Angela Hay

KNOX Action in Arabidopsis Is Mediated by Coordinate Regulation of Cytokinin and Gibberellin Activities

The shoot apical meristem (SAM) is a pluripotent group of cells that gives rise to the aerial parts of higher plants. Class-I KNOTTED1-like homeobox (KNOX) transcription factors promote meristem function partly through repression of biosynthesis of the growth regulator gibberellin (GA). However, regulation of GA activity cannot fully account for KNOX action. Here, we show that KNOX function is also mediated by cytokinin (CK), a growth regulator that promotes cell division and meristem function. We demonstrate that KNOX activity is sufficient to rapidly activate both CK biosynthetic gene expression and a SAM-localized CK-response regulator. We also show that CK signaling is necessary for SAM function in a weak hypomorphic allele of the KNOX gene SHOOTMERISTEMLESS (STM). Additionally, we provide evidence that a combination of constitutive GA signaling and reduced CK levels is detrimental to SAM function. Our results indicate that CK activity is both necessary and sufficient for stimulating GA catabolic gene expression, thus reinforcing the low-GA regime established by KNOX proteins in the SAM. We propose that KNOX proteins may act as general orchestrators of growth-regulator homeostasis at the shoot apex of Arabidopsis by simultaneously activating CK and repressing GA biosynthesis, thus promoting meristem activity.

The Gibberellin Pathway Mediates KNOTTED1-Type Homeobox Function in Plants with Different Body Plans

BACKGROUND The shoot apical meristem (SAM) is an indeterminate structure that gives rise to the aerial parts of higher plants. Leaves arise from the differentiation of cells at the flanks of the SAM. Current evidence suggests that the precise regulation of KNOTTED1-like homeobox (KNOX) transcription factors is central to the acquisition of leaf versus meristem identity in a wide spectrum of plant species. Factors required to repress KNOX gene expression in leaves have recently been identified. Additional factors such as the CHD3 chromatin remodeling factor PICKLE (PKL) act to restrict meristematic activity in Arabidopsis leaves without repressing KNOX gene expression. Less is known regarding downstream targets of KNOX function. Recent evidence, however, has suggested that growth regulators may mediate KNOX activity in a variety of plant species. RESULTS Here we show that reduced activity of the gibberellin (GA) growth regulator pathway promotes meristematic activity, both in the natural context of KNOX function in the SAM and upon ectopic KNOX expression in Arabidopsis leaves. We show that constitutive signaling through the GA pathway is detrimental to meristem maintenance. Furthermore, we provide evidence that one of the functions of the KNOX protein SHOOTMERISTEMLESS (STM) is to exclude transcription of the GA-biosynthesis gene AtGA20ox1 from the SAM. We also demonstrate that AtGA20ox1 transcript is reduced in the pkl mutant in a KNOX-independent manner. Moreover, we show a similar interaction between KNOX proteins and GA-biosynthesis gene expression in the tomato leaf and implicate this interaction in regulation of the dissected leaf form. CONCLUSIONS We suggest that repression of GA activity by KNOX transcription factors is a key component of meristem function. Transfer of the KNOX/GA regulatory module from the meristem to the leaf may have contributed to the generation of the diverse leaf morphologies observed in higher plants.

KNOX genes: versatile regulators of plant development and diversity

Knotted1-like homeobox (KNOX) proteins are homeodomain transcription factors that maintain an important pluripotent cell population called the shoot apical meristem, which generates the entire above-ground body of vascular plants. KNOX proteins regulate target genes that control hormone homeostasis in the meristem and interact with another subclass of homeodomain proteins called the BELL family. Studies in novel genetic systems, both at the base of the land plant phylogeny and in flowering plants, have uncovered novel roles for KNOX proteins in sculpting plant form and its diversity. Here, we discuss how KNOX proteins influence plant growth and development in a versatile context-dependent manner.

The genetic basis for differences in leaf form between Arabidopsis thaliana and its wild relative Cardamine hirsuta

A key question in biology is how differences in gene function or regulation produce new morphologies during evolution. Here we investigate the genetic basis for differences in leaf form between two closely related plant species, Arabidopsis thaliana and Cardamine hirsuta. We report that in C. hirsuta, class I KNOTTED1-like homeobox (KNOX) proteins are required in the leaf to delay cellular differentiation and produce a dissected leaf form, in contrast to A. thaliana, in which KNOX exclusion from leaves results in a simple leaf form. These differences in KNOX expression arise through changes in the activity of upstream gene regulatory sequences. The function of ASYMMETRIC LEAVES1/ROUGHSHEATH2/PHANTASTICA (ARP) proteins to repress KNOX expression is conserved between the two species, but in C. hirsuta the ARP-KNOX regulatory module controls new developmental processes in the leaf. Thus, evolutionary tinkering with KNOX regulation, constrained by ARP function, may have produced diverse leaf forms by modulating growth and differentiation patterns in developing leaf primordia.

A conserved molecular framework for compound leaf development.

Diversity in leaf shape is produced by alterations of the margin: for example, deep dissection leads to leaflet formation and less-pronounced incision results in serrations or lobes. By combining gene silencing and mutant analyses in four distantly related eudicot species, we show that reducing the function of NAM/CUC boundary genes (NO APICAL MERISTEM and CUP-SHAPED COTYLEDON) leads to a suppression of all marginal outgrowths and to fewer and fused leaflets. We propose that NAM/CUC genes promote formation of a boundary domain that delimits leaflets. This domain has a dual role promoting leaflet separation locally and leaflet formation at distance. In this manner, boundaries of compound leaves resemble boundaries functioning during animal development.

ASYMMETRIC LEAVES1 and auxin activities converge to repress BREVIPEDICELLUS expression and promote leaf development in Arabidopsis.

Leaf development in higher plants requires the specification of leaf initials at the flanks of a pluripotent structure termed the shoot apical meristem. In Arabidopsis, this process is facilitated by negative interactions between class I KNOTTED1-like homeobox (KNOX) and ASYMMETRIC LEAVES1 (AS1) transcription factors, such that KNOX proteins are confined to the meristem and AS1 to leaf initials. Sites of leaf inception are also defined by local accumulation of the hormone auxin; however, it is unknown how auxin and AS1 activities are integrated to control leaf development. Here, we show that auxin and AS1 pathways converge to repress expression of the KNOX gene BREVIPEDICELLUS (BP) and thus promote leaf fate. We also demonstrate that regulated auxin gradients control leaf shape in a KNOX-independent fashion and that inappropriate KNOX activity in leaves perturbs these gradients, hence altering leaf shape. We propose that regulatory interactions between auxin, AS1 and KNOX activities may both direct leaf initiation and sculpt leaf form.

A developmental framework for dissected leaf formation in the Arabidopsis relative Cardamine hirsuta.

The developmental basis for the generation of divergent leaf forms is largely unknown. Here we investigate this problem by studying processes that distinguish development of two related species: Arabidopsis thaliana, which has simple leaves, and Cardamine hirsuta, which has dissected leaves with individual leaflets. Using genetics, expression studies and cell lineage tracing, we show that lateral leaflet formation in C. hirsuta requires the establishment of growth foci that form after leaf initiation. These growth foci are recruited at the leaf margin in response to activity maxima of auxin, a hormone that polarizes growth in diverse developmental contexts. Class I KNOTTED1-like homeobox (KNOX) proteins also promote leaflet initiation in C. hirsuta, and here we provide evidence that this action of KNOX proteins is contingent on the ability to organize auxin maxima via the PINFORMED1 (PIN1) auxin efflux transporter. Thus, differential deployment of a fundamental mechanism polarizing cellular growth contributed to the diversification of leaf form during evolution.

Model for the regulation of Arabidopsis thaliana leaf margin development

Biological shapes are often produced by the iterative generation of repeated units. The mechanistic basis of such iteration is an area of intense investigation. Leaf development in the model plant Arabidopsis is one such example where the repeated generation of leaf margin protrusions, termed serrations, is a key feature of final shape. However, the regulatory logic underlying this process is unclear. Here, we use a combination of developmental genetics and computational modeling to show that serration development is the morphological read-out of a spatially distributed regulatory mechanism, which creates interspersed activity peaks of the growth-promoting hormone auxin and the CUP-SHAPED COTYLEDON2 (CUC2) transcription factor. This mechanism operates at the growing leaf margin via a regulatory module consisting of two feedback loops working in concert. The first loop relates the transport of auxin to its own distribution, via polar membrane localization of the PINFORMED1 (PIN1) efflux transporter. This loop captures the potential of auxin to generate self-organizing patterns in diverse developmental contexts. In the second loop, CUC2 promotes the generation of PIN1-dependent auxin activity maxima while auxin represses CUC2 expression. This CUC2-dependent loop regulates activity of the conserved auxin efflux module in leaf margins to generate stable serration patterns. Conceptualizing leaf margin development via this mechanism also helps to explain how other developmental regulators influence leaf shape.

SERRATE coordinates shoot meristem function and leaf axial patterning in Arabidopsis

Leaves of flowering plants are determinate organs produced by pluripotent structures termed shoot apical meristems. Once specified, leaves differentiate an adaxial (upper) side specialized for light capture, and an abaxial (lower) side specialized for gas exchange. A functional relationship between meristem activity and the differentiation of adaxial leaf fate has been recognized for over fifty years, but the molecular basis of this interaction is unclear. In Arabidopsis thaliana, activity of the class I KNOX (KNOTTED1-like homeobox) genes SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS (BP) is required for meristem function but excluded from leaves, whereas members of the HD-Zip III (class III homeodomain leucine zipper) protein family function to promote both meristem activity and adaxial leaf fate. Here we show that the zinc-finger protein SERRATE acts in a microRNA (miRNA) gene-silencing pathway to regulate expression of the HD-Zip III gene PHABULOSA (PHB) while also limiting the competence of shoot tissue to respond to KNOX expression. Thus, SERRATE acts to coordinately regulate meristem activity and leaf axial patterning.

MorphoGraphX: A platform for quantifying morphogenesis in 4D

Morphogenesis emerges from complex multiscale interactions between genetic and mechanical processes. To understand these processes, the evolution of cell shape, proliferation and gene expression must be quantified. This quantification is usually performed either in full 3D, which is computationally expensive and technically challenging, or on 2D planar projections, which introduces geometrical artifacts on highly curved organs. Here we present MorphoGraphX (www.MorphoGraphX.org), a software that bridges this gap by working directly with curved surface images extracted from 3D data. In addition to traditional 3D image analysis, we have developed algorithms to operate on curved surfaces, such as cell segmentation, lineage tracking and fluorescence signal quantification. The softwares modular design makes it easy to include existing libraries, or to implement new algorithms. Cell geometries extracted with MorphoGraphX can be exported and used as templates for simulation models, providing a powerful platform to investigate the interactions between shape, genes and growth. DOI: http://dx.doi.org/10.7554/eLife.05864.001