Negative regulation of <Emphasis Type="Italic">KNOX</Emphasis> expression in tomato leaves

Leaves of seed plants can be described as simple, where the leaf blade is entire, or dissected, where the blade is divided into distinct leaflets. Both simple and dissected leaves are initiated at the flanks of a pluripotent structure termed the shoot apical meristem (SAM). In simple-leafed species, expression of class I KNOTTED1-like homeobox (KNOX) proteins is confined to the meristem while in many dissected leaf plants, including tomato, KNOX expression persists in leaf primordia. Elevation of KNOX expression in tomato leaves can result in increased leaflet number, indicating that tight regulation of KNOX expression may help define the degree of leaf dissection in this species. To test this hypothesis and understand the mechanisms controlling leaf dissection in tomato, we studied the clausa (clau) and tripinnate (tp) mutants both of which condition increased leaflet number phenotypes. We show that TRIPINNATE and CLAUSA act together, to restrict the expression level and domain of the KNOX genes Tkn1 and LeT6/Tkn2 during tomato leaf development. Because loss of CLAU or TP activity results in increased KNOX expression predominantly on the adaxial (upper) leaf domain, our observations indicate that CLAU and TP may participate in a domain-specific KNOX repressive system that delimits the ability of the tomato leaf to generate leaflets.     

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.     

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.     

L1 division and differentiation patterns influence shoot apical meristem maintenance

Plant development requires regulation of both cell division and differentiation. The class 1 KNOTTED1-like homeobox (KNOX) genes such as knotted1 (kn1) in maize (Zea mays) and SHOOTMERISTEMLESS in Arabidopsis (Arabidopsis thaliana) play a role in maintaining shoot apical meristem indeterminacy, and their misexpression is sufficient to induce cell division and meristem formation. KNOX overexpression experiments have shown that these genes interact with the cytokinin, auxin, and gibberellin pathways. The L1 layer has been shown to be necessary for the maintenance of indeterminacy in the underlying meristem layers. This work explores the possibility that the L1 affects meristem function by disrupting hormone transport pathways. The semidominant Extra cell layers1 (Xcl1) mutation in maize leads to the production of multiple epidermal layers by overproduction of a normal gene product. Meristem size is reduced in mutant plants and more cells are incorporated into the incipient leaf primordium. Thus, Xcl1 may provide a link between L1 division patterns, hormonal pathways, and meristem maintenance. We used double mutants between Xcl1 and dominant KNOX mutants and showed that Xcl1 suppresses the Kn1 phenotype but has a synergistic interaction with gnarley1 and rough sheath1, possibly correlated with changes in gibberellin and auxin signaling. In addition, double mutants between Xcl1 and crinkly4 had defects in shoot meristem maintenance. Thus, proper L1 development is essential for meristem function, and XCL1 may act to coordinate hormonal effects with KNOX gene function at the shoot apex.     

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.     

Weeds of change: Cardamine hirsuta as a new model system for studying dissected leaf development

Cardamine hirsuta, a small crucifer closely related to the model organism Arabidopsis thaliana, offers high genetic tractability and has emerged as a powerful system for studying the genetic basis for diversification of plant form. Contrary to A. thaliana, which has simple leaves, C. hirsuta produces dissected leaves divided into individual units called leaflets. Leaflet formation requires activity of Class I KNOTTED1-like homeodomain (KNOX) proteins, which also promote function of the shoot apical meristem (SAM). In C. hirsuta, KNOX genes are expressed in the leaves whereas in A. thaliana their expression is confined to the SAM, and differences in expression arise through cis-regulatory divergence of KNOX regulation. KNOX activity in C. hirsuta leaves delays the transition from proliferative growth to differentiation thus facilitating the generation of lateral growth axes that give rise to leaflets. These axes reflect the sequential generation of cell division foci across the leaf proximodistal axis in response to auxin activity maxima, which are generated by the PINFORMED1 (PIN1) auxin efflux carriers in a process that resembles organogenesis at the SAM. Delimitation of C. hirsuta leaflets also requires the activity of CUP SHAPED COTYLEDON (CUC) genes, which direct formation of organ boundaries at the SAM. These observations show how species-specific deployment of fundamental shoot development networks may have sculpted simple versus dissected leaf forms. These studies also illustrate how extending developmental genetic studies to morphologically divergent relatives of model organisms can greatly help elucidate the mechanisms underlying the evolution of form.     

Coordination of leaf development via regulation of KNOX1 genes

Class I KNOTTED1-LIKE HOMEOBOX (KNOX1) genes are expressed in the shoot apical meristem (SAM) to effect its formation and maintenance. KNOX1 genes are also involved in leaf shape control throughout angiosperm evolution. Leaves can be classified as either simple or compound, and KNOX1 expression patterns in leaf primordia are highly correlated with leaf shape; in most simple-leafed species, KNOX1 genes are expressed only in the SAM but not in leaf primordia, while in compound-leafed species they are expressed both in the SAM and leaf primordia. How can KNOX1 expression be maintained to a high degree in the SAM, but simultaneously be so variable in leaves? This dichotomy suggests that the processes of leaf and SAM development have been compartmentalized during evolution. Here, we introduce our findings regarding the regulation of expression of SHOOT MERISTEMLESS, a KNOX1 gene, together with a brief review of KNOX1 genes from an evolutionary viewpoint. We also present our findings regarding another aspect of KNOX1 regulation via a protein–protein interaction network involved in the natural variation in leaf shape. Both aspects of KNOX1 regulation could be utilized for fine-tuning leaf morphology during evolution without affecting the essential function of KNOX genes in the shoot.     

Analysis of the competence to respond to KNOTTED1 activity in Arabidopsis leaves using a steroid induction system

Expression of KNOX (KNOTTED1-like homeobox) genes in the shoot apical meristem of Arabidopsis is required for maintenance of a functional meristem, whereas exclusion of KNOX gene expression from leaf primordia is required for the elaboration of normal leaf morphology. We have constructed a steroid-inducible system to regulate both the amount and timing of KN1 (KNOTTED1) misexpression in Arabidopsis leaves. We demonstrate that lobed leaf morphology is produced in a dose-dependent manner, indicating that the amount of KN1 quantitatively affects the severity of lobing. The KN1-glucocorticoid receptor fusion protein is not detected in leaves in the absence of steroid induction, suggesting that it is only stable when associated with steroid in an active state. By using a second inducible fusion protein to mark exposure of leaf primordia to the steroid, we determined the stage of leaf development that produces lobed leaves in response to KN1. Primordia as old as plastochron 7 and as young as plastochron 2 were competent to respond to KN1.     

The role of KNOX genes in the evolution of morphological novelty in Streptocarpus

The genus Streptocarpus comprises species with diverse body plans. Caulescent species produce leaves from a conventional shoot apical meristem (SAM), whereas acaulescent species lack a conventional SAM and produce only a single leaf (the unifoliate form) or clusters of leaves from the base of more mature leaves (the rosulate form). These distinct morphologies reflect fundamental differences in the role of the SAM and the process of leaf specification. A subfamily of KNOTTED-like homeobox (KNOX) genes are known to be important in regulating meristem function and leaf development in model species with conventional morphologies. To test the involvement of KNOX genes in Streptocarpus evolution, two parologous KNOX genes (SSTM1 and SSTM2) were isolated from species with different growth forms. Their phylogenetic analysis suggested a gene duplication before the subgeneric split of Streptocarpus and resolved species relationships, supporting multiple evolutionary origins of the rosulate and unifoliate morphologies. In S. saxorum, a caulescent species with a conventional SAM, KNOX proteins were expressed in the SAM and transiently downregulated in incipient leaf primordia. The ability of acaulescent species to initiate leaves from existing leaves was found to correlate with SSTM1 expression and KNOX protein accumulation in leaves and to reflect genetic differences at two loci. Neither locus corresponded to SSTM1, suggesting that cis-acting differences in SSTM1 regulation were not responsible for evolution of the rosulate and unifoliate forms. However, the involvement of KNOX proteins in leaf formation in rosulate species suggests that they have played an indirect role in the development of morphological diversity in Streptocarpus.