galactosidases and hence seem to represent two different α‐galactosidase genes in barley. Expression of the genes corresponding to HvSF11 and HvSF23 was analyzed on one hand in primary foliage leaves incubated in darkness to accelerate senescence and on the other hand in flag leaves from barley plants grown under field conditions. The transcript represented by the cDNA HvSF23 is detectable in fully expanded leaves and its level is slightly upregulated during dark induced senescence of primary foliage leaves as well as during natural senescence of flag leaves. In contrast, the level of the transcripts represented by the cDNA HvSF11 decreases in flag leaves during natural senescence and inversely increases in primary foliage leaves induced to senesce by darkness. Antibodies raised against overexpressed peptide sequences deduced from the two different partial cDNA sequences react with the same proteins. In extracts from primary foliage leaves induced to senesce by prolonged darkness two proteins were immunodecorated by these antibodies. In senescent flag leaves from a barley field an additional third protein was detected.

An α-galactosidase with an essential function during leaf development

“…Equal amounts of protein (20 g) from each fraction were separated by SDS-PAGE (Laemmli 1970) and were either stained with Coomassie R250 for protein visualization or transferred onto a OPTITRAN BA-S 85 membrane (Schleicher & Schuell, Germany). Blotting and incubation with primary antibodies raised against the HvSF11 protein were performed as described by Chrost and Krupinska (2000). As secondary antibody a peroxidase-coupled anti-rabbit serum allowing chemiluminescence detection (Amersham, Germany) was used for visualization of immunoreactive protein bands.…”

“…To control loading of RNA the membrane was stained with methylene blue. Hybridizations were done with radiolabelled cDNA fragments of HvSF11 as described by Chrost and Krupinska (2000).…”

Regulation of a-galactosidase gene expression in primary foliage leaves of barley ( Hordeum vulgare L.) during dark-induced senescence

“…To investigate whether the senescence-related changes in a-galactosidase activity are linked to one or both of the two a-galactosidase genes represented by the cDNA clones HvSF11 and HvSF23 (Chrost and Krupinska 2000), the expression of both genes was examined in parallel ( Fig. 1).…”

“…To control loading of RNA the membrane was stained with methylene blue. Hybridizations were done with radiolabelled cDNA fragments of HvSF23 and HvSF11 fragments, as described by Chrost and Krupinska (2000).…”

“…The corresponding genes are also expressed in primary foliage leaves induced to senesce by darkness. Sequence analyses revealed that both genes encode putative agalactosidase enzymes (Chrost and Krupinska 2000). In the present study we have investigated the expression of the two genes in primary foliage leaves of barley in response to sugar depletion in the course of dark-induced senescence and after application of sugars.…”