Discovery and characterization of KNOX proteins lacking a homeodomain, produced by alternative splicing of KNAT1-like genes in gymnosperms and angiosperms

Embryogenesis in Gymnosperms and Angiosperms while broadly similar differs in important respects. Despite these differences, the genes expressed in developing pine embryos show strong sequence similarity to those found in angiosperm genomes. The distinguishing features of conifer embryogenesis may be a consequence of the level, timing or localization of expression of certain regulatory genes perhaps supplemented by the activity of a few unique or highly differentiated proteins.;Homeobox genes encode homeodomain (HD) proteins which function as transcription factors and play an important role in plant and animal development by controlling cell specification and pattern formation. The KNOX (knotted-like homeobox) family of regulatory genes belongs to a homeobox gene family which exerts considerable influence upon plant development. Alteration in the expression of KNOX genes, through mutation or by increasing or decreasing mRNA levels in transgenic plants, produces profound changes in morphology and growth. The ability of these proteins to affect a broad range of cellular activities is explained in great measure by the presence within the KNOX proteins of distinct domains that can interact with a variety of other regulatory proteins. The HD region of KNOX proteins is a sequence-specific DNA-binding domain that can directly regulate expression of target genes. The KNOX1 and KNOX2 regions constitute the MEIKNOX domain which has been shown to homodimerize with KNOX family proteins or heterodimerize with members of the BELL family of TALE homeodomain proteins.;I have cloned (Knotted1 in Arabidopsis thaliana) KNAT1-like mRNAs which lack HD sequences from embryos of loblolly pine (Pinus taeda L.), hereafter referred as PtKN1(hd-). Production of PtKN1(hd-) mRNAs is developmentally regulated and their encoded protein is abundant in mature pine embryos. The PtKN1 gene has 5 exons; KNOX1 and KNOX2 domains are encoded by the first three exons, and the large exon 3 separates these from exons 4 and 5 which encode HD sequences. The PtKN1(hd-) mRNA has a unique 3'UTR which derives from the proximal region of 'exon 3'. Both forms of PtKN1 are produced by the same gene; the regulatory dynamic is between cleavage-polyadenylation within intron 3 to produce PtKN1 mRNA lacking HD sequences and splicing of exon 3 to exon 4 which excludes the 3'UTR/exon3 sequence to create an mRNA which encodes a HD. I have identified and characterized a KNAT1 mRNA in Arabidopsis which lacks HD sequences. While KNAT1 has been studied for many years, this is the first report of a KNAT1 mRNA lacking HD. The expression pattern of AtKNAT1(hd-) mRNA differs from that of the KNATM-B mRNA, which encodes a KNOX gene lacking HD sequences, implying a distinct role for these two genes. While KNATM appears unique to dicotyledons, I identified a KNAT1 mRNA lacking HD sequences for the RS1 gene of maize, a monocotyledon. This is the first report of splicing of KNAT1 genes to produce mRNAs lacking HD sequences. The phenomenon appears to be ubiquitous as it is observed in gymnosperms, and both dicotyledonous and monocotyledonous angiosperms.