Susan D. Johnson

AUXIN RESPONSE FACTOR8 Is a Negative Regulator of Fruit Initiation in Arabidopsis

Fruit and seed formation in plants is normally initiated after pollination and fertilization, and, in the absence of fertilization, flowers senesce. In the Arabidopsis thaliana mutant fruit without fertilization, a mutation in AUXIN RESPONSE FACTOR8 (ARF8) results in the uncoupling of fruit development from pollination and fertilization and gives rise to seedless (parthenocarpic) fruit. Parthenocarpy was confirmed in two additional recessive alleles and was caused by mutations within the coding region of ARF8. Genetic experiments indicate that ARF8 acts as an inhibitor to stop further carpel development in the absence of fertilization and the generation of signals required to initiate fruit and seed development. Expression of ARF8 was found to be regulated at multiple levels, and transcriptional autoregulation of ARF8 was observed. Analysis of plants transformed with a transcriptional PARF8:β-glucuronidase (GUS) construct or a translational ARF8:GUS fusion construct displayed distinct developmental regulation of the reporter in floral tissues involved in pollination and fertilization and in the carpel wall. After fertilization, the level of GUS activity declined in the developing seed, while in unfertilized ovules that are destined to senesce, ARF8:GUS expression spread throughout the ovule. This is consistent with a proposed role for ARF8 in restricting signal transduction processes in ovules and growth in pistils until the fruit initiation cue.

Expression of aberrant forms of AUXIN RESPONSE FACTOR8 stimulates parthenocarpy in Arabidopsis and tomato.

Fruit initiation in Arabidopsis (Arabidopsis thaliana) is generally repressed until fertilization occurs. However, mutations in AUXIN RESPONSE FACTOR8 (ARF8) uncouple fruit initiation from fertilization, resulting in the formation of seedless, parthenocarpic fruit. Here we induced parthenocarpy in wild-type Arabidopsis by introducing either the mutant genomic (g) Atarf8-4 sequence or gAtARF8:β-glucuronidase translational fusion constructs by plant transformation. Silencing of endogenous AtARF8 transcription was not observed, indicating that the introduced, aberrant ARF8 transcripts were compromising the function of endogenous ARF8 and/or associated factors involved in suppressing fruit initiation. To analyze the role of ARF8 in tomato (Solanum lycopersicum) we initially emasculated 23 tomato cultivars to test for background parthenocarpy. Surprisingly, all had a predisposition to initiate fertilization-independent fruit growth. Expression of gAtarf8-4 in transgenic tomato (‘Monalbo’) resulted in a significant increase in the number and size of parthenocarpic fruit. Isolation of tomato ARF8 cDNA indicated significant sequence conservation with AtARF8. SlARF8 may therefore control tomato fruit initiation in a similar manner as AtARF8 does in Arabidopsis. Two SlARF8 cDNAs differing in size by 5 bp were found, both arising from the same gene. The smaller cDNA is a splice variant and is also present in Arabidopsis. We propose that low endogenous levels of the splice variant products might interfere with efficient formation/function of a complex repressing fruit initiation, thereby providing an explanation for the observed ovary expansion in tomato and also Arabidopsis after emasculation. Increasing the levels of aberrant Atarf8-4 transcripts may further destabilize formation/function of the complex in a dosage-dependent manner enhancing tomato parthenocarpic fruit initiation frequency and size and mimicking the parthenocarpic dehiscent silique phenotype found in homozygous Atarf8-4 mutants. Collectively these data suggest that similar mechanisms involving auxin signaling exist to inhibit parthenocarpic fruit set in tomato and Arabidopsis.

Sexual and apomictic development in Hieracium

Abstract Most members of the genus Hieracium are apomictic and set seed without fertilization, but sexual forms also exist. A cytological study was conducted on an apomictic accession of H. aurantiacum (A3.4) and also H. piloselloides (D3) to precisely define the cellular basis for apomixis. The apomictic events were compared with the sexual events in a self-incompatible isolate of H. pilosella (P4). All plants were maintained as vegetatively propagated lines each derived from a single plant. Sexual P4 exhibited characteristic events of polygonum-type embryo sac formation, showed no latent apomitic tendencies, and depended upon fertilization to set seed. In contrast, D3 and A3.4 were autonomous aposporous apomicts, forming both embryo and endosperm spontaneously inside an unreduced embryo sac. The two apomicts exhibited distinct mechanisms, but variation was also observed within each apomictic line. Seeds from apomicts often contained more than one embryo. A degree of developmental instability was also observed amongst germinated seedlings and included variation in meristem and cotyledon number, altered phyllotaxis, callus formation, and seedling fusion. In most cases abnormal seedlings developed into normal plants. Such phenomena were not observed following germination of hybrid seeds derived from crosses between sexual P4 and the apomictic plants. The three plants can now be used in inheritance studies and also to investigate the molecular mechanisms controlling apomixis.

KNUCKLES (KNU) encodes a C2H2 zinc-finger protein that regulates development of basal pattern elements of the Arabidopsis gynoecium

Flowers of the parthenocarpic knuckles mutant are conditionally male sterile and contain ectopic stamens and carpels that originate from placental tissue within developing gynoecia. The mutation was mapped to a 123 Kb interval on chromosome 5 using molecular markers. All aspects of the knuckles phenotype could be complemented by a genomic fragment from the region which contained the annotated MAC12.2 gene. A guanine to adenine transition within a predicted C2H2 zinc finger-encoding region of MAC12.2 causes the second of two critical zinc-binding cysteine residues to be replaced by a tyrosine. Transgenic plants in which translational fusions of the GUS reporter to KNUCKLES were driven by the presumptive KNUCKLES promoter indicate that the gene is expressed first in developing carpel primordia, and later in stamens and ovules of flower buds. In situ hybridization experiments showed a broader pattern of transcript localization, suggesting that post-transcriptional regulatory mechanisms may limit KNUCKLES protein accumulation and localization. Based on genetic evidence and the presence of a carboxy-terminal motif demonstrated by others to function as an active repression domain, we propose that KNUCKLES might function as a transcriptional repressor of cellular proliferation that regulates floral determinacy and relative size of basal pattern elements along the proximo-distal axis of the developing Arabidopsis gynoecium.

Apomixis is not developmentally conserved in related, genetically characterized Hieracium plants of varying ploidy

Abstract Apomixis is facultative in characterized members of the genus Hieracium. The three components that comprise the apomictic mechanism include apospory followed by autonomous embryo and endosperm formation. The time of aposporous embryo sac initiation and mode of embryo sac formation are different in Hieracium piloselloides (D3) and Hieracium aurantiacum (A3.4). Genetic studies have shown that a single dominant locus encodes all three components of apomixis in both species (Bicknell et al. 2000). We histologically examined a range of related, genetically characterized apomictic Hieracium plants derived from D3 and A3.4 to assess conservation of the apomictic mechanism in different genetic backgrounds. The plants varied in ploidy, and also in the amount of DNA introduced from sexual Hieracium pilosella (P4). An apomictic hybrid from a cross between the two apomicts was also examined. The developmental processes observed in the parental apomicts were not conserved in the examined plants and alterations occurred in the components of apomixis. One plant also exhibited adventitious embryony. The results show that other genetic factors can modify apomixis with respect to time of initiation, spatial location, and mode of developmental progression. Both the apomictic locus and the modifiers are essential for efficient penetrance of the trait in Hieracium. Some of the findings in Hieracium correspond with observations in Ranunculus and this is discussed in terms of models for apomictic development and the control of apomixis in crops.

Sexual reproduction is the default mode in apomictic Hieracium subgenus Pilosella, in which two dominant loci function to enable apomixis

Asexual seed formation, or apomixis, in the Hieracium subgenus Pilosella is controlled by two dominant independent genetic loci, LOSS OF APOMEIOSIS (LOA) and LOSS OF PARTHENOGENESIS (LOP). We examined apomixis mutants that had lost function in one or both loci to establish their developmental roles during seed formation. In apomicts, sexual reproduction is initiated first. Somatic aposporous initial (AI) cells differentiate near meiotic cells, and the sexual pathway is terminated as AI cells undergo mitotic embryo sac formation. Seed initiation is fertilization-independent. Using a partially penetrant cytotoxic reporter to inhibit meioisis, we showed that developmental events leading to the completion of meiotic tetrad formation are required for AI cell formation. Sexual initiation may therefore stimulate activity of the LOA locus, which was found to be required for AI cell formation and subsequent suppression of the sexual pathway. AI cells undergo nuclear division to form embryo sacs, in which LOP functions gametophytically to stimulate fertilization-independent embryo and endosperm formation. Loss of function in either locus results in partial reversion to sexual reproduction, and loss of function in both loci results in total reversion to sexual reproduction. Therefore, in these apomicts, sexual reproduction is the default reproductive mode upon which apomixis is superimposed. These loci are unlikely to encode genes essential for sexual reproduction, but may function to recruit the sexual machinery at specific time points to enable apomixis.

Generation of Backbone-Free, Low Transgene Copy Plants by Launching T-DNA from the Agrobacterium Chromosome

In both applied and basic research, Agrobacterium-mediated transformation is commonly used to introduce genes into plants. We investigated the effect of three Agrobacterium tumefaciens strains and five transferred (T)-DNA origins of replication on transformation frequency, transgene copy number, and the frequency of integration of non-T-DNA portions of the T-DNA-containing vector (backbone) into the genome of Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). Launching T-DNA from the picA locus of the Agrobacterium chromosome increases the frequency of single transgene integration events and almost eliminates the presence of vector backbone sequences in transgenic plants. Along with novel Agrobacterium strains we have developed, our findings are useful for improving the quality of T-DNA integration events.

Sexual and Apomictic Seed Formation in Hieracium Requires the Plant Polycomb-Group Gene FERTILIZATION INDEPENDENT ENDOSPERM

A Polycomb-Group (PcG) complex, FERTILIZATION INDEPENDENT SEED (FIS), represses endosperm development in Arabidopsis thaliana until fertilization occurs. The Hieracium genus contains apomictic species that form viable seeds asexually. To investigate FIS function during apomictic seed formation, FERTILIZATION INDEPENDENT ENDOSPERM (FIE), encoding a WD-repeat member of the FIS complex, was isolated and downregulated in sexual and apomictic Hieracium species. General downregulation led to defects in leaf and seed development, consistent with a role in developmental transitions and cell fate. PcG-like activity of Hieracium FIE was also supported by its interaction in vitro with the Arabidopsis CURLY LEAF PcG protein. By contrast, specific downregulation of FIE in developing seeds of sexual Hieracium did not result in autonomous endosperm proliferation but led to seed abortion after cross-pollination. Furthermore, in apomictic Hieracium, specific FIE downregulation inhibited autonomous embryo and endosperm initiation, and most autonomous seeds displayed defective embryo and endosperm growth. Therefore, FIE is required for both apomictic and fertilization-induced seed initiation in Hieracium. Since Hieracium FIE failed to interact with FIS class proteins in vitro, its partner proteins might differ from those in the FIS complex of Arabidopsis. These differences in protein interaction were attributed to structural modifications predicted from comparisons of Arabidopsis and Hieracium FIE molecular models.

Expression of rolB in apomictic Hieracium piloselloides Vill. causes ectopic meristems in planta and changes in ovule formation, where apomixis initiates at higher frequency

Abstract. The effects of the Agrobacterium rhizogenesrolB oncogene on apomixis were examined in the facultative apomictic plant Hieracium piloselloides because the oncogene has been shown to alter plant growth, morphogenesis and cellular sensitivity to auxin. Introduction of rolB under the control of either its own promoter or the CaMV35S promoter induced ectopic meristem formation from the inflorescence, confirming in planta a meristem-inducing role for this oncogene previously observed only in tissue culture. These ectopic meristems formed vegetative rosettes and floral plant organs. Upon immersion in water these meristems generated roots, suggesting that meristem commitment towards the generation of a specific organ type is a separate and later event that is dependent upon the developmental context. Ovule identity and form was altered in ectopically induced florets in plants expressing the CaMV35S::rolB construct. In contrast to the ovules of untransformed apomictic plants, the sexual process ceased earlier, prior to meiosis, yet surprisingly, apomixis initiated from a greater number of cells, and embryos and endosperm continued to develop in the structurally altered ovules. The alternative possibilities that the effects on reproduction might result from rolB influencing cellular response to auxin, or from alterations in cell signaling caused by changes in ovule morphology that are induced because of the expression of the oncogene are discussed.

Enlarging Cells Initiating Apomixis in Hieracium praealtum Transition to an Embryo Sac Program prior to Entering Mitosis

Transcriptomic analyses show that apomixis-initiating cells embark on an embryo sac program prior to nuclear division. Hieracium praealtum forms seeds asexually by apomixis. During ovule development, sexual reproduction initiates with megaspore mother cell entry into meiosis and formation of a tetrad of haploid megaspores. The sexual pathway ceases when a diploid aposporous initial (AI) cell differentiates, enlarges, and undergoes mitosis, forming an aposporous embryo sac that displaces sexual structures. Embryo and endosperm development in aposporous embryo sacs is fertilization independent. Transcriptional data relating to apomixis initiation in Hieracium spp. ovules is scarce and the functional identity of the AI cell relative to other ovule cell types is unclear. Enlarging AI cells with undivided nuclei, early aposporous embryo sacs containing two to four nuclei, and random groups of sporophytic ovule cells not undergoing these events were collected by laser capture microdissection. Isolated amplified messenger RNA samples were sequenced using the 454 pyrosequencing platform and comparatively analyzed to establish indicative roles of the captured cell types. Transcriptome and protein motif analyses showed that approximately one-half of the assembled contigs identified homologous sequences in Arabidopsis (Arabidopsis thaliana), of which the vast majority were expressed during early Arabidopsis ovule development. The sporophytic ovule cells were enriched in signaling functions. Gene expression indicative of meiosis was notably absent in enlarging AI cells, consistent with subsequent aposporous embryo sac formation without meiosis. The AI cell transcriptome was most similar to the early aposporous embryo sac transcriptome when comparing known functional annotations and both shared expressed genes involved in gametophyte development, suggesting that the enlarging AI cell is already transitioning to an embryo sac program prior to mitotic division.