Editing of SlWRKY29 by CRISPR-activation promotes somatic embryogenesis in Solanum lycopersicum cv. Micro-Tom.

At present, the development of plants with improved traits like superior quality, high yield, or stress resistance, are highly desirable in agriculture. Accelerated crop improvement, however, must capitalize on revolutionary new plant breeding technologies, like genetically modified and gene-edited crops, to heighten food crop traits. Genome editing still faces ineffective methods for the transformation and regeneration of different plant species and must surpass the genotype dependency of the transformation process. Tomato is considered an alternative plant model system to rice and Arabidopsis, and a model organism for fleshy-fruited plants. Furthermore, tomato cultivars like Micro-Tom are excellent models for tomato research due to its short life cycle, small size, and capacity to grow at high density. Therefore, we developed an indirect somatic embryo protocol from cotyledonary tomato explants and used this to generate epigenetically edited tomato plants for the SlWRKY29 gene via CRISPR-activation (CRISPRa). We found that epigenetic reprogramming for SlWRKY29 establishes a transcriptionally permissive chromatin state, as determined by an enrichment of the H3K4me3 mark. A whole transcriptome analysis of CRISPRa-edited pro-embryogenic masses and mature somatic embryos allowed us to characterize the mechanism driving somatic embryo induction in the edited tomato cv. Micro-Tom. Furthermore, we show that enhanced embryo induction and maturation are influenced by the transcriptional effector employed during CRISPRa, as well as by the medium composition and in vitro environmental conditions such as osmotic components, plant growth regulators, and light intensity.

Plant genome information facilitates plant functional genomics.

MAIN CONCLUSION: In this review, we give an overview of plant sequencing efforts and how this impacts plant functional genomics research. Plant genome sequence information greatly facilitates the studies of plant biology, functional genomics, evolution of genomes and genes, domestication processes, phylogenetic relationships, among many others. More than two decades of sequencing efforts have boosted the number of available sequenced plant genomes. The first plant genome, of Arabidopsis, was published in the year 2000 and currently, 4604 plant genomes from 1482 plant species have been published. Various large sequence initiatives are running, which are planning to produce tens of thousands of sequenced plant genomes in the near future. In this review, we give an overview on the status of sequenced plant genomes and on the use of genome information in different research areas.

Two orthogonal differentiation gradients locally coordinate fruit morphogenesis.

Morphogenesis requires the coordination of cellular behaviors along developmental axes. In plants, gradients of growth and differentiation are typically established along a single longitudinal primordium axis to control global organ shape. Yet, it remains unclear how these gradients are locally adjusted to regulate the formation of complex organs that consist of diverse tissue types. Here we combine quantitative live imaging at cellular resolution with genetics, and chemical treatments to understand the formation of Arabidopsis thaliana female reproductive organ (gynoecium). We show that, contrary to other aerial organs, gynoecium shape is determined by two orthogonal, time-shifted differentiation gradients. An early mediolateral gradient controls valve morphogenesis while a late, longitudinal gradient regulates style differentiation. Local, tissue-dependent action of these gradients serves to fine-tune the common developmental program governing organ morphogenesis to ensure the specialized function of the gynoecium.

A common regulatory switch controls a suite of C4 traits in multiple cell types.

The C4 photosynthetic pathway provided a major advantage to plants growing in hot, dry environments, including the ancestors of our most productive crops. Two traits were essential for the evolution of this pathway: increased vein density and the functionalization of bundle sheath cells for photosynthesis. Although GRAS transcriptional regulators, including SHORT ROOT (SHR), have been implicated in mediating leaf patterning in both C3 and C4 species, little is known about what controls the specialized features of the cells that mediate C4 metabolism and physiology. We show in the model monocot, Setaria viridis, that SHR regulates components of multiple cell identities, including chloroplast biogenesis and photosynthetic gene expression in bundle sheath cells, a central feature of C4 plants. Furthermore, we found that it also contributes to the two-cell compartmentalization of the characteristic four-carbon shuttle pathway. Disruption of SHR function clearly reduced photosynthetic capacity and seed yield in mutant plants under heat stress. Together, these results show how cell identities are remodeled by SHR to host the suite of traits characteristic of C4 regulation, which are a main engineering target in non-C4 crops to improve climate resilience.

EXPANSIN15 is involved in flower and fruit development in Arabidopsis.

KEY MESSAGE: EXPANSIN15 is involved in petal cell morphology and size, the fusion of the medial tissues in the gynoecium and expansion of fruit valve cells. It genetically interacts with SPATULA and FRUITFULL. Cell expansion is fundamental for the formation of plant tissues and organs, contributing to their final shape and size during development. To better understand this process in flower and fruit development, we have studied the EXPANSIN15 (EXPA15) gene, which showed expression in petals and in the gynoecium. By analyzing expa15 mutant alleles, we found that EXPA15 is involved in petal shape and size determination, by affecting cell morphology and number. EXPA15 also has a function in fruit size, by affecting cell size and number. Furthermore, EXPA15 promotes fusion of the medial tissues in the gynoecium. In addition, we observed genetic interactions with the transcription factors SPATULA (SPT) and FRUITFULL (FUL) in gynoecium medial tissue fusion, style and stigma development and fruit development in Arabidopsis. These findings contribute to the importance of EXPANSINS in floral and fruit development in Arabidopsis.

A high-resolution gene expression map of the medial and lateral domains of the gynoecium of Arabidopsis.

Angiosperms are characterized by the formation of flowers, and in their inner floral whorl, one or various gynoecia are produced. These female reproductive structures are responsible for fruit and seed production, thus ensuring the reproductive competence of angiosperms. In Arabidopsis (Arabidopsis thaliana), the gynoecium is composed of two fused carpels with different tissues that need to develop and differentiate to form a mature gynoecium and thus the reproductive competence of Arabidopsis. For these reasons, they have become the object of study for floral and fruit development. However, due to the complexity of the gynoecium, specific spatio-temporal tissue expression patterns are still scarce. In this study, we used precise laser-assisted microdissection and high-throughput RNA sequencing to describe the transcriptional profiles of the medial and lateral domain tissues of the Arabidopsis gynoecium. We provide evidence that the method used is reliable and that, in addition to corroborating gene expression patterns of previously reported regulators of these tissues, we found genes whose expression dynamics point to being involved in cytokinin and auxin homeostasis and in cell cycle progression. Furthermore, based on differential gene expression analyses, we functionally characterized several genes and found that they are involved in gynoecium development. This resource is available via the Arabidopsis eFP browser and will serve the community in future studies on developmental and reproductive biology.

Organogenic events during gynoecium and fruit development in Arabidopsis.

Angiosperms are the most successful group of land plants. This success is mainly due to the gynoecium, the innermost whorl of the flower. In Arabidopsis, the gynoecium is a syncarpic structure formed by two congenitally fused carpels. At the fusion edges of the carpels, the carpel margin meristem forms. This quasi-meristem is important for medial-tissue development, including the ovules. After the double fertilization, both the seeds and fruit begin to develop. Due to the importance of seeds and fruits as major food sources worldwide, it has been an important task for the scientific community to study gynoecium development. In this review, we present the most recent advances in Arabidopsis gynoecium patterning, as well as some questions that remain unanswered.

Hormones and Flower Development in Arabidopsis.

Sexual reproduction requires the participation of two gametes, female and male. In angiosperms, gametes develop in specialized organs, pollen (containing the male gametes) develops in the stamens, and the ovule (containing the female gamete) develops in the gynoecium. In Arabidopsis thaliana, the female and male sexual organs are found within the same structure called flower, surrounded by the perianth, which is composed of petals and sepals. During flower development, different organs emerge in an established order and throughout their development distinct tissues within each organ are differentiated. All this requires the coordination and synchronization of several biological processes. To achieve this, hormones and genes work together. These components can interact at different levels generating hormonal interplay and both positive and negative feedback loops, which in turn, gives robustness, stability, and flexibility to flower development. Here, we summarize the progress made on elucidating the role of different hormonal pathways during flower development in Arabidopsis thaliana.

A NanoLuc-Based Transactivation Assay in Plants.

Protein-DNA interactions are determinant of the regulation of gene expression in living organisms. Luminescence studies have been used in a wide range of techniques to identify how gene transcription can be regulated by proteins such as transcription factors (TFs). Despite the great advances in the use of luciferases as reporters in the performance of this mechanism, some of them still have disadvantages that have been tried to be solved by the generation of new luciferases that induce a more stable and perfectly visualizable reaction. NanoLuc is a recently described luciferase that has been characterized by its efficient, stable, and powerful luminescence. These qualities have been considered to create a new and efficient reporter system to detect protein-DNA interactions. In this chapter, we take advantage of NanoLuc and describe its use in a reliable procedure to detect protein-DNA interactions in Nicotiana benthamiana extracts and entire leaves.

Hormone immunolabeling in resin-embedded Arabidopsis tissues.

Here, we present a protocol for immunolabeling of molecules in Arabidopsis tissues. We describe steps for tissue fixation and embedding in resin of microtome-derived sections, immunolabeling using fluorescent and non-fluorescent secondary antibodies, and visualization of cytokinin and auxin molecules. This protocol is suitable to study reproductive structures such as inflorescences, flowers, fruits, and tissue-culture-derived samples. This protocol is useful for studying the distribution of a wide range of molecules including hormones and cell wall components. For complete details on the use and execution of this protocol, please refer to Herrera-Ubaldo et al. (2019).1.

An evo-devo view of the gynoecium.

The appearance of the flower marks a key event in the evolutionary history of plants. Among the four types of floral organs, the gynoecium represents the major adaptive advantage of the flower. The gynoecium is an enclosing structure that protects and facilitates the fertilization of the ovules, which then mature as seeds. Upon fertilization, in many species, the gynoecium itself eventually becomes the fruit, which contributes to the dispersal of the seeds. However, despite its importance and the recent advances in our understanding of the genetic regulatory network guiding early gynoecium development, many questions remain to be resolved regarding the extent of the conservation of the molecular mechanisms for gynoecium development among different taxa, and how these mechanisms give origin and diversification to the gynoecium. In this review, we compile the existing knowledge about the evolution, development, and molecular mechanisms involved in the origin and evolution of the gynoecium.

CRISPRa-mediated transcriptional activation of the SlPR-1 gene in edited tomato plants.

With the continuous deterioration of arable land due to an ever-growing population, improvement of crops and crop protection have a fundamental role in maintaining and increasing crop productivity. Alternatives to the use of pesticides encompass the use of biological control agents, generation of new resistant crop cultivars, the application of plant activator agrochemicals to enhance plant defenses, and the use of gene editing techniques, like the CRISPR-Cas system. Here, we test the hypothesis that epigenome editing, via CRISPR activation (CRISPRa), activate tomato plant defense genes to confer resistance against pathogen attack. We provide evidence that edited tomato plants for the PATHOGENESIS-RELATED GENE 1 gene (SlPR-1) show enhanced disease resistance to Clavibacter michiganensis subsp. michiganensis infection. Resistance was assessed by evaluating disease progression and symptom appearance, pathogen accumulation, and changes in SlPR-1 gene expression at different time points. We determined that CRISPRa-edited plants develop enhanced disease-resistant to the pathogen without altering their agronomic characteristics and, above all, preventing the advancement of disease symptoms, stem canker, and plant death.

Novel Roles of SPATULA in the Control of Stomata and Trichome Number, and Anthocyanin Biosynthesis.

The bHLH transcription factor SPATULA (SPT) has been identified as a regulator during different stages of Arabidopsis development, including the control of leaf size. However, the mechanism via which it performs this function has not been elucidated. To better understand the role of SPT during leaf development, we used a transcriptomic approach to identify putative target genes. We found putative SPT target genes related to leaf development, and to stomata and trichome formation. Furthermore, genes related to anthocyanin biosynthesis. In this work, we demonstrate that SPT is a negative regulator of stomata number and a positive regulator of trichome number. In addition, SPT is required for sucrose-mediated anthocyanin biosynthesis.

The protein-protein interaction landscape of transcription factors during gynoecium development in Arabidopsis.

Flowers are composed of organs whose identity is defined by the combinatorial activity of transcription factors (TFs). The interactions between MADS-box TFs and protein complex formation have been schematized in the floral quartet model of flower development. The gynoecium is the flower's female reproductive part, crucial for fruit and seed production and, hence, for reproductive success. After the establishment of carpel identity, many tissues arise to form a mature gynoecium. TFs have been described as regulators of gynoecium development, and some interactions and complexes have been identified. However, broad knowledge about the interactions among these TFs and their participation during development remains scarce. In this study, we used a systems biology approach to understand the formation of a complex reproductive unit-as the gynoecium-by mapping binary interactions between well-characterized TFs. We analyzed almost 4500 combinations and detected more than 250 protein-protein interactions (PPIs), resulting in a process-specific interaction map. Topological analyses suggest hidden functions and novel roles for many TFs. In addition, we observed a close relationship between TFs involved in auxin and cytokinin-signaling pathways and other TFs. Furthermore, we analyzed the network by combining PPI data, expression, and genetic data, which helped us to dissect it into several dynamic spatio-temporal subnetworks related to gynoecium development processes. Finally, we generated an extended PPI network that predicts new players in gynoecium development. Taken together, all these results serve as a valuable resource for the plant community.

Twisting development, the birth of a potential new gene.

Evolution has long been considered to be a conservative process in which new genes arise from pre-existing genes through gene duplication, domain shuffling, horizontal transfer, overprinting, retrotransposition, etc. However, this view is changing as new genes originating from non-genic sequences are discovered in different organisms. Still, rather limited functional information is available. Here, we have identified TWISTED1 (TWT1), a possible de novo-originated protein-coding gene that modifies microtubule arrangement and causes helicoidal growth in Arabidopsis thaliana when its expression is increased. Interestingly, even though TWT1 is a likely recent gene, the lack of TWT1 function affects A. thaliana development. TWT1 seems to have originated from a non-genic sequence. If so, it would be one of the few examples to date of how during evolution de novo genes are integrated into developmental cellular and organismal processes.

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.

Gynoecium and fruit development in Arabidopsis.

Flowering plants produce flowers and one of the most complex floral structures is the pistil or the gynoecium. All the floral organs differentiate from the floral meristem. Various reviews exist on molecular mechanisms controlling reproductive development, but most focus on a short time window and there has been no recent review on the complete developmental time frame of gynoecium and fruit formation. Here, we highlight recent discoveries, including the players, interactions and mechanisms that govern gynoecium and fruit development in Arabidopsis. We also present the currently known gene regulatory networks from gynoecium initiation until fruit maturation.

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.

Developmental Signals in the 21st Century; New Tools and Advances in Plant Signaling.

This special issue includes different research papers and reviews that studied the role of signaling cascades controlling both plant developmental processes and plant response mechanisms to biotic and abiotic stresses [...].

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.