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Parental environments can influence offspring traits. However, the magnitude of the impact of parental environments on offspring molecular phenotypes is poorly understood. Here, we test the direct effects and intergenerational effects of jasmonic acid (JA) treatment, which is involved in herbivory-induced defense signaling, on transcriptomes and metabolomes in apomictic common dandelion (Taraxacum officinale). In a full factorial crossed design with parental and offspring JA and control treatments, we performed leaf RNA-seq gene expression analysis, LC-MS metabolomics and total phenolics assays in offspring plants. Expression analysis, leveraged by a de novo assembled transcriptome, revealed an induced response to JA exposure that is consistent with known JA effects. The intergenerational effect of treatment was considerable: 307 of 858 detected JA-responsive transcripts were affected by parental JA treatment. In terms of the numbers of metabolites affected, the magnitude of the chemical response to parental JA exposure was c. 10% of the direct JA treatment response. Transcriptome and metabolome analyses both identified the phosphatidylinositol signaling pathway as a target of intergenerational JA effects. Our results highlight that parental environments can have substantial effects in offspring generations. Transcriptome and metabolome assays provide a basis for zooming in on the potential mechanisms of inherited JA effects.

The inheritance of glyphosate resistance in two Amaranthus palmeri populations (R1 and R2) was examined in reciprocal crosses (RC) and second reciprocal crosses (2RC) between glyphosate-resistant (R) and -susceptible (S) parents of this dioecious species. R populations and Female-R × Male-S crosses contain higher 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene copy numbers than the S population. EPSPS expression, EPSPS enzyme activity, EPSPS protein quantity, and level of resistance to glyphosate correlated positively with genomic EPSPS relative copy number. Transfer of resistance was more influenced by the female than the male parent in spite of the fact that the multiple copies of EPSPS are amplified in the nuclear genome. This led us to hypothesize that this perplexing pattern of inheritance may result from apomictic seed production in A. palmeri. We confirmed that reproductively isolated R and S female plants produced seeds, indicating that A. palmeri can produce seeds both sexually and apomictically (facultative apomixis). This apomictic trait accounts for the low copy number inheritance in the Female-S × Male-R offsprings. Apomixis may also enhance the stability of the glyphosate resistance trait in the R populations in the absence of reproductive partners.

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.

DNA methylation can cause heritable phenotypic modifications in the absence of changes in DNA sequence. Environmental stresses can trigger methylation changes and this may have evolutionary consequences, even in the absence of sequence variation. However, it remains largely unknown to what extent environmentally induced methylation changes are transmitted to offspring, and whether observed methylation variation is truly independent or a downstream consequence of genetic variation between individuals. Genetically identical apomictic dandelion (Taraxacum officinale) plants were exposed to different ecological stresses, and apomictic offspring were raised in a common unstressed environment. We used methylation-sensitive amplified fragment length polymorphism markers to screen genome-wide methylation alterations triggered by stress treatments and to assess the heritability of induced changes. Various stresses, most notably chemical induction of herbivore and pathogen defenses, triggered considerable methylation variation throughout the genome. Many modifications were faithfully transmitted to offspring. Stresses caused some epigenetic divergence between treatment and controls, but also increased epigenetic variation among plants within treatments. These results show the following. First, stress-induced methylation changes are common and are mostly heritable. Second, sequence-independent, autonomous methylation variation is readily generated. This highlights the potential of epigenetic inheritance to play an independent role in evolutionary processes, which is superimposed on the system of genetic inheritance.

Apomixis, a clonal plant reproduction by seeds, is controlled in Paspalum spp. by a single locus which is blocked in terms of recombination. Partial sequence analysis of the apomixis locus revealed structural features of heterochromatin, namely the presence of repetitive elements, gene degeneration, and de-regulation. To test the epigenetic control of apomixis, a study on the distribution of cytosine methylation at the apomixis locus and the effect of artificial DNA demethylation on the mode of reproduction was undertaken in two apomictic Paspalum species. The 5-methylcytosine distribution in the apomixis-controlling genomic region was studied in P. simplex by methylation-sensitive restriction fragment length polymorphism (RFLP) analysis and in P. notatum by fluorescene in situ hybridization (FISH). The effect of DNA demethylation was studied on the mode of reproduction of P. simplex by progeny test analysis of apomictic plants treated with the demethylating agent 5’-azacytidine. A high level of cytosine methylation was detected at the apomixis-controlling genomic region in both species. By analysing a total of 374 open pollination progeny, it was found that artificial demethylation had little or no effect on apospory, whereas it induced a significant depression of parthenogenesis. The results suggested that factors controlling repression of parthenogenesis might be inactivated in apomictic Paspalum by DNA methylation. Results presented in this work show for the first time partial phenotype reversion in a natural apomictic plant by an artificial epigenetic landscape modification.

The effects of the Agrobacterium rhizogenes rolB 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.

Silver fern (Pityrogramma calomelanos L.) is a terrestrial or lithophytic herbaceous fern used for ornamental and medicinal purposes. In its farina it produces the cytotoxic and anticancer compound dihydrochalcone. In vitro induction of apospory and apogamy, and direct field establishment of aposporous gametophytes and subsequent sporophyte development has been accomplished. Half-strength Murashige and Skoog (MS) medium with 3.33 μM N⁶-benzyladenine (BA) and 2.32 μM kinetin (Kn) showed earlier development and produced higher numbers of aposporous gametophytes than half-strength MS basal medium. Crozier explants developed higher numbers (mean value 29.2) of gametophytes, but were slower than frond explants (mean value 23.2). The gametophytes originated from the epidermal hairs progressed from uniseriate filamentous to cordate through bi-, tri- and multiseriate and spatulate stage with the development of antheridia. Reduction in the nutrient and sucrose concentrations in the media favoured apogamy. Sucrose-free 1/10 strength MS medium and agar plates developed a mean of 30.4 and 29.9 sporophytes, respectively in the light. The greenhouse-established gametophytes developed sporophytes. The established sporophytes ex vitro showed 95% survival rate. Apogamous sporophytes and the source plant showed the same chromosome numbers (2n=116). The established protocol accomplishes apogamy and apospory in silver fern, and the aposporous gametophytes can be used for genetic transformation and development of transgenic silver fern.

This work showed for the first time the relationship between the effect of exogenous auxins and gibberellins on apogamy in Dryopteris affinis (Lowe) Fraser-Jenkins sp. affinis and its endogenous contents during early apogamic events. The addition of NAA (0.53 and 5.37 micromolar) or GA3 2.8 micromolar to an MS solid medium significantly increased apogamous sporophyte formation. BA induced brown callus that regenerated sporophytes in a hormone-free medium. The endogenous contents of GA1, GA3, GA4, GA7, GA9 and IAA were determined by GC-MS in gametophytes cultured on MS solid medium, before and during early stages of apogamous embryo development. The accumulation of both GA9 and IAA before embryo development was evident as high levels of GA4 in the earliest analysed stage of embryo development and high levels of GA3 in elongating shoots were found. The role of gibberellins on apogamy was also supported by data showing a decrease in the percentage of gametophytes developing embryos because of the addition of flurprimidol to the culture medium.

Taraxacum officinale L. (dandelion) is a vigorous weed in Europe with diploid sexual populations in the southern regions and partially overlapping populations of diploid sexuals and triploid or tetraploid apomicts in the central and northern regions. Previous studies have demonstrated unexpectedly high levels of genetic variation in the apomictic populations, suggesting the occurrence of genetic segregation in the apomicts and (or) hybridization between sexual and apomictic individuals. In this study we analysed meiosis in both sexual diploid and apomictic triploid plants to find mechanisms that could account for the high levels of genetic variation in the apomicts. Microscopic study of microsporocytes in the triploid apomicts revealed that the levels of chromosome pairing and chiasma formation at meiotic prophase I were lower than in that of the sexual diploids, but still sufficient to assume recombination between the homologues. Nomarski DIC (differential interference contrast) microscopy of optically cleared megasporocytes in the apomicts demonstrated incidental formation of tetrads, which suggests that hybridization can occur in triploid apomicts.Key words: apomixis, autosegregation, diplospory, meiotic recombination, meiotic restitution, subsexual reproduction.

Eleven Dichanthium annulatum (Forssk) plants were regenerated from embryogenic callus co-transformed with two plasmids encoding either the hygromycin phosphotransferase gene (hph) or the beta-glucuronidase (GUS) gene (uidA). Analysis of these putative transformants showed that three plants were transformed with the hph gene, showed the presence of the hph transcript and expressed hygromycin resistance after transfer to soil. Two of these also contained the uidA gene but did not express GUS and were shown to be the same transformation event. All three of the transformants set seed. Hygromycin resistance varied from 68-100% in the progeny of the three transformants. Transgene transmission appeared to have been mainly through apomixis.