Stuart F. Baum

Radial patterning of Arabidopsis Shoots by class III HD-ZIP and KANADI genes

BACKGROUND Shoots of all land plants have a radial pattern that can be considered to have an adaxial (central)-abaxial (peripheral) polarity. In Arabidopsis, gain-of-function alleles of PHAVOLUTA and PHABULOSA, members of the class III HD-ZIP gene family, result in adaxialization of lateral organs. Conversely, loss-of-function alleles of the KANADI genes cause an adaxialization of lateral organs. Thus, the class III HD-ZIP and KANADI genes comprise a genetic system that patterns abaxial-adaxial polarity in lateral organs produced from the apical meristem. RESULTS We show that gain-of-function alleles of REVOLUTA, another member of the class III HD-ZIP gene family, are characterized by adaxialized lateral organs and alterations in the radial patterning of vascular bundles in the stem. The gain-of-function phenotype can be obtained by changing only the REVOLUTA mRNA sequence and without changing the protein sequence; this finding indicates that this phenotype is likely mediated through an interference with microRNA binding. Loss of KANADI activity results in similar alterations in vascular patterning as compared to REVOLUTA gain-of-function alleles. Simultaneous loss-of-function of PHABULOSA, PHAVOLUTA, and REVOLUTA abaxializes cotyledons, abolishes the formation of the primary apical meristem, and in severe cases, eliminates bilateral symmetry; these phenotypes implicate these three genes in radial patterning of both embryonic and postembryonic growth. CONCLUSIONS Based on complementary vascular and leaf phenotypes of class III HD-ZIP and KANADI mutants, we propose that a common genetic program dependent upon miRNAs governs adaxial-abaxial patterning of leaves and radial patterning of stems in the angiosperm shoot. This finding implies that a common patterning mechanism is shared between apical and vascular meristems.

Establishment of polarity in lateral organs of plants

BACKGROUND Asymmetric development of plant lateral organs initiates by partitioning of organ primordia into distinct domains along their adaxial/abaxial axis. A recent model proposes that a meristem-born signal, acting in a concentration-dependent manner, differentially activates PHABULOSA-like genes, which in turn suppress abaxial-promoting factors. As yet, no abaxial factors have been identified that when compromised give rise to adaxialized organs. RESULTS Single mutants in either of the closely related genes KANADI1 (KAN1) or KANADI2 (KAN2) have little or no effect on plant morphology. However, in kan1 kan2 double mutant plants, there is a replacement of abaxial cell types by adaxial ones in most lateral organs. The alterations in polarity establishment are associated with expansion in the expression domain of the PHB-like genes and reduction in the expression of the previously described abaxial-promoting YABBY genes. Ectopic expression of either of the KANADI genes throughout leaf primordia results in dramatic transformation of adaxial cell types into abaxial ones, failure of lateral blade expansion, and vascular tissue formation. CONCLUSION The phenotypes of KANADI loss- and gain-of-function alleles suggest that fine regulation of these genes is at the core of polarity establishment. As such, they are likely to be targets of the PHB-mediated meristem-born signaling that patterns lateral organ primordia. PHB-like genes and the abaxial-promoting KANADI and YABBY genes appear to be expressed throughout primordia anlagen before becoming confined to their corresponding domains as primordia arise. This suggests that the establishment of polarity in plant lateral organs occurs via mutual repression interactions between ab/ad factors after primordium emergence, consistent with the results of classical dissection experiments.

Distinct Mechanisms Promote Polarity Establishment in Carpels of Arabidopsis

Lateral organs of plants display asymmetry with abaxial identity being specified by members of the Arabidopsis YABBY gene family. Mutations in CRABS CLAW, the founding family member, display ectopic formation of adaxial carpel tissues only when the functions of other genes, such as GYMNOS or KANADI, are also compromised. Mutations in these genes alone do not result in loss of polar differentiation, and therefore, they act redundantly with CRABS CLAW to establish polarity. As GYMNOS encodes a uniformly expressed homolog of the chromatin-remodeling protein, Mi2, we argue that the unique genetic interactions do not reflect a molecular redundancy. Rather, CRABS CLAW regulates transcription spatially, whereas GYMNOS regulates downstream targets temporally to ensure proper differentiation of the carpels.

Asymmetric leaf development and blade expansion in Arabidopsis are mediated by KANADI and YABBY activities

Asymmetric development of plant lateral organs is initiated by a partitioning of organ primordia into distinct domains along their adaxial/abaxial axis. Two primary determinants of abaxial cell fate are members of the KANADI and YABBY gene families. Progressive loss of KANADI activity in loss-of-function mutants results in progressive transformation of abaxial cell types into adaxial ones and a correlated loss of lamina formation. Novel, localized planes of blade expansion occur in some kanadi loss-of-function genotypes and these ectopic lamina outgrowths are YABBY dependent. We propose that the initial asymmetric leaf development is regulated primarily by mutual antagonism between KANADI and PHB-like genes, which is translated into polar YABBY expression. Subsequently, polar YABBY expression contributes both to abaxial cell fate and to abaxial/adaxial juxtaposition-mediated lamina expansion.

Establishment of polarity in angiosperm lateral organs

In seed plants, lateral organs such as leaves and floral organs are formed from the flanks of apical meristems. Therefore, they have an inherent positional relationship: organ primordia have an adaxial side next to the meristem, and an abaxial one away from the meristem. Surgical and genetic studies suggest that a morphogenetic gradient, which originates in the meristem, converts the inherent polarity into a functional one. Once an adaxial-abaxial axis of polarity is established within organ primordia, it provides cues for proper lamina growth and asymmetrical development. Several key participants in this process have been identified, and analyses of these genes support and refine our views of axis formation in plants. The complex relationships between and within various members of these plant-specific gene families (class III HD-ZIPs, YABBYs and KANADIs) might account for a substantial part of the morphological variation in lateral organs of seed plants.

Molecular genetics of gynoecium development in Arabidopsis.

Carpels are the ovule-bearing structural units in angiosperms. In Arabidopsis, the specification of carpel identity is achieved by at least two separate pathways: a pathway mediated by the C class gene AG and an AG-independent pathway. Both pathways are negatively regulated by A class genes. Two genes, SPT and CRC, can promote differentiation of carpel tissue independently of AG and are thus components of the AG-independent pathway. CRC and SPT appear to act in a redundant manner to promote the differentiation of subsets of carpel tissues. The carpel primordium is subdivided into regional domains, both medial versus lateral and abaxial versus adaxial. Based on morphological and gene expression analyses, it appears likely that these domains define developmental compartments. The medial domain appears fated to differentiate into the marginal tissue types of the carpel (septum with transmitting tract and placenta with ovules), whereas the lateral domain gives rise to the ovary walls. The expression of ETT defines the abaxial domain, and this gene is involved in the abaxial-adaxial and, possibly, the apical-basal patterning of tissues in the carpel. Once regional domains have been established, the differentiation of tissue and cell types occurs. The MADS-box gene FUL and AGLI/5 are involved in the differentiation of specific tissue types in the valves and valve margins. Thus, the genes identified can be arranged in a functional hierarchy: specification of carpel identity, patterning of the carpel primordium and directing the differentiation of the specialized tissues of the carpel.

Recruitment of CRABS CLAW to promote nectary development within the eudicot clade

Nectaries are secretory organs that are widely present in flowering plants that function to attract floral pollinators. Owing to diversity in nectary positions and structures, they are thought to have originated multiple times during angiosperm evolution, with their potential contribution to the diversification of flowering plants and pollinating animals being considerable. We investigated the genetic basis of diverse nectary forms in eudicot angiosperm species using CRABS CLAW (CRC), a gene required for nectaries in Arabidopsis. CRC expression is conserved in morphologically different nectaries from several core eudicot species and is required for nectary development in both rosids and asterids, two major phylogenetic lineages of eudicots. However, in a basal eudicot species, no evidence of CRC expression in nectaries was found. Considering the phylogenetic distribution of nectary positions and CRC expression analyses in eudicots, we propose that diverse nectaries in core eudicots share conserved CRC gene regulation, and that derived nectary positions in eudicots have altered regulation of CRC. As the ancestral function of CRC lies in the regulation of carpel development, it may have been co-opted as a regulator of nectary development within the eudicots, concomitant with the association of nectaries with reproductive organs in derived lineages.

Apical organization and maturation of the cortex and vascular cylinder inArabidopsis thaliana (Brassicaceae) roots

Developmental and physiological studies of roots are frequently limited to a post-germination stage. In Arabidopsis, a developmental change in the root meristem architecture during plant ontogenesis has not previously been studied and is addressed presently. Arabidopsis thaliana have closed root apical organization, in which all cell file lineages connect directly to one of three distinct initial tiers. The root meristem organization is dynamic and changes as the root ages from 1 to 4 wk post-germination. During the ontogeny of the root, the number of cells within the root apical meristem (RAM) increases and then decreases due to changes in the number of cortical layers and number of cell files within a central cylinder. The architecture of the initial tiers also changes as the root meristem ages. Included in the RAMs ontogeny is a pattern associated with the periclinal divisions that give rise to the middle cortex and endodermis; the three-dimensional arrangement of periclinally dividing derivative cells resembles one gyre of a helix. Four- or 5-wk-old roots exhibit a disorganized array of vacuolated initial cells that are a manifestation of the determinate nature of the meristem. Vascular cambium is formed via coordinated divisions of vascular parenchyma and pericycle cells. The phellogen is the last meristem to complete its development, and it is derived from pericycle cells that delineate the outer boundary of the root.

Activation of CRABS CLAW in the Nectaries and Carpels of Arabidopsis

CRABS CLAW (CRC), a member of the YABBY gene family, is required for nectary and carpel development. To further understand CRC regulation in Arabidopsis thaliana, we performed phylogenetic footprinting analyses of 5′ upstream regions of CRC orthologs from three Brassicaceae species, including Arabidopsis. Phylogenetic footprinting efficiently identified functionally important regulatory regions (modules), indicating that CRC expression is regulated by a combination of positive and negative regulatory elements in the modules. Within the conserved modules, we identified putative binding sites of LEAFY and MADS box proteins, and functional in vivo analyses revealed their importance for CRC expression. Both expression and genetic studies demonstrate that potential binding sites for MADS box proteins within the conserved regions are functionally significant for the transcriptional regulation of CRC in nectaries. We propose that in wild-type flowers, a combination of floral homeotic gene activities, specifically the B class genes APETALA3 and PISTILLATA and the C class gene AGAMOUS act redundantly with each other and in combination with SEPALLATA genes to activate CRC in the nectaries and carpels. In the absence of B and C class gene activities, other genes such as SHATTERPROOF1/2 can substitute if they are ectopically expressed, as in an A class mutant background (apetala2). These MADS box proteins may provide general floral factors that must work in conjunction with specific factors in the activation of CRC in the nectaries and carpels.

Root apical organization in Arabidopsis thaliana ecotype 'WS' and a comment on root cap structure

Arabidopsis thaliana roots have closed apical organization with three initial tiers. The dermatogen/calyptrogen tier consists of two parts-the central initials form the columella root cap, and the peripheral initial cells form the protoderm (epidermis) and the peripheral root cap. These peripheral initials divide in a sequence to form a root cap consisting of interconnected cones. the periblem initial tier forms the ground meristem (cortex). For the first week after germination the periblem consists of one layer of initial cells. The peripheral cells of the tier divide periclinally and then anticlinally (a T-division) to form the two-layered cortex (outer cortex and endodermis). After about one week, all the peripheral cells have divided periclinally forming two initials; the outermost produces the outer cortex while the inner initial produces the endodermis and middle cortex layer. The latter two cells arise via a periclinal division. During this time, other cells within the tier divide periclinally to form a two-layered tier. The plerome forms the cells of the procambium (vascular cylinder) by simple anticlinal divisions followed by longitudinal divisions to fill out the cell files of the vascular cylinder. A survey (27 dicot species in 17 families) of roots with closed apical organization revealed that there are three different types of root cap-concentric cylinders of cells (e.g.Linum), interconnecting cones (e.g.Arabidopsis) or overlapping arcs (e.g.Gossypium). H Lambers Section editor