Selective interaction of plant homeodomain proteins mediates high DNA-binding affinity

Abstract

Understanding molecular mechanisms that control cell fate in the shoot apical meristem is a fundamental question in plant development. Genetic and molecular studies demonstrate that maize KNOTTED1 (KN1) of the TALE (3-aa acid loop extension) class of homeodomain (HD) proteins is involved in shoot apical meristem function. We show that KN1 interacts with knotted interacting protein (KIP), a BEL1-like TALE HD protein. Interaction between KN1 and KIP is mediated by conserved domains in the N termini of both proteins. The KN1 DNA-binding sequence, TGACAG(G͞C)T, was biochemically identified, and in vitro DNA-binding assays show that individually KN1 and the HD of KIP bind specifically to this motif with low affinity. The KN1-KIP complex, however, binds specifically to this DNA-binding motif with high affinity, indicating that the association of KN1 and KIP may function in transcriptional regulation. In plants, development of the aerial shoot is controlled by a group of cells in the shoot apex called the shoot apical meristem (SAM). The SAM is the site where organogenesis is initiated and from which leaves, flowers, or other determinate structures are produced. Because plant development is a continuous process, the SAM must balance the process of differentiation and maintenance to allow growth.Maize knotted1 (kn1) and the Arabidopsis ortholog SHOOT-MERISTEMLESS (STM) are members of the knox family of genes that are required for SAM function (1). The expression of kn1 and STM is restricted to the indeterminate cells of the SAM. Moreover, this pattern of expression is excluded from the zone of the SAM that gives rise to organs (2, 3). Loss-of-function mutations in kn1 and STM produce a shootless phenotype, where the SAM prematurely terminates after initiating cotyledons, demonstrating that kn1 and STM function in meristem maintenance (3-5). In backgrounds where some shoot formation occurs, organ production is reduced, indicating that vegetative and inflorescence meristems are compromised (4,(6)(7)(8). In addition to maintenance functions, class 1 knox genes, including kn1, are capable of respecifying cell fates when ectopically expressed in the leaf (1). Studies that support this hypothesis come from analysis of dominant mutations in the maize knox genes, kn1, rough sheath1 (rs1), liguleless3 (lg3), and gnarley1 (gn1) (9-12). Each of these phenotypes is caused by ectopic leaf expression, resulting in proximal leaf cell identities transposed distally into the blade (1, 13).The knox gene family was the first group of plant genes identified that encodes homeodomain (HD) proteins (14). In animals, HD-containing proteins function as transcription factors that are important regulators of animal cell fate and development (15). The HD is a conserved structure that contains three ␣ helices, and a sequence motif in the third helix recognizes and binds to the appropriate DNA sequence (15). Amino acid sequence comparison studies have subdivided the HD family into two classes of proteins: the typical HD class and the TALE (3-aa loop...

KNOX genes: versatile regulators of plant development and diversity

“…KNOX proteins interact with another group of TALE proteins, the BEL1-like homeodomain family (BELL or BLH, see Table 2), in a highly connected, complex network that determines not only high-affinity KNOX target selection but also their subcellular localization (Bellaoui et al, 2001;Bhatt et al, 2004;Cole et al, 2006;Hackbusch et al, 2005;Smith et al, 2002). These interactions depend on the KNOX MEINOX domain, and each homeodomain of the two proteins binds to target DNA as a protein heterodimer (Box 3).…”

“…TALE protein interactions are selective between specific members of the KNOX and BELL protein families, and these interactions are required for high-affinity DNA binding (Smith et al, 2002 …”

Transcriptomic Analyses Indicate That Maize Ligule Development Recapitulates Gene Expression Patterns That Occur during Lateral Organ Initiation

“…Given the proposed role of KNOX in leaf proximal-distal patterning, we investigated genes that are DE in our data and are also bound and modulated by KN1 protein (Bolduc et al, 2012b). In Arabidopsis, BEL1-like homeodomain (BELL) proteins form heterodimers with KNOX proteins in combinations that determine target selection and subcellular localization (Bellaoui Smith et al, 2002;Bhatt et al, 2004;Hackbusch et al, 2005;Cole et al, 2006). Our data show that maize bel12 (GRMZM2G154641) is significantly upregulated in the preligule region, whereas bel14 (GRMZM2G125976) is expressed at low levels in the blade, moderate levels in the sheath, and most highly in the ligule region ( Figure 4G; Supplemental Table 4).…”

Ontogeny of the Maize Shoot Apical Meristem

“…Transcription factors implicated in stem cell maintenance include six CLASS I KNOTTED1-LIKE HOMEOBOX (KNOX) genes and three BEL-LIKE (BELL) HOMEOBOX genes; CLASS I KNOX and BELL proteins form heterodimers that regulate stem cell identity (see Supplemental Data Set 3 online) Bellaoui et al, 2001;Müller et al, 2001;Smith et al, 2002;Mukherjee et al, 2009;reviewed in Hay and Tsiantis, 2010). At least two gene transcripts encoding WUSCHEL-RELATED HOMEODOMAIN (WOX) proteins (WOX2A and WOX9B) were upregulated in the proembryo.…”