Asexual Female Gametogenesis Involves Contact with a Sexually-Fated Megaspore in Apomictic Hieracium.

Apomixis results in asexual seed formation where progeny are identical to the maternal plant. In ovules of apomictic species of the Hieracium subgenus Pilosella, meiosis of the megaspore mother cell generates four megaspores. Aposporous initial (AI) cells form during meiosis in most ovules. The sexual pathway terminates during functional megaspore (FM) differentiation, when an enlarged AI undergoes mitosis to form an aposporous female gametophyte. Then, the mitotically programmed FM dies along with the three other megaspores by unknown mechanisms. Transcriptomes of laser-dissected AIs, ovule cells, and ovaries from apomicts and AI-deficient mutants were analyzed to understand the pathways involved. The steps leading to AI mitosis and sexual pathway termination were determined using antibodies against arabinogalactan protein epitopes found to mark both sexual and aposporous female gametophyte lineages at inception. At most, four AIs differentiated near developing megaspores. The first expanding AI cell to contact the FM formed a functional AI that underwent mitosis soon after megaspore degeneration. Transcriptome analyses indicated that the enlarged, laser-captured AIs were arrested in the S/G2 phase of the cell cycle and were metabolically active. Further comparisons with AI-deficient mutants showed that AIs were enriched in transcripts encoding homologs of genes involved in, and potentially antagonistic to, known FM specification pathways. We propose that AI and FM cell contact provides cues required for AI mitosis and megaspore degeneration. Specific candidates to further interrogate AI-FM interactions were identified here and include Hieracium arabinogalactan protein family genes.

Genetic analyses of the inheritance and expressivity of autonomous endosperm formation in Hieracium with different modes of embryo sac and seed formation.

Background and Aims: Apomixis, or asexual seed formation, in polyploid Hieracium subgenus Pilosella species results in clonal progeny with a maternal genotype. An aposporous embryo sac forms mitotically from a somatic cell, without prior meiosis, while embryo and endosperm formation is fertilization independent (autonomous). The latter two developmental components are tightly linked in Hieracium . Recently, two plants, AutE196 and AutE24, were identified from two different crosses. Both form embryo sacs via the sexual route by undergoing meiosis, and embryo development requires fertilization; however, 18 % of embryo sacs can undergo autonomous endosperm (AutE) formation. This study investigated the qualitative and quantitative inheritance of the AutE trait and factors influencing phenotype expressivity. An additional focus was to identify the linkage group bearing the AutE locus in AutE196. Methods: Crosses and cytology were used to examine the inheritance of AutE from AutE24 and AutE196, and to reintroduce apomictic components into AutE plants, thereby changing the ploidy of developing embryo sacs and increasing the dosage of AutE loci. Markers from a Hieracium apomict linkage map were examined within a backcrossed AutE196 mapping population to identify the linkage group containing the AutE196 locus. Key Results: Qualitative autonomous endosperm in the AutE24 line was conferred by a single dominant locus, and the trait was transmitted through male and female gametes in AutE196 and AutE24. Expressivity of the trait did not significantly increase when AutE loci from AutE196 and AutE24 were both present in the progeny, within embryo sacs formed via apospory, or sexually derived embryo sacs with increased ploidy. It remains unclear if these are identical loci. Conclusions: The qualitative trait of autonomous endosperm formation is conferred by single dominant loci in AutE196 and AutE24. High expressivity of autonomous endosperm formation observed in apomicts requires additional genetic factors. Potential candidates may be signals arising from fertilization-independent embryo formation.

Generation of an integrated Hieracium genomic and transcriptomic resource enables exploration of small RNA pathways during apomixis initiation.

BACKGROUND: Application of apomixis, or asexual seed formation, in crop breeding would allow rapid fixation of complex traits, economizing improved crop delivery. Identification of apomixis genes is confounded by the polyploid nature, high genome complexity and lack of genomic sequence integration with reproductive tissue transcriptomes in most apomicts. RESULTS: A genomic and transcriptomic resource was developed for Hieracium subgenus Pilosella (Asteraceae) which incorporates characterized sexual, apomictic and mutant apomict plants exhibiting reversion to sexual reproduction. Apomicts develop additional female gametogenic cells that suppress the sexual pathway in ovules. Disrupting small RNA pathways in sexual Arabidopsis also induces extra female gametogenic cells; therefore, the resource was used to examine if changes in small RNA pathways correlate with apomixis initiation. An initial characterization of small RNA pathway genes within Hieracium was undertaken, and ovary-expressed ARGONAUTE genes were identified and cloned. Comparisons of whole ovary transcriptomes from mutant apomicts, relative to the parental apomict, revealed that differentially expressed genes were enriched for processes involved in small RNA biogenesis and chromatin silencing. Small RNA profiles within mutant ovaries did not reveal large-scale alterations in composition or length distributions; however, a small number of differentially expressed, putative small RNA targets were identified. CONCLUSIONS: The established Hieracium resource represents a substantial contribution towards the investigation of early sexual and apomictic female gamete development, and the generation of new candidate genes and markers. Observed changes in small RNA targets and biogenesis pathways within sexual and apomictic ovaries will underlie future functional research into apomixis initiation in Hieracium.

A reference genetic linkage map of apomictic Hieracium species based on expressed markers derived from developing ovule transcripts.

BACKGROUND AND AIMS: Apomixis in plants generates clonal progeny with a maternal genotype through asexual seed formation. Hieracium subgenus Pilosella (Asteraceae) contains polyploid, highly heterozygous apomictic and sexual species. Within apomictic Hieracium, dominant genetic loci independently regulate the qualitative developmental components of apomixis. In H. praealtum, LOSS OF APOMEIOSIS (LOA) enables formation of embryo sacs without meiosis and LOSS OF PARTHENOGENESIS (LOP) enables fertilization-independent seed formation. A locus required for fertilization-independent endosperm formation (AutE) has been identified in H. piloselloides. Additional quantitative loci appear to influence the penetrance of the qualitative loci, although the controlling genes remain unknown. This study aimed to develop the first genetic linkage maps for sexual and apomictic Hieracium species using simple sequence repeat (SSR) markers derived from expressed transcripts within the developing ovaries. METHODS: RNA from microdissected Hieracium ovule cell types and ovaries was sequenced and SSRs were identified. Two different F1 mapping populations were created to overcome difficulties associated with genome complexity and asexual reproduction. SSR markers were analysed within each mapping population to generate draft linkage maps for apomictic and sexual Hieracium species. KEY RESULTS: A collection of 14 684 Hieracium expressed SSR markers were developed and linkage maps were constructed for Hieracium species using a subset of the SSR markers. Both the LOA and LOP loci were successfully assigned to linkage groups; however, AutE could not be mapped using the current populations. Comparisons with lettuce (Lactuca sativa) revealed partial macrosynteny between the two Asteraceae species. CONCLUSIONS: A collection of SSR markers and draft linkage maps were developed for two apomictic and one sexual Hieracium species. These maps will support cloning of controlling genes at LOA and LOP loci in Hieracium and should also assist with identification of quantitative loci that affect the expressivity of apomixis. Future work will focus on mapping AutE using alternative populations.

The LOSS OF APOMEIOSIS (LOA) locus in Hieracium praealtum can function independently of the associated large-scale repetitive chromosomal structure.

Apomixis or asexual seed formation in Hieracium praealtum (Asteraceae) is controlled by two independent dominant loci. One of these, the LOSS OF APOMEIOSIS (LOA) locus, controls apomixis initiation, mitotic embryo sac formation (apospory) and suppression of the sexual pathway. The LOA locus is found near the end of a hemizygous chromosome surrounded by extensive repeats extending along the chromosome arm. Similar apomixis-carrying chromosome structures have been found in some apomictic grasses, suggesting that the extensive repetitive sequences may be functionally relevant to apomixis. Fluorescence in situ hybridization (FISH) was used to examine chromosomes of apomeiosis deletion mutants and rare recombinants in the critical LOA region arising from a cross between sexual Hieracium pilosella and apomictic H. praealtum. The combined analyses of aposporous and nonaposporous recombinant progeny and chromosomal karyotypes were used to determine that the functional LOA locus can be genetically separated from the very extensive repeat regions found on the LOA-carrying chromosome. The large-scale repetitive sequences associated with the LOA locus in H. praealtum are not essential for apospory or suppression of sexual megasporogenesis (female meiosis).

Enlarging cells initiating apomixis in Hieracium praealtum transition to an embryo sac program prior to entering mitosis.

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.

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.

Chromosomes carrying meiotic avoidance loci in three apomictic eudicot Hieracium subgenus Pilosella species share structural features with two monocot apomicts.

The LOSS OF APOMEIOSIS (LOA) locus is one of two dominant loci known to control apomixis in the eudicot Hieracium praealtum. LOA stimulates the differentiation of somatic aposporous initial cells after the initiation of meiosis in ovules. Aposporous initial cells undergo nuclear proliferation close to sexual megaspores, forming unreduced aposporous embryo sacs, and the sexual program ceases. LOA-linked genetic markers were used to isolate 1.2 Mb of LOA-associated DNAs from H. praealtum. Physical mapping defined the genomic region essential for LOA function between two markers, flanking 400 kb of identified sequence and central unknown sequences. Cytogenetic and sequence analyses revealed that the LOA locus is located on a single chromosome near the tip of the long arm and surrounded by extensive, abundant complex repeat and transposon sequences. Chromosomal features and LOA-linked markers are conserved in aposporous Hieracium caespitosum and Hieracium piloselloides but absent in sexual Hieracium pilosella. Their absence in apomictic Hieracium aurantiacum suggests that meiotic avoidance may have evolved independently in aposporous subgenus Pilosella species. The structure of the hemizygous chromosomal region containing the LOA locus in the three Hieracium subgenus Pilosella species resembles that of the hemizygous apospory-specific genomic regions in monocot Pennisetum squamulatum and Cenchrus ciliaris. Analyses of partial DNA sequences at these loci show no obvious conservation, indicating that they are unlikely to share a common ancestral origin. This suggests convergent evolution of repeat-rich hemizygous chromosomal regions containing apospory loci in these monocot and eudicot species, which may be required for the function and maintenance of the trait.

Sporophytic ovule tissues modulate the initiation and progression of apomixis in Hieracium.

Apomixis in Hieracium subgenus Pilosella initiates in ovules when sporophytic cells termed aposporous initial (AI) cells enlarge near sexual cells undergoing meiosis. AI cells displace the sexual structures and divide by mitosis to form unreduced embryo sac(s) without meiosis (apomeiosis) that initiate fertilization-independent embryo and endosperm development. In some Hieracium subgenus Pilosella species, these events are controlled by the dominant LOSS OF APOMEIOSIS (LOA) and LOSS OF PARTHENOGENESIS (LOP) loci. In H. praealtum and H. piloselloides, which both contain the same core LOA locus, the timing and frequency of AI cell formation is altered in derived mutants exhibiting abnormal funiculus growth and in transgenic plants expressing rolB which alters cellular sensitivity to auxin. The impact on apomictic and sexual reproduction was examined here when a chimeric RNAse gene was targeted to the funiculus and basal portions of the ovule, and also when polar auxin transport was inhibited during ovule development following N-1-naphthylphthalamic acid (NPA) application. Both treatments led to ovule deformity in the funiculus and distal parts of the ovule and LOA-dependent alterations in the timing, position, and frequency of AI cell formation. In the case of NPA treatment, this correlated with increased expression of DR5:GFP in the ovule, which marks the accumulation of the plant hormone auxin. Our results show that sporophytic information potentiated by funiculus growth and polar auxin transport influences ovule development, the initiation of apomixis, and the progression of embryo sac development in Hieracium. Signals associated with ovule pattern formation and auxin distribution or perception may influence the capacity of sporophytic ovule cells to respond to LOA.

Apomixis in hawkweed: Mendel's experimental nemesis.

Mendel used hawkweeds and other plants to verify the laws of inheritance he discovered using Pisum. Trait segregation was not evident in hawkweeds because many form seeds asexually by apomixis. Meiosis does not occur during female gametophyte formation and the mitotically formed embryo sacs do not require fertilization for seed development. The resulting progeny retain a maternal genotype. Hawkweeds in Hieracium subgenus Pilosella form mitotic embryo sacs by apospory. The initiation of sexual reproduction is required to stimulate apospory in ovules and to promote the function of the dominant locus, LOSS OF APOMEIOSIS, which stimulates the differentiation of somatic aposporous initial (AI) cells near sexually programmed cells. As AI cells undergo nuclear mitosis the sexual pathway terminates. The function of the dominant locus LOSS OF PARTHENOGENESIS in aposporous embryo sacs enables fertilization-independent embryo and endosperm development. Deletion of either locus results in partial reversion to sexual reproduction, and loss of function in both loci results in reversion to sexual development. In these apomicts, sexual reproduction is therefore the default reproductive mode upon which apomixis is superimposed. These loci are unlikely to encode factors critical for sexual reproduction but might recruit the sexual pathway to enable apomixis. Incomplete functional penetrance of these dominant loci is likely to lead to the generation of rare sexual progeny also derived from these facultative apomicts.

Phenotypic plasticity of aposporous embryo sac development in Hieracium praealtum.

Apomixis in Hieracium praealtum follows a developmental pathway of apospory, where an unreduced embryo sac develops from a somatic ovule cell without meiosis. The avoidance of meiosis together with fertilization-independent seed formation leads to clonal progeny genetically identical to the maternal plant. We have previously described the initial developmental steps of aposporous embryo sac formation in H. praealtum and here, we cytologically observed more than 500 ovules with a focus on the later stages of embryo sac maturation. Aposporous embryo sac maturation is a stochastic process in H. praealtum with single or multiple embryo sacs formed, in addition to off-types and embryo sac abortion. The frequency of twin embryo sacs growing at the same rate is a rare event and, in most ovules, the additional embryo sac undergoes developmental arrest suggesting dominance or growth promotion of a single embryo sac. Observed deviations from the Polygonum-type embryo sac in H. praealtum indicate developmental plasticity during embryo sac maturation. Nevertheless, fertilization-independent seed formation successfully occurs.

Assembled genomic and tissue-specific transcriptomic data resources for two genetically distinct lines of Cowpea ( Vigna unguiculata (L.) Walp).

Cowpea ( Vigna unguiculata (L.) Walp) is an important legume crop for food security in areas of low-input and smallholder farming throughout Africa and Asia. Genetic improvements are required to increase yield and resilience to biotic and abiotic stress and to enhance cowpea crop performance. An integrated cowpea genomic and gene expression data resource has the potential to greatly accelerate breeding and the delivery of novel genetic traits for cowpea. Extensive genomic resources for cowpea have been absent from the public domain; however, a recent early release reference genome for IT97K-499-35 ( Vigna unguiculata v1.0, NSF, UCR, USAID, DOE-JGI, http://phytozome.jgi.doe.gov/) has now been established in a collaboration between the Joint Genome Institute (JGI) and University California (UC) Riverside. Here we release supporting genomic and transcriptomic data for two transformable cowpea varieties, IT97K-499-35 and IT86D-1010. The transcriptome resource includes six tissue-specific datasets for each variety, with particular emphasis on reproductive tissues that extend and support the V. unguiculata v1.0 reference. Annotations have been included in our resource to allow direct mapping to the v1.0 cowpea reference. The resource described here is supported by downloadable raw and assembled sequence data.

New observations on gametogenic development and reproductive experimental tools to support seed yield improvement in cowpea [Vigna unguiculata (L.) Walp].

KEY MESSAGE: Cowpea reproductive tools. Vigna unguiculata L. Walp. (cowpea) is recognized as a major legume food crop in Africa, but seed yields remain low in most varieties adapted to local conditions. The development of hybrid cowpea seed that could be saved after each generation, enabling significant yield increases, will require manipulation of reproductive development from a sexual to an asexual mode. To develop new technologies that could support the biotechnological manipulation of reproductive development in cowpea, we examined gametogenesis and seed formation in two transformable, African-adapted, day-length-insensitive varieties. Here, we show that these two varieties exhibit distinct morphological and phenological traits but share a common developmental sequence in terms of ovule formation and gametogenesis. We present a reproductive calendar that allows prediction of male and female gametogenesis on the basis of sporophytic parameters related to floral bud size and reproductive organ development, determining that gametogenesis occurs more rapidly in the anther than in the ovule. We also show that the mode of megagametogenesis is of the Polygonum-type and not Oenothera-type, as previously reported. Finally, we developed a whole-mount immunolocalization protocol and applied it to detect meiotic proteins in the cowpea megaspore mother cell, opening opportunities for comparing the dynamics of protein localization during male and female meiosis, as well as other reproductive events in this emerging legume model system.

Parental Genome Imbalance Causes Post-Zygotic Seed Lethality and Deregulates Imprinting in Rice.

BACKGROUND: Reproductive isolation between rice of different ploidy levels is manifested as endosperm and embryo abortion in seeds produced by interploidy crosses. Genomic imprinting is considered to be the underlying mechanism establishing the post-zygotic hybridization barrier. We characterized disrupted seed development in reciprocal crosses between a diploid Japonica rice and a tetraploid Indica rice. RESULTS: Triploid seeds from these crosses had aborted development and could not germinate in soil but could be rescued in culture medium with significantly more seeds developing to seedlings in the 4n × 2n (♀-♂) cross with excess maternal genomes than in the 2n × 4n cross with excess paternal genome. Consistent with previous findings, precocious endosperm cellularization and bigger embryos were observed in the seeds from the maternal excess cross, whereas absence of cellularization and arrested globular embryos were found in the seeds from the paternal excess cross, supporting the idea that endosperm cellularization is an important transition for embryo development. Moreover, we found that starch granules were persistently deposited in the pericarp parenchyma cells of the paternal excess cross, while pericarp starch gradually decreased and relocated to the developing endosperm in balanced and maternal excess crosses in which cellularization and starch deposition occur in endosperm, suggesting that parental genome balance influences pericarp starch relocation via cellularization and starch deposition. Loss of imprinting, or altered expression of imprinted genes and epigenetic regulators, OsFIE2 and OsMET1b were observed, implying the potential role of imprinting and epigenetic mechanisms in regulating the differential parental genome dosage effects on endosperm development. CONCLUSIONS: Our results support the hypothesis that the maternal genome dosage promotes endosperm cellularization and the paternal genome dosage delays or inhibits cellularization via contributing different sets of imprinted genes.

Genetic separation of autonomous endosperm formation (AutE) from the two other components of apomixis in Hieracium.

In apomictic Hieracium subgenus Pilosella species, embryo sacs develop in ovules without meiosis. Embryo and endosperm formation then occur without fertilization, producing seeds with a maternal genotype encased in a fruit (achene). Genetic analyses in H. praealtum indicate a dominant locus (LOA) controls meiotic avoidance, and another dominant locus (LOP) controls both fertilization-independent embryogenesis and endosperm formation. While cytologically examining developmental events in ovules of progeny from crosses between different wild-type and mutant Hieracium apomicts, and a sexual Hieracium species, we identified two plants, AutE196 and AutE24, which have lost the capacity for meiotic avoidance and fertilization-independent embryo formation. AutE196 and AutE24 exhibit autonomous endosperm formation and set parthenocarpic, seedless achenes at a penetrance of 18 %. Viable seed form after pollination. Cytological examination of 102 progeny from a backcross of AutE196 with sexual H. pilosella showed that autonomous endosperm formation is a heritable, dominant, qualitative trait, detected in 51 % of progeny. Variation in quantitative trait penetrance indicates other factors influence its expression. The correlation between autonomous endosperm development and mature parthenocarpic achene formation suggests the former is sufficient to trigger fruit maturation in Hieracium. The developmental component of autonomous endosperm formation is therefore genetically separable from those controlling meiotic avoidance and autonomous embryogenesis in Hieracium and has been denoted as AutE. We postulate that tight linkage of AutE and genes controlling autonomous embryogenesis at the LOP locus in H. praealtum may explain why inheritance of autonomous seed formation is typically observed as a single component.

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 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:beta-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.

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 P(ARF8):beta-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.

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.