The interaction of two homeobox genes,BREVIPEDICELLUS and PENNYWISE,regulates internode patterning in the Arabidopsis inflorescence

Plant architecture results from the activity of the shoot apical meristem, which initiates leaves, internodes, and axillary meristems. KNOTTED1-like homeobox (KNOX) genes are expressed in specific patterns in the shoot apical meristem and play important roles in plant architecture. KNOX proteins interact with BEL1-like (BELL) homeodomain proteins and together bind a target sequence with high affinity. We have obtained a mutation in one of the Arabidopsis BELL genes, PENNYWISE (PNY), that appears phenotypically similar to the KNOX mutant brevipedicellus (bp). Both bp and pny have randomly shorter internodes and display a slight increase in the number of axillary branches. The double mutant shows a synergistic phenotype of extremely short internodes interspersed with long internodes and increased branching. PNY is expressed in inflorescence and floral meristems and overlaps with BP in a discrete domain of the inflorescence meristem where we propose the internode is patterned. The physical association of the PNY and BP proteins suggests that they participate in a complex that regulates early patterning events in the inflorescence meristem.     

Regulatory networks that function to specify flower meristems require the function of homeobox genes PENNYWISE and POUND-FOOLISH in Arabidopsis

Flowering is a major developmental phase change that transforms the fate of the shoot apical meristem (SAM) from a leaf-bearing vegetative meristem to that of a flower-producing inflorescence meristem. In Arabidopsis, floral meristems are specified on the periphery of the inflorescence meristem by the combined activities of the FLOWERING LOCUS T (FT)–FD complex and the flower meristem identity gene, LEAFY ( LFY ). Two redundant functioning homeobox genes, PENNYWISE ( PNY ) and POUND-FOOLISH ( PNF ), which are expressed in the vegetative and inflorescence SAM, regulate patterning events during reproductive development, including floral specification. To determine the role of PNY and PNF in the floral specification network, we characterized the genetic relationship of these homeobox genes with LFY and FT . Results from this study demonstrate that LFY functions downstream of PNY and PNF. Ectopic expression of LFY promotes flower formation in pny pnf plants, while the flower specification activity of ectopic FT is severely attenuated. Genetic analysis shows that when mutations in pny and pnf genes are combined with lfy , a synergistic phenotype is displayed that significantly reduces floral specification and alters inflorescence patterning events. In conclusion, results from this study support a model in which PNY and PNF promote LFY expression during reproductive development. At the same time, the flower formation activity of FT is dependent upon the function of PNY and PNF.     

Requirement of KNAT1/BP for the Development of Abscission Zones in Arabidopsis thaliana

The KNAT1 gene is a member of the Class I KNOXhomeobox gene family and is thought to play an important role in meristem development and leaf morphogenesis. Recent studies have demonstrated that KNAT1/BP regulates the architecture of the inflorescence by affecting pedicle development in Arabidopsis thaliana. Herein, we report the characterization of an Arabidopsis T-DNA insertion mutant that shares considerable phenotypic similarity to the previously identified mutant brevipedicle （bp）. Molecular and genetic analyses showed that the mutant is allelic to bp and that the T-DNA is located within the first helix of the KNAT1 homeodomain （HD）. Although the mutation causes a typical abnormality of short pedicles, propendent siliques, and semidwarfism, no obvious defects are observed in the vegetative stage. A study on cell morphology showed that asymmetrical division and inhibition of cell elongation contribute to the downward-pointing and shorter pedicle phenotype. Loss of KNAT/BPfunction results in the abnormal development of abscission zones. Mlcroarray analysis of gene expression profiling suggests that KNAT1/BP may regulate abscission zone development through hormone signaling and hormone metabolism in Arabidopsis.     

KNAT6: an Arabidopsis homeobox gene involved in meristem activity and organ separation

The homeobox gene family plays a crucial role during the development of multicellular organisms. The KNOTTED-like genes from Arabidopsis thaliana (KNAT6 and KNAT2) are close relatives of the meristematic genes SHOOT MERISTEMLESS (STM) and BREVIPEDICELLUS, but their function is not currently known. To investigate their role, we identified null alleles of KNAT6 and KNAT2. We demonstrate that KNAT6 contributes redundantly with STM to the maintenance of the shoot apical meristem (SAM) and organ separation. Consistent with this role, the expression domain of KNAT6 in the SAM marks the boundaries between the SAM and cotyledons. The lack of meristematic activity in the knat6 stm-2 double mutant and the fusion of cotyledons were linked to the modulation of CUP-SHAPED COTYLEDON (CUC) activity. During embryogenesis, KNAT6 is expressed later than STM and CUC. In agreement with this fact, CUC1 and CUC2 were redundantly required for KNAT6 expression. These data provide the basis for a model in which KNAT6 contributes to SAM maintenance and boundary establishment in the embryo via the STM/CUC pathway. KNAT2, although the closest related member of the family to KNAT6, did not have such a function.     

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.     

KNAT2: evidence for a link between knotted-like genes and carpel development

The KNAT2 (for KNOTTED-like from Arabidopsis thaliana 2) homeobox gene is expressed in the vegetative apical meristem. It is also active during flower development, suggesting a function in the structuring of flowers. To investigate its role, we used a DEXAMETHASONE (DEX)-inducible system to generate transgenic plants that overexpressed a fusion of KNAT2 with the hormone binding domain of the glucocorticoid receptor. DEX-induced plants were similar to plants overexpressing the closely related KNAT1 gene, indicating overlapping functions, although we observed differences as well. In particular, KNAT2-GR activation induced ectopic carpel features. First, KNAT2 induced the homeotic conversion of nucellus into carpel-like structures. Second, KNAT2 induced stigmatic papillae on rosette leaves in the ap2-5 background. Third, ectopic expression of the carpel identity gene AGAMOUS (AG) was observed in carpels and ovules. Interestingly, the homeotic conversion was not dependent on AG activity, because it was maintained in the ag-1 ap2-5 double mutant. Therefore, our data indicate that KNAT2 also must activate other carpel regulators. Together, these results suggest that KNAT2 plays a role in carpel development.     

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.     

Gene TAENIATA is a novel negative regulator of the Arabidopsis thaliana homeobox genes KNAT1, KNAT2, KNAT6, and STM

Mutant Arabidopsis thaliana taeniata (tae) plants are characterized by an altered morphology of leaves and the inflorescence. At the beginning of flowering, the inflorescence produces fertile flowers morphologically intermediate between a shoot and a flower. The recessive mutation tae also causes the formation of ectopic meristems and shoot rosettes on leaves. The expressivity of the mutant characters depend on the temperature and photoperiod. Analysis of the activity of KNOX class I genes in the leaves of the tae mutant has demonstrated the expression of genes KNAT2 and STM and an increase in the expression of genes KNAT1 and KNAT6 compared to wild-type leaves. These data indicate that the TAE gene negatively regulates the KNAT1, KNAT2, KNAT6, and STM genes.     

Shoot apical meristem function in Arabidopsis requires the combined activities of three BEL1-like homeodomain proteins

In plants, most of the above-ground body is formed post-embryonically by the continuous organogenic potential of the shoot apical meristem (SAM). Proper function of the SAM requires maintenance of a delicate balance between the depletion of stem cell daughters into developing primordia and proliferation of the central stem cell population. Here we show that initiation and maintenance of the Arabidopsis SAM, including that of floral meristems, requires the combinatorial action of three members of the BELL-family of TALE homeodomain proteins, ARABIDOPSIS THALIANA HOMEOBOX 1 (ATH1), PENNYWISE (PNY) and POUND-FOOLISH (PNF). All three proteins interact with the KNOX TALE homeodomain protein STM, and combined lesions in ATH1 , PNY and PNF result in a phenocopy of stm mutations. Therefore, we propose that ath1 pny pnf meristem defects result from loss of combinatorial BELL-STM control. Further, we demonstrate that heterodimerization-controlled cellular localization of BELL and KNOX proteins involves a CRM1/exportin-1-mediated nuclear exclusion mechanism that is probably generic to control the activity of BELL and KNOX combinations. We conclude that in animals and plants corresponding mechanisms regulate the activity of TALE homeodomain proteins through controlled nuclear-cytosolic distribution of these proteins.     

The role of PENNYWISE and POUND-FOOLISH in the maintenance of the shoot apical meristem in Arabidopsis

Growth of the aerial part of the plant is dependent upon the maintenance of the shoot apical meristem (SAM). A balance between the self-renewing stem cells in the central zone (CZ) and organogenesis in the peripheral zone (PZ) is essential for the integrity, function, and maintenance of the SAM. Understanding how the SAM maintains a balance between stem cell perpetuation and organogenesis is a central question in plant biology. Two related BELL1-like homeodomain proteins, PENNYWISE (PNY) and POUND-FOOLISH (PNF), act to specify floral meristems during reproductive development. However, genetic studies also show that PNY and PNF regulate the maintenance of the SAM. To understand the role of PNY and PNF in meristem maintenance, the expression patterns for genes that specifically localize to the peripheral and central regions of the SAM were examined in Arabidopsis (Arabidopsis thaliana). Results from these experiments indicate that the integrity of the CZ is impaired in pny pnf plants, which alters the balance of stem cell renewal and organogenesis. As a result, pools of CZ cells may be allocated into initiating leaf primordia. Consistent with these results, the integrity of the central region of pny pnf SAMs can be partially restored by increasing the size of the CZ. Interestingly, flower specification is also reestablished by augmenting the size of the SAM in pny pnf plants. Taken together, we propose that PNY and PNF act to restrict organogenesis to the PZ by maintaining a boundary between the CZ and PZ.     

Specification of reproductive meristems requires the combined function of SHOOT MERISTEMLESS and floral integrators FLOWERING LOCUS T and FD during Arabidopsis inflorescence development

In Arabidopsis floral meristems are specified on the periphery of the inflorescence meristem by the combined activities of the FLOWERING LOCUS T (FT)-FD complex and the flower meristem identity gene LEAFY. The floral specification activity of FT is dependent upon two related BELL1-like homeobox (BLH) genes PENNYWISE (PNY) and POUND-FOOLISH (PNF) which are required for floral evocation. PNY and PNF interact with a subset of KNOTTED1-LIKE homeobox proteins including SHOOT MERISTEMLESS (STM). Genetic analyses show that these BLH proteins function with STM to specify flowers and internodes during inflorescence development. In this study, experimental evidence demonstrates that the specification of flower and coflorescence meristems requires the combined activities of FT-FD and STM. FT and FD also regulate meristem maintenance during inflorescence development. In plants with reduced STM function, ectopic FT and FD promote the formation of axillary meristems during inflorescence development. Lastly, gene expression studies indicate that STM functions with FT-FD and AGAMOUS-LIKE 24 (AGL24)-SUPPRESSOR OF OVEREXPRESSION OF CONTANS1 (SOC1) complexes to up-regulate flower meristem identity genes during inflorescence development.