The function of RNAi in plant development.

The morphological phenotype of mutations in genes required for posttranscriptional gene silencing (PTGS) or RNA interference (RNAi) in Arabidopsis demonstrates that this process is critical for normal development. One way in which RNAi contributes to gene regulation is through its involvement in the biogenesis of trans-acting small interfering RNAs (siRNAs). These endogenous siRNAs are derived from noncoding transcripts that are cleaved by a microRNA (miRNA) and mediate the silencing of protein-coding transcripts. Some protein-coding genes are also subject to miRNA-initiated transitive silencing. Several developmentally important transcription factors regulated by these silencing mechanisms have been identified.

KANADI regulates organ polarity in Arabidopsis.

Leaves and floral organs are polarized along their adaxial-abaxial (dorsal-ventral) axis. In Arabidopsis, this difference is particularly obvious in the first two rosette leaves, which possess trichomes (leaf hairs) on their adaxial surface but not their abaxial surface. Mutant alleles of KANADI (KAN) were identified in a screen for mutants that produce abaxial trichomes on these first two leaves. kan mutations were originally identified as enhancers of the mutant floral phenotype of crabs claw (crc), a gene that specifies abaxial identity in carpels. Here we show that KAN is required for abaxial identity in both leaves and carpels, and encodes a nuclear-localized protein in the GARP family of putative transcription factors. The expression pattern of KAN messenger RNA and the effect of ectopically expressing KAN under the regulation of the cauliflower mosaic virus (CAMV) 35S promoter indicate that KAN may also specify peripheral identity in the developing embryo.

Regulation of vegetative phase change in Arabidopsis thaliana by cyclophilin 40.

During its development, a plant shoot progresses from a juvenile to an adult phase of vegetative growth and from a reproductively incompetent to a reproductively competent state. In Arabidopsis, loss-of-function mutations in SQUINT (SQN) reduced the number of juvenile leaves and had subtle effects on inflorescence morphology but had no effect on flowering time or on reproductive competence. SQN encodes the Arabidopsis homolog of cyclophilin 40 (CyP40), a protein found in association with the Hsp90 chaperone complex in yeast, mammals, and plants. Thus, in Arabidopsis, CyP40 is specifically required for the vegetative but not the reproductive maturation of the shoot.

Phase identity of the maize leaf is determined after leaf initiation.

The vegetative development of the maize shoot can be divided into juvenile and adult phases based on the types of leaves produced at different times in shoot development. Models for the regulation of phase change make explicit predictions about when the identity of these types of leaves is determined. To test these models, we examined the timing of leaf type determination in maize. Clones induced in transition leaf primordia demonstrated that the juvenile and adult regions of these leaves do not become clonally distinct until after the primordium is 700 microm in length, implying that these cell fates were undetermined at this stage of leaf development. Adult shoot apices were cultured in vitro to induce rejuvenation. We found that leaf primordia as large as 3 mm in length can be at least partially rejuvenated by this treatment, and the location of rejuvenated tissue is correlated with the maturation pattern of the leaf. The amount and distribution of juvenile tissue in rejuvenated leaves suggests that rejuvenation occurs nearly simultaneously in all leaf primordia. In vitro culture rejuvenated existing leaf primordia and the P0 primordium, but did not change the identity of subsequent primordia or the total number of leaves produced by the shoot. This result suggests that leaf identity can be regulated independently of the identity of the shoot apical meristem, and it implies that vegetative phase change is not initiated by a change in the identity of the shoot apical meristem.

HASTY: a gene that regulates the timing of shoot maturation in Arabidopsis thaliana.

In Arabidopsis thaliana, leaves produced at different stages of shoot development can be distinguished by the distribution of trichomes on the abaxial and adaxial surfaces. Leaves produced early in the development of the rosette (juvenile leaves) have trichomes on their adaxial, but not their abaxial surface, whereas leaves produced later in rosette development (adult leaves) have trichomes on both surfaces. In order to identify genes that regulate the transition between these developmental phases we screened for mutations that accelerate the production of leaves with abaxial trichomes. 9 alleles of the HASTY gene were recovered in this screen. In addition to accelerating the appearance of adult leaves these mutations also accelerate the loss of adaxial trichomes (a trait typical of bracts), reduce the total number of leaves produced by the shoot, and have a number of other effects on shoot morphology. The basis for this phenotype was examined by testing the interaction between hasty and genes that affect flowering time (35S::LEAFY, 35S::APETALA1, terminal flower1), gibberellin production (ga1-3) or perception (gai), and floral morphogenesis (leafy, apetala1, agamous). We found that hasty increased the reproductive competence of the shoot, and that its does not require gibberellin or a gibberellin response for its effect on vegetative or reproductive development. The phenotype of hasty is not suppressed by leafy, apetala1 and agamous, demonstrating that this phenotype does not result from the inappropriate expression of these genes. We suggest that HASTY promotes a juvenile pattern of vegetative development and inhibits flowering by reducing the competence of the shoot to respond to LEAFY and APETALA1.

The specification of leaf identity during shoot development.

A single plant produces several different types of leaves or leaf-like organs during its life span. This phenomenon, which is termed heteroblasty, is an invariant feature of shoot development but is also regulated by environmental factors that affect the physiology of the plant. Invariant patterns of heteroblastic development reflect global changes in the developmental status of the shoot, such as the progression from embryogenesis through juvenile and adult phases of vegetative development, culminating in the production of reproductive structures. Genes that regulate these phase-specific aspects of leaf identity have been identified by mutational analysis in both maize and Arabidopsis. These mutations have revealed that leaf production is regulated independently of leaf identity, implying that the identity of a leaf at a particular position on the shoot may depend on when the leaf was initiated in relation to a temporal program of shoot development.

Mutations of Arabidopsis thaliana that transform leaves into cotyledons.

We describe mutations of three genes in Arabidopsis thaliana-extra cotyledon1 (xtc1), extra cotyledon2 (xtc2), and altered meristem programming1 (amp1)-that transform leaves into cotyledons. In all three of these mutations, this transformation is associated with a change in the timing of events in embryogenesis. xtc1 and xtc2 delay the morphogenesis of the embryo proper at the globular-to-heart transition but permit the shoot apex to develop to an unusually advanced stage late in embryogenesis. Both mutations have little or no effect on seed maturation and do not affect the viability of the shoot or the rate of leaf initiation after germination. amp1 perturbs the pattern of cell division at an early globular stage, dramatically increases the size of the shoot apex and, like xtc1 and xtc2, produces enlarged leaf primordia during seed development. These unusual phenotypes suggest that these genes play important regulatory roles in embryogenesis and demonstrate that the development of the shoot apical meristem and the development of the embryo proper are regulated by independent processes that must be temporally coordinated to ensure normal organ identity.

Phase change and the regulation of trichome distribution in Arabidopsis thaliana.

Higher plants pass through several phases of shoot growth during which they may produce morphologically distinct vegetative structures. In Arabidopsis thaliana this phenomenon is apparent in the distribution of trichomes on the leaf surface. Leaves produced early in rosette development lack trichomes on their abaxial (lower) surface, leaves produced later have trichomes on both surfaces, and leaves in the inflorescence (bracts) may have few or no trichomes on their adaxial (upper) surface. Here we describe some of the factors that regulate this distribution pattern. We found that the timing of abaxial trichome production and the extent to which bracts lack adaxial trichomes varies in different ecotypes. The production of abaxial trichomes appears to be regulated by the age, rather than the size of the plant. This conclusion is based on the observation that mutations that affect either the rate (altered meristem programming1) or onset (paused) of leaf initiation respectively increase or decrease the number of leaves that lack abaxial trichomes, but have only a minor effect on the time at which the first leaf with abaxial trichomes is produced. The production of abaxial trichomes is coordinated with the reproductive development of the shoot as this trait is delayed by photoperiodic conditions and some mutations that delay flowering. The loss of adaxial trichomes is likely to be a consequence of floral induction, and is accelerated by terminal flower1-10, a mutation that accelerates inflorescence development. We demonstrate that gibberellins promote trichome production in Arabidopsis and present evidence indicating that abaxial trichome production is regulated by both the level of a trichome inducer and the competence of the abaxial epidermis to respond to this inducer.

Shoot development in plants: time for a change.

The shoot system in plants progresses through several discrete phases during its development. Changes in the timing of these phases have important consequences for the morphogenesis of the shoot and are likely to be important in plant evolution. Genetic analysis of phase change in herbaceous plants, such as maize and Arabidopsis, has defined some of the genes involved in this phenomenon and has suggested a model for the regulation of this key feature of plant development.

Gibberellins promote vegetative phase change and reproductive maturity in maize.

Postembryonic shoot development in maize (Zea mays L.) is divided into a juvenile vegetative phase, an adult vegetative phase, and a reproductive phase that differ in the expression of many morphological traits. A reduction in the endogenous levels of bioactive gibberellins (GAs) conditioned by any one of the dwarf1, dwarf3, dwarf5, or anther ear1 mutations in maize delays the transition from juvenile vegetative to adult vegetative development and from adult vegetative to reproductive development. Mutant plants cease producing juvenile traits (e.g. epicuticular wax) and begin producing adult traits (e.g. epidermal hairs) later than wild-type plants. They also cease producing leaves and begin producing reproductive structures later than wild-type plants. These mutations greatly enhance most aspects of the phenotype of Teopod1 and Teopod2, suggesting that GAs suppress part but not all of the Teopod phenotype. Application of GA3 to Teopod2 mutants and Teopod1, dwarf3 double mutants confirms this result. We conclude that GAs act in conjunction with several other factors to promote both vegetative and reproductive maturation but affect different developmental phases unequally. Furthermore, the GAs that regulate vegetative and reproductive maturation, like those responsible for stem elongation, are downstream of GA20 in the GA biosynthetic pathway.

Heterochronic effects of glossy15 mutations on epidermal cell identity in maize.

Vegetative development in maize is divided into a juvenile phase and an adult phase that differ in the expression of a large number of morphological, anatomical, and biochemical traits. Recessive mutations of Glossy15 cause a premature switch in the expression of some of these phase-specific traits. Mutant plants cease producing juvenile traits (e.g. epicuticular wax) and begin to produce adult traits (e.g. epidermal hairs) significantly earlier than their wild-type siblings. In glossy15-1 plants this switch generally occurs at leaf 2 or 3 rather than at the normal position of leaf 6 or 7. An analysis of the effect of glossy15 mutations on a variety of vegetative and reproductive traits revealed that these mutations only affect the character of the epidermis. They have no effect on the overall vegetative morphology of the plant, or on its reproductive development. This phenotype is the opposite of that of the gain-of-function mutations Teopod1, Teopod2 and Teopod3, all of which prolong the expression of a large number of juvenile traits. Double mutants between glossy15 and Teopod1 or Teopod2 indicate that Glossy15 is required for the effect of Teopod1 and Teopod2 on epidermal traits but not for other aspects of the Teopod phenotype. We conclude that Glossy15 initiates or maintains the expression of juvenile epidermal traits and suppresses the expression of adult epidermal traits, and that it acts downstream of the Teopod genes.

The heterochronic Teopod1 and Teopod2 mutations of maize are expressed non-cell-autonomously.

Teopod1 and Teopod2 are dominant, unlinked mutations in maize that cause dramatic morphological abnormalities, including inappropriate expression of juvenile traits in adult vegetative phytomers and the transformation of reproductive structures into vegetative ones. These phenotypes are consistent with the constitutive expression of a juvenile phase of development throughout shoot growth. To investigate the basis of the Tp1 and Tp2 phenotypes we have analyzed their cell-autonomy in mosaic Teopod:wild-type plants. Mosaic plants were generated by three different mechanisms. Tp1 has previously been shown to be non-cell-autonomous; to verify and extend these results, large wild-type sectors were generated on Tp1 plants by the spontaneous loss of a B-A translocation chromosome containing the Tp1 gene. Analysis of Tp2 cell-autonomy was complicated by a lack of useful markers on chromosome 10L proximal to Tp2. To circumvent this problem two strategies were used. A reciprocal translocation was used to link Tp2 the wild-type allele of lw2. Sectors were induced in plants of this type by irradiation of imbibed seeds. Also, a chromosome-breaking Ds element located proximal to Tp2 was used to generate somatic sectors that uncovered w2, an albino mutation distal to Tp2. Our results demonstrate conclusively that both Tp1 and Tp2 are non-cell-autonomous. The general use of these techniques for clonal analysis in plants and the potential role of a diffusible factor in regulating the juvenile phase of development in maize are discussed.

Heterochronic Effects of Teopod 2 on the Growth and Photosensitivity of the Maize Shoot.

Teopod 2 (Tp2) is a semidominant mutation of maize that prolongs the expression of juvenile vegetative traits, increases the total number of leaves produced by the shoot, and transforms reproductive structures into vegetative ones. Here, we show that Tp2 prolongs the duration of vegetative growth without prolonging the overall duration of shoot growth. Mutant shoots produce leaves at the same rate as wild-type plants and continue to produce leaves after wild-type plants have initiated a tassel. Although Tp2/+ plants initiate a tassel later than their wild-type siblings, this mutant tassel ceases differentiation at the same time as, or shortly before, the primary meristem of a wild-type tassel completes its development. To investigate the relationship between the vegetative and reproductive development of the shoot, Tp2/+ and wild-type plants were exposed to floral inductive short day (SD) treatments at various stages of shoot growth. Tassel initiation in wild-type plants (which normally produced 18 to 19 leaves) was maximally sensitive to SD between plastochrons 15 and 16, whereas tassel branching was maximally sensitive to SD between plastochrons 15 and 18. Tassel initiation and tassel morphology in Tp2/+ plants (which normally produced 21 to 26 leaves) were both maximally sensitive to SD between plastochrons 15 and 18. Thus, the constitutive expression of a juvenile vegetative program in Tp2/+ plants does not significantly delay the reproductive maturation of the shoot.

The effect of a heterochronic mutation, Teopod2, on the cell lineage of the maize shoot.

Teopod2 (Tp2) is a semi-dominant mutation of maize that prolongs the expression of characteristics normally confined to the juvenile phase of development. Two of the many dramatic morphological effects of this mutation are an increase in the number of vegetative nodes, and a reduction in the overall size of the shoot. To determine the cellular basis of these phenotypes, the technique of clonal analysis was used to compare the cell division patterns of wild-type and Tp2 plants. Our results indicate that Tp2 increases the number of vegetative nodes produced by the apicalmost cells in the meristem but does not affect the cell lineage of the basal, juvenile, part of the shoot. This result demonstrates that Tp2 does not act uniquely in a 'juvenile' domain of the meristem, but instead causes cells that are normally destined to produce adult structures to express juvenile traits inappropriately. Clonal analysis also demonstrates that Tp2 does not affect the size of the meristem prior to germination, nor does it affect the cell lineage of the basic structural unit of the stem, the phytomer. Thus the effects of this mutation on the size of the shoot are the result of changes in cell fate late in development.

Phase change and the regulation of shoot morphogenesis in plants.

The shoot system of higher plants passes through several different phases during its development. Each of these phases is characterized by a unique set of morphological and physiological attributes. The intermediate character of the structures produced during phase changes and the phenotypes of mutations that affect this process demonstrate that these phases are specified by independently regulated, overlapping developmental programs. Transitions between phases appear to be initiated by factors extrinsic to the shoot apical meristem; the ability of the shoot to respond to such factors and to remain in a particular phase of development is regulated by factors intrinsic to the meristem. The possibility that developmental phases are maintained by epigenetic cell states and the role of DNA methylation in this process are discussed.

The liguleless-1 gene acts tissue specifically in maize leaf development.

The liguleless-1 (lg1) gene affects maize leaf development. In a normal maize leaf, a ligule and auricles separate the blade and sheath. The recessive lg1 mutation prevents formation of ligules and auricles during leaf development. To determine the timing and site of lg1 gene action, we compared development of wild-type and lg1 mutant leaves, and analyzed genetic mosaics composed of wild-type and lg1 mutant cells. In wild-type leaves the first sign of differentiation of the ligular region is a series of specialized anticlinal divisions in the adaxial epidermis. This establishes a distinct band of cells, from which the ligule arises via periclinal divisions. The anticlinal divisions preceding ligule formation are altered in the mutant; therefore, the gene acts early in development, before the periclinal divisions, and possibly during basipetal vascularization. Genetic mosaic analysis indicates that the lg1 gene has at least two functions with different tissue specificities: The Lg1+ wild-type allele acts autonomously in the adaxial epidermis for normal ligule development, and in internal tissues for auricle formation. Wild-type internal tissue in direct contact with lg1 epidermis appears able to induce the mutant epidermis to form a rudimentary ligule. The results indicate that the lg1 gene acts tissue specifically in an early step of ligule and auricle initiation.

Heterochronic mutations affecting shoot development in maize.

Three semidominant, nonallelic mutations of maize, Teopod 1 (Tp1), Teopod 2 (Tp2) and Teopod 3 (Tp3), have a profound effect on both vegetative and reproductive development. Although each mutation is phenotypically distinct, they all (1) increase the number of vegetative phytomers; (2) increase the number of phytomers producing ears, tillers and prop roots; (3) increase the number of leaves bearing epidermal wax; (4) decrease the size of leaves and internodes; (5) decrease the size of both the ear and tassel; and (6) transform reproductive structures into vegetative ones. The analysis presented here suggests that this phenotype reflects the prolonged expression of a juvenile, vegetative developmental program which overlaps with the reproductive developmental program. The expression of these mutations is different in each of the four inbred backgrounds used in this study. Tp1 and Tp2 have similar phenotypes and are more highly expressed in the A632 and Oh51a inbred backgrounds than in W23 and Mo17. Tp3 has less extreme effects than either of these mutations and has the opposite modification pattern; i.e., it is more highly expressed in W23 and Mo17 than in A632 and Oh51a. The expression of Tp1 and Tp2 in the presence of varying doses of their wild-type alleles indicate that both are gain-of-function mutations. The phenotypes of Tp1 and Tp2 and the nature of their response to variation in gene dose suggest that they control related, but nonidentical functions. The developmental and evolutionary implications of the heterochronic phenotype of these mutations is discussed.

Cell-lineage patterns in the shoot apical meristem of the germinating maize embryo.

A fate map for the shoot apical meristem of Zea mays L. at the time of germination was constructed by examining somatic sectors (clones) induced by γ-rays. The shoot apical meristem produced stem, leaves, and reproductive structures above leaf 6 after germination and the analysis here concerns their formation. On 160 adult plants which had produced 17 or 18 leaves, 277 anthocyanin-deficient sectors were scored for size and position. Sectors found on the ear shoot or in the tassel most often extended into the vegetative part of the plant. Sectors ranged from one to six internodes in length and some sectors of more than one internode were observed at all positions on the plant. Single-internode sectors predominated in the basal internodes (7,8,9) while longer sectors were common in the middle and upper internodes. The apparent number of cells which gave rise to a particular internode was variable and sectors were not restricted to the lineage unit: a leaf, the internode below it, and the axillary bud and prophyll at the base of the internode. These observations established two major features of meristem activity: 1) at the time of germination the developmental fate of any cell or group of cells was not fixed, and 2) at the time of germination cells at the same location in a meristem could produce greatly different amounts of tissue in the adult plant. Consequently, the developmental fate of specific cells in the germinating meristem could only be assigned in a general way.