Molecular and cellular studies of early endosperm development in barley (Hordeum vulgare L.)

Abstract

Barley grain is an important commercial crop, being used mainly as an animal feed and in the production of malt for the brewing and distilling industries. The protein and carbohydrate composition of the endosperm (the major storage tissue) determines the grain quality and suitability for different end uses. The differentiation and maturation stages of endosperm development have been extensively studied. However, little is known about the cellular and molecular biology of the syncytial and cellularisation stages of development which occur within the first 8 days post anthesis (DPA). Events occurring during this period of development are particularly important as the overall pattern for the development and structure of the grain is laid down. Patterns of gene expression during the syncytial and cellularisation stages were investigated. A cDNA library was constructed from whole caryopses aged between 1 and 10 DPA. This cDNA library was then differentially screened using mRNA from 3 and 10 DPA caryopses. Northern and dot blot analysis led to the isolation of a number of clones which appear to show variation in level of expression. Partial sequencing of some of these clones and FASTA analysis (Genetics Computer Group, 1991) has shown four clones to have significant identity to sequences in the databases. These clones were clone 27B which showed identity to Ketol acid reductoisomerase (KARI) sequences, clone 16D which showed identity to Caffeoyl CoA-O-methyltransferase (CCoAOMT) sequences, clone 3B which showed identity to sucrose synthase sequences and clone 16B which showed identity to blue copper-binding protein sequences. A further 4 clones which were sequenced showed no significant identity to data base entries following FASTA analysis (Genetics Computer Group, 1991). The temporal and spatial distribution of these clones within tissues of barley caryopses was then analysed by in-situ hybrdisation. None were found to be associated uniquely with the endosperm tissues of barley caryopses. However, there were indications that the expression of the genes represented by the cDNA clones might vary during the course of development. Immunolocation studies utilising a set of JIM (John Innes Monoclonal) antibodies (and MAC207), which recognise carbohydrate epitopes of arabinogalactan proteins (AGP) were also carried out. AGPs have been associated with the plant cell surface and have been ascribed a number of possible functions related to developmental processes. The temporal and spatial distribution of AGPs within barley endosperm was analysed using sections from fixed and embedded barley caryopses and immunolocalisation techniques at the light microscope level. This revealed that at least one AGP epitope, recognised by JIM13, was expressed during early barley grain formation. JIM 13 binding was observed in developing barley caryopses at the beginning of endosperm cellularisation. It was localised to the first anticlinal and then periclinal endosperm cell walls, to the crease region and the nucellar/endosperm boundary. It was not observed in any caryopsis tissue at the earlier stage of syncytial endosperm and unfortunately its distribution could not be studied at later stages of endosperm development because of poor structural integrity within the sections.