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.     

Common regulatory networks in leaf and fruit patterning revealed by mutations in the Arabidopsis ASYMMETRIC LEAVES1 gene

Carpels and leaves are evolutionarily related organs, as the former are thought to be modified leaves. Therefore, developmental pathways that play crucial roles in patterning both organs are presumably conserved. In leaf primordia of Arabidopsis thaliana, the ASYMMETRIC LEAVES1 (AS1) gene interacts with AS2 to repress the class I KNOTTED1-like homeobox (KNOX) genes BREVIPEDICELLUS (BP), KNAT2 and KNAT6, restricting the expression of these genes to the meristem. In this report, we describe how AS1, presumably in collaboration with AS2, patterns the Arabidopsis gynoecium by repressing BP, which is expressed in the replum and valve margin, interacts in the replum with REPLUMLESS (RPL), an essential gene for replum development, and positively regulates the expression of this gene. Misexpression of BP in the gynoecium causes an increase in replum size, while the valve width is slightly reduced, and enhances the effect of mutations in FRUITFULL (FUL), a gene with an important function in valve development. Altogether, these findings strongly suggest that BP plays a crucial role in replum development. We propose a model for pattern formation along the mediolateral axis of the ovary, whereby three domains (replum, valve margin and valve) are specified by the opposing gradients of two antagonistic factors, valve factors and replum factors, the class I KNOX genes working as the latter.     

Genetic networks regulated by ASYMMETRIC LEAVES1 (AS1) and AS2 in leaf development in Arabidopsis thaliana: KNOX genes control five morphological events

The asymmetric leaves 1 ( as1 ) and as2 mutants of Arabidopsis thaliana exhibit pleiotropic phenotypes. Expression of a number of genes, including three class-1 KNOTTED -like homeobox ( KNOX ) genes ( BP , KNAT2 and KNAT6 ) and ETTIN / ARF3 , is enhanced in these mutants. In the present study, we attempted to identify the phenotypic features of as1 and as2 mutants that were generated by ectopic expression of KNOX genes, using multiple loss-of-function mutations of KNOX genes as well as as1 and as2 . Our results revealed that the ectopic expression of class-1 KNOX genes resulted in reductions in the sizes of leaves, reductions in the size of sepals and petals, the formation of a less prominent midvein, the repression of adventitious root formation and late flowering. Our results also revealed that the reduction in leaf size and late flowering were caused by the repression, by KNOX genes, of a gibberellin (GA) pathway in as1 and as2 plants. The formation of a less prominent midvein and the repression of adventitious root formation were not, however, related to the GA pathway. The asymmetric formation of leaf lobes, the lower complexity of higher-ordered veins, and the elevated frequency of adventitious shoot formation on leaves of as1 and as2 plants were not rescued by multiple mutations in KNOX genes. These features must, therefore, be controlled by other genes in which expression is enhanced in the as1 and as2 mutants.     

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.     

FIE and CURLY LEAF polycomb proteins interact in the regulation of homeobox gene expression during sporophyte development

The Arabidopsis FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) polycomb group (PcG) protein, a WD40 homologue of Drosophila extra sex comb (ESC), regulates endosperm and embryo development and represses flowering during embryo and seedling development. As fie alleles are not transmitted maternally, homozygous mutant plants cannot be obtained. To study FIE function during the entire plant life cycle, we used Arabidopsis FIE co-suppressed plants. Low FIE level in these plants produced dramatic morphological aberrations, including loss of apical dominance, curled leaves, early flowering and homeotic conversion of leaves, flower organs and ovules into carpel-like structures. These morphological aberrations are similar to those exhibited by plants overexpressing AGAMOUS (AG) or CURLY LEAF (clf) mutants. Furthermore, the aberrant leaf morphology of FIE-silenced and clf plants correlates with de-repression of the class I KNOTTED-like homeobox (KNOX) genes including KNOTTED-like from Arabidopsis thaliana 2 (KNAT2) and SHOOTMERISTEMLESS (STM), whereas BREVIPEDICELLUS (BP) was upregulated in FIE-silenced plants, but not in the clf mutant. Thus, FIE is essential for the control of shoot and leaf development. Yeast two-hybrid and pull-down assays demonstrate that FIE interacts with CLF. Collectively, the morphological characteristics, together with the molecular and biochemical data presented in this work, strongly suggest that in plants, as in mammals and insects, PcG proteins control expression of homeobox genes. Our findings demonstrate that the versatility of the plant FIE function, which is derived from association with different SET (SU (VAR)3-9, E (Z), Trithorax) domain PcG proteins, results in differential regulation of gene expression throughout the plant life cycle.     

Overexpression of a gibberellin inactivation gene alters seed development, KNOX gene expression, and plant development in Arabidopsis

We have examined the role of gibberellins (GAs) in plant development by expression of the pea GA 2-oxidase2 ( PsGA2ox2 ) cDNA, which encodes a GA inactivating enzyme, under the control of the MEDEA (MEA) promoter. Expression of MEA:PsGA2ox2 in Arabidopsis caused seed abortion, demonstrating that active GAs in the endosperm are essential for normal seed development. MEA:PsGA2ox2 plants had reduced ovule number per ovary and exhibited defects in phyllotaxy and leaf morphology which were partly suppressed by GA treatment. The leaf architecture and phyllotaxy defects of MEA:PsGA2ox2 plants were also restored by sly1-d which reduces DELLA protein stability to increase GA response. MEA:PsGA2ox2 seedlings had increased expression of the KNOTTED1 -like homeobox (KNOX) genes, BP , KNAT2 and KNAT6 , which are known to control plant architecture. The expression of KNOX genes is also altered in wild-type plants treated with GA. These results support the conclusion that GAs can suppress the effects of elevated KNOX gene expression, and raise the possibility that localized changes in GA levels caused by PsGA2ox2 alter the expression of KNOX genes to modify plant architecture.     

Regulation of compound leaf development in Medicago truncatula by fused compound leaf1, a class M KNOX gene

Medicago truncatula is a legume species belonging to the inverted repeat lacking clade (IRLC) with trifoliolate compound leaves. However, the regulatory mechanisms underlying development of trifoliolate leaves in legumes remain largely unknown. Here, we report isolation and characterization of fused compound leaf1 (fcl1) mutants of M. truncatula. Phenotypic analysis suggests that FCL1 plays a positive role in boundary separation and proximal-distal axis development of compound leaves. Map-based cloning indicates that FCL1 encodes a class M KNOX protein that harbors the MEINOX domain but lacks the homeodomain. Yeast two-hybrid assays show that FCL1 interacts with a subset of Arabidopsis thaliana BEL1-like proteins with slightly different substrate specificities from the Arabidopsis homolog KNATM-B. Double mutant analyses with M. truncatula single leaflet1 (sgl1) and palmate-like pentafoliata1 (palm1) leaf mutants show that fcl1 is epistatic to palm1 and sgl1 is epistatic to fcl1 in terms of leaf complexity and that SGL1 and FCL1 act additively and are required for petiole development. Previous studies have shown that the canonical KNOX proteins are not involved in compound leaf development in IRLC legumes. The identification of FCL1 supports the role of a truncated KNOX protein in compound leaf development in M. truncatula.     

Ectopic expression of soybean GmKNT1 in Arabidopsis results in altered leaf morphology and flower identity

Plant morphology is specified by leaves and flowers, and the shoot apical meristem (SAM) defines the architecture of plant leaves and flowers. Here, we reported the characterization of a soybean KNOX gene GmKNT1, which was highly homologous to Arabidopsis STM. The GmKNT1 was strongly expressed in roots, flowers and developing seeds. Its expression could be induced by IAA, ABA and JA, but inhibited by GA or cytokinin. Staining of the transgenic plants overexpressing GmKNT1-GUS fusion protein revealed that the GmKNT1 was mainly expressed at lobe region, SAM of young leaves, sepal and carpel, not in seed and mature leaves. Scanning electron micros- copy (SEM) disclosed multiple changes in morphology of the epidermal cells and stigma. The transgenic Arabidopsis plants overexpress- ing the GmKNT1 showed small and lobed leaves, shortened internodes and small clustered inflorescence. The lobed leaves might result from the function of the meristems located at the boundary of the leaf. Compared with wild type plants, transgenic plants had higher ex- pression of the SAM-related genes including the CUP, WUS, CUC1, KNAT2 and KNAT6. These results indicated that the GmKNT1 could affect multiple aspects of plant growth and development by regulation of downstream genes expression.     

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.     

Direct repression of KNOX loci by the ASYMMETRIC LEAVES1 complex of Arabidopsis

KNOTTED1-like homeobox (KNOX) genes promote stem cell activity and must be repressed to form determinate lateral organs. Stable KNOX gene silencing during organogenesis is known to involve the predicted DNA binding proteins ASYMMETRIC LEAVES1 (AS1) and AS2 as well as the chromatin-remodeling factor HIRA. However, the mechanism of silencing is unknown. Here, we show that AS1 and AS2 form a repressor complex that binds directly to the regulatory motifs CWGTTD and KMKTTGAHW present at two sites in the promoters of the KNOX genes BREVIPEDICELLUS (BP) and KNAT2. The two binding sites act nonredundantly, and interaction between AS1-AS2 complexes at these sites is required to repress BP. Promoter deletion analysis further indicates that enhancer elements required for BP expression in the leaf are located between the AS1-AS2 complex binding sites. We propose that AS1-AS2 complexes interact to create a loop in the KNOX promoter and, likely through recruitment of HIRA, form a repressive chromatin state that blocks enhancer activity during organogenesis. Our model for AS1-AS2-mediated KNOX gene silencing is conceptually similar to the action of an insulator. This regulatory mechanism may be conserved in simple leafed species of monocot and dicot lineages and constitutes a potential key determinant in the evolution of compound leaves.     

KNOX homeobox genes potentially have similar function in both diploid unicellular and multicellular meristems, but not in haploid meristems

Members of the class 1 knotted-like homeobox (KNOX) gene family are important regulators of shoot apical meristem development in angiosperms. To determine whether they function similarly in seedless plants, three KNOX genes (two class 1 genes and one class 2 gene) from the fern Ceratopteris richardii were characterized. Expression of both class 1 genes was detected in the shoot apical cell, leaf primordia, marginal part of the leaves, and vascular bundles by in situ hybridization, a pattern that closely resembles that of class 1 KNOX genes in angiosperms with compound leaves. The fern class 2 gene was expressed in all sporophyte tissues examined, which is characteristic of class 2 gene expression in angiosperms. All three CRKNOX genes were not detected in gametophyte tissues by RNA gel blot analysis. Arabidopsis plants overexpressing the fern class 1 genes resembled plants that overexpress seed plant class 1 KNOX genes in leaf morphology. Ectopic expression of the class 2 gene in Arabidopsis did not result in any unusual phenotypes. Taken together with phylogenetic analysis, our results suggest that (a) the class 1 and 2 KNOX genes diverged prior to the divergence of fern and seed plant lineages, (b) the class 1 KNOX genes function similarly in seed plant and fern sporophyte meristem development despite their differences in structure, (c) KNOX gene expression is not required for the development of the fern gametophyte, and (d) the sporophyte and gametophyte meristems of ferns are not regulated by the same developmental mechanisms at the molecular level.     

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 BLADE-ON-PETIOLE 1 gene controls leaf pattern formation through the modulation of meristematic activity in Arabidopsis

The plant leaf provides an ideal system to study the mechanisms of organ formation and morphogenesis. The key factors that control leaf morphogenesis include the timing, location and extent of meristematic activity during cell division and differentiation. We identified an Arabidopsis mutant in which the regulation of meristematic activities in leaves was aberrant. The recessive mutant allele blade-on-petiole1-1 (bop1-1) produced ectopic, lobed blades along the adaxial side of petioles of the cotyledon and rosette leaves. The ectopic organ, which has some of the characteristics of rosette leaf blades with formation of trichomes in a dorsoventrally dependent manner, was generated by prolonged and clustered cell division in the mutant petioles. Ectopic, lobed blades were also formed on the proximal part of cauline leaves that lacked a petiole. Thus, BOP1 regulates the meristematic activity of leaf cells in a proximodistally dependent manner. Manifestation of the phenotypes in the mutant leaves was dependent on the leaf position. Thus, BOP1 controls leaf morphogenesis through control of the ectopic meristematic activity but within the context of the leaf proximodistality, dorsoventrality and heteroblasty. BOP1 appears to regulate meristematic activity in organs other than leaves, since the mutation also causes some ectopic outgrowths on stem surfaces and at the base of floral organs. Three class I knox genes, i.e., KNAT1, KNAT2 and KNAT6, were expressed aberrantly in the leaves of the bop1-1 mutant. Furthermore, the bop1-1 mutation showed some synergistic effect in double mutants with as1-1 or as2-2 mutation that is known to be defective in the regulation of meristematic activity and class I knox gene expression in leaves. The bop1-1 mutation also showed a synergistic effect with the stm-1 mutation, a strong mutant allele of a class I knox gene, STM. We, thus, suggest that BOP1 promotes or maintains a developmentally determinate state in leaf cells through the regulation of class I knox genes.     

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.