Genetic and molecular mechanisms underlying the parthenocarpic fruit mutation in tomato.

Parthenocarpy allows fruit set independently of fertilization. In parthenocarpic-prone tomato genotypes, fruit set can be achieved under pollen-limiting environmental conditions and in sterile mutants. Parthenocarpy is also regarded as a quality-related trait, when seedlessness is associated with positive fruit quality aspects. Among the different sources of genetic parthenocarpy described in tomato, the parthenocarpic fruit (pat) mutation is of particular interest because of its strong expressivity, high fruit set, and enhanced fruit quality. The complexity of the pat "syndrome" associates a strong competence for parthenocarpy with a complex floral phenotype involving stamen and ovule developmental aberrations. To understand the genetic basis of the phenotype, we mapped the pat locus within a 0.19-cM window of Chr3, comprising nine coding loci. A non-tolerated missense mutation found in the 14th exon of Solyc03g120910, the tomato ortholog of the Arabidopsis HD-Zip III transcription factor HB15 (SlHB15), cosegregated with the pat phenotype. The role of SlHB15 in tomato reproductive development was supported by its expression in developing ovules. The link between pat and SlHB15 was validated by complementation and knock out experiments by co-suppression and CRISPR/Cas9 approaches. Comparing the phenotypes of pat and those of Arabidopsis HB15 mutants, we argued that the gene plays similar functions in species with fleshy and dry fruits, supporting a conserved mechanism of fruit set regulation in plants.

HISTONE DEACETYLASE19 Controls Ovule Number Determination and Transmitting Tract Differentiation.

The gynoecium is critical for the reproduction of flowering plants as it contains the ovules and the tissues that foster pollen germination, growth, and guidance. These tissues, known as the reproductive tract (ReT), comprise the stigma, style, and transmitting tract (TT). The ReT and ovules originate from the carpel margin meristem (CMM) within the pistil. SHOOT MERISTEMLESS (STM) is a key transcription factor for meristem formation and maintenance. In all above-ground meristems, including the CMM, local STM downregulation is required for organ formation. However, how this downregulation is achieved in the CMM is unknown. Here, we have studied the role of HISTONE DEACETYLASE 19 (HDA19) in Arabidopsis (Arabidopsis thaliana) during ovule and ReT differentiation based on the observation that the hda19-3 mutant displays a reduced ovule number and fails to differentiate the TT properly. Fluorescence-activated cell sorting (FACS) coupled with RNA-sequencing (RNA-seq) revealed that in the CMM of hda19-3 mutants, genes promoting organ development are downregulated while meristematic markers, including STM, are upregulated. HDA19 was essential to downregulate STM in the CMM, thereby allowing ovule formation and TT differentiation. STM is ectopically expressed in hda19-3 at intermediate stages of pistil development, and its downregulation by RNA interference alleviated the hda19-3 phenotype. Chromatin immunoprecipitation assays indicated that STM is a direct target of HDA19 during pistil development and that the transcription factor SEEDSTICK is also required to regulate STM via histone acetylation. Thus, we identified factors required for the downregulation of STM in the CMM, which is necessary for organogenesis and tissue differentiation.

Maternal control of triploid seed development by the TRANSPARENT TESTA 8 (TT8) transcription factor in Arabidopsis thaliana.

The balance between parental genome dosage is critical to offspring development in both animals and plants. In some angiosperm species, despite the imbalance between maternally and paternally inherited chromosome sets, crosses between parental lines of different ploidy may result in viable offspring. However, many plant species, like Arabidopsis thaliana, present a post-zygotic reproductive barrier, known as triploid block which results in the inability of crosses between individuals of different ploidy to generate viable seeds but also, in defective development of the seed. Several paternal regulators have been proposed as active players in establishing the triploid block. Maternal regulators known to be involved in this process are some flavonoid biosynthetic (FB) genes, expressed in the innermost layer of the seed coat. Here we explore the role of selected flavonoid pathway genes in triploid block, including TRANSPARENT TESTA 4 (TT4), TRANSPARENT TESTA 7 (TT7), SEEDSTICK (STK), TRANSPARENT TESTA 16 (TT16), TT8 and TRANSPARENT TESTA 13 (TT13). This approach allowed us to detect that TT8, a bHLH transcription factor, member of this FB pathway is required for the paternal genome dosage, as loss of function tt8, leads to complete rescue of the triploid block to seed development.

Game-changing alternatives to conventional fungicides: small RNAs and short peptides.

Fungicide use is one of the core elements of intensive agriculture because it is necessary to fight pathogens that would otherwise cause large production losses. Oomycete and fungal pathogens are kept under control using several active compounds, some of which are predicted to be banned in the near future owing to serious concerns about their impact on the environment, non-targeted organisms, and human health. To avoid detrimental repercussions for food security, it is essential to develop new biomolecules that control existing and emerging pathogens but are innocuous to human health and the environment. This review presents and discusses the use of novel low-risk biological compounds based on small RNAs and short peptides that are attractive alternatives to current contentious fungicides.

Genetic Interaction of SEEDSTICK, GORDITA and AUXIN RESPONSE FACTOR 2 during Seed Development.

Seed development is under the control of complex and coordinated molecular networks required for the formation of its different components. The seed coat development largely determines final seed size and shape, in addition to playing a crucial role in protecting the embryo and promoting germination. In this study, we investigated the role of three transcription factors known to be active during seed development in Arabidopsis thaliana: SEEDSTICK (STK) and GORDITA (GOA), two MADS-domain proteins, and AUXIN RESPONSE FACTOR 2 (ARF2), belonging to the ARF family. Through a reverse genetic approach, we characterized the seed phenotypes of all the single, double and triple loss-of-function mutants in relation to seed size/shape and the effects on metabolic pathways occurring in the seed coat. This approach revealed that dynamic networks involving these TFs are active throughout ovule and seed development, affecting the formation of the seed coat. Notably, while the genetic interaction among these genes results in synergies that control the promotion of cell expansion in the seed coat upon pollination and production of proanthocyanidins, functional antagonists arise in the control of cell proliferation and release of mucilage.

The NAC side of the fruit: tuning of fruit development and maturation.

Fruits and seeds resulting from fertilization of flowers, represent an incredible evolutionary advantage in angiosperms and have seen them become a critical element in our food supply.Many studies have been conducted to reveal how fruit matures while protecting growing seeds and ensuring their dispersal. As result, several transcription factors involved in fruit maturation and senescence have been isolated both in model and crop plants. These regulators modulate several cellular processes that occur during fruit ripening such as chlorophyll breakdown, tissue softening, carbohydrates and pigments accumulation.The NAC superfamily of transcription factors is known to be involved in almost all these aspects of fruit development and maturation. In this review, we summarise the current knowledge regarding NACs that modulate fruit ripening in model species (Arabidopsis thaliana and Solanum lycopersicum) and in crops of commercial interest (Oryza sativa, Malus domestica, Fragaria genus, Citrus sinensis and Musa acuminata).

The Arabidopsis MADS-Domain Transcription Factor SEEDSTICK Controls Seed Size via Direct Activation of E2Fa.

Seed size is the result of complex molecular networks controlling the development of the seed coat (of maternal origin) and the two fertilization products, the embryo and the endosperm. In this study we characterized the role of Arabidopsis thaliana MADS-domain transcription factor SEEDSTICK (STK) in seed size control. STK is known to regulate the differentiation of the seed coat as well as the structural and mechanical properties of cell walls in developing seeds. In particular, we further characterized stk mutant seeds. Genetic evidence (reciprocal crosses) of the inheritance of the small-seed phenotype, together with the provided analysis of cell division activity (flow cytometry), demonstrate that STK acts in the earlier phases of seed development as a maternal activator of growth. Moreover, we describe a molecular mechanism underlying this activity by reporting how STK positively regulates cell cycle progression via directly activating the expression of E2Fa, a key regulator of the cell cycle. Altogether, our results unveil a new genetic network active in the maternal control of seed size in Arabidopsis.

NoPv1: a synthetic antimicrobial peptide aptamer targeting the causal agents of grapevine downy mildew and potato late blight.

Grapevine (Vitis vinifera L.) is a crop of major economic importance. However, grapevine yield is guaranteed by the massive use of pesticides to counteract pathogen infections. Under temperate-humid climate conditions, downy mildew is a primary threat for viticulture. Downy mildew is caused by the biotrophic oomycete Plasmopara viticola Berl. & de Toni, which can attack grapevine green tissues. In lack of treatments and with favourable weather conditions, downy mildew can devastate up to 75% of grape cultivation in one season and weaken newly born shoots, causing serious economic losses. Nevertheless, the repeated and massive use of some fungicides can lead to environmental pollution, negative impact on non-targeted organisms, development of resistance, residual toxicity and can foster human health concerns. In this manuscript, we provide an innovative approach to obtain specific pathogen protection for plants. By using the yeast two-hybrid approach and the P. viticola cellulose synthase 2 (PvCesA2), as target enzyme, we screened a combinatorial 8 amino acid peptide library with the aim to identify interacting peptides, potentially able to inhibit PvCesa2. Here, we demonstrate that the NoPv1 peptide aptamer prevents P. viticola germ tube formation and grapevine leaf infection without affecting the growth of non-target organisms and without being toxic for human cells. Furthermore, NoPv1 is also able to counteract Phytophthora infestans growth, the causal agent of late blight in potato and tomato, possibly as a consequence of the high amino acid sequence similarity between P. viticola and P. infestans cellulose synthase enzymes.

Barley's Second Spring as A Model Organism for Chloroplast Research.

Barley (Hordeum vulgare) has been widely used as a model crop for studying molecular and physiological processes such as chloroplast development and photosynthesis. During the second half of the 20th century, mutants such as albostrians led to the discovery of the nuclear-encoded, plastid-localized RNA polymerase and the retrograde (chloroplast-to-nucleus) signalling communication pathway, while chlorina-f2 and xantha mutants helped to shed light on the chlorophyll biosynthetic pathway, on the light-harvesting proteins and on the organization of the photosynthetic apparatus. However, during the last 30 years, a large fraction of chloroplast research has switched to the more "user-friendly" model species Arabidopsis thaliana, the first plant species whose genome was sequenced and published at the end of 2000. Despite its many advantages, Arabidopsis has some important limitations compared to barley, including the lack of a real canopy and the absence of the proplastid-to-chloroplast developmental gradient across the leaf blade. These features, together with the availability of large collections of natural genetic diversity and mutant populations for barley, a complete genome assembly and protocols for genetic transformation and gene editing, have relaunched barley as an ideal model species for chloroplast research. In this review, we provide an update on the genomics tools now available for barley, and review the biotechnological strategies reported to increase photosynthesis efficiency in model species, which deserve to be validated in barley.

HEBE, a novel positive regulator of senescence in Solanum lycopersicum.

Leaf senescence and plant aging are traits of great interest for breeders. Senescing cells undergo important physiological and biochemical changes, while cellular structures such as chloroplasts are degraded with dramatic metabolic consequences for the whole plant. The possibility of prolonging the photosynthetic ability of leaves could positively impact the plant's life span with benefits for biomass production and metabolite accumulation; plants with these characteristics display a stay-green phenotype. A group of plant transcription factors known as NAC play a pivotal role in controlling senescence: here we describe the involvement of the tomato NAC transcription factor Solyc12g036480, which transcript is present in leaves and floral buds. Since its silencing delays leaf senescence and prevents plants from ageing, we renamed Solyc12g0364 HEBE, for the Greek goddess of youth. In this manuscript we describe how HEB downregulation negatively affects the progression of senescence, resulting in changes in transcription of senescence-promoting genes, as well as the activity of enzymes involved in chlorophyll degradation, thereby explaining the stay-green phenotype.

Emergent Ascomycetes in Viticulture: An Interdisciplinary Overview.

The reduction of pesticide usage is a current imperative and the implementation of sustainable viticulture is an urgent necessity. A potential solution, which is being increasingly adopted, is offered by the use of grapevine cultivars resistant to its main pathogenic threats. This, however, has contributed to changes in defense strategies resulting in the occurrence of secondary diseases, which were previously controlled. Concomitantly, the ongoing climate crisis is contributing to destabilizing the increasingly dynamic viticultural context. In this review, we explore the available knowledge on three Ascomycetes which are considered emergent and causal agents of powdery mildew, black rot and anthracnose. We also aim to provide a survey on methods for phenotyping disease symptoms in fields, greenhouse and lab conditions, and for disease control underlying the insurgence of pathogen resistance to fungicide. Thus, we discuss fungal genetic variability, highlighting the usage and development of molecular markers and barcoding, coupled with genome sequencing. Moreover, we extensively report on the current knowledge available on grapevine-ascomycete interactions, as well as the mechanisms developed by the host to counteract the attack. Indeed, to better understand these resistance mechanisms, it is relevant to identify pathogen effectors which are involved in the infection process and how grapevine resistance genes function and impact the downstream cascade. Dealing with such a wealth of information on both pathogens and the host, the horizon is now represented by multidisciplinary approaches, combining traditional and innovative methods of cultivation. This will support the translation from theory to practice, in an attempt to understand biology very deeply and manage the spread of these Ascomycetes.

Fruit ripening: the role of hormones, cell wall modifications, and their relationship with pathogens.

Fruits result from complex biological processes that begin soon after fertilization. Among these processes are cell division and expansion, accumulation of secondary metabolites, and an increase in carbohydrate biosynthesis. Later fruit ripening is accomplished by chlorophyll degradation and cell wall lysis. Fruit maturation is an essential step to optimize seed dispersal, and is controlled by a complex network of transcription factors and genetic regulators that are strongly influenced by phytohormones. Abscisic acid (ABA) and ethylene are the major regulators of ripening and senescence in both dry and fleshy fruits, as demonstrated by numerous ripening-defective mutants, effects of exogenous hormone application, and transcriptome analyses. While ethylene is the best characterized player in the final step of a fruit's life, ABA also has a key regulatory role, promoting ethylene production and acting as a stress-related hormone in response to drought and pathogen attack. In this review, we focus on the role of ABA and ethylene in relation to the interconnected biotic and abiotic phenomena that affect ripening and senescence. We integrate and discuss the most recent data available regarding these biological processes, which are crucial for post-harvest fruit conservation and for food safety.

Time-Course Transcriptome Analysis of Arabidopsis Siliques Discloses Genes Essential for Fruit Development and Maturation.

Fruits protect the developing seeds of angiosperms and actively contribute to seed dispersion. Furthermore, fruit and seed development are highly synchronized and require exchange of information between the mother plant and the developing generations. To explore the mechanisms controlling fruit formation and maturation, we performed a transcriptomic analysis on the valve tissue of the Arabidopsis (Arabidopsis thaliana) silique using RNA sequencing. In doing so, we have generated a data set of differentially regulated genes that will help to elucidate the molecular mechanisms that underpin the initial phase of fruit growth and, subsequently, trigger fruit maturation. The robustness of our data set has been tested by functional genomic studies. Using a reverse genetics approach, we selected 10 differentially expressed genes and explored the consequences of their disruption for both silique growth and senescence. We found that genes contained in our data set play essential roles in different stages of silique development and maturation, indicating that our transcriptome-based gene list is a powerful tool for the elucidation of the molecular mechanisms controlling fruit formation in Arabidopsis.

Trans-splicing of plastid rps12 transcripts, mediated by AtPPR4, is essential for embryo patterning in Arabidopsis thaliana.

MAIN CONCLUSION: AtPPR4-mediated trans-splicing of plastid rps12 transcripts is essential for key embryo morphogenetic events such as development of cotyledons, determination of provascular tissue, and organization of the shoot apical meristem (SAM), but not for the formation of the protodermal layer. Members of the pentatricopeptide repeat (PPR) containing protein family have emerged as key regulators of the organelle post-transcriptional processing and to be essential for proper plant embryo development. In this study, we report the functional characterization of the AtPPR4 (At5g04810) gene encoding a plastid nucleoid PPR protein. In-situ hybridization analysis reveals the presence of AtPPR4 transcripts already at the transition stage of embryo development. As a consequence, embryos lacking the AtPPR4 protein arrest their development at the transition/early-heart stages and show defects in the determination of the provascular tissue and organization of SAM. This complex phenotype is due to the specific role of AtPPR4 in the trans-splicing of the plastid rps12 transcripts, as shown by northern and slot-blot hybridizations, and the consequent defect in 70S ribosome accumulation and plastid protein synthesis, in agreement with the role proposed for the maize orthologue, ZmPPR4.

ERAMOSA controls lateral branching in snapdragon.

Plant forms display a wide variety of architectures, depending on the number of lateral branches, internode elongation and phyllotaxy. These are in turn determined by the number, the position and the fate of the Axillary Meristems (AMs). Mutants that affect AM determination during the vegetative phase have been isolated in several model plants. Among these genes, the GRAS transcription factor LATERAL SUPPRESSOR (Ls) plays a pivotal role in AM determination during the vegetative phase. Hereby we characterize the phylogenetic orthologue of Ls in Antirrhinum, ERAMOSA (ERA). Our data supported ERA control of AM formation during both the vegetative and the reproductive phase in snapdragon. A phylogenetic analysis combined with an analysis of the synteny of Ls in several species strongly supported the hypothesis that ERA is a phylogenetic orthologue of Ls, although it plays a broader role. During the reproductive phase ERA promotes the establishment of the stem niche at the bract axis but, after the reproductive transition, it is antagonized by the MADS box transcription factor SQUAMOSA (SQUA). Surprisingly double mutant era squa plants display a squa phenotype developing axillary meristems, which can eventually turn into inflorescences or flowers.

SUPPRESSOR OF FRIGIDA (SUF4) Supports Gamete Fusion via Regulating Arabidopsis EC1 Gene Expression.

The EGG CELL1 (EC1) gene family of Arabidopsis (Arabidopsis thaliana) comprises five members that are specifically expressed in the egg cell and redundantly control gamete fusion during double fertilization. We investigated the activity of all five EC1 promoters in promoter-deletion studies and identified SUF4 (SUPPRESSOR OF FRIGIDA4), a C2H2 transcription factor, as a direct regulator of the EC1 gene expression. In particular, we demonstrated that SUF4 binds to all five Arabidopsis EC1 promoters, thus regulating their expression. The down-regulation of SUF4 in homozygous suf4-1 ovules results in reduced EC1 expression and delayed sperm fusion, which can be rescued by expressing SUF4-ß-glucuronidase under the control of the SUF4 promoter. To identify more gene products able to regulate EC1 expression together with SUF4, we performed coexpression studies that led to the identification of MOM1 (MORPHEUS' MOLECULE1), a component of a silencing mechanism that is independent of DNA methylation marks. In mom1-3 ovules, both SUF4 and EC1 genes are down-regulated, and EC1 genes show higher levels of histone 3 lysine-9 acetylation, suggesting that MOM1 contributes to the regulation of SUF4 and EC1 gene expression.

The Developmental Regulator SEEDSTICK Controls Structural and Mechanical Properties of the Arabidopsis Seed Coat.

Although many transcription factors involved in cell wall morphogenesis have been identified and studied, it is still unknown how genetic and molecular regulation of cell wall biosynthesis is integrated into developmental programs. We demonstrate by molecular genetic studies that SEEDSTICK (STK), a transcription factor controlling ovule and seed integument identity, directly regulates PMEI6 and other genes involved in the biogenesis of the cellulose-pectin matrix of the cell wall. Based on atomic force microscopy, immunocytochemistry, and chemical analyses, we propose that structural modifications of the cell wall matrix in the stk mutant contribute to defects in mucilage release and seed germination under water-stress conditions. Our studies reveal a molecular network controlled by STK that regulates cell wall properties of the seed coat, demonstrating that developmental regulators controlling organ identity also coordinate specific aspects of cell wall characteristics.

Peptide aptamers: The versatile role of specific protein function inhibitors in plant biotechnology.

In recent years, peptide aptamers have emerged as novel molecular tools that have attracted the attention of researchers in various fields of basic and applied science, ranging from medicine to analytical chemistry. These artificial short peptides are able to specifically bind, track, and inhibit a given target molecule with high affinity, even molecules with poor immunogenicity or high toxicity, and represent a remarkable alternative to antibodies in many different applications. Their use is on the rise, driven mainly by the medical and pharmaceutical sector. Here we discuss the enormous potential of peptide aptamers in both basic and applied aspects of plant biotechnology and food safety. The different peptide aptamer selection methods available both in vivo and in vitro are introduced, and the most important possible applications in plant biotechnology are illustrated. In particular, we discuss the generation of broad-based virus resistance in crops, "reverse genetics" and aptasensors in bioassays for detecting contaminations in food and feed. Furthermore, we suggest an alternative to the transfer of peptide aptamers into plant cells via genetic transformation, based on the use of cell-penetrating peptides that overcome the limits imposed by both crop transformation and Genetically Modified Organism commercialization.

SEEDSTICK is a master regulator of development and metabolism in the Arabidopsis seed coat.

The role of secondary metabolites in the determination of cell identity has been an area of particular interest over recent years, and studies strongly indicate a connection between cell fate and the regulation of enzymes involved in secondary metabolism. In Arabidopsis thaliana, the maternally derived seed coat plays pivotal roles in both the protection of the developing embryo and the first steps of germination. In this regard, a characteristic feature of seed coat development is the accumulation of proanthocyanidins (PAs - a class of phenylpropanoid metabolites) in the innermost layer of the seed coat. Our genome-wide transcriptomic analysis suggests that the ovule identity factor SEEDSTICK (STK) is involved in the regulation of several metabolic processes, providing a strong basis for a connection between cell fate determination, development and metabolism. Using phenotypic, genetic, biochemical and transcriptomic approaches, we have focused specifically on the role of STK in PA biosynthesis. Our results indicate that STK exerts its effect by direct regulation of the gene encoding BANYULS/ANTHOCYANIDIN REDUCTASE (BAN/ANR), which converts anthocyanidins into their corresponding 2,3-cis-flavan-3-ols. Our study also demonstrates that the levels of H3K9ac chromatin modification directly correlate with the active state of BAN in an STK-dependent way. This is consistent with the idea that MADS-domain proteins control the expression of their target genes through the modification of chromatin states. STK might thus recruit or regulate histone modifying factors to control their activity. In addition, we show that STK is able to regulate other BAN regulators. Our study demonstrates for the first time how a floral homeotic gene controls tissue identity through the regulation of a wide range of processes including the accumulation of secondary metabolites.

Genetic regulation and structural changes during tomato fruit development and ripening.

Fruits are an important evolutionary acquisition of angiosperms, which afford protection for seeds and ensure their optimal dispersal in the environment. Fruits can be divided into dry or fleshy. Dry fruits are the more ancient and provide for mechanical seed dispersal. In contrast, fleshy fruits develop soft tissues in which flavor compounds and pigments accumulate during the ripening process. These serve to attract animals that eat them and disseminate the indigestible seeds. Fruit maturation is accompanied by several striking cytological modifications. In particular, plastids undergo significant structural alterations, including the dedifferentiation of chloroplasts into chromoplasts. Chloroplast biogenesis, their remodeling in response to environmental constraints and their conversion into alternative plastid types are known to require communication between plastids and the nucleus in order to coordinate the expression of their respective genomes. In this review, we discuss the role of plastid modifications in the context of fruit maturation and ripening, and consider the possible involvement of organelle-nucleus crosstalk via retrograde (plastid to nucleus) and anterograde (nucleus to plastid) signaling in the process.