The Arabidopsis embryonic shoot fate map.

A fate map has been constructed for the shoot apical region of the embryo of the dicotyledonous plant Arabidopsis thaliana using spontaneously arising clonal albino sectors caused by the chloroplast mutator 1-2 mutation. Chimeric seedlings exhibiting albino sectors shared between the cotyledons and first true leaves revealed patterns of organ inclusion and exclusion. Frequencies of clone sharing were used to calculate developmental distances between organs based on the frequency of clonal sectors failing to extend between different organs. The resulting fate map shows asymmetry in the developmental distances between the cotyledons (embryonic leaves) which in turn predicts the location of the first post-germination leaf and the handedness of the spiral of leaf placement around the central stem axis in later development. The map suggests that embryonic leaf fate specification in the cotyledons may represent a developmental ground state necessary for the formation of the shoot apical meristem.

Preservation of duplicate genes by complementary, degenerative mutations.

The origin of organismal complexity is generally thought to be tightly coupled to the evolution of new gene functions arising subsequent to gene duplication. Under the classical model for the evolution of duplicate genes, one member of the duplicated pair usually degenerates within a few million years by accumulating deleterious mutations, while the other duplicate retains the original function. This model further predicts that on rare occasions, one duplicate may acquire a new adaptive function, resulting in the preservation of both members of the pair, one with the new function and the other retaining the old. However, empirical data suggest that a much greater proportion of gene duplicates is preserved than predicted by the classical model. Here we present a new conceptual framework for understanding the evolution of duplicate genes that may help explain this conundrum. Focusing on the regulatory complexity of eukaryotic genes, we show how complementary degenerative mutations in different regulatory elements of duplicated genes can facilitate the preservation of both duplicates, thereby increasing long-term opportunities for the evolution of new gene functions. The duplication-degeneration-complementation (DDC) model predicts that (1) degenerative mutations in regulatory elements can increase rather than reduce the probability of duplicate gene preservation and (2) the usual mechanism of duplicate gene preservation is the partitioning of ancestral functions rather than the evolution of new functions. We present several examples (including analysis of a new engrailed gene in zebrafish) that appear to be consistent with the DDC model, and we suggest several analytical and experimental approaches for determining whether the complementary loss of gene subfunctions or the acquisition of novel functions are likely to be the primary mechanisms for the preservation of gene duplicates. For a newly duplicated paralog, survival depends on the outcome of the race between entropic decay and chance acquisition of an advantageous regulatory mutation. Sidow 1996(p. 717) On one hand, it may fix an advantageous allele giving it a slightly different, and selectable, function from its original copy. This initial fixation provides substantial protection against future fixation of null mutations, allowing additional mutations to accumulate that refine functional differentiation. Alternatively, a duplicate locus can instead first fix a null allele, becoming a pseudogene. Walsh 1995 (p. 426) Duplicated genes persist only if mutations create new and essential protein functions, an event that is predicted to occur rarely. Nadeau and Sankoff 1997 (p. 1259) Thus overall, with complex metazoans, the major mechanism for retention of ancient gene duplicates would appear to have been the acquisition of novel expression sites for developmental genes, with its accompanying opportunity for new gene roles underlying the progressive extension of development itself. Cooke et al. 1997 (p. 362)

Temperature-sensitive mutations that arrest Arabidopsis shoot development.

To identify genes involved in meristem function we have designed a screen for temperature-sensitive mutations that cause a conditional arrest of early shoot development in Arabidopsis. We describe the characterization of three mutations, arrested development (add) 1, 2 and 3. At the restrictive temperature the add1 and add2 mutations disrupt apical meristem function as assayed by leaf initiation. Furthermore, add1 and add2 plants exhibit defects in leaf morphogenesis following upshift from permissive to restrictive temperature. This result suggests that proximity to a functional meristem is required for the completion of normal leaf morphogenesis. The add3 mutation does not have a dramatic effect on the production of leaves by the apical meristem; however, add3 prevents the expansion of leaf blades at high temperature. Thus, in this mutant the temperature-dependent arrest of epicotyl development is due to a failure of normal leaf development rather than new leaf initiation. While all add mutants have a reduced rate of root growth in comparison to wild-type plants, the mutants do not display a temperature-dependent arrest of root development. All add mutants display some developmental defects at low temperature, suggesting that these mutations affect genes involved in inherently temperature-sensitive developmental processes.

Mutations in the AXR3 gene of Arabidopsis result in altered auxin response including ectopic expression from the SAUR-AC1 promoter.

A new auxin response gene in Arabidopsis called AXR3 has been identified. This gene is defined by two semi-dominant mutations which affect many auxin-regulated developmental processes. Auxin has been shown to maintain apical dominance, inhibit root elongation, stimulate adventitious rooting, mediate root gravitropism, and stimulate transcription from the SAUR-AC1 promoter. Mutant axr3 plants show enhanced apical dominance, reduced root elongation, increased adventitious rooting, no root gravitropism, and ectopic expression from the SAUR-AC1 promoter. These phenotypes suggest an increased auxin response in the mutants. In support of this hypothesis, many of the phenotypes are partially restored to wild-type by exogenous cytokinin, a treatment that could restore a more wild-type auxin to cytokinin ratio.

The AXR1 and AUX1 genes of Arabidopsis function in separate auxin-response pathways.

The recessive mutations aux1 and axr1 of Arabidopsis confer resistance to the plant hormone auxin. The axr1 mutants display a variety of morphological defects. In contrast, the only morphological defect observed in aux1 mutants is a loss of root gravitropism. To learn more about the function of these genes in auxin response, the expression of the auxin-regulated gene SAUR-AC1 in mutant and wild-type plants has been examined. It has been found that axr1 plants display a pronounced deficiency in auxin-induced accumulation of SAUR-AC1 mRNA in seedlings as well as rosette leaves and mature roots. In contrast, the aux1 mutation has a modest effect on auxin induction of SAUR-AC1. To determine if the AUX1 and AXR1 genes interact to facilitate auxin response, plants which are homozygous for both aux1 and axr1 mutations have been constructed and characterized. The two mutations are additive in their effects on auxin response, suggesting that each mutation confers resistance by a different mechanism. However, the morphology of double mutant plants indicates that there is an inter-action between the AXR1 and AUX1 genes. In mature plants, the aux1-7 mutation acts to partially suppress the morphological defects conferred by the axr1-12 mutation. This suppression is not accompanied by an increase in auxin response, as measured by SAUR-AC1 expression, suggesting that the interaction between the AUX1 and AXR1 genes is indirect.

The FLO10 Gene Product Regulates the Expression Domain of Homeotic Genes AP3 and PI in Arabidopsis Flowers.

We describe a novel mutant of Arabidopsis, Flo10, which is the result of a recessive allele, flo10, in the nuclear gene FLO10. The first three organ whorls (sepals, petals, and stamens) of Flo10 flowers are normal, but the fourth, gynoecial whorl is replaced by two to eight stamens or stamen-carpel intermediate organs. Studies of ontogeny suggest that the position of the first six of these fourth-whorl organs often resembles that of the wild-type third-whorl organs. To determine the interaction of the FLO10 gene with the floral organ homeotic genes APETALA2 (AP2), PISTILLATA (PI), AP3, and AGAMOUS (AG), we generated lines homozygous for flo10 and heterozygous or homozygous for a recessive allele of the homeotic genes. On the basis of our data, we suggest that FLO10 functions to prevent the expression of the AP3/PI developmental pathway in the gynoecial (fourth) whorl.

The aux1 Mutation of Arabidopsis Confers Both Auxin and Ethylene Resistance.

Mutagenized populations of Arabidopsis thaliana seedlings were screened for plants capable of root growth on inhibitory concentrations of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. Four of the mutant lines recovered from this screen display a defect in root gravitropism as well as hormone resistance. The aerial portions of these plants are similar to wild-type in appearance. Genetic analysis of these four mutants demonstrated that hormone resistance segregated as a recessive trait and that all four mutations were alleles of the auxin-resistant mutation aux1 [Maher HP, Martindale SJB (1980) Biochem Genet 18: 1041-1053]. These new mutants have been designated aux1-7, 1-12, 1-15, and 1-19. The sensitivity of wild-type and aux1-7 roots to indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid, and ethylene was determined. The results of these assays show that aux1-7 plants require a 12-fold (indole-3-acetic acid) or 18-fold (2,4-dichlorophenoxyacetic acid) higher concentration of auxin than wild-type for a 50% inhibition of root growth. In addition, ethylene inhibition of root growth in aux1-7 plants is approximately 30% that of wild-type at saturating ethylene concentrations. These results indicate that aux1 plants are resistant to both auxin and ethylene. We have also determined the effect of ethylene treatment on chlorophyll loss and peroxidase activity in the leaves of aux1 and wild-type plants. No difference between mutant and wild-type plants was observed in these experiments, indicating that hormone resistance in aux1 plants may be limited to root growth. Our studies suggest that the AUX1 gene may have a specific function in the hormonal regulation of gravitropism.

A dominant mutation in Arabidopsis confers resistance to auxin, ethylene and abscisic acid.

We have screened a large population of M2 seeds of Arabidopsis thaliana for plants which are resistant to exogenously applied indole-acetic acid (IAA). One of the resistant lines identified in this screen carries a dominant mutation which we have named axr2. Linkage analysis indicates that the axr2 gene lies on chromosome 3. Plants carrying the axr2 mutation are severe dwarfs and display defects in growth orientation of both the shoot and root suggesting that the mutation affects some aspect of gravitropic growth. In addition, the roots of axr2 plants lack root hairs. Growth inhibition experiments indicate that the roots of axr2 plants are resistant to ethylene and abscisic acid as well as auxin.