Functional analysis of the conserved domains of a rice KNOX homeodomain protein, OSH15

The rice KNOX protein OSH15 consists of four conserved domains: the MEINOX domain, which can be divided into two subdomains (KNOX1 and KNOX2); the GSE domain; the ELK domain; and the homeodomain (HD). To investigate the function of each domain, we generated 10 truncated proteins with deletions in the conserved domains and four proteins with mutations in the conserved amino acids in the HD. Transgenic analysis suggested that KNOX2 and HD are essential for inducing the abnormal phenotype and that the KNOX1 and ELK domains affect phenotype severity. We also found that both KNOX2 and HD are necessary for homodimerization and that only HD is needed for binding of OSH15 to its target sequence. Transactivation studies suggested that both the KNOX1 and ELK domains play a role in suppressing target gene expression. On the basis of these findings, we propose that overproduced OSH15 probably acts as a dimer and may ectopically suppress the expression of target genes that induce abnormal morphology in transgenic plants.     

Over-expression of tobacco knotted1-type class1 homeobox genes alters various leaf morphology

We compared the phenotypes of transgenic tobacco plants over-expressing various knotted1-type class1 homeobox genes. All transformants showed abnormal leaf morphology, with the degree of abnormality depending upon the Nicotiana tabacum homeobox (NTH) gene that was over-expressed. Tobacco plants over-expressing NTH1 or NTH9 showed a relatively weak phenotype, while NTH15 and NTH20 over-expressing plants exhibited severe alterations, with occasional ectopic shoot formation on the leaves. Plants over-expressing NTH22 had a relatively severe phenotype, but did not form any ectopic shoots. These results indicate that all of the NTH genes can influence leaf development from the shoot apical meristem, but that the effect varies with the gene. Based on phylogenetic analysis of the NTH genes and comparison of the phenotypes of plants over-expressing them, we suggest that the kn1-type class1 family can be divided into two subgroups, and that the differences in their ability to induce the abnormal phenotype corresponds to the structures of their conserved domains.     

Domain exchanges between KNAT3 and KNAT1 suggest specificity of the kn1-like homeodomains requires sequences outside of the third helix and N-terminal arm of the homeodomain

Domain exchange constructs that traded regions surrounding the homeodomain were constructed for two kn1 -like genes, KNAT1 and KNAT3, and introduced into Arabidopsis thaliana under the control of the 35S CaMV promoter. The kn1-like homeodomain proteins all have the homeodomain located near the C-terminus of the protein, and also share a second conserved domain (the ELK domain) immediately N-terminal to the homeodomain. Progeny were scored for the appearance of the KNAT1 overexpression phenotype. A construct containing the KNAT3 N-terminus and the KNAT1 ELK- and homeodomain resulted in a KNAT1 overexpression phenotype, indicating that specificity mainly resides within the ELK- and homeodomain region. Further exchanges demonstrated that specificity probably does not arise from a single region within the ELK and/or homeodomain but rather requires sequences both N-terminal and C-terminal to residue 23 of the homeodomain. Further, in contrast to some animal homeodomains, KNAT1 does not utilize the residues of the N-terminal arm of the homeodomain for specificity.     

The expression of tobacco knotted1-type class 1 homeobox genes correspond to regions predicted by the cytohistological zonation model

We have isolated and characterized four tobacco homeobox genes, NTH1, NTH9, NTH20, NTH22 (Nicotiana tabacum homeobox) which belong to the class 1 knotted1-type family of homeobox genes. Comparison of the inferred amino acid sequences of the ELK homeodomains of these genes and previously reported kn1-type class 1 proteins has revealed that the four new tobacco genes belong to distinct subclasses, suggesting that each NTH gene may have distinct functions. Using in situ hybridization and by analysing the distribution of GUS activity in tobacco plants transformed with NTH promoter::GUS constructs, localized expression of the three NTH genes was observed in the shoot apical meristem (SAM). In the vegetative SAM, NTH1 and NTH15 showed overlapping expression in the corpus, NTH20 was expressed in the peripheral zone, and NTH9 was predominantly expressed in the rib zone. The expression patterns of the different NTH genes correspond to regions predicted by the cytohistological zonation model, suggesting that each NTH gene specifies the function of the SAM zone with which it is associated.     

Over-expression of a tobacco homeobox gene, NTH15 , decreases the expression of a gibberellin biosynthetic gene encoding GA 20-oxidase

Ectopic expression of the homeobox gene, NTH15 ( Nicotiana tabacum homeobox 15) in transgenic tobacco leads to abnormal leaf and flower morphology, accompanied by a decrease in the content of the active gibberellin, GA₁. Quantitative analysis of intermediates in the GA biosynthetic pathway revealed that the step from GA₁₉ to GA₂₀ was blocked in transgenic tobacco plants overexpressing NTH15 . To investigate the relationship between the expression of NTH15 and genes involved in GA biosynthesis, we isolated three cDNA clones from tobacco encoding two types of GA 20-oxidase and a 3β-hydroxylase. RNA gel blot analysis revealed that the expression of one gene ( Ntc12 , encoding GA 20-oxidase), which in wild-type tobacco plants was abundantly expressed in leaves, was strongly suppressed in the transformants. The expression level of Ntc12 decreased with increasing severity of phenotype of transgenic tobacco leaves. The abnormal leaf morphology was largely overcome by treatment with GA₂₀ or GA₁ but not by GA₁₉. These data strongly suggest that overexpression of NTH15 inhibits the expression of Ntc12 , resulting in reduced levels of active GA and abnormal leaf morphology in transgenic tobacco plants. In situ hybridization in wild-type tobacco revealed that expression of Ntc12 occurred mainly in the rib meristem, cells surrounding the procambium and in leaf primordia. Expression was not seen in the tunica, corpus and procambium, tissues in which NTH15 was predominantly expressed. The contrasting expression patterns of these genes may reflect their antagonistic functions in the formation of lateral organs from the shoot apical meristem.     

Characterization of a novel class of plant homeodomain proteins that bind to the C4 phosphoenolpyruvate carboxylase gene of Flaveria trinervia

We are interested in the regulatory mechanisms responsible for the mesophyll-specific expression of C₄ phosphoenolpyruvate carboxylase (PEPCase). A one-hybrid screen resulted in the cloning of four different members of a novel class of plant homeodomain proteins, which are most likely involved in the mesophyll-specific expression of the C₄ PEPCase gene in C₄ species of the genus Flaveria. Inspection of the homeodomains of the four proteins reveals that they share many common features with homeodomains described so far, but there are also significant differences. Interestingly, this class of homeodomain proteins occurs also in Arabidopsis thaliana and other C₃ plants. One-hybrid experiments as well as in vitro DNA binding studies confirmed that these novel homeodomain proteins specifically interact with the proximal region of the C₄ PEPCase gene. The N-terminal domains of the homeodomain proteins contain highly conserved sequence motifs. Two-hybrid experiments show that these motifs are sufficient to confer homo- or heterodimer formation between the proteins. Mutagenesis of conserved cysteine residues within the dimerization domain indicates that these residues are essential for dimer formation. Therefore, we designate this novel class of homeobox proteins ZF-HD, for zinc finger homeodomain protein. Our data suggest that the ZF-HD class of homeodomain proteins may be involved in the establishment of the characteristic expression pattern of the C₄ PEPCase gene.     

Transgenic tobacco over-expressing a homeobox gene shows a developmental interaction between leaf morphogenesis and phyllotaxy.

The tobacco gene, NTH1, encodes a polypeptide of 326 amino acids and is a member of the class1 KN1-type family of homeobox genes. Expression of NTH1 has mainly been observed in vegetative and reproductive shoot apices, not observed in roots or expanded leaves. Over-expression of NTH1 in transgenic plants caused abnormal leaf morphology, consisting of wrinkling and curvature. Interestingly, the direction of leaf curvature tended to be conserved among almost all of the leaves in any given transformant. In transgenic plants exhibiting clockwise or anticlockwise phyllotaxy, leaves curved to the right or left, respectively, when looking from the shoot apex toward the base. Micro-surgical experiments demonstrated that the presence of the shoot apex is necessary for the development of leaf curvature, indicating that the order of formation of leaves on the stem (the generative spiral) affects leaf development. We found a correlation between the severity of leaf curvature and the value of the plastochron ratio, a parameter of phyllotaxy. Transformants with more severe phenotypes had larger plastochron ratios. From these findings, we discuss the possibility that an increase in the plastochron ratio, caused by over-expression of NTH1 in the shoot apex, may be involved in leaf curvature.     

Interaction of the BELL-like protein ATH1 with DNA: role of homeodomain residue 54 in specifying the different binding properties of BELL and KNOX proteins

We have studied the interaction of the BELL-like Arabidopsis homeodomain protein ATH1 with DNA. Analysis of oligonucleotides selected by the ATH1 homeodomain from a random mixture suggests that ATH1 preferentially binds the sequence TGACAGGT. Single nucleotide replacements at positions 2 or 3 of this sequence abolish binding, while changes at position 4 are more tolerated. Changes outside this core differentially affect binding, depending on the position. Hydroxyl radical footprinting and missing nucleoside experiments showed that ATH1 interacts with a 7-bp region of the strand carrying the GAC core. On the other strand, protection was observed over a 7-bp region, comprising one additional nucleotide complementary to T in position 1. A comparative analysis of the binding preferences of the homeodomains of ATH1 and STM (a KNOX homeodomain protein) indicated that they bind similar sequences, but with differences in affinity and specificity. The decreased affinity displayed by the ATH1 homeodomain correlates with the presence of valine (instead of lysine as in STM) at position 54. This difference also explains the decreased and increased selectivities, respectively, at positions 4 and 5. Our results point to an essential role of residue 54 in determining the different binding properties of BELL and KNOX homeodomains.