Constitutive Expression Exposes Functional Redundancy between the Arabidopsis Histone H2A Gene HTA1 and Other H2A Gene Family Members

Abstract

The Arabidopsis thaliana histone H2A gene HTA1 is essential for efficient transformation of Arabidopsis roots by Agrobacterium tumefaciens. Disruption of this gene in the rat5 mutant results in decreased transformation. In Arabidopsis, histone H2A proteins are encoded by a 13-member gene family. RNA encoded by these genes accumulates to differing levels in roots and whole plants; HTA1 transcripts accumulate to levels up to 1000-fold lower than do transcripts of other HTA genes. We examined the extent to which other HTA genes or cDNAs could compensate for loss of HTA1 activity when overexpressed in rat5 mutant plants. Overexpression of all tested HTA cDNAs restored transformation competence to the rat5 mutant. However, only the HTA1 gene, but not other HTA genes, could phenotypically complement rat5 mutant plants when expressed from their native promoters. Expression analysis of HTA promoters indicated that they had distinct but somewhat overlapping patterns of expression in mature plants. However, only the HTA1 promoter was induced by wounding or by Agrobacterium infection of root segments. Our data suggest that, with respect to Agrobacterium-mediated transformation, all tested histone H2A proteins are functionally redundant. However, this functional redundancy is not normally evidenced because of the different expression patterns of the HTA genes.

Overexpression of SeveralArabidopsisHistone Genes IncreasesAgrobacterium-Mediated Transformation and Transgene Expression in Plants

“…Mutant Arabidopsis lines containing T-DNA insertions in or near several other core histone genes, including HTA2, HTA3, HTA11, HTB5, HTB6, HTR5/4, HFO3, and HFO4, also reduced transformation frequency to various extents (Zhu et al, 2003b), although we have not yet determined the specific stage of transformation that was disrupted. Our observations that overexpression of HTA1 increased transformation frequency (Mysore et al, 2000a;Yi et al, 2006) suggested to us that increased T-DNA integration may be the cause of this hypersusceptibility to Agrobacterium-mediated transformation. However, the results of this study and other work (L.-Y.…”

“…Increasing evidence indicates that these histone variants have specialized functions; therefore, their incorporation into chromatin at specific regions of the genome or loci may confer specific regulatory or structural features (Kamakaka and Beggins, 2005;March-Diaz et al, 2008;Jin et al, 2009). Histone H2A has the largest number of variants, which include H2A-X (Arabidopsis HTA3 and HTA5) and H2A-Z (Arabidopsis HTA8, HTA9, and HTA11; Raisner and Madhani, 2006;Yi et al, 2006). In plants, as in other eukaryotes, histone H3 variants include H3.2 and H3.3 (Arabidopsis HTR12), which is found in the centromeric region.…”

“…Expression of HTA1 in plants coincides with the tissues and cell types that are most susceptible to transformation (Yi et al, 2002). Overexpression of HTA1 genomic or cDNAs results in increased transformation of Arabidopsis root segments (Mysore et al, 2000a;Yi et al, 2006).…”

“…cDNAs for the histone genes HTA1 (E1701), HTA6 (E2272), and HTA8 (E2267) were excised from their respective pBluescript vectors (Yi et al, 2006) using EcoRI and XbaI and cloned into the same sites of the T-DNA binary vector pE2786 (see below). cDNAs for the histone genes HTB1 (C00142), HTB3 (U14625), HTR4 (U12616), and HFO3 (U1196) were obtained from the Arabidopsis Stock Center (The Ohio State University), excised from their respective vectors using EcoRI and XbaI, and cloned into these same sites of pE2786.…”

“…Expression of HTA1 in plants coincides with the tissues and cell types that are most susceptible to transformation (Yi et al, 2002). Overexpression of HTA1 genomic or cDNAs results in increased transformation of Arabidopsis root segments (Mysore et al, 2000a;Yi et al, 2006).The Arabidopsis genome comprises 47 genes that encode 33 different core histone proteins (13 HTA genes that encode 13 different H2A proteins, 11 HTB genes that encode 11 different H2B proteins, 15 HTR genes that encode eight different H3 proteins, and eight HFO genes that encode 1 H4 protein; www. chromdb.org; see Supplemental Figure 1 online).…”