The sheathing leaf found in grasses and other monocots is an evolutionary innovation, yet its origin has been a subject of long-standing debate. Here, we revisit the problem in the light of developmental genetics and computational modeling. We show that the sheathing leaf likely arose through WOX-gene-dependent extension of a primordial zone straddling concentric domains around the shoot apex. Patterned growth within this zone, oriented by two polarity fields, accounts for wild-type, mutant and mosaic grass leaf development, whereas zone contraction and growth remodeling accounts for eudicot leaf development. In contrast to the prevailing view, our results suggest that the sheath derives from petiole, whereas the blade derives from the lamina of the eudicot leaf, consistent with homologies proposed in the 19th century.

Plant architecture is determined by the iterative building of phytomer units, each containing a portion of stem, an organ and an axillary meristem. Each phytomer can follow different developmental paths underpinning the complexity, and plasticity, of plant form. Auxin plays a central role in the coordination of phytomer development, regulating organ initiation and patterning across all axes. This diversity in auxin function results from changes in the activities and expression of auxin signaling components, including the AUX/IAA repressors. Higher land plants have multigene AUX/IAA families, which leads to functional redundancy. Dominant mutations, which prevent AUX/IAA degradation in response to auxin, have highlighted the importance of these proteins in fine-tuning organ development. Here we report a new dominant AUX/IAA mutant in maize, Hoja loca1 ( Oja ). Oja has a mutation in the degron motif of ZmIAA28 and specifically affects aerial organ initiation and leaf medio-lateral patterning, but phytomer initiation remains unchanged. Mutant phenotypes are variable and transcriptional profiling of individual meristems identified clusters of genes that may underpin the phenotypic differences. The unique phenotype of Oja provides evidence of species-specific sub-functionalization of the AUX/IAAs, and illustrates the crucial role of auxin signaling in the tight coordination of phytomer unit development.

Auxin plays a vital role in plant organ development, influencing organ initiation and patterning across all axes. The diversity in auxin patterning results from changes in the activities and expression of auxin signaling components, including the AUX/IAA repressors. Higher land plants have multigene AUX/IAA families, which leads to functional redundancy and a lack of phenotype in loss of function mutants. Instead, dominant mutations, which prevent AUX/IAA degradation in response to auxin, have highlighted the importance of these proteins in organ development. Here we report a new dominant AUX/IAA mutant in maize, (). has a mutation in the degron motif of ZmIAA28 and affects aerial organ initiation and medio-lateral patterning in the leaf. These phenotypes contrast with other maize AUX/IAA mutants that affect the root or inflorescence only. illustrates the role of auxin signaling in the tight coordination of phytomer unit development and provides evidence of species-specific sub-functionalization of the AUX/IAAs.

The knotted1 ( kn1 ) homeobox ( knox ) gene rough sheath1 plays a critical, dosage-dependent role in shoot apical meristem establishment and maintenance, stem elongation and inflorescence development that is only uncovered in a knotted1 mutant background. ABSTRACT The knotted1 ( kn1 ) homeobox ( knox ) gene family was first identified through gain-of-function dominant mutants in maize. Class I knox members are expressed in meristems but excluded from leaves. In maize, a loss-of-function phenotype has only been characterized for kn1 . To assess the function of another knox member, we characterized a loss-of-function mutation of rough sheath1 ( rs1 ). rs1-mum1 has no phenotype alone but exacerbates several aspects of the kn1 phenotype. In permissive backgrounds in which kn1 mutants grow to maturity, loss of a single copy of rs1 enhances the tassel branch reduction phenotype while loss of both copies results in limited shoots. In less introgressed lines, double mutants can grow to maturity but are shorter. Using a KNOX antibody, we demonstrate that RS1 binds in vivo to some of the KN1 target genes, which could partially explain why KN1 binds many genes, but modulates few. Our results demonstrate an unequal redundancy between knox genes, with a role for rs1 only revealed in the complete absence of kn1 .

Leaf development presents a tremendous resource for tackling the question of patterning in biology. Leaves can be simple or highly dissected. They may have elaborated parts such as the tendrils of a pea leaf or the rolled blade of a carnivorous pitcher plant. Despite the variation in size, shape, and function, all leaves initiate in the same manner: from the flanks of a meristem. The maize leaf is useful for analysis of patterning due to the wealth of mutants and the distinct tissues along the proximal distal axis. The blade is distal, the sheath is proximal, and the ligule forms at the blade/sheath boundary. Establishment of this boundary involves the transcription factors LIGULELESS1 and LIGULELESS2 and the kinase LIGULELESS NARROW. The meristem-specific protein KNOTTED1 (KN1) binds and modulates the lg2 gene. Given the localization of KN1 at the proximal end of the leaf from the time of inception, we hypothesize that KN1 has a role in establishing the very proximal end of the leaf, whereas an auxin maximum guides the growing distal tip.

KNOTTED1 (KN1)-like homeobox (KNOX) transcription factors are involved in the establishment and maintenance of plant meristems; however, few direct targets of KNOX proteins have been recognized. Using a combination of double mutant analysis and biochemistry, we found that in maize (Zea mays), KN1 negatively modulates the accumulation of gibberellin (GA) through the control of ga2ox1, which codes for an enzyme that inactivates GA. The ga2ox1 mRNA level is elevated in immature leaves of dominant KNOX mutants and downregulated in reproductive meristems of the null allele kn1-e1. KN1 binds in vivo to an intron of ga2ox1 through a cis-regulatory element containing two TGAC motifs. VP16-KN1 activates transcription in planta from a chimeric promoter containing this binding site. The domains of expression of kn1 and ga2ox1 mRNAs overlap at the base of the shoot apical meristem and the base of newly initiated leaves, suggesting that KN1-mediated activation of ga2ox1 maintains a boundary between meristem cell identity and rapidly elongating cells of the shoot. The KN1 binding site is conserved in ga2ox1 genes of different grasses, suggesting that the local regulation of bioactive GA levels through KNOX proteins is a common theme in grasses.

Organogenesis in plants is controlled by meristems. Axillary meristems, which give rise to branches and flowers, play a critical role in plant architecture and reproduction. Maize (Zea mays) and rice (Oryza sativa) have additional types of axillary meristems in the inflorescence compared to Arabidopsis (Arabidopsis thaliana) and thus provide an excellent model system to study axillary meristem initiation. Previously, we characterized the barren inflorescence2 (bif2) mutant in maize and showed that bif2 plays a key role in axillary meristem and lateral primordia initiation in the inflorescence. In this article, we cloned bif2 by transposon tagging. Isolation of bif2-like genes from seven other grasses, along with phylogenetic analysis, showed that bif2 is a co-ortholog of PINOID (PID), which regulates auxin transport in Arabidopsis. Expression analysis showed that bif2 is expressed in all axillary meristems and lateral primordia during inflorescence and vegetative development in maize and rice. Further phenotypic analysis of bif2 mutants in maize illustrates additional roles of bif2 during vegetative development. We propose that bif2/PID sequence and expression are conserved between grasses and Arabidopsis, attesting to the important role they play in development. We provide further support that bif2, and by analogy PID, is required for initiation of both axillary meristems and lateral primordia.

Genetic control of grass inflorescence architecture is critical given that cereal seeds provide most of the world's food. Seeds are borne on axillary branches, which arise from groups of stem cells in axils of leaves and whose branching patterns dictate most of the variation in plant form. Normal maize (Zea mays) ears are unbranched, and tassels have long branches only at their base. The ramosa2 (ra2) mutant of maize has increased branching with short branches replaced by long, indeterminate ones. ra2 was cloned by chromosome walking and shown to encode a LATERAL ORGAN BOUNDARY domain transcription factor. ra2 is transiently expressed in a group of cells that predicts the position of axillary meristem formation in inflorescences. Expression in different mutant backgrounds places ra2 upstream of other genes that regulate branch formation. The early expression of ra2 suggests that it functions in the patterning of stem cells in axillary meristems. Alignment of ra2-like sequences reveals a grass-specific domain in the C terminus that is not found in Arabidopsis thaliana. The ra2-dm allele suggests this domain is required for transcriptional activation of ra1. The ra2 expression pattern is conserved in rice (Oryza sativa), barley (Hordeum vulgare), sorghum (Sorghum bicolor), and maize, suggesting that ra2 is critical for shaping the initial steps of grass inflorescence architecture.

Positional cloning has been and remains a powerful method for gene identification in Arabidopsis. With the completion of the rice genome sequence, positional cloning in rice also took off, including the cloning of several quantitative trait loci. Positional cloning in cereals such as maize whose genomes are much larger than that of rice was considered near impossible because of the vast amounts of repetitive DNA. However, conservation of synteny across the cereal genomes, in combination with new maize resources, has now made positional cloning in maize feasible. In fact, a chromosomal walk is usually much faster than the more traditional method of gene isolation in maize by transposon tagging.

Spectacular variety in plant form and adaptation can be seen throughout the Angiosperms. Even basal plants display complex morphologies as a result Of their genetic programs. Both the shoot and root arise from a fixed number of stem cells in the respective shoot and root apical meristems. These meristems are controlled by gene net-works that in turn are influenced by events that occur externally - a necessity due to the sessile nature of plants. Recent studies suggest chat the key regulatory pathways controlling maintenance of stem cell Populations and their transition into organs have largely been conserved since the divergence of monocots and dicots, 134 million years ago (WIKSTROM et al., 2001). The regulation of these path-ways may help explain differences in morphology between these two major plant subdivisions. Maize has comparatively large meristems and initiates a series of inflorescence meristems before producing determinant floral meristems. This expanded developmental program, along with abundant genetic resources, makes maize an ideal model for meristem studies. Several pathways have been elucidated in Arabidopsis that control organ generation and meristem maintenance. Maize orthologues have been identified that are conserved in protein sequence and function. Examples of these shared networks include those of the clavata and knox pathways. Evolution Of unique structures particular to the grasses, such as the spikelet, has also Occurred. In this case, no Arabidopsis orthologs have been identified, rather different genes have been co-opted for unique functions. Such examples are the adaptation of ERF proteins for spikelet meristem determinacy. We present a comparison of the regulation of key meristem pathways in the dicot model plant, Arabidopsis, and maize.

Development in higher plants depends on the activity of meristems, formative regions that continuously initiate new organs at their flanks. Meristems must maintain a balance between stem cell renewal and organ initiation. In fasciated mutants, organ initiation fails to keep pace with meristem proliferation. The thick tassel dwarf1 (td1) mutation of maize affects both male and female inflorescence development. The female inflorescence, which results in the ear, is fasciated, with extra rows of kernels. The male inflorescence, or tassel, shows an increase in spikelet density. Floral meristems are also affected in td1 mutants; for example, male florets have an increase in stamen number. These results suggest that td1 functions in the inflorescence to limit meristem size. In addition, td1 mutants are slightly shorter than normal siblings, indicating that td1 also plays a role in vegetative development. td1 encodes a leucine-rich repeat receptor-like kinase (LRR-RLK) that is a putative ortholog of the Arabidopsis CLAVATA1 protein. These results complement previous work showing that fasciated ear2 encodes a CLAVATA2-like protein, and suggest that the CLAVATA signaling pathway is conserved in monocots. td1 maps in the vicinity of quantitative trait loci that affect seed row number, spikelet density and plant height. We discuss the possible selection pressures on td1 during maize domestication.

Plants undergo a series of profound developmental changes throughout their lifetimes in response to both external environmental factors and internal intrinsic ones. When these changes are abrupt and dramatic, the process is referred to as phase change. Recently, several genes have been discovered that play a role in these developmental transitions. Their sequence and expression patterns shed new light on the mechanisms of phase change, and provide a link between the external and internal factors that control them. Examples of these transitions include changes from juvenile to adult leaf formation, vegetative to inflorescence meristem development, and inflorescence to floral meristem initiation.

The INDETERMINATE protein, ID1, plays a key role in regulating the transition to flowering in maize. ID1 is the founding member of a plant-specific zinc finger protein family that is defined by a highly conserved amino sequence called the ID domain. The ID domain includes a cluster of three different types of zinc fingers separated from a fourth C2H2 finger by a long spacer; ID1 is distinct from other ID domain proteins by having a much longer spacer. In vitro DNA selection and amplification binding assays and DNA binding experiments showed that ID1 binds selectively to an 11 bp consensus motif via the ID domain. Unexpectedly, site-directed mutagenesis of the ID1 protein showed that zinc fingers located at each end of the ID domain are not required for binding to the consensus motif despite the fact that one of these zinc fingers is a canonical C2H2 DNA binding domain. In addition, an ID1 in vitro deletion mutant that lacks the extra spacer between zinc fingers binds the same 11 bp motif as normal ID1, suggesting that all ID domain-containing proteins recognize the same DNA target sequence. Our results demonstrate that maize ID1 and ID domain proteins have novel zinc finger configurations with unique DNA binding properties.

Meristems require a myriad of intercellular signaling pathways for coordination of cell division within and between functional zones and clonal cell layers. This control of cell division ensures a constant availability of stem cells throughout the life span of the meristem while limiting overproliferation of meristematic cells and maintaining the meristem structure. We have undertaken a genetic screen to identify additional components of meristem signaling pathways. We identified pluripetala (plp) mutants based on their dramatically larger meristems and increased floral organ number. PLURIPETALA encodes the alpha-subunit shared between protein farnesyltransferase and protein geranylgeranyltransferase-I. plp mutants also have altered abscisic acid responses and overall much slower growth rate. plp is epistatic to mutations in the beta-subunit of farnesyltransferase and shows a synergistic interaction with clavata3 mutants. plp mutants lead to insights into the mechanism of meristem homeostasis and provide a unique in vivo system for studying the functional role of prenylation in eukaryotes.

The maize leaf consists of four distinct tissues along its proximodistal axis: sheath, ligule, auricle and blade. liguleless1 (lg1)functions cell autonomously to specify ligule and auricle, and may propagate a signal that correctly positions the blade-sheath boundary. The dominant Wavy auricle in blade (Wab1) mutation disrupts both the mediolateral and proximodistal axes of the maize leaf. Wab1 leaf blades are narrow and ectopic auricle and sheath extend into the blade. The recessive lg1-R mutation exacerbates the Wab1 phenotype; in the double mutants, most of the proximal blade is deleted and sheath tissue extends along the residual blade. We show that lg1 is misexpressed in Wab1 leaves. Our results suggest that the Wab1 defect is partially compensated for by lg1 expression. A mosaic analysis of Wab1 was conducted in Lg1+ and lg1-R backgrounds to determine if Wab1 affects leaf development in a cell-autonomous manner. Normal tissue identity was restored in all wab1+/–sectors in a lg1-R mutant background, and in three quarters of sectors in a Lg1+ background. These results suggest that lg1can influence the autonomy of Wab1. In both genotypes, leaf-halves with wab1+/– sectors were significantly wider than non-sectored leaf-halves, suggesting that Wab1 acts cell-autonomously to affect lateral growth. The mosaic analysis, lg1 expression data and comparison of mutant leaf shapes reveal previously unreported functions of lg1 in both normal leaf development and in the dominant Wab1mutant.

Mature maize leaves have defined cell types along the proximal distal and medial lateral axes. The patterning events that establish these axes take place early in leaf initiation. We have identified a new dominant mutation, Wavy auricle in blade1 (Wab1), which affects patterning in both axes in a dose-dependent manner. Wab1 leaves are narrower than normal leaves and displace proximal tissues distally. We show that the proximal distal patterning defects are not due to misexpression of knox genes. Genetic analyses suggest that the action of dominant Wab1 alleles is localized to a lateral domain of the leaf, located between the midvein and the marginal domain that is determined by narrow sheath function. Thus, Wab1 defines a knox-independent pathway that affects specification of the proximal distal axis of the maize leaf. We suggest that failure to elaborate a normal lateral domain in the Wab1 leaf is responsible for disrupting patterning of the proximal distal axis.

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.

Plant architecture results from the activity of the shoot apical meristem, which initiates leaves, internodes, and axillary meristems. KNOTTED1-like homeobox (KNOX) genes are expressed in specific patterns in the shoot apical meristem and play important roles in plant architecture. KNOX proteins interact with BEL1-like (BELL) homeodomain proteins and together bind a target sequence with high affinity. We have obtained a mutation in one of the Arabidopsis BELL genes, PENNYWISE (PNY), that appears phenotypically similar to the KNOX mutant brevipedicellus (bp). Both bp and pny have randomly shorter internodes and display a slight increase in the number of axillary branches. The double mutant shows a synergistic phenotype of extremely short internodes interspersed with long internodes and increased branching. PNY is expressed in inflorescence and floral meristems and overlaps with BP in a discrete domain of the inflorescence meristem where we propose the internode is patterned. The physical association of the PNY and BP proteins suggests that they participate in a complex that regulates early patterning events in the inflorescence meristem.

Members of theKNOXgene family have important roles in plant meristems by regulating cell division and differentiation.BREVIPEDICELLUS(BP), one of sevenKNOXgenes inArabidopsis, has a primary role in internode patterning. We carried out a comparison of RNA expression profiles between wild-type seedlings andbpmutants at a developmental stage prior to a visible phenotypic difference. Transcript differences were found for a number of genes in cell wall biosynthesis, especially genes in the lignin pathway. The regulation of lignin biosynthesis byBPwas demonstrated by observing increased lignin deposition inbpmutants following bolting, decreased lignification in plants overexpressingBP, and aberrant lignin deposition in discrete regions of thebpstem. Furthermore, we showed that BP binds promoters of some genes in the lignin pathway. Our results provide a metabolic fingerprint forBPand identify the lignin pathway as one of the coordinate processes thatBPregulates.