Mechanisms that control knox gene expression in the Arabidopsis shoot

Knotted1-like homeobox (knox) genes are expressed in specific patterns within shoot meristems and play an important role in meristem maintenance. Misexpression of the knox genes, KNAT1 or KNAT2, in Arabidopsis produces a variety of phenotypes, including lobed leaves and ectopic stipules and meristems in the sinus, the region between lobes. We sought to determine the mechanisms that control knox gene expression in the shoot by examining recessive mutants that share phenotypic characteristics with 35S::KNAT1 plants. Double mutants of serrate (se) with either asymmetric1 (as1) or asymmetric2 (as2) showed lobed leaves, ectopic stipules in the sinuses and defects in the timely elongation of sepals, petals and stamens, similar to 35S::KNAT1 plants. Ectopic stipules and in rare cases, ectopic meristems, were detected in the sinuses on plants that were mutant for pickle and either as1 or as2. KNAT1 and KNAT2 were misexpressed in the leaves and flowers of single as1 and as2 mutants and in the sinuses of leaves of the different double mutants, but not in se or pickle single mutants. These results suggest that AS1 and AS2 promote leaf differentiation through repression of knox expression in leaves, and that SE and PKL globally restrict the competence to respond to genes that promote morphogenesis.     

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.     

Three knotted1-like homeobox genes in Arabidopsis

Five arabidopsis kn1-like homeobox genes were cloned through low-stringency screening of Arabidopsis cDNA libraries with the kn1 homeobox from maize. These five genes were named KNAT1-5 (for kn1-like Arabidopsis thaliana). An analysis of KNAT1 and 2 has been presented previously [19]. Here we present an analysis of the genes KNAT3, 4 and 5. On the basis of sequence and expression patterns, these three genes belong to the class II subfamily of kn1-like homeobox genes [16]. Low-stringency Southern analysis suggests several additional members of the class II genes exist in the Arabidopsis genome. The predicted amino acid sequences of the three genes share extensive homology outside of the homeodomain, including 84% between KNAT3 and KNAT4. Northern analysis shows that although all three genes are expressed in all tissues examined, the level of KNAT3 RNA is highest in young siliques, inflorescences and roots, KNAT4 RNA level is strongest in leaves and young siliques, and KNAT5 RNA level is highest in roots. The specificity of these patterns was confirmed by RNA fingerprint analysis. KNAT3 and 4 are light-regulated as they show reduced expression in etiolated seedlings and also in hy3, cop1 and det1 mutant backgrounds.     

ATH1 and KNAT2 proteins act together in regulation of plant inflorescence architecture

The inflorescence of flowering plants is a highly organized structure, not only contributing to plant reproductive processes, but also constituting an important part of the entire plant morphology. Previous studies have revealed that the class-I KNOTTED1-like homeobox (KNOX) genes BREVIPEDICELLUS (BP or KNAT1), KNAT2, and KNAT6 play essential roles in inflorescence architecture. Pedicel morphology is known to contribute greatly to inflorescence architecture, and BP negatively regulates KNAT2 and KNAT6 to ensure that pedicels have a normal upward-pointing orientation. These findings indicate that a genetic network exists in controlling pedicel orientation, but how this network functions in the developmental process remains elusive. Here it is reported that the ARABIDOPSIS THALIANA HOMEOBOX GENE1 (ATH1) gene, which belongs to the BELL1-like homeodomain gene family, is a new member participating in regulating pedicel orientation in the class-I KNOX network. In a genetic screening for suppressors of isoginchaku-2D, a gain-of-function ASYMMETRIC LEAVES2 mutant that displays downward-pointing pedicels, a suppressor mutant was obtained. Characterization of this mutant revealed that the mutation corresponds to ATH1. Genetic analysis indicated that ATH1 acts mainly in the KNAT2 pathway. Yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated that ATH1 physically interacts with KNAT2. The data indicate that the ATH1-KNAT2 complex acts redundantly with KNAT6, both of which are negatively regulated by BP during pedicel development.     

The KNAT2 homeodomain protein interacts with ethylene and cytokinin signaling

Using a transgenic line that overexpresses a fusion of the KNAT2 (KNOTTED-like Arabidopsis) homeodomain protein and the hormone-binding domain of the glucocorticoid receptor (GR), we have investigated the possible relations between KNAT2 and various hormones. Upon activation of the KNAT2-GR fusion, we observed a delayed senescence of the leaves and a higher rate of shoot initiation, two processes that are also induced by cytokinins and inhibited by ethylene. Furthermore, the activation of the KNAT2-GR fusion induced lobing of the leaves. This feature was partially suppressed by treatment with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid, or by the constitutive ethylene response ctr1 mutation. Conversely, some phenotypic traits of the ctr1 mutant were suppressed by the activation of the KNAT2-GR fusion. These data suggest that KNAT2 acts synergistically with cytokinins and antagonistically with ethylene. In the shoot apical meristem, the KNAT2 gene is expressed in the L3 layer and the rib zone. 1-Aminocyclopropane-1-carboxylic acid treatment restricted the KNAT2 expression domain in the shoot apical meristem and reduced the number of cells in the L3. The latter effect was suppressed by the activation of the KNAT2-GR construct. Conversely, the KNAT2 gene expression domain was enlarged in the ethylene-resistant etr1-1 mutant or in response to cytokinin treatment. These data suggest that ethylene and cytokinins act antagonistically in the meristem via KNAT2 to regulate the meristem activity.     

KNAT1 induces lobed leaves with ectopic meristems when overexpressed in Arabidopsis.

Plant development depends on the activity of apical meristems, which are groups of indeterminate cells whose derivatives elaborate the organs of the mature plant. Studies of knotted1 (kn1) and related gene family members have determined potential roles for homeobox genes in the function of shoot meristems. The Arabidopsis kn1-like gene, KNAT1, is expressed in the shoot apical meristem and not in determinate organs. Here, we show that ectopic expression of KNAT1 in Arabidopsis transforms simple leaves into lobed leaves. The lobes initiate in the position of serrations yet have features of leaves, such as stipules, which form in the sinus, the region at the base of two lobes. Ectopic meristems also arise in the sinus region close to veins. Identity of the meristem, that is, vegetative or floral, depends on whether the meristem develops on a rosette or cauline leaf, respectively. Using in situ hybridization, we analyzed the expression of KNAT1 and another kn1-like homeobox gene, SHOOT MERISTEMLESS, in cauliflower mosaic virus 35S::KNAT1 transformants. KNAT1 expression is strong in vasculature, possibly explaining the proximity of the ectopic meristems to veins. After leaf cells have formed a layered meristem, SHOOT MERISTEMLESS expression begins in only a subset of these cells, demonstrating that KNAT1 is sufficient to induce meristems in the leaf. The shootlike features of the lobed leaves are consistent with the normal domain of KNAT1's expression and further suggest that kn1-related genes may have played a role in the evolution of leaf diversity.     

Investigation of systemic control of plant cell division and differentiation in the model of tumor growth in radish

The study addresses the control of plant cell division and differentiation using the model of tumor-forming lines of radish. Expression of the genes involved in control of the cell cycle (CycD3), maintenance of meristematic cell activity (STM, WUS, and KNAT1), and primary response to cytokinin (ARR) was studied in inbred radish lines characterized by tumor growth at different stages of development. The influence of exogenic cytokinin on the expression of the genes of interest is analyzed. The possible role of the CycD3, KNAT1, STM, WUS, and ARR5 in tumor formation in radish is discussed.     

Interaction of KNAT6 and KNAT2 with BREVIPEDICELLUS and PENNYWISE in Arabidopsis inflorescences

The three amino acid loop extension (TALE) homeodomain superfamily, which comprises the KNOTTED-like and BEL1-like families, plays a critical role in regulating meristem activity. We previously demonstrated a function for KNAT6 (for KNOTTED-like from Arabidopsis thaliana 6) in shoot apical meristem and boundary maintenance during embryogenesis. KNAT2, the gene most closely related to KNAT6, does not play such a role. To investigate the contribution of KNAT6 and KNAT2 to inflorescence development, we examined their interactions with two TALE genes that regulate internode patterning, BREVIPEDICELLUS (BP) and PENNYWISE (PNY). Our data revealed distinct and overlapping interactions of KNAT6 and KNAT2 during inflorescence development. Removal of KNAT6 activity suppressed the pny phenotype and partially rescued the bp phenotype. Removal of KNAT2 activity had an effect only in the absence of both BP and KNAT6 or in the absence of both BP and PNY. Consistent with this, KNAT6 and KNAT2 expression patterns were enlarged in both bp and pny mutants. Thus, the defects seen in pny and bp are attributable mainly to the misexpression of KNAT6 and to a lesser extent of KNAT2. Hence, our data showed that BP and PNY restrict KNAT6 and KNAT2 expression to promote correct inflorescence development. This interaction was also revealed in the carpel.     

Combining enhanced root and shoot growth reveals cross talk between pathways that control plant organ size in Arabidopsis

Functionally distinct Arabidopsis (Arabidopsis thaliana) genes that positively affect root or shoot growth when ectopically expressed were combined to explore the feasibility of enhanced biomass production. Enhanced root growth resulting from cytokinin deficiency was obtained by overexpressing CYTOKININ OXIDASE/DEHYDROGENASE3 (CKX3) under the control of the root-specific PYK10 promoter. Plants harboring the PYK10-CKX3 construct were crossed with four different transgenic lines showing enhanced leaf growth. For all combinations, the phenotypic traits of the individual lines could be combined, resulting in an overall growth increase. Unexpectedly, three out of four combinations had more than additive effects. Both leaf and root growth were synergistically enhanced in plants ectopically expressing CKX3 and BRASSINOSTEROID INSENSITIVE1, indicating cross talk between cytokinins and brassinosteroids. In agreement, treatment of PYK10-CKX3 plants with brassinolide resulted in a dramatic increase in lateral root growth that could not be observed in wild-type plants. Coexpression of CKX3 and the GROWTH-REGULATING FACTOR5 (GRF5) antagonized the effects of GRF5 overexpression, revealing an interplay between cytokinins and GRF5 during leaf cell proliferation. The combined overexpression of CKX3 and GIBBERELLIN 20-OXIDASE1 led to a synergistic increase in leaf growth, suggesting an antagonistic growth control by cytokinins and gibberellins. Only additive effects on root and shoot growth were visible in plants ectopically expressing both CKX3 and ARABIDOPSIS VACUOLAR PYROPHOSPHATASE1, hinting at an independent action mode. Our results show new interactions and contribute to the molecular and physiological understanding of biomass production at the whole plant level.     

Cell-fate switch of synergid to egg cell in Arabidopsis eostre mutant embryo sacs arises from misexpression of the BEL1-like homeodomain gene BLH1

In Arabidopsis thaliana, the female gametophyte is a highly polarized structure consisting of four cell types: one egg cell and two synergids, one central cell, and three antipodal cells. In this report, we describe the characterization of a novel female gametophyte mutant, eostre, which affects establishment of cell fates in the mature embryo sac. The eostre phenotype is caused by misexpression of the homeodomain gene BEL1-like homeodomain 1 (BLH1) in the embryo sac. It is known that BELL-KNAT proteins function as heterodimers whose activities are regulated by the Arabidopsis ovate family proteins (OFPs). We show that the phenotypic effect of BLH1 overexpression is dependent upon the class II knox gene KNAT3, suggesting that KNAT3 must be expressed and functional during megagametogenesis. Moreover, disruption of At OFP5, a known interactor of KNAT3 and BLH1, partially phenocopies the eostre mutation. Our study indicates that suppression of ectopic activity of BELL-KNOX TALE complexes, which might be mediated by At OFP5, is essential for normal development and cell specification in the Arabidopsis embryo sac. As eostre-1 embryo sacs also show nuclear migration abnormalities, this study suggests that a positional mechanism might be directing establishment of cell fates in early megagametophyte development.     

Hormonal control of cell proliferation requires PASTICCINO genes

PASTICCINO (PAS) genes are required for coordinated cell division and differentiation during plant development. In loss-of-function pas mutants, plant aerial tissues showed ectopic cell division that was specifically enhanced by cytokinins, leading to disorganized tumor-like tissue. To determine the role of the PAS genes in controlling cell proliferation, we first analyzed the expression profiles of several genes involved in cell division and meristem function. Differentiated and meristematic cells of the pas mutants were more competent for cell division as illustrated by the ectopic and enlarged expression profiles of CYCLIN-DEPENDENT KINASE A and CYCLIN B1. The expression of meristematic homeobox genes KNOTTED-LIKE IN ARABIDOPSIS (KNAT2, KNAT6), and SHOOT MERISTEMLESS was also increased in pas mutants. Moreover, the loss of meristem function caused by shoot meristemless mutation can be suppressed by pas2. The KNAT2 expression pattern defines an enlarged meristematic zone in pas mutants that can be mimicked in wild type by cytokinin treatment. Cytokinin induction of the primary cytokinin response markers, ARABIDOPSIS RESPONSE REGULATOR (ARR5 and ARR6), was enhanced and lasted longer in pas mutants, suggesting that PAS genes in wild type repress cytokinin responses. The expression of the cytokinin-regulated cyclin D, cyclin D3.1, was nonetheless not modified in pas mutants. However, primary auxin response genes were down-regulated in pas mutants, as shown by a lower auxin induction of IAA4 and IAA1 genes, demonstrating that the auxin response was also modified. Altogether, our results suggest that PAS genes are involved in the hormonal control of cell division and differentiation.     

Characterization of the KNOX class homeobox genes Oskn2 and Oskn3 identified in a collection of cDNA libraries covering the early stages of rice embryogenesis

For identification of genes involved in embryogenesis in the model cereal rice, we have constructed a collection of cDNA libraries of well-defined stages of embryo development before, during and after organ differentiation. Here, we focus on the possible role of KNOX (maize Knotted1-like) class homeobox genes in regulation of rice embryogenesis. Three types of KNOX clones were identified in libraries of early zygotic embryos. Two of these, Oskn2 and Oskn3, encode newly described KNOX genes, whereas the third (Oskn1) corresponds to the previously described OSH1 gene. In situ hybridizations showed that during the early stages of embryo development, all three KNOX genes are expressed in the region where the shoot apical meristem (SAM) is organizing, suggesting that these genes are involved in regulating SAM formation. Whereas OSH1 was previously proposed to function also in SAM maintenance, Oskn3 may be involved in patterning organ positions, as its expression was found to mark the boundaries of different embryonic organs following SAM formation. The expression pattern of Oskn2 suggested an additional role in scutellum and epiblast development. Transgenic expression of Oskn2 and Oskn3 in tobacco further supported their involvement in cell fate determination, like previously reported for Knotted1 and OSH1 ectopic expression. Whereas Oskn3 transformants showed the most pronounced phenotypic effects during vegetative development, Oskn2 transformants showed relatively mild alterations in the vegetative phase but a more severly affected flower morphology. The observation that the KNOX genes produce similar though distinct phenotypic reponses in tobacco, indicates that their gene products act on overlapping but different sets of target genes, or that cell-type specific factors determine their precise action.     

Investigation of systemic control of plant cell division and differentiation in the model of tumor growth in radish

The study addresses the control of plant cell division and differentiation using the model of tumor-forming lines of radish. Expression of the genes involved in control of the cell cycle (CycD3), maintenance of meristematic cell activity (STM, WUS, and KNAT1), and primary response to cytokinin (ARR) was studied in inbred radish lines characterized by tumor growth at different stages of development. The influence of exogenic cytokinin on the expression of the genes of interest is analyzed. The possible role of the CycD3, KNAT1, tSTM, WUS, and ARR5 in tumor formation in radish is discussed.