Site specificity of the Arabidopsis METI DNA methyltransferase demonstrated through hypermethylation of the superman locus.

Plants with low levels of DNA methylation show a range of developmental abnormalities including homeotic transformation of floral organs. Two independent DNA METHYLTRANSFERASEI (METI) antisense transformants with low levels of DNA methylation had flowers with increased numbers of stamens which resembled flowers seen on the loss-of-function superman (sup) mutant plants and on transgenic plants that ectopically express APETALA3 (AP3). These METI antisense plants have both increased and decreased methylation in and around the sup gene, compared with untransformed controls. DNA from the antisense plants was demethylated at least 4 kb upstream of the sup gene, while there was dense methylation around the start of transcription and within the coding region of this gene; these regions were unmethylated in control DNA. Methylation within the sup gene was correlated with an absence of SUP transcripts. The pattern and density of methylation was heterogeneous among different DNA molecules from the same plant, with some molecules being completely unmethylated. Methylcytosine occurred in asymmetric sites and in symmetric CpA/TpG but rarely in CpG dinucleotides in the antisense plants. In contrast, segregants lacking the METI antisense construct and epimutants with a hypermethylated allele of sup (clark kent 3), both of which have active METI genes, showed a higher frequency of methylation of CpG dinucleotides and of asymmetric cytosines. We conclude that METI is the predominant CpG methyltransferase and directly or indirectly affects asymmetric methylation.

Overexpression of a gene encoding a cytochrome P450, CYP78A9, induces large and seedless fruit in arabidopsis.

An activation tagging screen in which the cauliflower mosaic virus 35S enhancer was inserted randomly into an Arabidopsis genome homozygous for the floral homeotic mutation apetala2-1 (ap2-1) resulted in a line (28-5) with extraordinarily wide, heart-shaped ovaries. The ovary of the 28-5 ap2-1 mutant shows an oval shape because of increased numbers of enlarged cells. When the ap2-1 mutation is crossed out of the genetic background, more elongated rather than wider fruits are obtained. Normally, Arabidopsis fruits will develop to a normal size only when the ovules are present and fertilized. In the 28-5 single mutant, the siliques keep growing despite failure of fertilization and can reach nearly normal size. When wild-type pollen was used to pollinate the mutant pistil, the pollinated 28-5 silique became >10% longer and 40% wider than a wild-type silique, although producing very few seeds. The enhancer insertion in line 28-5 acts by hyperactivating a cytochrome P450 gene, CYP78A9. The pistil of 28-5 ap2-1 mutant flowers shows a structure similar to that of Capsella bursa-pastoris, a distant mustard relative of Arabidopsis, suggesting that the processes regulated by the CYP78A9-encoded protein may be involved in evolutionary control of carpel shape.

Regulation of SUP expression identifies multiple regulators involved in arabidopsis floral meristem development.

During the course of flower development, floral homeotic genes are expressed in defined concentric regions of floral meristems called whorls. The SUPERMAN (SUP, also called FLO10) gene, which encodes a C2H2-type zinc finger protein, is involved in maintenance of the stamen/carpel whorl boundary (the boundary between whorl 3 and whorl 4) in Arabidopsis. Here, we show that the regulation of SUP expression in floral meristems is complex, consisting of two distinct phases, initiation and maintenance. The floral meristem identity gene LEAFY (LFY) plays a role in the initiation phase through at least two pathways, which differ from each other in the involvement of two homeotic genes, APETALA3 (AP3) and PISTILLATA (PI). AP3, PI, and another homeotic gene, AGAMOUS (AG), are further required for SUP expression in the later maintenance phase. Aside from these genes, there are other as yet unidentified genes that control both the temporal and spatial patterns of SUP expression in whorl 3 floral meristems. SUP appears to act transiently, probably functioning to trigger a genetic circuit that creates the correct position of the whorl 3/whorl 4 boundary.

Dependence of stem cell fate in Arabidopsis on a feedback loop regulated by CLV3 activity.

The fate of stem cells in plant meristems is governed by directional signaling systems that are regulated by negative feedback. In Arabidopsis thaliana, the CLAVATA (CLV) genes encode the essential components of a negative, stem cell-restricting pathway. We used transgenic plants overexpressing CLV3 to show that meristem cell accumulation and fate depends directly on the level of CLV3 activity and that CLV3 signaling occurs exclusively through a CLV1/CLV2 receptor kinase complex. We also demonstrate that the CLV pathway acts by repressing the activity of the transcription factor WUSCHEL, an element of the positive, stem cell-promoting pathway.

Cloning of the Arabidopsis WIGGUM gene identifies a role for farnesylation in meristem development.

Control of cellular proliferation in plant meristems is important for maintaining the correct number and position of developing organs. One of the genes identified in the control of floral and apical meristem size and floral organ number in Arabidopsis thaliana is WIGGUM. In wiggum mutants, one of the most striking phenotypes is an increase in floral organ number, particularly in the sepals and petals, correlating with an increase in the width of young floral meristems. Additional phenotypes include reduced and delayed germination, delayed flowering, maturation, and senescence, decreased internode elongation, shortened roots, aberrant phyllotaxy of flowers, aberrant sepal development, floral buds that open precociously, and occasional apical meristem fasciation. As a first step in determining a molecular function for WIGGUM, we used positional cloning to identify the gene. DNA sequencing revealed that WIGGUM is identical to ERA1 (enhanced response to abscisic acid), a previously identified farnesyltransferase beta-subunit gene of Arabidopsis. This finding provides a link between protein modification by farnesylation and the control of meristem size. Using in situ hybridization, we examined the expression of ERA1 throughout development and found it to be nearly ubiquitous. This extensive expression domain is consistent with the pleiotropic nature of wiggum mutants and highlights a broad utility for farnesylation in plant growth and development.

Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana.

We investigated the potential of double-stranded RNA interference (RNAi) with gene activity in Arabidopsis thaliana. To construct transformation vectors that produce RNAs capable of duplex formation, gene-specific sequences in the sense and antisense orientations were linked and placed under the control of a strong viral promoter. When introduced into the genome of A. thaliana by Agrobacterium-mediated transformation, double-stranded RNA-expressing constructs corresponding to four genes, AGAMOUS (AG), CLAVATA3, APETALA1, and PERIANTHIA, caused specific and heritable genetic interference. The severity of phenotypes varied between transgenic lines. In situ hybridization revealed a correlation between a declining AG mRNA accumulation and increasingly severe phenotypes in AG (RNAi) mutants, suggesting that endogenous mRNA is the target of double-stranded RNA-mediated genetic interference. The ability to generate stably heritable RNAi and the resultant specific phenotypes allows us to selectively reduce gene function in A. thaliana.

Ectopic hypermethylation of flower-specific genes in Arabidopsis.

BACKGROUND: Arabidopsis mutations causing genome-wide hypomethylation are viable but display a number of specific developmental abnormalities, including some that resemble known floral homeotic mutations. We previously showed that one of the developmental abnormalities present in an antisense-METHYLTRANSFERASEI (METI) transgenic line resulted from ectopic hypermethylation of the SUPERMAN gene. RESULTS: Here, we investigate the extent to which hypermethylation of SUPERMAN occurs in several hypomethylation mutants, and describe methylation effects at a second gene, AGAMOUS. SUPERMAN gene hypermethylation occurred at a high frequency in several mutants that cause overall decreases in genomic DNA methylation. The hypermethylation pattern was largely similar in the different mutant backgrounds. Genetic analysis suggests that hypermethylation most likely arose either during meiosis or somatically in small sectors of the plant. A second floral development gene, AGAMOUS, also became hypermethylated and silenced in an Arabidopsis antisense-METI line. CONCLUSIONS: These results suggest that ectopic hypermethylation of specific genes in mutant backgrounds that show overall decreases in methylation may be a widespread phenomenon that could explain many of the developmental defects seen in Arabidopsis methylation mutants. This resembles a phenomenon seen in cancer cells, which can simultaneously show genome-wide hypomethylation and hypermethylation of specific genes. Comparison of the methylated sequences in SUPERMAN and AGAMOUS suggests that hypermethylation could involve DNA secondary structures formed by pyrimidine-rich sequences.

Cell signaling within the shoot meristem.

Shoot apical meristems are self-renewing stem cell populations that generate all of the above-ground organs (i.e. stems, leaves and flowers) of higher plants. Recent studies have identified new molecular components required for proper shoot meristem activity, and they have revealed that complex, intercellular communication pathways play important roles in coordinating meristem function.

The plant plan: multicellular life in the other Kingdom.

One must conclude, then, that plants and animals may have evolved in quite different fashions. There is no doubt that they have independently evolved development, and this is demonstrated by the nonhomology of genes serving identical developmental functions in prepattern formation. There is nonetheless also no doubt that plants and animals have evolved from a common eukaryotic ancestor, as indicated by the clear homology of the genes that control the chromatin level of gene regulation. There is also little doubt that some developmentally important genes of plants, such as the ethylene and red light receptors, have derived from an event of horizontal evolutionary transfer specific to plants. And it is at least possible to think that the variation on which Darwinian evolution acts in plants results in part from phenomena that are not seen in animals, namely, the controlled appearance and Mendelian inheritance of epigenetically silenced genes. Genomic and genetic analyses of plants thus reveal a type of organism with familiar features, but profound differences from the more-studied animals. Only by further study of plants and of animals can we fully understand the differences between plants and animals, and consequently distinguish between those features of developmental pattern formation and cellular signaling that are necessary aspects of complex organisms, and those that are accidents of evolutionary history.

Plants, animals and the logic of development.

Multicellular plants and animals have evolved independently from a unicellular, last common ancestor. Each lineage started with a common toolkit of functioning genes and evolved to complex, multicellular forms. Comparison of the genes used to serve similar functions shows how organisms can use different genes for similar ends and thereby reveals the principles of development.

Disruption of an RNA helicase/RNAse III gene in Arabidopsis causes unregulated cell division in floral meristems.

Arabidopsis thaliana floral meristems are determinate structures that produce a defined number of organs, after which cell division ceases. A new recessive mutant, carpel factory (caf), converts the floral meristems to an indeterminate state. They produce extra whorls of stamens, and an indefinite number of carpels. Thus, CAF appears to suppress cell division in floral meristems. The function of CAF is partially redundant with the function of the CLAVATA (CLV) and SUPERMAN (SUP) genes, as caf clv and caf sup double mutants show dramatically enhanced floral meristem over-proliferation. caf mutant plants also show other defects, including absence of axillary inflorescence meristems, and abnormally shaped leaves and floral organs. The CAF gene was cloned and found to encode a putative protein of 1909 amino acids containing an N-terminal DExH/DEAD-box type RNA helicase domain attached to a C-terminal RNaseIII-like domain. A very similar protein of unknown function is encoded by a fungal and an animal genome. Helicase proteins are involved in a number of processes, including specific mRNA localization and mRNA splicing. RNase III proteins are involved in the processing of rRNA and some mRNA molecules. Thus CAF may act through some type of RNA processing event(s). CAF gives rise to two major transcripts of 2.5 and 6.2 kb. In situ hybridization experiments show that CAF RNA is expressed throughout all shoot tissues.

Non-AUG initiation of AGAMOUS mRNA translation in Arabidopsis thaliana.

The MADS box organ identity gene AGAMOUS (AG) controls several steps during Arabidopsis thaliana flower development. AG cDNA contains an open reading frame that lacks an ATG triplet to function as the translation initiation codon, and the actual amino terminus of the AG protein remains uncharacterized. We have considered the possibility that AG translation can be initiated at a non-AUG codon. Two possible non-AUG initiation codons, CUG and ACG, are present in the 5' region of AG mRNA preceding the highly conserved MADS box sequence. We prepared a series of AG genomic constructs in which these codons are mutated and assayed their activity in phenotypic rescue experiments by introducing them as transgenes into ag mutant plants. Alteration of the CTG codon to render it unsuitable for acting as a translation initiation site does not affect complementation of the ag-3 mutation in transgenic plants. However, a similar mutation of the downstream ACG codon prevents the rescue of the ag-3 mutant phenotype. Conversely, if an ATG is introduced immediately 5' to the disrupted ACG codon, the resulting construct fully complements the ag-3 mutation. The AG protein synthesized in vitro by initiating translation at the ACG position is active in DNA binding and is of the same size as the AG protein detected from floral tissues, whereas AG polypeptides with additional amino-terminal residues do not appear to bind DNA. These results indicate that translation of AG is initiated exclusively at an ACG codon and prove that non-AUG triplets may be efficiently used as the sole translation initiation site in some plant cellular mRNAs.

Transcriptional activation of APETALA1 by LEAFY.

Plants produce new appendages reiteratively from groups of stem cells called shoot apical meristems. LEAFY (LFY) and APETALA1 (AP1) are pivotal for the switch to the reproductive phase, where instead of leaves the shoot apical meristem produces flowers. Use of steroid-inducible activation of LFY demonstrated that early expression of AP1 is a result of transcriptional induction by LFY. This AP1 induction is independent of protein synthesis and occurs specifically in the tissues and at the developmental stage in which floral fate is assumed. Later expression of AP1 appears to be only indirectly affected by LFY.

HUA1 and HUA2 are two members of the floral homeotic AGAMOUS pathway.

The identities of the four floral organ types in an Arabidopsis flower are specified by the combinatorial activities of the floral homeotic A, B, and C function genes; AGAMOUS is the only known C function gene. We have identified two genes that interact with AG in the specification of floral structure, HUA1 and HUA2, from a screen for enhancers of a weak ag allele, ag-4. HUA1 and HUA2 are involved in all aspects of AG function. HUA2 encodes a novel protein that contains nuclear localization signals and signature motifs that suggest HUA2, like AG, may be a transcription factor. Molecular analyses suggest that HUA2 (and possibly HUA1) acts to facilitate AG action at the same hierarchical level as AG.

Genetic and physical characterization of a region of Arabidopsis chromosome 1 containing the CLAVATA1 gene.

With the advance of Arabidopsis as a model system for understanding plant genetics, development and biochemistry, a detailed description of the genome is necessary. As such, focused projects are underway to map and sequence the Arabidopsis nuclear genome. We have characterized a region of chromosome 1, surrounding the CLAVATA1 (CLV1) locus. Three (RFLP) clones were mapped relative to clv1-1, and were used to construct an ca. 700 kb yeast artificial chromosome (YAC) contig. Three cosmids spanning the CLV1 locus were analyzed and ca. 24 kb of genomic DNA was sequenced, including a continuous stretch of 18 kb. In addition to generating clones in this region of chromosome 1, we have analyzed the size, spacing and organization of several contiguous genes.

Signaling of cell fate decisions by CLAVATA3 in Arabidopsis shoot meristems.

In higher plants, organogenesis occurs continuously from self-renewing apical meristems. Arabidopsis thaliana plants with loss-of-function mutations in the CLAVATA (CLV1, 2, and 3) genes have enlarged meristems and generate extra floral organs. Genetic analysis indicates that CLV1, which encodes a receptor kinase, acts with CLV3 to control the balance between meristem cell proliferation and differentiation. CLV3 encodes a small, predicted extracellular protein. CLV3 acts nonautonomously in meristems and is expressed at the meristem surface overlying the CLV1 domain. These proteins may act as a ligand-receptor pair in a signal transduction pathway, coordinating growth between adjacent meristematic regions.

The PERIANTHIA gene encodes a bZIP protein involved in the determination of floral organ number in Arabidopsis thaliana.

Mutations in the PERIANTHIA (PAN) gene of Arabidopsis thaliana specifically transform flowers from tetramerous to largely pentamerous, which is a characteristic of flowers of ancestral plants. We have cloned the PAN gene and here we show that it encodes a member of the basic region/leucine zipper class of transcription factors. Immunohistochemical analysis shows that the encoded protein is present in the apical meristem, the floral meristem, each whorl of organ primordia, and in ovule primordia during wild-type flower development. PAN expression occurs independently of genes affecting floral meristem identity, floral meristem size, or floral organ number. The near absence of a phenotype in transgenic plants overexpressing PAN and the contrast between the broad expression of PAN and the specificity of its mutant phenotype suggest that its activity may be regulated post-translationally or by the presence of partner proteins. Based on these results and on data reported previously, we propose models for the role of PAN in the evolution of flower pattern in the mustard family.

Non-autonomy of AGAMOUS function in flower development: use of a Cre/loxP method for mosaic analysis in Arabidopsis.

Angiosperms use a multi-layered meristem (typically L1, L2 and L3) to produce primordia that then develop into plant organs. A number of experiments show that communication between the cell layers is important for normal development. We examined whether the function of the flower developmental control gene AGAMOUS involves communication across these layers. We developed a mosaic strategy using the Cre/loxP site-specific recombinase system, and identified the sector structure for mosaics that produced mutant flowers. The major conclusions were that (1) AGAMOUS must be active in the L2 for staminoid and carpelloid tissues, (2) that AGAMOUS must be active in the L2 and the L3 for floral meristem determinacy, and (3) that epidermal cell identity can be communicated by the L2 to the L1 layer.