Hordein Gene Expression in a Low Protein Barley Cultivar

Hordein Gene Expression in a Low Protein Barley Cultivar1 Received for publication September 28, 1987 and in revised form May 9, 1988

JESSICA E. DAILEY2, DAVID M. PETERSON*, AND THOMAS C. OSBORN Department ofAgronomy, University of Wisconsin, Madison, Wisconsin 53706 (J.E.D., D.M.P., T.C.O.), and U.S. Department ofAgriculture, Agricultural Research Service, 501 N. Walnut St., Madison, Wisconsin 53705 (D.M.P.) ABSTRACT The low protein barley (Hordeum vulgare L.) cultivar 'Karl' is deficient in hordeins, the major storage protein fraction, but has normal levels of other endosperm protein fractions. We compared hordein gene expression in Karl with that in 'Traill,' a related cultivar that contains normal amounts of hordein. In plants grown in controlled environmental conditions, hordein accumulation in Karl was 55% of that in Traill. The amount of hordein synthesized at all developmental stages tested was lower in Karl, as shown by pulse labeling of excised spikes with I3H]eucine. To determine whether levels of hordein mRNAs were related to amounts of hordein synthesis, total RNA preparations from endosperms at three or four developmental stages were hybridized to cDNA probes corresponding to B and C hordein genes. Both B and C hordein mRNA levels were significantly lower in Karl than in Traill at intermediate developmental stages. It was concluded that the low-hordein character of Karl was regulated by the size of the hordein mRNA pool. Transcriptional and/or post-transcriptional controls may be involved in the regulation of hordein mRNA levels.

normal protein and lysine levels. Low protein and high lysine are desirable characteristics for malting and nutritive value, respectively, and in Karl these traits are not accompanied by a shrivelled kernel and low seed yields, as in other high lysine mutants (9). Karl has low levels of hordein, and this apparently is responsible for lower percentage protein and higher lysine as percentage of total protein (4). Although these traits are highly heritable (4), the genetic control and mechanism of regulation are not known. In this study, we investigated hordein gene expression in Karl, and compared it to that in Trail, its normal protein parent. We compared the synthesis and accumulation of hordeins and the levels of Horl and Hor2 gene transcripts at various developmental stages.

MATERIALS AND METHODS Plant Material. Barley plants (Hordeum vulgare L. cvs. 'Karl' and 'Traill') were grown in pots in a 1:1 peat:vermiculite mixture in a growth chamber programmed for a 16 h photoperiod with 600 MuE m-2s-' of 400 to 700 nm light at the top of the plants and a day/night temperature regime of 19/16C. Pots were irrigated with a complete nutrient solution. Spikes were tagged at the approximate anthesis date, harvested 8 to 34 d later, and stored at -80C. The regulation of seed storage protein synthesis is amenable Frozen spikes, minus the awns, were weighed and lyophilized. for study because these proteins are generally specific to endo- Because of the difficulty of precisely noting anthesis date of each sperm and/or embryo tissues, the expression of their genes is floret, which may vary by several days within a spike, relative developmentally regulated, and the proteins have been exten- stages of spike development were based on percent moisture of sively characterized. In barley, the major endosperm storage a single central kernel. We found this to be a more precise proteins are the alcohol-soluble hordeins. These proteins are measure of endosperm development. Moistures were approxiclassified into three groups of polypeptides called B, C, and D mately 70 to 75% at 8 d after anthesis, declining steadily to 40 hordeins, and each group is synthesized from a family of struc- to 50% at 34 d. tural genes, Hor2, Horl, and Hor3, respectively (23). The B, C, Hordein Extraction and Quantification. Freeze-dried kernels and D hordeins account for approximately 80 to 90%, 10 to were ground in a Wiley Mill3 to pass a 20-mesh sieve. Following 20%, and