M2WISH: An easy and efficient protocol for whole-mount mRNA in situ hybridization that allows 3D cell resolution of gene expression in Arabidopsis thaliana.

Gene expression analysis is essential for understanding the mechanisms involved in plant development. Here, we developed M2WISH, a protocol based on MicroWave treatment for Wholemount mRNA In Situ Hybridization in Arabidopsis. By permeabilizing tissues without damaging cellular organization this protocol results in high and homogeneous hybridization yields that enable systematic analysis of gene expression dynamics. Moreover, when combined with cellular histochemical staining, M2WISH successfully provides a cellular resolution of gene expression. Thus, we demonstrate the robustness of M2WISH with 10 genes on roots, aerial meristems, leaves, and embryos in the seed. We applied M2WISH to study the spatial dynamics of WUSCHEL (WUS) and CLAVATA3 (CLV3) expression during in vitro meristematic conversion of roots into shoot apical meristems. Thus, we showed that shoot apical meristems could arise from two different types of root structures that differed by their CLV3 gene expression patterns. We constructed 3D cellular representations of WUS and CLV3 gene co-expression pattern and stressed the variability inherent to meristem conversion. Thus, this protocol generates a large amount of data on the localization of gene expression, which can be used to model complex systems.

Ethylene produced in carpel primordia controls CmHB40 expression to inhibit stamen development.

Sex determination evolved to control the development of unisexual flowers. In agriculture, it conditions how plants are cultivated and bred. We investigated how female flowers develop in monoecious cucurbits. We discovered in melon, Cucumis melo, a mechanism in which ethylene produced in the carpel is perceived in the stamen primordia through spatially differentially expressed ethylene receptors. Subsequently, the CmEIN3/CmEIL1 ethylene signalling module, in stamen primordia, activates the expression of CmHB40, a transcription factor that downregulates genes required for stamen development and upregulates genes associated with organ senescence. Investigation of melon genetic biodiversity revealed a haplotype, originating in Africa, altered in EIN3/EIL1 binding to CmHB40 promoter and associated with bisexual flower development. In contrast to other bisexual mutants in cucurbits, CmHB40 mutations do not alter fruit shape. By disentangling fruit shape and sex-determination pathways, our work opens up new avenues in plant breeding.

<b>The control of carpel determinacy pathway leads to sex determination</b> <b>in cucurbits</b>.

Male and female unisexual flowers evolved from hermaphroditic ancestors, and control of flower sex is useful for plant breeding. We isolated a female-to-male sex transition mutant in melon and identified the causal gene as the carpel identity gene <i>CRABS CLAW (CRC)</i>. We show that the master regulator of sex determination in cucurbits, the transcription factor <i>WIP1</i> whose expression orchestrates male flower development, recruits the corepressor TOPLESS to the <i>CRC</i> promoter to suppress its expression through histone deacetylation. Impairing TOPLESS-WIP1 physical interaction leads to <i>CRC</i> expression, carpel determination, and consequently the expression of the stamina inhibitor, the aminocyclopropane-1-carboxylic acid synthase 7 (<i>CmACS7</i>), leading to female flower development. Our findings suggest that sex genes evolved to interfere with flower meristematic function, leading to unisexual flower development.

Precision Phenotyping of Nectar-Related Traits Using X-ray Micro Computed Tomography.

Flower morphologies shape the accessibility to nectar and pollen, two major traits that determine plant-pollinator interactions and reproductive success. Melon is an economically important crop whose reproduction is completely pollinator-dependent and, as such, is a valuable model for studying crop-ecological functions. High-resolution imaging techniques, such as micro-computed tomography (micro-CT), have recently become popular for phenotyping in plant science. Here, we implemented micro-CT to study floral morphology and honey bees in the context of nectar-related traits without a sample preparation to improve the phenotyping precision and quality. We generated high-quality 3D models of melon male and female flowers and compared the geometric measures. Micro-CT allowed for a relatively easy and rapid generation of 3D volumetric data on nectar, nectary, flower, and honey bee body sizes. A comparative analysis of male and female flowers showed a strong positive correlation between the nectar gland volume and the volume of the secreted nectar. We modeled the nectar level inside the flower and reconstructed a 3D model of the accessibility by honey bees. By combining data on flower morphology, the honey bee size and nectar volume, this protocol can be used to assess the flower accessibility to pollinators in a high resolution, and can readily carry out genotypes comparative analysis to identify nectar-pollination-related traits.

Ethylene plays a dual role in sex determination and fruit shape in cucurbits.

Shapes of vegetables and fruits are the result of adaptive evolution and human selection. Modules controlling organ shape have been identified. However, little is known about signals coordinating organ development and shape. Here, we describe the characterization of a melon mutation rf1, leading to round fruit. Histological analysis of rf1 flower and fruits revealed fruit shape is determined at flower stage 8, after sex determination and before flower fertilization. Using positional cloning, we identified the causal gene as the monoecy sex determination gene CmACS7, and survey of melon germplasms showed strong association between fruit shape and sexual types. We show that CmACS7-mediated ethylene production in carpel primordia enhances cell expansion and represses cell division, leading to elongated fruit. Cell size is known to rise as a result of endoreduplication. At stage 8 and anthesis, we found no variation in ploidy levels between female and hermaphrodite flowers, ruling out endoreduplication as a factor in fruit shape determination. To pinpoint the gene networks controlling elongated versus round fruit phenotype, we analyzed the transcriptomes of laser capture microdissected carpels of wild-type and rf1 mutant. These high-resolution spatiotemporal gene expression dynamics revealed the implication of two regulatory modules. The first module implicates E2F-DP transcription factors, controlling cell elongation versus cell division. The second module implicates OVATE- and TRM5-related proteins, controlling cell division patterns. Our finding highlights the dual role of ethylene in the inhibition of the stamina development and the elongation of ovary and fruit in cucurbits.

One-Week Scutellar Somatic Embryogenesis in the Monocot Brachypodium distachyon.

Plant somatic embryogenesis (SE) is a natural process of vegetative propagation. It can be induced in tissue cultures to investigate developmental transitions, to create transgenic or edited lines, or to multiply valuable crops. We studied the induction of SE in the scutellum of monocots with Brachypodium distachyon as a model system. Towards the in-depth analysis of SE initiation, we determined the earliest stages at which somatic scutellar cells acquired an embryogenic fate, then switched to a morphogenetic mode in a regeneration sequence involving treatments with exogenous hormones: first an auxin (2,4-D) then a cytokinin (kinetin). Our observations indicated that secondary somatic embryos could already develop in the proliferative calli derived from immature zygotic embryo tissues within one week from the start of in vitro culture. Cell states and tissue identity were deduced from detailed histological examination, and in situ hybridization was performed to map the expression of key developmental genes. The fast SE induction method we describe here facilitates the mechanistic study of the processes involved and may significantly shorten the production of transgenic or gene-edited plants.

In Situ Gene Expression in Native Cryofixed Bone Tissue.

Bone is a very complex tissue that is constantly changing throughout the lifespan. The precise mechanism of bone regeneration remains poorly understood. Large bone defects can be caused by gunshot injury, trauma, accidents, congenital anomalies and tissue resection due to cancer. Therefore, understanding bone homeostasis and regeneration has considerable clinical and scientific importance in the development of bone therapy. Macrophages are well known innate immune cells secreting different combinations of cytokines and their role in bone regeneration during bone healing is essential. Here, we present a method to identify mRNA transcripts in cryosections of non-decalcified rat bone using in situ hybridization and hybridization chain reaction to explore gene expression in situ for better understanding the gene expression of the bone tissues.

Sclareol and linalyl acetate are produced by glandular trichomes through the MEP pathway.

Sclareol, an antifungal specialized metabolite produced by clary sage, Salvia sclarea, is the starting plant natural molecule used for the hemisynthesis of the perfume ingredient ambroxide. Sclareol is mainly produced in clary sage flower calyces; however, the cellular localization of the sclareol biosynthesis remains unknown. To elucidate the site of sclareol biosynthesis, we analyzed its spatial distribution in the clary sage calyx epidermis using laser desorption/ionization mass spectrometry imaging (LDI-FTICR-MSI) and investigated the expression profile of sclareol biosynthesis genes in isolated glandular trichomes (GTs). We showed that sclareol specifically accumulates in GTs' gland cells in which sclareol biosynthesis genes are strongly expressed. We next isolated a glabrous beardless mutant and demonstrate that more than 90% of the sclareol is produced by the large capitate GTs. Feeding experiments, using 1-13C-glucose, and specific enzyme inhibitors further revealed that the methylerythritol-phosphate (MEP) biosynthetic pathway is the main source of isopentenyl diphosphate (IPP) precursor used for the biosynthesis of sclareol. Our findings demonstrate that sclareol is an MEP-derived diterpene produced by large capitate GTs in clary sage emphasing the role of GTs as biofactories dedicated to the production of specialized metabolites.

The miR166-SlHB15A regulatory module controls ovule development and parthenocarpic fruit set under adverse temperatures in tomato.

Fruit set is inhibited by adverse temperatures, with consequences on yield. We isolated a tomato mutant producing fruits under non-permissive hot temperatures and identified the causal gene as SlHB15A, belonging to class III homeodomain leucine-zipper transcription factors. SlHB15A loss-of-function mutants display aberrant ovule development that mimics transcriptional changes occurring in fertilized ovules and leads to parthenocarpic fruit set under optimal and non-permissive temperatures, in field and greenhouse conditions. Under cold growing conditions, SlHB15A is subjected to conditional haploinsufficiency and recessive dosage sensitivity controlled by microRNA 166 (miR166). Knockdown of SlHB15A alleles by miR166 leads to a continuum of aberrant ovules correlating with parthenocarpic fruit set. Consistent with this, plants harboring an Slhb15a-miRNA166-resistant allele developed normal ovules and were unable to set parthenocarpic fruit under cold conditions. DNA affinity purification sequencing and RNA-sequencing analyses revealed that SlHB15A is a bifunctional transcription factor expressed in the ovule integument. SlHB15A binds to the promoters of auxin-related genes to repress auxin signaling and to the promoters of ethylene-related genes to activate their expression. A survey of tomato genetic biodiversity identified pat and pat-1, two historical parthenocarpic mutants, as alleles of SlHB15A. Taken together, our findings demonstrate the role of SlHB15A as a sentinel to prevent fruit set in the absence of fertilization and provide a mean to enhance fruiting under extreme temperatures.

The Genetic Control of Nectary Development.

Nectar is the most important reward offered by flowering plants to pollinators for pollination services. Since pollinator decline has emerged as a major threat for agriculture, and the food demand is growing globally, studying the nectar gland is of utmost importance. Although the genetic mechanisms that control the development of angiosperm flowers have been quite well understood for many years, the development and maturation of the nectar gland and the secretion of nectar in synchrony with the maturation of the sexual organs appears to be one of the flower's best-kept secrets. Here we review key findings controlling these processes. We also raise key questions that need to be addressed to develop crop ecological functions that take into consideration pollinators' needs.

Hybridization-chain-reaction is a relevant method for in situ detection of M2d-like macrophages in a mini-pig model.

Macrophages are a heterogeneous population of cells with an important role in innate immunity and tissue regeneration. Based on in vitro experiments, macrophages have been subdivided into five distinct subtypes named M1, M2a, M2b, M2c, and M2d, depending on the means of their activation and the cell surface markers they display. Whether all subtypes can be detected in vivo is still unclear. The identification of macrophages in vivo in the regenerating muscle could be used as a new diagnostic tool to monitor therapeutic strategies for tissue repair. The use of classical immunolabeling techniques is unable to discriminate between different M2 macrophages and a functional characterization of these macrophages is lacking. Using in situ hybridization coupled with hybridization-chain-reaction detection (HCR), we achieved the identification of M2d-like macrophages within regenerating muscle and applied this technique to understand the role of M2 macrophages in the regeneration of irradiated pig-muscle after adipose tissue stem cell treatment. Our work highlights the limits of immunolabeling and the usefulness of HCR analysis to provide valuable information for macrophage characterization.

Roles of BdUNICULME4 and BdLAXATUM-A in the non-domesticated grass Brachypodium distachyon.

In cultivated grasses, tillering, spike architecture and seed shattering represent major agronomical traits. In barley, maize and rice, the NOOT-BOP-COCH-LIKE (NBCL) genes play important roles in development, especially in ligule development, tillering and flower identity. However, compared with dicots, the role of grass NBCL genes is underinvestigated. To better understand the role of grass NBCLs and to overcome any effects of domestication that might conceal their original functions, we studied TILLING nbcl mutants in the non-domesticated grass Brachypodium distachyon. In B. distachyon, the NBCL genes BdUNICULME4 (CUL4) and BdLAXATUM-A (LAXA) are orthologous, respectively, to the barley HvUniculme4 and HvLaxatum-a, to the maize Zmtassels replace upper ears1 and Zmtassels replace upper ears2 and to the rice OsBLADE-ON-PETIOLE1 and OsBLADE-ON-PETIOLE2/3. In B. distachyon, our reverse genetics study shows that CUL4 is not essential for the establishment of the blade-sheath boundary but is necessary for the development of the ligule and auricles. We report that CUL4 also exerts a positive role in tillering and a negative role in spikelet meristem activity. On the other hand, we demonstrate that LAXA plays a negative role in tillering, positively participates in spikelet development and contributes to the control of floral organ number and identity. In this work, we functionally characterized two new NBCL genes in a context of non-domesticated grass and highlighted original roles for grass NBCL genes that are related to important agronomical traits.

The gynoecious CmWIP1 transcription factor interacts with CmbZIP48 to inhibit carpel development.

In angiosperms, sex determination leads to development of unisexual flowers. In Cucumis melo, development of unisexual male flowers results from the expression of the sex determination gene, CmWIP1, in carpel primordia. To bring new insight on the molecular mechanisms through which CmWIP1 leads to carpel abortion in male flowers, we used the yeast two-hybrid approach to look for CmWIP1-interacting proteins. We found that CmWIP1 physically interacts with an S2 bZIP transcription factor, CmbZIP48. We further determined the region mediating the interaction and showed that it involves the N-terminal part of CmWIP1. Using laser capture microdissection coupled with quantitative real-time gene expression analysis, we demonstrated that CmWIP1 and CmbZIP48 share a similar spatiotemporal expression pattern, providing the plant organ context for the CmWIP1-CmbZIP48 protein interaction. Using sex transition mutants, we demonstrated that the expression of the male promoting gene CmWIP1 correlates with the expression of CmbZIP48. Altogether, our data support a model in which the coexpression and the physical interaction of CmWIP1 and CmbZIP48 trigger carpel primordia abortion, leading to the development of unisexual male flowers.

Gibberellins negatively regulate the development of Medicago truncatula root system.

The root system displays a remarkable plasticity that enables plants to adapt to changing environmental conditions. This plasticity is tightly linked to the activity of root apical meristems (RAMs) and to the formation of lateral roots, both controlled by related hormonal crosstalks. In Arabidopsis thaliana, gibberellins (GAs) were shown to positively control RAM growth and the formation of lateral roots. However, we showed in Medicago truncatula that GAs negatively regulate root growth and RAM size as well as the number of lateral roots depending at least on the MtDELLA1 protein. By using confocal microscopy and molecular analyses, we showed that GAs primarily regulate RAM size by affecting cortical cell expansion and additionally negatively regulate a subset of cytokinin-induced root expansin encoding genes. Moreover, GAs reduce the number of cortical cell layers, resulting in the formation of both shorter and thinner roots. These results suggest contrasting effects of GA regulations on the root system architecture depending on plant species.

Three cytosolic glutamine synthetase isoforms localized in different-order veins act together for N remobilization and seed filling in Arabidopsis.

Glutamine synthetase (GS) is central for ammonium assimilation and consists of cytosolic (GS1) and chloroplastic (GS2) isoenzymes. During plant ageing, GS2 protein decreases due to chloroplast degradation, and GS1 activity increases to support glutamine biosynthesis and N remobilization from senescing leaves. The role of the different Arabidopsis GS1 isoforms in nitrogen remobilization was examined using 15N tracing experiments. Only the gln1;1-gln1;2-gln1;3 triple-mutation affecting the three GLN1;1, GLN1;2, and GLN1;3 genes significantly reduced N remobilization, total seed yield, individual seed weight, harvest index, and vegetative biomass. The triple-mutant accumulated a large amount of ammonium that could not be assimilated by GS1. Alternative ammonium assimilation through asparagine biosynthesis was increased and was related to higher ASN2 asparagine synthetase transcript levels. The GS2 transcript, protein, and activity levels were also increased to compensate for the lack of GS1-related glutamine biosynthesis. Localization of the different GLN1 genes showed that they were all expressed in the phloem companion cells but in veins of different order. Our results demonstrate that glutamine biosynthesis for N-remobilization occurs in veins of all orders (major and minor) in leaves, it is mainly catalysed by the three major GS1 isoforms (GLN1;1, GLN1;2, and GLN1;3), and it is alternatively supported by AS2 in the veins and GS2 in the mesophyll cells.

An Integrated "Multi-Omics" Comparison of Embryo and Endosperm Tissue-Specific Features and Their Impact on Rice Seed Quality.

Although rice is a key crop species, few studies have addressed both rice seed physiological and nutritional quality, especially at the tissue level. In this study, an exhaustive "multi-omics" dataset on the mature rice seed was obtained by combining transcriptomics, label-free shotgun proteomics and metabolomics from embryo and endosperm, independently. These high-throughput analyses provide a new insight on the tissue-specificity related to rice seed quality. Foremost, we pinpointed that extensive post-transcriptional regulations occur at the end of rice seed development such that the embryo proteome becomes much more diversified than the endosperm proteome. Secondly, we observed that survival in the dry state in each seed compartment depends on contrasted metabolic and enzymatic apparatus in the embryo and the endosperm, respectively. Thirdly, it was remarkable to identify two different sets of starch biosynthesis enzymes as well as seed storage proteins (glutelins) in both embryo and endosperm consistently with the supernumerary embryo hypothesis origin of the endosperm. The presence of a putative new glutelin with a possible embryonic favored abundance is described here for the first time. Finally, we quantified the rate of mRNA translation into proteins. Consistently, the embryonic panel of protein translation initiation factors is much more diverse than that of the endosperm. This work emphasizes the value of tissue-specificity-centered "multi-omics" study in the seed to highlight new features even from well-characterized pathways. It paves the way for future studies of critical genetic determinants of rice seed physiological and nutritional quality.

Natural and induced loss of function mutations in SlMBP21 MADS-box gene led to jointless-2 phenotype in tomato.

Abscission is the mechanism by which plants disconnect unfertilized flowers, ripe fruits, senescent or diseased organs from the plant. In tomato, pedicel abscission is an important agronomic factor that controls yield and post-harvest fruit quality. Two non-allelic mutations, jointless (j) and jointless-2 (j-2), controlling pedicel abscission zone formation have been documented but only j-2 has been extensively used in breeding. J was shown to encode a MADS-box protein. Using a combination of physical mapping and gene expression analysis we identified a positional candidate, Solyc12g038510, associated with j-2 phenotype. Targeted knockout of Solyc12g038510, using CRISPR/Cas9 system, validated our hypothesis. Solyc12g038510 encodes the MADS-box protein SlMBP21. Molecular analysis of j-2 natural variation revealed two independent loss-of-function mutants. The first results of an insertion of a Rider retrotransposable element. The second results of a stop codon mutation that leads to a truncated protein form. To bring new insights into the role of J and J-2 in abscission zone formation, we phenotyped the single and the double mutants and the engineered alleles. We showed that J is epistatic to J-2 and that the branched inflorescences and the leafy sepals observed in accessions harboring j-2 alleles are likely the consequences of linkage drags.

A cucurbit androecy gene reveals how unisexual flowers develop and dioecy emerges.

Understanding the evolution of sex determination in plants requires identifying the mechanisms underlying the transition from monoecious plants, where male and female flowers coexist, to unisexual individuals found in dioecious species. We show that in melon and cucumber, the androecy gene controls female flower development and encodes a limiting enzyme of ethylene biosynthesis, ACS11. ACS11 is expressed in phloem cells connected to flowers programmed to become female, and ACS11 loss-of-function mutants lead to male plants (androecy). CmACS11 represses the expression of the male promoting gene CmWIP1 to control the development and the coexistence of male and female flowers in monoecious species. Because monoecy can lead to dioecy, we show how a combination of alleles of CmACS11 and CmWIP1 can create artificial dioecy.

Repression of Lateral Organ Boundary Genes by PENNYWISE and POUND-FOOLISH Is Essential for Meristem Maintenance and Flowering in Arabidopsis.

In the model plant Arabidopsis (Arabidopsis thaliana), endogenous and environmental signals acting on the shoot apical meristem cause acquisition of inflorescence meristem fate. This results in changed patterns of aerial development seen as the transition from making leaves to the production of flowers separated by elongated internodes. Two related BEL1-like homeobox genes, PENNYWISE (PNY) and POUND-FOOLISH (PNF), fulfill this transition. Loss of function of these genes impairs stem cell maintenance and blocks internode elongation and flowering. We show here that pny pnf apices misexpress lateral organ boundary genes BLADE-ON-PETIOLE1/2 (BOP1/2) and KNOTTED-LIKE FROM ARABIDOPSIS THALIANA6 (KNAT6) together with ARABIDOPSIS THALIANA HOMEOBOX GENE1 (ATH1). Inactivation of genes in this module fully rescues pny pnf defects. We further show that BOP1 directly activates ATH1, whereas activation of KNAT6 is indirect. The pny pnf restoration correlates with renewed accumulation of transcripts conferring floral meristem identity, including FD, SQUAMOSA PROMOTER-BINDING PROTEIN LIKE genes, LEAFY, and APETALA1. To gain insight into how this module blocks flowering, we analyzed the transcriptome of BOP1-overexpressing plants. Our data suggest a central role for the microRNA156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE-microRNA172 module in integrating stress signals conferred in part by promotion of jasmonic acid biosynthesis. These data reveal a potential mechanism by which repression of lateral organ boundary genes by PNY-PNF is essential for flowering.

A conserved role for CUP-SHAPED COTYLEDON genes during ovule development.

The evolution of plant reproductive strategies has led to a remarkable diversity of structures, especially within the flower, a structure characteristic of the angiosperms. In flowering plants, sexual reproduction depends notably on the development of the gynoecium that produces and protects the ovules. In Arabidopsis thaliana, ovule initiation is promoted by the concerted action of auxin with CUC1 (CUP-SHAPED COTYLEDON1) and CUC2, two genes that encode transcription factors of the NAC family (NAM/ATAF1,2/CUC). Here we highlight an additional role for CUC2 and CUC3 in Arabidopsis thaliana ovule separation. While CUC1 and CUC2 are broadly expressed in the medial tissue of the gynoecium, CUC2 and CUC3 are expressed in the placental tissue between developing ovules. Consistent with the partial overlap between CUC1, CUC2 and CUC3 expression patterns, we show that CUC proteins can physically interact, both in yeast cells and in planta. We found that the cuc2;cuc3 double mutant specifically harbours defects in ovule separation, producing fused seeds that share the seed coat, and suggesting that CUC2 and CUC3 promote ovule separation in a partially redundant manner. Functional analyses show that CUC transcription factors are also involved in ovule development in Cardamine hirsuta. Additionally we show a conserved expression pattern of CUC orthologues between ovule primordia in other phylogenetically distant species with different gynoecium architectures. Taken together these results suggest an ancient role for CUC transcription factors in ovule separation, and shed light on the conservation of mechanisms involved in the development of innovative structures.