Volatile communication in plants relies on a KAI2-mediated signaling pathway.

Plants are constantly exposed to volatile organic compounds (VOCs) that are released during plant-plant communication, within-plant self-signaling, and plant-microbe interactions. Therefore, understanding VOC perception and downstream signaling is vital for unraveling the mechanisms behind information exchange in plants, which remain largely unexplored. Using the hormone-like function of volatile terpenoids in reproductive organ development as a system with a visual marker for communication, we demonstrate that a petunia karrikin-insensitive receptor, PhKAI2ia, stereospecifically perceives the (-)-germacrene D signal, triggering a KAI2-mediated signaling cascade and affecting plant fitness. This study uncovers the role(s) of the intermediate clade of KAI2 receptors, illuminates the involvement of a KAI2ia-dependent signaling pathway in volatile communication, and provides new insights into plant olfaction and the long-standing question about the nature of potential endogenous KAI2 ligand(s).

Phosphate Suppression of Arbuscular Mycorrhizal Symbiosis Involves Gibberellic Acid Signaling.

Most land plants entertain a mutualistic symbiosis known as arbuscular mycorrhiza with fungi (Glomeromycota) that provide them with essential mineral nutrients, in particular phosphate (Pi), and protect them from biotic and abiotic stress. Arbuscular mycorrhizal (AM) symbiosis increases plant productivity and biodiversity and is therefore relevant for both natural plant communities and crop production. However, AM fungal populations suffer from intense farming practices in agricultural soils, in particular Pi fertilization. The dilemma between natural fertilization from AM symbiosis and chemical fertilization has raised major concern and emphasizes the need to better understand the mechanisms by which Pi suppresses AM symbiosis. Here, we test the hypothesis that Pi may interfere with AM symbiosis via the phytohormone gibberellic acid (GA) in the Solanaceous model systems Petunia hybrida and Nicotiana tabacum. Indeed, we find that GA is inhibitory to AM symbiosis and that Pi may cause GA levels to increase in mycorrhizal roots. Consistent with a role of endogenous GA as an inhibitor of AM development, GA-defective N. tabacum lines expressing a GA-metabolizing enzyme (GA methyltransferase-GAMT) are colonized more quickly by the AM fungus Rhizoglomus irregulare, and exogenous Pi is less effective in inhibiting AM colonization in these lines. Systematic gene expression analysis of GA-related genes reveals a complex picture, in which GA degradation by GA2 oxidase plays a prominent role. These findings reveal potential targets for crop breeding that could reduce Pi suppression of AM symbiosis, thereby reconciling the advantages of Pi fertilization with the diverse benefits of AM symbiosis.

Spatial patterning of scent in petunia corolla is discriminated by bees and involves the ABCG1 transporter.

Floral guides are patterned cues that direct the pollinator to the plant reproductive organs. The spatial distribution of showy visual and olfactory traits allows efficient plant-pollinator interactions. Data on the mechanisms underlying floral volatile patterns or their interactions with pollinators are lacking. Here we characterize the spatial emission patterns of volatiles from the corolla of the model plant Petunia × hybrida and reveal the ability of honeybees to distinguish these patterns. Along the adaxial epidermis, in correlation with cell density, the petal base adjacent to reproductive organs emitted significantly higher levels of volatiles than the distal petal rim. Volatile emission could also be differentiated between the two epidermal surfaces: emission from the adaxial side was significantly higher than that from the abaxial side. Similar emission patterns were also observed in other petunias, Dianthus caryophyllus (carnation) and Argyranthemum frutescens (Marguerite daisy). Analyses of transcripts involved in volatile production/emission revealed lower levels of the plasma-membrane transporter ABCG1 in the abaxial versus adaxial epidermis. Transient overexpression of ABCG1 enhanced emission from the abaxial epidermis to the level of the adaxial epidermis, suggesting its involvement in spatial emission patterns in the epidermal layers. Proboscis extension response experiments showed that differences in emission levels along the adaxial epidermis, that is, petal base versus rim, detected by GC-MS are also discernible by honeybees.

miR156/157 Targets SPLs to Regulate Flowering Transition, Plant Architecture and Flower Organ Size in Petunia.

miR156/157 plays multiple pivotal roles during plant growth and development. In this study, we identified 11 miR156- and 5 miR157-encoding loci from the genome of Petunia axillaris and Petunia inflata, designated as PaMIR0156/157s and PiMIR0156/157s, respectively. Real-time quantitative reverse transcription PCR (qRT-PCR) analysis indicated that PhmiR156/157 was expressed predominantly in cotyledons, germinating seeds, flower buds, young fruits and seedlings. PhmiR156/157 levels declined in shoot apical buds and leaves of petunia before flowering as the plant ages; moreover, the temporal expression patterns of most miR156/157-targeted PhSPLs were complementary to that of PhmiR156/157. Ectopic expression of PhMIR0157a in Arabidopsis and petunia resulted in delayed flowering, dwarf plant stature, increased branches and reduced organ size. However, PhMIR0156f-overexpressing Arabidopsis and petunia plants showed only delayed flowering. In addition, downregulation of PhmiR156/157 level by overexpressing STTM156/157 led to taller plants with less branches, longer internodes and precocious flowering. qRT-PCR analysis indicated that PhmiR156/157 modulates these traits mainly by downregulating their PhSPL targets and subsequently decreasing the expression of flowering regulatory genes. Our results demonstrate that the PhmiR156/157-PhSPL module has conserved but also divergent functions in growth and development, which will help us decipher the genetic basis for the improvement of flower transition, plant architecture and organ development in petunia.

Simultaneous targeting of duplicated genes in Petunia protoplasts for flower color modification via CRISPR-Cas9 ribonucleoproteins.

KEY MESSAGE: We obtained a complete mutant line of Petunia having mutations in both F3H genes via Cas9-ribonucleoproteins delivery, which exhibited a pale purplish pink flower color. The CRISPR-Cas system is now revolutionizing agriculture by allowing researchers to generate various desired mutations in plants at will. In particular, DNA-free genome editing via Cas9-ribonucleoproteins (RNPs) delivery has many advantages in plants; it does not require codon optimization or specific promoters for expression in plant cells; furthermore, it can bypass GMO regulations in some countries. Here, we have performed site-specific mutagenesis in Petunia to engineer flower color modifications. We determined that the commercial Petunia cultivar 'Madness Midnight' has two F3H coding genes and designed one guide RNA that targets both F3H genes at once. Among 67 T0 plants regenerated from Cas9-RNP transfected protoplasts, we obtained seven mutant lines that contain mutations in either F3HA or F3HB gene and one complete mutant line having mutations in both F3H genes without any selectable markers. It is noteworthy that only the f3ha f3hb exhibited a clearly modified, pale purplish pink flower color (RHS 69D), whereas the others, including the single copy gene knock-out plants, displayed purple violet (RHS 93A) flowers similar to the wild-type Petunia. To the best of our knowledge, we demonstrated a precedent of ornamental crop engineering by DNA-free CRISPR method for the first time, which will greatly accelerate a transition from a laboratory to a farmer's field.

Sequence analysis of the Petunia inflata S-locus region containing 17 S-Locus F-Box genes and the S-RNase gene involved in self-incompatibility.

Self-incompatibility in Petunia is controlled by the polymorphic S-locus, which contains S-RNase encoding the pistil determinant and 16-20 S-locus F-box (SLF) genes collectively encoding the pollen determinant. Here we sequenced and assembled approximately 3.1 Mb of the S2 -haplotype of the S-locus in Petunia inflata using bacterial artificial chromosome clones collectively containing all 17 SLF genes, SLFLike1, and S-RNase. Two SLF pseudogenes and 28 potential protein-coding genes were identified, 20 of which were also found at the S-loci of both the S6a -haplotype of P. inflata and the SN -haplotype of self-compatible Petunia axillaris, but not in the S-locus remnants of self-compatible potato (Solanum tuberosum) and tomato (Solanum lycopersicum). Comparative analyses of S-locus sequences of these three S-haplotypes revealed potential genetic exchange in the flanking regions of SLF genes, resulting in highly similar flanking regions between different types of SLF and between alleles of the same type of SLF of different S-haplotypes. The high degree of sequence similarity in the flanking regions could often be explained by the presence of similar long terminal repeat retroelements, which were enriched at the S-loci of all three S-haplotypes and in the flanking regions of all S-locus genes examined. We also found evidence of the association of transposable elements with SLF pseudogenes. Based on the hypothesis that SLF genes were derived by retrotransposition, we identified 10 F-box genes as putative SLF parent genes. Our results shed light on the importance of non-coding sequences in the evolution of the S-locus, and on possible evolutionary mechanisms of generation, proliferation, and deletion of SLF genes.

Identification of transcription factors controlling floral morphology in wild Petunia species with contrasting pollination syndromes.

Adaptation to different pollinators is an important driver of speciation in the angiosperms. Genetic approaches such as QTL mapping have been successfully used to identify the underlying speciation genes. However, these methods are often limited by widespread suppression of recombination due to divergence between species. While the mutations that caused the interspecific differences in floral color and scent have been elucidated in a variety of plant genera, the genes that are responsible for morphological differences remain mostly unknown. Differences in floral organ length determine the pollination efficiency of hawkmoths and hummingbirds, and therefore the genes that control these differences are potential speciation genes. Identifying such genes is challenging, especially in non-model species and when studying complex traits for which little prior genetic and biochemical knowledge is available. Here we combine transcriptomics with detailed growth analysis to identify candidate transcription factors underlying interspecific variation in the styles of Petunia flowers. Starting from a set of 2284 genes, stepwise filtering for expression in styles, differential expression between species, correlation with growth-related traits, allele-specific expression in interspecific hybrids, and/or high-impact polymorphisms resulted in a set of 43 candidate speciation genes. Validation by virus-induced gene silencing identified two MYB transcription factors, EOBI and EOBII, that were previously shown to regulate floral scent emission, a trait associated with pollination by hawkmoths.

Sodium butyrate induces genotoxic stress in function of photoperiod variations and differentially modulates the expression of genes involved in chromatin modification and DNA repair in Petunia hybrida seedlings.

MAIN CONCLUSION: Sodium butyrate applied to Petunia hybrida seeds under a long-day photoperiod has a negative impact (reduced seedling length, decreased production of photosynthetic pigments, and accumulation of DNA damage) on early seedling development, whereas its administration under dark/light conditions (complete dark conditions for 5 days followed by exposure to long-day photoperiod for 5 days) bypasses some of the adverse effects. Genotoxic stress impairs plant development. To circumvent DNA damage, plants activate DNA repair pathways in concert with chromatin dynamics. These are essential during seed germination and seedling establishment, and may be influenced by photoperiod variations. To assess this interplay, an experimental design was developed in Petunia hybrida, a relevant horticultural crop and model species. Seeds were treated with different doses of sodium butyrate (NaB, 1 mM and 5 mM) as a stress agent applied under different light/dark conditions throughout a time period of 10 days. Phenotypic (germination percentage and speed, seedling length, and photosynthetic pigments) and molecular (DNA damage and gene expression profiles) analyses were performed to monitor the response to the imposed conditions. Seed germination was not affected by the treatments. Seedling development was hampered by increasing NaB concentrations applied under a long-day photoperiod (L) as reflected by the decreased seedling length accompanied by increased DNA damage. When seedlings were grown under dark conditions for 5 days and then exposed to long-day photoperiod for the remaining 5 days (D/L), the damaging effects of NaB were circumvented. NaB exposure under L conditions resulted in enhanced expression of HAT/HDAC (HISTONE ACETYLTRANSFERASES/HISTONE DEACTEYLASES) genes along with repression of genes involved in DNA repair. Differently, under D/L conditions, the expression of DNA repair genes was increased by NaB treatment and this was associated with lower levels of DNA damage. The observed DNA damage and gene expression profiles suggest the involvement of chromatin modification- and DNA repair-associated pathways in response to NaB and dark/light exposure during seedling development.

The N1-Methyladenosine Methylome of Petunia mRNA.

N1-methyladenosine is a unique type of base methylation in that it blocks Watson-Crick base pairing and introduces a positive charge. m1A is prevalent in yeast and mammalian mRNA and plays a functional role. However, little is known about the abundance, dynamics, and topology of this modification in plant mRNA. Dot blotting and liquid chromatography tandem mass spectrometry analyses revealed a dynamic pattern of m1A mRNA modification in various tissues and at different developmental stages in petunia (Petunia hybrida), a model system for plant growth and development. We performed transcriptome-wide profiling of m1A in petunia mRNA by m1A mRNA immunoprecipitation followed by a deep-sequencing approach (m1A-seq, using an m1A-specific antibody). m1A-seq analysis identified 4,993 m1A peaks in 3,231 genes expressed in petunia corollas; there were 251 m1A peaks in which A residues were partly replaced by thymine and/or reverse transcription stopped at an adenine site. m1A was enriched in coding sequences, with single peaks located immediately after start codons. Ethylene treatment upregulated 400 m1A peaks in 375 mRNAs and downregulated 603 m1A peaks in 530 mRNAs in petunia corollas; 975 m1A peaks in mRNA were only detected in corollas treated with air and 430 were only detected in corollas treated with ethylene. Silencing of petunia tRNA-specific methyltransferase 61A (PhTRMT61A) reduced the m1A level in mRNA in vivo and in vitro. In addition, PhTRMT61A silencing caused abnormal leaf development, and the PhTRMT61A protein was localized to the nucleus. Thus, m1A in mRNA is an important epitranscriptome marker and plays a role in plant growth and development.

Aminooxyacetic acid (АОА), inhibitor of 1-aminocyclopropane-1-carboxilic acid (AСС) synthesis, suppresses self-incompatibility-induced programmed cell death in self-incompatible Petunia hybrida L. pollen tubes.

Self-incompatibility (SI) is genetically determined reproductive barrier preventing inbreeding and thereby providing the maintenance of plant species diversity. At present, active studies of molecular bases of SI mechanisms are underway. S-RNAse-based SI in Petunia hybrida L. is a self-/non-self recognition system that allows the pistil to reject self pollen and to accept non-self pollen for outcrossing. In the present work, using fluorescent methods including the TUNEL method allowed us to reveal the presence of markers of programmed cell death (PCD), such as DNA fragmentation, in growing in vivo petunia pollen tubes during the passage of the SI reaction. The results of statistical analysis reliably proved that PCD is the factor of S-RNAse-based SI. It was found that preliminary treatment before self-pollination of stigmas of petunia self-incompatible line with aminooxyacetic acid (AOA), inhibitor of ACC synthesis, led to stimulation of pollen tubes growth when the latter did not exhibit any hallmarks of PCD. These data argue in favor of assumption that ethylene controls the passage of PCD in incompatible pollen tubes in the course of S-RNAse-based SI functioning. The involvement of the hormonal regulation in SI mechanism in P. hybrida L. is the finding observed by us for the first time.

Identification and expression analysis of NAC transcription factors potentially involved in leaf and petal senescence in Petunia hybrida.

Progression of leaf senescence depends on several families of transcription factors. In Arabidopsis, the NAC family plays crucial roles in the modulation of leaf senescence; however, the mechanisms involved in this NAC-mediated regulation have not been extensively explored in agronomic species. Petunia hybrida is an ornamental plant that is commonly found worldwide. Decreasing the rate of leaf and petal senescence in P. hybrida is essential for maintaining plant quality. In this study, we examined the NAC-mediated networks involved in regulating senescence in this species. From 41 NAC genes, the expression of which changed in Arabidopsis during leaf senescence, we identified 29 putative orthologs in P. hybrida. Analysis using quantitative real-time-PCR indicated that 24 genes in P. hybrida changed their transcript levels during natural leaf senescence. Leaf-expressed genes were subsequently assessed in petals undergoing natural and pollination-induced senescence. Expression data and phylogenetic analysis were used to generate a list of 10-15 candidate genes; 7 of these were considered key regulatory candidates in senescence because of their consistent upregulation in the three senescence processes examined. Altogether, we identified common and distinct patterns of gene expression at different stages of leaf and petal development and during progression of senescence. The results obtained in this study will contribute to the understanding of NAC-mediated regulatory networks in petunia.

Identification of Quantitative Trait Loci for Component Traits of Flowering Capacity Across Temperature in Petunia.

For ornamental annual bedding plants, flowering performance is critical. Flowering performance includes the length of the flowering period, the longevity of individual flowers, and the number of flowers produced during the flowering period, or flowering capacity. Flowering capacity is a function of several component traits, including the number of branches producing flowers, the number of inflorescences per flowering branch, and the number of flower buds per inflorescence. We employed an F7Petunia axillaris × P. exserta recombinant inbred line population to identify QTL for flowering capacity component traits. The population was phenotyped at 14, 17, and 20° over two years. Fifteen robust QTL (rQTL; QTL detected in two or more temperatures/years) were identified across six of the seven Petunia chromosomes (Chr) for total flower bud number (FlBud), branch number (Branch), flowering branch number (FlBranch), and primary shoot flower bud number (FlBudPS). The largest effect QTL explained up to 28.8, 34.9, 36, and 23.1% of the phenotypic variation for FlBub, FlBudPS, Branch, and FlBranch, respectively. rQTL for FlBud and FlBranch co-localized on Chr 1, and rQTL for FlBud, FlBudPS, and FlBranch co-localized on Chr 4. These regions in particular should be useful for identifying genes controlling flowering capacity of this important ornamental plant.

Thermal imaging as a noninvasive technique for analyzing circadian rhythms in plants.

Endogenous (˜24 circadian) rhythms control an enormously diverse range of processes in plants and are, increasingly, the target of studies aimed at understanding plant performance. Although in the previous few decades most plant circadian research has focused on Arabidopsis, there is a pressing need for low-cost, high-throughput tools for analyzing rhythms in a wider variety of species. The present contribution investigates using circadian temperature oscillations as a novel marker for assaying plant circadian rhythms. A thermal imaging platform was set up to measure diel and circadian rhythms in different plant species, in wild-type and circadian mutant plants, and in leaves and flowers. Results from the thermal imaging technique were compared with those from other established circadian assay techniques. All of the dicot and monocot species examined showed robust circadian rhythms of leaf surface temperature; the effects of circadian mutations on thermocycles were similar to those reported using other techniques. In Petunia × atkinsiana plants circadian oscillations were observed in both leaves and flowers. Thermal imaging is an extremely useful technique for analyzing circadian rhythms in plants. It is predicted that the ability to make very high temporal resolution measurements may facilitate the discovery of novel aspects of circadian control.

Petunia PLEIOTROPIC DRUG RESISTANCE 1 Is a Strigolactone Short-Distance Transporter with Long-Distance Outcomes.

Phytohormones of the strigolactone (SL) family have been characterized as negative regulators of lateral bud outgrowth and triggers of symbioses between plants and mycorrhizal fungi. SLs and their precursors are synthesized in root tips as well as along shoot and root vasculature; they either move shoot-wards and regulate plant architecture or are exuded from roots into the soil to establish mycorrhizal symbiosis. Owing to the difficulty in quantification of SL in shoot tissues because of low abundance, it is not yet clear how SL distribution in plants is regulated at short- and long-distances from SL biosynthetic and target tissues. To address this question, we grafted wild-type scions and rootstocks from different petunia mutants for SL biosynthesis/transport and investigated SL activity by quantifying lateral bud outgrowth in the main shoot. Based on these results, we show that (i) the previously reported petunia SL transporter PLEIOTROPIC DRUG RESISTANCE 1 (PDR1) directly accounts for short-distance SL transport and (ii) long-distance transport of SLs seems to be partially and not directly dependent on PDR1. These data suggest that the root-to-shoot transport of SLs occurs either via the vasculature bundle through transporters other than PDR1 or involves SL precursors that are not substrates of PDR1.

Pseudogenization and Resurrection of a Speciation Gene.

A persistent question in evolutionary biology is how complex phenotypes evolve and whether phenotypic transitions are reversible. Multiple losses of floral pigmentation have been documented in the angiosperms, but color re-gain has not yet been described, supporting that re-gain is unlikely. Pollinator-mediated selection in Petunia has resulted in several color shifts comprised of both losses and gains of color. The R2R3-MYB transcription factor AN2 has been identified as a major locus responsible for shifts in pollinator preference. Whereas the loss of visible color has previously been attributed to repeated pseudogenization of AN2, here, we describe the mechanism of an independent re-gain of floral color via AN2 evolution. In P. secreta, purple color is restored through the improbable resurrection of AN2 gene function from a non-functional AN2-ancestor by a single reading-frame-restoring mutation. Thus, floral color evolution in Petunia is mechanistically dependent on AN2 functionality, highlighting its role as a hotspot in color transitions and a speciation gene for the genus.

S-Locus F-Box Proteins Are Solely Responsible for S-RNase-Based Self-Incompatibility of Petunia Pollen.

Self-incompatibility (SI) in Petunia is regulated by a polymorphic S-locus. For each S-haplotype, the S-locus contains a pistil-specific S-RNase gene and multiple pollen-specific S-locus F-box (SLF) genes. Both gain-of-function and loss-of-function experiments have shown that S-RNase alone regulates pistil specificity in SI. Gain-of-function experiments on SLF genes suggest that the entire suite of encoded proteins constitute the pollen specificity determinant. However, clear-cut loss-of-function experiments must be performed to determine if SLF proteins are essential for SI of pollen. Here, we used CRISPR/Cas9 to generate two frame-shift indel alleles of S2 -SLF1 (SLF1 of S2 -haplotype) in S2S3 plants of P. inflata and examined the effect on the SI behavior of S2 pollen. In the absence of a functional S2-SLF1, S2 pollen was either rejected by or remained compatible with pistils carrying one of eight normally compatible S-haplotypes. All results are consistent with interaction relationships between the 17 SLF proteins of S2 -haplotype and these eight S-RNases that had been determined by gain-of-function experiments performed previously or in this work. Our loss-of-function results provide definitive evidence that SLF proteins are solely responsible for SI of pollen, and they reveal their diverse and complex interaction relationships with S-RNases to maintain SI while ensuring cross-compatibility.

Spectral effects of light-emitting diodes on plant growth, visual color quality, and photosynthetic photon efficacy: White versus blue plus red radiation.

Arrays of blue (B, 400-500 nm) and red (R, 600-700 nm) light-emitting diodes (LEDs) used for plant growth applications make visual assessment of plants difficult compared to a broad (white, W) spectrum. Although W LEDs are sometimes used in horticultural lighting fixtures, little research has been published using them for sole-source lighting. We grew seedlings of begonia (Begonia ×semperflorens), geranium (Pelargonium ×horturum), petunia (Petunia ×hybrida), and snapdragon (Antirrhinum majus) at 20°C under six sole-source LED lighting treatments with a photosynthetic photon flux density (PPFD) of 160 µmol∙m-2∙s-1 using B (peak = 447 nm), green (G, peak = 531 nm), R (peak = 660 nm), and/or mint W (MW, peak = 558 nm) LEDs that emitted 15% B, 59% G, and 26% R plus 6 µmol∙m-2∙s-1 of far-red radiation. The lighting treatments (with percentage from each LED in subscript) were MW100, MW75R25, MW45R55, MW25R75, B15R85, and B20G40R40. At the transplant stage, total leaf area, and fresh and dry weight were similar among treatments in all species. Surprisingly, when petunia seedlings were grown longer (beyond the transplant stage) under sole-source lighting treatments, the primary stem elongated and had flower buds earlier under MW100 and MW75R25 compared to under B15R85. The color rendering index of MW75R25 and MW45R55 were 72, and 77, respectively, which was higher than those of other treatments, which were ≤64. While photosynthetic photon efficacy of B15R85 (2.25 µmol∙J-1) was higher than the W light treatments (1.51-2.13 µmol∙J-1), the dry weight gain per unit electric energy consumption (in g∙kWh-1) of B15R85 was similar to those of MW25R75, MW45R55, and MW75R25 in three species. We conclude that compared to B+R radiation, W radiation had generally similar effects on seedling growth at the same PPFD with similar electric energy consumption, and improved the visual color quality of sole-source lighting.

TM8 represses developmental timing in Nicotiana benthamiana and has functionally diversified in angiosperms.

BACKGROUND: MADS-box genes are key regulators of plant reproductive development and members of most lineages of this gene family have been extensively studied. However, the function and diversification of the ancient TM8 lineage remains elusive to date. The available data suggest a possible function in flower development in tomato and fast evolution through numerous gene loss events in flowering plants. RESULTS: We show the broad conservation of TM8 within angiosperms and find that in contrast to other MADS-box gene lineages, no gene duplicates have been retained after major whole genome duplication events. Through knock-down of NbTM8 by virus induced gene silencing in Nicotiana benthamiana, we show that NbTM8 represses miR172 together with another MADS-box gene, SHORT VEGETATIVE PHASE (NbSVP). In the closely related species Petunia hybrida, PhTM8 is not expressed under the conditions we investigated and consistent with this, a knock-out mutant did not show a phenotype. Finally, we generated transgenic tomato plants in which TM8 was silenced or ectopically expressed, but these plants did not display a clear phenotype. Therefore, no clear function could be confirmed for Solanum lycopersium. CONCLUSIONS: While the presence of TM8 is generally conserved, it remains difficult to propose a general function in angiosperms. Based on all the available data to date, supplemented with our own results, TM8 function seems to have diversified quickly throughout angiosperms and acts as repressor of miR172 in Nicotiana benthamiana, together with NbSVP.

CRISPR/Cas9-mediated knockout of PiSSK1 reveals essential role of S-locus F-box protein-containing SCF complexes in recognition of non-self S-RNases during cross-compatible pollination in self-incompatible Petunia inflata.

KEY MESSAGE: Function of Petunia PiSSK1. Self-incompatibility (SI), an inbreeding-preventing mechanism, is regulated in Petunia inflata by the polymorphic S-locus, which houses multiple pollen-specific S-locus F-box (SLF) genes and a single pistil-specific S-RNase gene. S 2-haplotype and S 3-haplotype possess the same 17 polymorphic SLF genes (named SLF1 to SLF17), and each SLF protein produced in pollen is assembled into an SCF (Skp1-Cullin1-F-box) E3 ubiquitin ligase complex. A complete suite of SLF proteins is thought to collectively interact with all non-self S-RNases to mediate their ubiquitination and degradation by the 26S proteasome, allowing cross-compatible pollination. For each SCFSLF complex, the Cullin1 subunit (named PiCUL1-P) and Skp1 subunit (named PiSSK1), like the F-box protein subunits (SLFs), are pollen-specific, raising the possibility that they also evolved specifically to function in SI. Here we used CRISPR/Cas9-meditated genome editing to generate frame-shift indel mutations in PiSSK1 and examined the SI behavior of a T 0 plant (S 2 S 3) with biallelic mutations in the pollen genome and two progeny plants (S 2 S 2) each homozygous for one of the indel alleles and not carrying the Cas9-containing T-DNA. Their pollen was completely incompatible with pistils of seven otherwise-compatible S-genotypes, but fully compatible with pistils of an S 3 S 3 transgenic plant in which production of S3-RNase was completely suppressed by an antisense S 3-RNase gene, and with pistils of immature flower buds, which produce little S-RNase. These results suggest that PiSSK1 specifically functions in SI and support the hypothesis that SLF-containing SCF complexes are essential for compatible pollination.

ABA and IAA control microsporogenesis in Petunia hybrida L.

The formation of fertile male gametophyte is known to require timely degeneration of polyfunctional tapetum tissue. The last process caused by the programmed cell death (PCD) is a part of the anther program maturation which leads to sequential anther tissue destruction coordinated with pollen differentiation. In the present work, distribution of abscisic acid (ABA) and indole-3-acetic acid (IAA) in developing anthers of male-fertile and male-sterile lines of petunia (Petunia hybrida L.) was analyzed by using the immunohistochemical method. It was established that the development of fertile male gametophyte was accompanied by monotonous elevation of ABA and IAA levels in reproductive cells and, in contrast, their monotonous lowering in tapetum cells and the middle layers. Abortion of microsporocytes in the meiosis prophase in the sterile line caused by premature tapetum degeneration along with complete maintenance of the middle layers was accompanied by dramatic, twofold elevation in the levels of both the phytohormones in reproductive cells. The data obtained allowed us to conclude that at the meiosis stage ABA and IAA are involved in the PCD of microsporocytes.