Question

What role do KNOX proteins have in maintaining the apical meristem?

Answer 1

Summary

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.

Introduction

The maize knotted1 (kn1) gene was isolated two decades ago through transposon tagging in a mutant with striking ‘knotted’ leaves, and revealed that the predicted gene product encoded a member of the homeodomain superfamily of transcriptional regulators. This discovery generated considerable excitement among developmental biologists, not least because the isolation of animal homeobox genes a few years earlier had revolutionized our understanding of the molecular basis of metazoan development and evolution. The cloning of the first member of a KNOX gene family in maize led to a similar explosion of new research in plant development (Hake et al., 1995). Today, a substantial body of information exists on the function of KNOX proteins in both model and non-model plants. These studies have revealed parallels with the mechanistic action of animal TALE homeodomain proteins, and have helped us to understand how these proteins influence plant development and to unravel key aspects of the logic that underpins cell fate allocation and tissue differentiation in the ‘green branch’ of the tree of life.

The discovery of KNOX genes provided the first molecular insights into the function of the shoot apical meristem. The nuclear expression of KN1 protein in the maize shoot is confined to meristem cells and is excluded from leaf founder cells, thus providing the earliest marker for meristem versus lateral organ cell fate (Smith et al., 1992). The SAM is established at the shoot pole during embryogenesis and harbours a stem cell population that allows continued organogenesis throughout the life of a plant. This continuous development contrasts with how animals develop and allows for plasticity in plant form, such that plants (which are sessile organisms) can readily modify their development in response to environmental cues. KN1 activity is required to prevent pluripotent cells in the maize SAM from adopting differentiated cell fates, as does the related protein SHOOT MERISTEMLESS (STM) in Arabidopsis, such that stm and kn1 loss-of-function mutants fail to establish and maintain a SAM (Long et al., 1996; Vollbrecht et al., 2000).

KNOX genes comprise a small family of TALE homeobox genes that are found in all green plant lineages and fall into two subclasses on the basis of sequence similarity within the homeodomain, intron position, expression pattern and phylogenetic analysis (Kerstetter et al., 1994; Mukherjee et al., 2009). Class I KNOX genes are most similar to kn1 and are expressed in overlapping domains within the SAMs of both monocot and eudicot plants (Hake et al., 2004; Jackson et al., 1994). Class II KNOX genes form a monophyletic group that is distinct from Class I genes, and show diverse expression patterns and few known functions (Zhong et al., 2008). We therefore focus this review on Class I KNOX genes.

Although the first plant homeobox gene was cloned over 20 years ago, only recently are we beginning to understand how KNOX genes function in diverse developmental contexts, and how these functions relate to developmental transitions during land plant evolution. In this review, we discuss the functions of Class I KNOX proteins during development and the advances in understanding of KNOX gene regulatory networks, including upstream regulators, protein partners and downstream effectors of KNOX function. We highlight the discovery of a novel class of KNOX genes that lack a homeobox and insights into KN1 protein trafficking in plants. We also discuss recent work in the unicellular green alga Chlamydomonas reinhardtii that suggests ancestral KNOX genes controlled diploid development and diversified in land plants in order to control multicellular body plans (Lee et al., 2008). Finally, we consider how evolutionary changes in KNOX gene regulation may have influenced plant diversity

Class I KNOX genes in meristem and compound leaf development

The Arabidopsis genome contains four Class I KNOX genes: STM, BREVIPEDICELLUS (BP), Kn1-like in Arabidopsis thaliana2 (KNAT2) and KNAT6 . These genes participate in various developmental processes during the plant life cycle; however, their expression in specific domains of the SAM maintains the activity of the SAM and of its lateral organ and stem boundaries throughout development. STM is the first KNOX gene expressed during early embryogenesis and its expression marks the entire SAM (Long et al., 1996). KNAT6 is expressed in the embryonic SAM once bilateral symmetry is established and later marks the SAM boundaries (Belles-Boix et al., 2006). BP shares aspects of both these gene expression patterns in the SAM during post-embryonic development (its embryonic expression marks the hypocotyl), whereas KNAT2 is expressed during embryogenesis and marks the base of the SAM (Byrne et al., 2002; Dockx et al., 1995). Genetic redundancy masks the contribution of KNAT6 and BP, which act, in addition to STM, to maintain SAM activity and organ separation (Belles-Boix et al., 2006; Byrne et al., 2002).

  

The architecture of bp mutants differs markedly from wild-type plants owing to their reduced height, irregularly shortened internodes and reduced apical dominance (Byrne et al., 2003; Douglas et al., 2002; Smith and Hake, 2003; Venglat et al., 2002). Plant architecture consists of repeating modules produced at the SAM called phytomers, each containing an internode, leaf and axillary meristem. In Arabidopsis inflorescences, growth of the leaf is suppressed such that only the floral meristem develops. BP expression at the boundary of the inflorescence SAM might, therefore, regulate cell allocation between floral primordia and internodes, explaining why the flowers are positioned aberrantly along bp mutant stems. Defective internode patterning is also observed in rice plants in which the related gene Oryza sativa homeobox15 (OSH15) is mutated (Sato et al., 1999). However, the flower stalks, or pedicels, are specifically shortened and curved downwards in bp mutants, owing to defects in cell division, cell elongation and cell differentiation (Douglas et al., 2002; Venglat et al., 2002), and it remains unclear which aspects of BP function are shared between this tissue and the SAM. KNAT6 and KNAT2 are part of a segmental chromosomal duplication in Arabidopsis, and single mutations in these genes do not affect shoot development (Belles-Boix et al., 2006; Byrne et al., 2002). However, KNAT6 and KNAT2 gene expression is expanded in bp mutants, and mutations in each of these genes show antagonistic genetic interactions with bp mutants, suggesting that the role of BP in inflorescence development is mediated at least in part by repression of KNAT6 and KNAT2 (Ragni et al., 2008).

In model organisms with simple leaves, such as Arabidopsis, maize and tobacco, KNOX gene expression is confined to the shoot meristem and stem, and leaf development is dramatically altered by the ectopic expression of KNOX genes in leaves (Lincoln et al., 1994; Sinha et al., 1993; Vollbrecht et al., 1991). For example, transgenic expression of any Class I KNOX gene ectopically in the leaves of Arabidopsis can dramatically alter the simple leaf margin to produce a highly lobed shape (Shani et al., 2009). These studies suggested that ectopic KNOX gene expression within a simple leaf development programme produced differential growth at the leaf margin, owing to the creation of novel ‘meristem-leaf’ boundaries. The ectopic initiation of shoot meristems in transgenic tobacco and Arabidopsis leaves supported this idea (Chuck et al., 1996; Sinha et al., 1993). Strikingly, this correlation between the expression of KNOX genes in the leaf and a more complex leaf shape was shown to hold for tomato and many other plant species with naturally compound leaves (also known as dissected leaves; (Bharathan et al., 2002; Hareven et al., 1996). It is now clear that KNOX gene expression is reactivated following leaf initiation in order to facilitate leaflet formation in both tomato and the Arabidopsis relative Cardamine hirsuta (Hay and Tsiantis, 2006; Shani et al., 2009). Gain of KNOX function in these species where KNOX genes are part of a compound leaf development programme can produce a striking reiteration of leaflets upon leaflets (Hareven et al., 1996; Hay and Tsiantis, 2006). Therefore, Class I KNOX genes have specific functions in compound leaf development that are distinct from their ability to induce shoot meristem formation.