Arabidopsis inflorescence architecture requires the activities of KNOX-BELL homeodomain heterodimers

In flowering plants, post-embryonic development is mediated by the activity of shoot and root apical meristems. Shoot architecture results from activity of the shoot apical meristem (SAM), which initiates primordia, including leaves, internodes and axillary meristems, repetitively from its flanks. Axillary meristems can develop into secondary shoots or flowers. In Arabidopsis, two paralogous BEL1-like (BELL) homeobox genes, PENNYWISE (PNY) and POUND-FOOLISH (PNF), expressed in the SAM, encode DNA-binding proteins that are essential for specifying floral primordia and establishing early internode patterning events during inflorescence development. Biochemical studies show that PNY associates with the knotted1-like homeobox (KNOX) proteins, SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS (BP). PNY-BP heterodimers are essential for establishing early internode patterning events, while PNY-STM heterodimers are critical for SAM function. In this report, we examined the role of PNY, PNF and STM during development. First, we show that PNF interacts with STM and BP indicating that PNY and PNF are redundant functioning proteins. Inflorescence development, but not vegetative development, is sensitive to the dosage levels of PNY, PNF and STM. Characterization of stm-10, a weak allele in the Columbia ecotype, indicates that STM is also involved in floral specification and internode development. Our examination of the genetic requirements for PNY, PNF and STM demonstrates that these KNOX–BELL heterodimers control floral specification, internode patterning and the maintenance of boundaries between initiating floral primordia and the inflorescence meristem.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

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.     

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.     

Expression of HtKNOT1, a class I KNOX gene, overlaps cell layers and development compartments of differentiating cells in stems and flowers of Helianthus tuberosus

In plant, post-embryonic development relies on the activities of indeterminate cell populations termed meristems, spatially clustered cell lineages, wherein a subset divides indeterminately. For correct growth, the plant must maintain a constant flow of cells through the meristem, where the input of dividing pluripotent cells offsets the output of differentiating cells. KNOTTED1-like homeobox (KNOX) genes are expressed in specific patterns in the plant meristems and play important roles in maintaining meristematic cell identity. We have analyzed the expression pattern of HtKNOT1, a class I KNOX gene of Helianthus tuberosus, in stems, inflorescence meristems, floral meristems and floral organs. HtKNOT1 is expressed in cambial cells, phloem cells and xylematic parenchyma within apical stem internodes, while in basal internodes HtKNOT1 expression was restricted to the presumptive initials and recently derived phloem cells. In the reproductive phase, HtKNOT1 mRNAs were detected in both the inflorescence and floral meristems as well within lateral organ primordia (i.e. floral bracts, petals, stamens and carpels). In more differentiated flowers, the expression of HtKNOT1 was restricted to developing ovules and pollen mother cells. HtKNOT1 may play a dual role being required to maintain the meristem initials as well as initiating differentiation and/or conferring new cell identity. In particular, it is possible that HtKNOT1 cooperates at floral level with additional factors that more specifically control floral organs and pollen development in H. tuberosus.     

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.     

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.     

Genetic and morphological characterization of the barley <Emphasis Type="Italic">uniculm2</Emphasis> (<Emphasis Type="Italic">cul2</Emphasis>) mutant

Axillary meristem growth and development help define plant architecture in barley (Hordeum vulgare L). Plants carrying the recessive uniculm2 (cul2) mutation initiate vegetative axillary meristem development but fail to develop tillers. In addition, inflorescence axillary meristems develop into spikelets, but the spikelets at the distal end of the inflorescence have an altered phyllotaxy and are sometimes absent. Double mutant combinations of cul2 and nine other recessive mutations that exhibit low to high tiller number phenotypes resulted in a uniculm vegetative phenotype. One exception was the occasional multiple shoots produced in combination with granum-a; a high tillering mutant that occasionally produces two shoot apical meristems. These results show that the CUL2 gene product plays a role in the development of axillary meristems into tillers but does not regulate the development of vegetative apical meristems. Moreover, novel double-mutant inflorescence phenotypes were observed with cul2 in combination with the other mutants. These data show that the wild-type CUL2 gene product is involved in controlling proper inflorescence development and that it functions in combination with some of the other genes that affect branching. Our genetic analysis indicates that there are genetically separate but not distinct regulatory controls on vegetative and inflorescence axillary development. Finally, to facilitate future positionally cloning of cul2, we positioned cul2 on chromosome 6(6H) of the barley RFLP map.     

A novel role of BELL1-like homeobox genes, PENNYWISE and POUND-FOOLISH, in floral patterning

Flowers are determinate shoots comprised of perianth and reproductive organs displayed in a whorled phyllotactic pattern. Floral organ identity genes display region-specific expression patterns in the developing flower. In Arabidopsis, floral organ identity genes are activated by LEAFY (LFY), which functions with region-specific co-regulators, UNUSUAL FLORAL ORGANS (UFO) and WUSCHEL (WUS), to up-regulate homeotic genes in specific whorls of the flower. PENNYWISE (PNY) and POUND-FOOLISH (PNF) are redundant functioning BELL1-like homeodomain proteins that are expressed in shoot and floral meristems. During flower development, PNY functions with a co-repressor complex to down-regulate the homeotic gene, AGAMOUS (AG), in the outer whorls of the flower. However, the function of PNY as well as PNF in regulating floral organ identity in the central whorls of the flower is not known. In this report, we show that combining mutations in PNY and PNF enhance the floral patterning phenotypes of weak and strong alleles of lfy, indicating that these BELL1-like homeodomain proteins play a role in the specification of petals, stamens and carpels during flower development. Expression studies show that PNY and PNF positively regulate the homeotic genes, APETALA3 and AG, in the inner whorls of the flower. Moreover, PNY and PNF function in parallel with LFY, UFO and WUS to regulate homeotic gene expression. Since PNY and PNF interact with the KNOTTED1-like homeodomain proteins, SHOOTMERISTEMLESS (STM) and KNOTTED-LIKE from ARABIDOPSIS THALIANA2 (KNAT2) that regulate floral development, we propose that PNY/PNF-STM and PNY/PNF-KNAT2 complexes function in the inner whorls to regulate flower patterning events.     

Developmental disaster1: A novel mutation causing defects during vegetative and inflorescence development in maize (Zea mays, Poaceae)

Axillary meristems, which give rise to branches and flowers, play a critical role in plant architecture and reproduction. To understand how axillary meristems initiate, we have screened for mutants with defects in axillary meristem initiation to uncover the genes controlling this process. These mutants, called the barren class of mutants in maize (Zea mays), have defects in axillary meristem initiation during both vegetative and reproductive development. Here, we identify and characterize a new member of the barren class of mutants named Developmental disaster1 (Dvd1), due to the pleiotropic effects of the mutation. Similar to the barren mutants, Dvd1 mutants have fewer branches, spikelets, florets, and floral organs in the inflorescence due to defects in the initiation of axillary meristems. Furthermore, double mutant analysis with teosinte branched1 shows that dvd1 also functions in axillary meristems during vegetative development. However, unlike the barren mutants, Dvd1 mutants are semidwarf due to the production of shorter internodes, and they produce leaves in the inflorescence due to the outgrowth of bract leaf primordia. The suite of defects seen in Dvd1 mutants, together with the genetic interaction of Dvd1 with barren inflorescence2, suggests that dvd1 is a novel regulator of axillary meristem and internode development.     

barren inflorescence2 regulates axillary meristem development in the maize inflorescence

Organogenesis in plants is controlled by meristems. Shoot apical meristems form at the apex of the plant and produce leaf primordia on their flanks. Axillary meristems, which form in the axils of leaf primordia, give rise to branches and flowers and therefore play a critical role in plant architecture and reproduction. To understand how axillary meristems are initiated and maintained, we characterized the barren inflorescence2 mutant, which affects axillary meristems in the maize inflorescence. Scanning electron microscopy, histology and RNA in situ hybridization using knotted1 as a marker for meristematic tissue show that barren inflorescence2 mutants make fewer branches owing to a defect in branch meristem initiation. The construction of the double mutant between barren inflorescence2 and tasselsheath reveals that the function of barren inflorescence2 is specific to the formation of branch meristems rather than bract leaf primordia. Normal maize inflorescences sequentially produce three types of axillary meristem: branch meristem, spikelet meristem and floral meristem. Introgression of the barren inflorescence2 mutant into genetic backgrounds in which the phenotype was weaker illustrates additional roles of barren inflorescence2 in these axillary meristems. Branch, spikelet and floral meristems that form in these lines are defective, resulting in the production of fewer floral structures. Because the defects involve the number of organs produced at each stage of development, we conclude that barren inflorescence2 is required for maintenance of all types of axillary meristem in the inflorescence. This defect allows us to infer the sequence of events that takes place during maize inflorescence development. Furthermore, the defect in branch meristem formation provides insight into the role of knotted1 and barren inflorescence2 in axillary meristem initiation.     

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.     

The Arabidopsis BEL1-LIKE HOMEODOMAIN proteins SAW1 and SAW2 act redundantly to regulate KNOX expression spatially in leaf margins

In Arabidopsis thaliana, the BEL1-like TALE homeodomain protein family consists of 13 members that form heterodimeric complexes with the Class 1 KNOX TALE homeodomain proteins, including SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS (BP). The BEL1-like protein BELLRINGER (BLR) functions together with STM and BP in the shoot apex to regulate meristem identity and function and to promote correct shoot architecture. We have characterized two additional BEL1-LIKE HOMEODOMAIN (BLH) proteins, SAWTOOTH1 (BLH2/SAW1) and SAWTOOTH2 (BLH4/SAW2) that, in contrast with BLR, are expressed in lateral organs and negatively regulate BP expression. saw1 and saw2 single mutants have no obvious phenotype, but the saw1 saw2 double mutant has increased leaf serrations and revolute margins, indicating that SAW1 and SAW2 act redundantly to limit leaf margin growth. Consistent with this hypothesis, overexpression of SAW1 suppresses overall growth of the plant shoot. BP is ectopically expressed in the leaf serrations of saw1 saw2 double mutants. Ectopic expression of Class 1 KNOX genes in leaves has been observed previously in loss-of-function mutants of ASYMMETRIC LEAVES (AS1). Overexpression of SAW1 in an as1 mutant suppresses the as1 leaf phenotype and reduces ectopic BP leaf expression. Taken together, our data suggest that BLH2/SAW1 and BLH4/SAW2 establish leaf shape by repressing growth in specific subdomains of the leaf at least in part by repressing expression of one or more of the KNOX genes.     

Herbivory could unlock mutations sequestered in stratified shoot apices of genetic mosaics

Many higher plants have shoot apical meristems that possess discrete cell layers, only one of which normally gives rise to gametes following the transition from vegetative meristem to floral meristem. Consequently, when mutations occur in the meristems of sexually reproducing plants, they may or may not have an evolutionary impact, depending on the apical layer in which they reside. In order to determine whether developmentally sequestered mutations could be released by herbivory (i.e., meristem destruction), a characterized genetic mosaic was subjected to simulated herbivory. Many plants develop two shoot meristems in the leaf axils of some nodes, here referred to as the primary and secondary axillary meristems. Destruction of the terminal and primary axillary meristems led to the outgrowth of secondary axillary meristems. Seed derived from secondary axillary meristems was not always descended from the second apical cell layer of the terminal shoot meristem as is expected for terminal and primary shoot meristems. Vegetative and reproductive analysis indicated that secondary meristems did not maintain the same order of cell layers present in the terminal shoot meristem. In secondary meristems reproductively sequestered cell layers possessing mutant cells can be repositioned into gamete-forming cell layers, thereby adding mutant genes into the gene pool. Herbivores feeding on shoot tips may influence plant evolution by causing the outgrowth of secondary axillary meristems.     

barren inflorescence2 Encodes a co-ortholog of the PINOID serine/threonine kinase and is required for organogenesis during inflorescence and vegetative development in maize

Organogenesis in plants is controlled by meristems. Axillary meristems, which give rise to branches and flowers, play a critical role in plant architecture and reproduction. Maize (Zea mays) and rice (Oryza sativa) have additional types of axillary meristems in the inflorescence compared to Arabidopsis (Arabidopsis thaliana) and thus provide an excellent model system to study axillary meristem initiation. Previously, we characterized the barren inflorescence2 (bif2) mutant in maize and showed that bif2 plays a key role in axillary meristem and lateral primordia initiation in the inflorescence. In this article, we cloned bif2 by transposon tagging. Isolation of bif2-like genes from seven other grasses, along with phylogenetic analysis, showed that bif2 is a co-ortholog of PINOID (PID), which regulates auxin transport in Arabidopsis. Expression analysis showed that bif2 is expressed in all axillary meristems and lateral primordia during inflorescence and vegetative development in maize and rice. Further phenotypic analysis of bif2 mutants in maize illustrates additional roles of bif2 during vegetative development. We propose that bif2/PID sequence and expression are conserved between grasses and Arabidopsis, attesting to the important role they play in development. We provide further support that bif2, and by analogy PID, is required for initiation of both axillary meristems and lateral primordia.     

The filifolium1 mutation perturbs shoot architecture in Zea mays (Poaceae)

Plant architecture is elaborated through the activity of shoot apical meristems (SAMs), which produce repeating units known as phytomers, that are comprised of leaf, node, internode, and axillary bud. Insight into how SAMs function and how individual phytomer components are related to each other can been obtained through characterization of recessive mutants with perturbed shoot development. In this study, we characterized a new mutant to further understand mechanisms underlying shoot development in maize. The filifolium1-0 (ffm1-0) mutants develop narrow leaves on dwarfed shoots. Shoot growth often terminates at the seedling stage from depletion of the SAM, but if plants survive to maturity they are invariably bushy. KN1-like homeobox (KNOX) proteins are inappropriately regulated in mutant apices, adaxial identity is not specified in mutant leaves, and axillary meristems develop precociously. We propose that FFM1 acts to demarcate zones within the SAM so that appropriate fates can be conferred on cells within those zones by other factors. On the basis of the mutant phenotype, we also speculate about different relationships between phytomer components in maize and Arabidopsis.     

Arabidopsis JAGGED LATERAL ORGANS Acts with ASYMMETRIC LEAVES2 to Coordinate KNOX and PIN Expression in Shoot and Root Meristems

Organ initiation requires the specification of a group of founder cells at the flanks of the shoot apical meristem and the creation of a functional boundary that separates the incipient primordia from the remainder of the meristem. Organ development is closely linked to the downregulation of class I KNOTTED1 LIKE HOMEOBOX (KNOX) genes and accumulation of auxin at sites of primordia initiation. Here, we show that Arabidopsis thaliana JAGGED LATERAL ORGANS (JLO), a member of the LATERAL ORGAN BOUNDARY DOMAIN (LBD) gene family, is required for coordinated organ development in shoot and floral meristems. Loss of JLO function results in ectopic expression of the KNOX genes SHOOT MERISTEMLESS and BREVIPEDICELLUS (BP), indicating that JLO acts to restrict KNOX expression. JLO acts in a trimeric protein complex with ASYMMETRIC LEAVES2 (AS2), another LBD protein, and AS1 to suppress BP expression in lateral organs. In addition to its role in KNOX regulation, we identified a role for AS2 in regulating PINFORMED (PIN) expression and auxin transport from embryogenesis onwards together with JLO. We propose that different JLO and AS2 protein complexes, possibly also comprising other LBD proteins, coordinate auxin distribution and meristem function through the regulation of KNOX and PIN expression during Arabidopsis 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.