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.

Development of pollinated and unpollinated ovules in Ginkgo biloba: unravelling pollen's role in ovule tissue maturation.

In gymnosperms such as Ginkgo biloba, the pollen's arrival plays a key role in ovule development, before fertilization occurs. Accordingly, G. biloba female plants geographically isolated from male plants aborted all their ovules after the pollination drop emission, which is the event that allows the ovule to capture pollen grains. To decipher the mechanism induced by pollination required to avoid ovule senescence and then abortion, we compared the transcriptomic of pollinated and unpollinated ovules at three time points after the end of the emission of pollination drops. Transcriptomic and in situ expression analyses revealed that several key genes involved in programmed cell death such as senescence and apoptosis, DNA replication, and cell cycle were differentially expressed in unpollinated ovules compared to pollinated ones. Interestingly, we provided evidence that the pollen captured by the pollination drop affects auxin local accumulation and might cause the deregulation of key genes required for ovule's programmed cell death and activating both the cell cycle and DNA replication genes.

The Invisible Discrimination: Biases in the Clinical Approach Regarding Migrants: A Study to Help Ethnopsychology Services and Clinicians.

The complexity of migration flows across the world has led to a redefinition of psychological and social services users. The access of migrants from different cultural backgrounds to clinical services or social health services has diversified the demand for concomitant help. Biases and misinterpretations have been created by unaccustomed professionals in this field, which could lead to serious consequences and invalidate diagnostic and treatment procedures. The purpose of this study is to summarize the evidence about errors or prejudices observed in clinical practices regarding the provision of social health services to people from different cultural backgrounds. Results show three main types of biases: racial stereotype activation, ethnocentrism and micro-aggressions. Some implications on the clinical setting were discussed, as being aware of these biases can help mental health professionals manage communication more consciously with users.

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 coupled with RNA-sequencing 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.

Evolutionary studies of the bHLH transcription factors belonging to MBW complex: their role in seed development.

BACKGROUND AND AIMS: The MBW complex consist of proteins belonging to three major families (MYB, bHLH and WDR) involved in various processes throughout plant development: epidermal cell development, mucilage secretory cells and flavonoid biosynthesis. Recently, it has been reported that TT8, encoding a bHLH transcription factor, is involved in the biosynthesis of flavonoids in the seed coat and it also plays a role in bypassing the postzygotic barrier resulting from an unbalance in genetic loads of the parental lines. Here, we focus on the functional evolution, in seed development, of the bHLH proteins that are part of the MBW complex, complemented with a literature review. METHODS: Phylogenetic analyses performed across seed plants and expression analyses in the reproductive tissues of four selected angiosperms (Arabidopsis thaliana, Brassica napus, Capsella rubella and Solanum lycopersicum) allow us to hypothesize on the evolution of its functions. KEY RESULTS: TT8 expression in the innermost layer of the seed coat is conserved in the selected angiosperms. However, except for Arabidopsis, TT8 is also expressed in ovules, carpels and fruits. The homologues belonging to the sister clade of TT8, EGL3/GL3, involved in trichome development, are expressed in the outermost layer of the seed coat, suggesting potential roles in mucilage. CONCLUSIONS: The ancestral function of these genes appears to be flavonoid biosynthesis, and the conservation of TT8 expression patterns in the innermost layer of the seed coat in angiosperms suggests that their function in postzygotic barriers might also be conserved. Moreover, the literature review and the results of the present study suggest a sophisticated association, linking the mechanisms of action of these genes to the cross-communication activity between the different tissues of the seed. Thus, it provides avenues to study the mechanisms of action of TT8 in the postzygotic triploid block, which is crucial because it impacts seed development in unbalanced crosses.

Assessing the role of REM13, REM34 and REM46 during the transition to the reproductive phase in Arabidopsis thaliana.

REM (reproductive meristem) transcription factors have been proposed as regulators of plant reproductive development mainly based on their specific expression patterns in reproductive structures, but their roles are still largely unknown probably because of their redundancy. We selected three REM genes (REM13, REM34 and REM46) for functional analysis, based on their genome position and/or co-expression data.Our results suggest that these genes have a role in flowering time regulation and may modulate cell cycle progression. In addition, protein interaction experiments revealed that REM34 and REM46 interact with each other, suggesting that they might work cooperatively to regulate cell division during inflorescence meristem commitment.Previous attempts of using co-expression data as a guide for functional analysis of REMs were limited by the transcriptomic data available at the time. Our results uncover previously unknown functions of three members of the REM family of Arabidopsis thaliana and open the door to more comprehensive studies of the REM family, where the combination of co-expression analysis followed by functional studies might contribute to uncovering the biological roles of these proteins and the relationship among them.

Identification of IPT9 in Brachiaria brizantha (syn. Urochloa brizantha) and expression analyses during ovule development in sexual and apomictic plants.

BACKGROUND: In Brachiaria sexual reproduction, during ovule development, a nucellar cell differentiates into a megaspore mother cell (MMC) that, through meiosis and mitosis, gives rise to a reduced embryo sac. In aposporic apomictic Brachiaria, next to the MMC, other nucellar cells differentiate into aposporic initials that enter mitosis directly forming an unreduced embryo sac. The IPT (isopentenyltransferase) family comprises key genes in the cytokinin (CK) pathway which are expressed in Arabidopsis during ovule development. BbrizIPT9, a B. brizantha (syn. Urochloa brizantha) IPT9 gene, highly similar to genes of other Poaceae plants, also shows similarity with Arabidopsis IPT9, AtIPT9. In this work, we aimed to investigate association of BbrizIPT9 with ovule development in sexual and apomictic plants. METHODS AND RESULTS: RT-qPCR showed higher BbrizIPT9 expression in the ovaries of sexual than in the apomictic B. brizantha. Results of in-situ hybridization showed strong signal of BbrizIPT9 in the MMC of both plants, at the onset of megasporogenesis. By analyzing AtIPT9 knockdown mutants, we verified enlarged nucellar cell, next to the MMC, in a percentage significantly higher than in the wild type, suggesting that knockout of AtIPT9 gene triggered the differentiation of extra MMC-like cells. CONCLUSIONS: Our results indicate that AtIPT9 might be involved in the proper differentiation of a single MMC during ovule development. The expression of a BbrizIPT9, localized in male and female sporocytes, and lower in apomicts than in sexuals, and effect of IPT9 knockout in Arabidopsis, suggest involvement of IPT9 in early ovule development.

AGPs as molecular determinants of reproductive development.

BACKGROUND AND AIMS: Morphogenesis occurs through accurate interaction between essential players to generate highly specialized plant organs. Fruit structure and function are triggered by a neat transcriptional control involving distinct regulator genes encoding transcription factors (TFs) or signalling proteins, such as the C2H2/C2HC zinc-finger NO TRANSMITTING TRACT (NTT) or the MADS-box protein SEEDSTICK (STK), which are important in setting plant reproductive competence, feasibly by affecting cell wall polysaccharide and lipid distribution. Arabinogalactan proteins (AGPs) are major components of the cell wall and are thought to be involved in the reproductive process as important players in specific stages of development. The detection of AGPs epitopes in reproductive tissues of NTT and other fruit development-related TFs, such as MADS-box proteins including SHATTERPROOF1 (SHP1), SHP2 and STK, was the focus of this study. METHODS: We used fluorescence microscopy to perform immunolocalization analyses on stk and ntt single mutants, on the ntt stk double mutant and on the stk shp1 shp2 triple mutant using specific anti-AGP monoclonal antibodies. In these mutants, the expression levels of selected AGP genes were also measured by quantitative real-time PCR and compared with the respective expression in wild-type (WT) plants. KEY RESULTS: The present immunolocalization study collects information on the distribution patterns of specific AGPs in Arabidopsis female reproductive tissues, complemented by the quantification of AGP expression levels, comparing WT, stk and ntt single mutants, the ntt stk double mutant and the stk shp1 shp2 triple mutant. CONCLUSIONS: These findings reveal distinct AGP distribution patterns in different developmental mutants related to the female reproductive unit in Arabidopsis. The value of the immunofluorescence labelling technique is highlighted in this study as an invaluable tool to dissect the remodelling nature of the cell wall in developmental processes.

Asexual reproduction through seeds: the complex case of diplosporous apomixis.

Apomixis is considered a potentially revolutionary tool to generate high-quality food at a lower cost and shorter developmental time due to clonal seed production through apomeiosis and parthenogenesis. In the diplosporous type of apomixis, meiotic recombination and reduction are circumvented either by avoiding or failing meiosis or by a mitotic-like division. Here, we review the literature on diplospory, from early cytological studies dating back to the late 19th century to recent genetic findings. We discuss diplosporous developmental mechanisms, including their inheritance. Furthermore, we compare the strategies adopted to isolate the genes controlling diplospory with those to produce mutants forming unreduced gametes. Nowadays, the dramatically improved technologies of long-read sequencing and targeted CRISPR/Cas mutagenesis justify the expectation that natural diplospory genes will soon be identified. Their identification will answer questions such as how the apomictic phenotype can be superimposed upon the sexual pathway and how diplospory genes have evolved. This knowledge will contribute to the application of apomixis in agriculture.

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.

The Genetic Control of SEEDSTICK and LEUNIG-HOMOLOG in Seed and Fruit Development: New Insights into Cell Wall Control.

Although much is known about seed and fruit development at the molecular level, many gaps remain in our understanding of how cell wall modifications can impact developmental processes in plants, as well as how biomechanical alterations influence seed and fruit growth. Mutants of Arabidopsis thaliana constitute an excellent tool to study the function of gene families devoted to cell wall biogenesis. We have characterized a collection of lines carrying mutations in representative cell wall-related genes for seed and fruit size developmental defects, as well as altered germination rates. We have linked these studies to cell wall composition and structure. Interestingly, we have found that disruption of genes involved in pectin maturation and hemicellulose deposition strongly influence germination dynamics. Finally, we focused on two transcriptional regulators, SEEDSTICK (STK) and LEUNIG-HOMOLOG (LUH), which positively regulate seed growth. Herein, we demonstrate that these factors regulate specific aspects of cell wall properties such as pectin distribution. We propose a model wherein changes in seed coat structure due to alterations in the xyloglucan-cellulose matrix deposition and pectin maturation are critical for organ growth and germination. The results demonstrate the importance of cell wall properties and remodeling of polysaccharides as major factors responsible for seed development.

How is vowel production in Italian affected by geminate consonants and stress patterns?

Italian vowels have a shorter duration before a geminate than before a singleton consonant, but a longer duration in syllables carrying stress. We asked whether children can produce the differentiation in vowel duration in singleton/geminate contexts reported for adults and whether their production changes depending on position of primary stress. Italian children (three-to-six-year-olds) and adults performed a nonword repetition. Each nonword appeared in four contexts, with the stressed/unstressed vowel preceding/following the singleton/geminate: /pa'paso/, /pap'paso/, 'papaso/, /'pappaso/. Acoustic analyses on the duration of the vowel preceding (V1) and following (V2) the medial consonant showed a type of consonant by age group interaction: the difference in vowel duration between children and adults was greater for geminate than singleton contexts, and was greater when the vowel carried stress. When V1 carried stress, its duration was shorter in the geminate than in the singleton in adults and older children, not in younger children.

Pivotal role of STIP in ovule pattern formation and female germline development in Arabidopsis thaliana.

In spermatophytes the sporophytic (diploid) and the gametophytic (haploid) generations co-exist in ovules, and the coordination of their developmental programs is of pivotal importance for plant reproduction. To achieve efficient fertilization, the haploid female gametophyte and the diploid ovule structures must coordinate their development to form a functional and correctly shaped ovule. WUSCHEL-RELATED HOMEOBOX (WOX) genes encode a family of transcription factors that share important roles in a wide range of processes throughout plant development. Here, we show that STIP is required for the correct patterning and curvature of the ovule in Arabidopsis thaliana. The knockout mutant stip-2 is characterized by a radialized ovule phenotype due to severe defects in outer integument development. In addition, alteration of STIP expression affects the correct differentiation and progression of the female germline. Finally, our results reveal that STIP is required to tightly regulate the key ovule factors INNER NO OUTER, PHABULOSA and WUSCHEL, and they define a novel genetic interplay in the regulatory networks determining ovule development.

Consonant and vowel transposition effects during reading development: A study on Italian children and adults.

Recently, Colombo, Spinelli, and Lupker, using a masked transposed letter (TL) priming paradigm, investigated whether consonant/vowel (CV) status is important early in orthographic processing. In four experiments with Italian and English adults, they found equivalent TL priming effects for CC, CV, and VC transpositions. Here, we investigated that question with younger readers (aged 7-10) and adults, as well as whether masked TL priming effects might have a phonological basis. That is, because young children are likely to use phonological recoding in reading, the question was whether they would show TL priming that is affected by CV status. In Experiment 1, target words were preceded by primes in which two letters (either CV, VC, or CC) were transposed versus substituted (SL). We found significant TL priming effects, with an increasing developmental trend but, again, no letter type by priming interaction. In Experiment 2, the transpositions/substitutions involved only pairs of vowels with those vowels having either diphthong or hiatus status. The difference between these two types of vowel clusters is only phonological; thus, the question was, "Would TL priming interact with this factor?" TL priming was again found with an increasing trend with age, but there was no vowel cluster by priming interaction. There was, however, an overall vowel cluster effect (slower responding to words with hiatuses) which decreased with age. The results suggest that TL priming only taps the orthographic level, and that CV status only becomes important at a later phonological level.

Cell wall modifications by α-XYLOSIDASE1 are required for control of seed and fruit size in Arabidopsis.

Cell wall modifications are of pivotal importance during plant development. Among cell wall components, xyloglucans are the major hemicellulose polysaccharide in primary cell walls of dicots and non-graminaceous monocots. They can connect the cellulose microfibril surface to affect cell wall mechanical properties. Changes in xyloglucan structure are known to play an important role in regulating cell growth. Therefore, the degradation of xyloglucan is an important modification that alters the cell wall. The α-XYLOSIDASE1 (XYL1) gene encodes the only α-xylosidase acting on xyloglucans in Arabidopsis thaliana. Here, we showed that mutation of XYL1 strongly influences seed size, seed germination, and fruit elongation. We found that the expression of XYL1 is directly regulated in developing seeds and fruit by the MADS-box transcription factor SEEDSTICK. We demonstrated that XYL1 complements the stk smaller seed phenotype. Finally, by atomic force microscopy, we investigated the role of XYL1 activity in maintaining cell stiffness and growth, confirming the importance of cell wall modulation in shaping organs.

Expression and Functional Analyses of Nymphaea caerulea MADS-Box Genes Contribute to Clarify the Complex Flower Patterning of Water Lilies.

Nymphaeaceae are early diverging angiosperms with large flowers characterized by showy petals and stamens not clearly whorled but presenting a gradual morphological transition from the outer elements to the inner stamens. Such flower structure makes these plant species relevant for studying flower evolution. MADS-domain transcription factors are crucial components of the molecular network that controls flower development. We therefore isolated and characterized MADS-box genes from the water lily Nymphaea caerulea. RNA-seq experiments on floral buds have been performed to obtain the transcript sequences of floral organ identity MADS-box genes. Maximum Likelihood phylogenetic analyses confirmed their belonging to specific MADS-box gene subfamilies. Their expression was quantified by RT-qPCR in all floral organs at two stages of development. Protein interactions among these transcription factors were investigated by yeast-two-hybrid assays. We found especially interesting the involvement of two different AGAMOUS-like genes (NycAG1 and NycAG2) in the water lily floral components. They were therefore functionally characterized by complementing Arabidopsis ag and shp1 shp2 mutants. The expression analysis of MADS-box genes across flower development in N. caerulea described a complex scenario made of numerous genes in numerous floral components. Their expression profiles in some cases were in line with what was expected from the ABC model of flower development and its extensions, while in other cases presented new and interesting gene expression patterns, as for instance the involvement of NycAGL6 and NycFL. Although sharing a high level of sequence similarity, the two AGAMOUS-like genes NycAG1 and NycAG2 could have undergone subfunctionalization or neofunctionalization, as only one of them could partially restore the euAG function in Arabidopsis ag-3 mutants. The hereby illustrated N. caerulea MADS-box gene expression pattern might mirror the morphological transition from the outer to the inner floral organs, and the presence of transition organs such as the petaloid stamens. This study is intended to broaden knowledge on the role and evolution of floral organ identity genes and the genetic mechanisms causing biodiversity in angiosperm flowers.

The role of pollination in controlling Ginkgo biloba ovule development.

Generally, in gymnosperms, pollination and fertilization events are temporally separated and the developmental processes leading the switch from ovule integument into seed coat are still unknown. The single ovule integument of Ginkgo biloba acquires the typical characteristics of the seed coat long before the fertilization event. In this study, we investigated whether pollination triggers the transformation of the ovule integument into the seed coat. Transcriptomics and metabolomics analyses performed on ovules just prior and after pollination lead to the identification of changes occurring in Ginkgo ovules during this specific time. A morphological atlas describing the developmental stages of ovule development is presented. The metabolic pathways involved in the lignin biosynthesis and in the production of fatty acids are activated upon pollination, suggesting that the ovule integument starts its differentiation into a seed coat before the fertilization. Omics analyses allowed an accurate description of the main changes that occur in Ginkgo ovules during the pollination time frame, suggesting the crucial role of the pollen arrival on the progression of ovule development.

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 emerging role of small RNAs in ovule development, a kind of magic.

In plants, small RNAs have been recognized as key genetic and epigenetic regulators of development. Small RNAs are usually 20 to 30 nucleotides in length and they control, in a sequence specific manner, the transcriptional or post-transcriptional expression of genes. In this review, we present a comprehensive overview of the most recent findings about the function of small RNAs in ovule development, including megasporogenesis and megagametogenesis, both in sexual and apomictic plants. We discuss recent studies on the role of miRNAs, siRNAs and trans-acting RNAs (ta-siRNAs) in early female germline differentiation. The mechanistic complexity and unique regulatory features are reviewed, and possible directions for future research are provided.