Phosphorylation of wheat (Triticum aestivum) and alfalfa (Medicago sativa) H2A histone variants was examined during early seedling growth. The C-terminal regions of wheat H2A variants contain multiple S-P tetrapeptides (senne-proline adjacent to a pair of basic amino acids) which resemble known phosphorylation sites in histones from other species. Phosphorylation of nucleosomal core histones was assessed by autoradiography of proteins labeled in vivo with 32pi and resolved by two-dimensional polyacrylamide gel electrophoresis, and phosphorylation sites were mapped by cleaving in vivo labeled H2A variants with Nbromosuccinimide. Essentially all phosphorylation of nucleosomal core histones in wheat and alfalfa seedlings occurred within the C-terminal peptides obtained from wheat and alfalfa H2A variants. A hypothesis accounting for the presence of large H2A and H2B histone variants in plants and phosphorylation of plant H2A C-terminal regions is proposed. The utility of S-P tetrapeptides for modulation of DNA-protein interactions is discussed.H2A and H2B histone variants from wheat contain long N-and C-terminal regions, rich in basic amino acids, hydroxyamino acids, proline and alanine (5,17,18). These regions resemble the DNA-binding 'head' and 'tail' regions of HI histones (1). Located periodically in the C-terminal regions of wheat H2A variants are two or three S-P tetrapeptides (serineproline adjacent to a pair of basic amino acids) (17, 18). Similar S-P tetrapeptides are known phosphorylation sites in the C-terminal regions of vertebrate HI histones (reviewed in Poccia [ 13]) and in the long N-terminal regions of sea urchin sperm HI and H2B histone variants, Sp HI and Sp H2B (7, 16).These observations prompted a study of nucleosomal core histone phosphorylation in early seedlings, during a period of rapid cell growth which follows a protracted period of nuclear quiescence. The results show that nearly all nucleosomal histone phosphorylation occurs within C-terminal peptides obtained from H2A histone variants, suggesting that S-P tetrapeptides are the primary sites ofphosphorylation, as they are in the sea urchin sperm variants Sp HI and Sp H2B. By analogy with Sp H1 and Sp H2B, we propose a hypothesis which accounts for the presence of large H2A and H2B histone variants in plants and C-terminal phosphorylation of the H2A variants. MATERIALS AND METHODSGermination, Seedling Growth, and Radiolabeling Seeds ofalfalfa (Medicago sativa) and hard red spring wheat (Triticum aestivum) were purchased from local grocery stores and germinated in Petri dishes on filter paper wetted with distilled water. After 48 h at 22°C, germinated seedlings (2 g, 2-4 mm in length) were transferred to fresh filter paper and wetted with 0.1 mCi/mL 32Pi (orthophosphate in carrier free aqueous solution; Amersham). Seedlings were incubated for an additional 24 h, after which endosperm or cotyledon tissues were removed. The seedlings were washed with cold water to remove residual 32Pi. Subsequent steps were at OC. Histone IsolationW...

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