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.

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.

The bHLH transcription factor SPATULA enables cytokinin signaling, and both activate auxin biosynthesis and transport genes at the medial domain of the gynoecium.

Fruits and seeds are the major food source on earth. Both derive from the gynoecium and, therefore, it is crucial to understand the mechanisms that guide the development of this organ of angiosperm species. In Arabidopsis, the gynoecium is composed of two congenitally fused carpels, where two domains: medial and lateral, can be distinguished. The medial domain includes the carpel margin meristem (CMM) that is key for the production of the internal tissues involved in fertilization, such as septum, ovules, and transmitting tract. Interestingly, the medial domain shows a high cytokinin signaling output, in contrast to the lateral domain, where it is hardly detected. While it is known that cytokinin provides meristematic properties, understanding on the mechanisms that underlie the cytokinin signaling pattern in the young gynoecium is lacking. Moreover, in other tissues, the cytokinin pathway is often connected to the auxin pathway, but we also lack knowledge about these connections in the young gynoecium. Our results reveal that cytokinin signaling, that can provide meristematic properties required for CMM activity and growth, is enabled by the transcription factor SPATULA (SPT) in the medial domain. Meanwhile, cytokinin signaling is confined to the medial domain by the cytokinin response repressor ARABIDOPSIS HISTIDINE PHOSPHOTRANSFERASE 6 (AHP6), and perhaps by ARR16 (a type-A ARR) as well, both present in the lateral domains (presumptive valves) of the developing gynoecia. Moreover, SPT and cytokinin, probably together, promote the expression of the auxin biosynthetic gene TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) and the gene encoding the auxin efflux transporter PIN-FORMED 3 (PIN3), likely creating auxin drainage important for gynoecium growth. This study provides novel insights in the spatiotemporal determination of the cytokinin signaling pattern and its connection to the auxin pathway in the young gynoecium.

Selection of Reference Genes for Quantitative Real-Time RT-PCR Studies in Tomato Fruit of the Genotype MT-Rg1.

Quantitative real-time RT-PCR (qRT-PCR) has become one of the most widely used methods for accurate quantification of gene expression. Since there are no universal reference genes for normalization, the optimal strategy to normalize raw qRT-PCR data is to perform an initial comparison of a set of independent reference genes to assess the most stable ones in each biological model. Normalization of a qRT-PCR experiment helps to ensure that the results are both statistically significant and biologically meaningful. Tomato is the model of choice to study fleshy fruit development. The miniature tomato (Solanum lycopersicum L.) cultivar Micro-Tom (MT) is considered a model system for tomato genetics and functional genomics. A new genotype, containing the Rg1 allele, improves tomato in vitro regeneration. In this work, we evaluated the expression stability of four tomato reference genes, namely CAC, SAND, Expressed, and ACTIN2. We showed that the genes CAC and Exp are the best reference genes of the four we tested during fruit development in the MT-Rg1 genotype. Furthermore, we validated the reference genes by showing that the expression profiles of the transcription factors FRUITFULL1 and APETALA2c during fruit development are comparable to previous reports using other tomato cultivars.

ARACNe-based inference, using curated microarray data, of Arabidopsis thaliana root transcriptional regulatory networks.

BACKGROUND: Uncovering the complex transcriptional regulatory networks (TRNs) that underlie plant and animal development remains a challenge. However, a vast amount of data from public microarray experiments is available, which can be subject to inference algorithms in order to recover reliable TRN architectures. RESULTS: In this study we present a simple bioinformatics methodology that uses public, carefully curated microarray data and the mutual information algorithm ARACNe in order to obtain a database of transcriptional interactions. We used data from Arabidopsis thaliana root samples to show that the transcriptional regulatory networks derived from this database successfully recover previously identified root transcriptional modules and to propose new transcription factors for the SHORT ROOT/SCARECROW and PLETHORA pathways. We further show that these networks are a powerful tool to integrate and analyze high-throughput expression data, as exemplified by our analysis of a SHORT ROOT induction time-course microarray dataset, and are a reliable source for the prediction of novel root gene functions. In particular, we used our database to predict novel genes involved in root secondary cell-wall synthesis and identified the MADS-box TF XAL1/AGL12 as an unexpected participant in this process. CONCLUSIONS: This study demonstrates that network inference using carefully curated microarray data yields reliable TRN architectures. In contrast to previous efforts to obtain root TRNs, that have focused on particular functional modules or tissues, our root transcriptional interactions provide an overview of the transcriptional pathways present in Arabidopsis thaliana roots and will likely yield a plethora of novel hypotheses to be tested experimentally.

An efficient flat-surface collar-free grafting method for Arabidopsis thaliana seedlings.

BACKGROUND: Grafting procedures are an excellent tool to study long range signalling processes within a plant. In the last decade, suitable flat-surface grafting procedures for young Arabidopsis seedlings using a collar to support the graft have been developed, allowing the study of long-range signals from a molecular perspective. RESULTS: In the modification presented here, scion and stock are put together on the medium without supporting elements, while cotyledons are removed from the scion, resulting in increased grafting success that can reach up to 100%. At the same time, the protocol enables to process as many as 36 seedlings per hour, which combined with the high success percentage represents increased efficiency per time unit. CONCLUSIONS: Growing cotyledons usually push the scion and the rootstock away in the absence of a supporting element. Removing them at the grafting step greatly improved success rate and reduced post-grafting manipulations.