A transcriptional complex of NGATHA and bHLH transcription factors directs stigma development in Arabidopsis.

The stigma is an angiosperm-specific tissue that is essential for pollination. In the last two decades, several transcription factors with key roles in stigma development in Arabidopsis thaliana have been identified. However, genetic analyses have thus far been unable to unravel the precise regulatory interactions among these transcription factors or the molecular basis for their selective roles in different spatial and temporal domains. Here, we show that the NGATHA (NGA) and HECATE (HEC) transcription factors, which are involved in different developmental processes but are both essential for stigma development, require each other to perform this function. This relationship is likely mediated by their physical interaction in the apical gynoecium. NGA/HEC transcription factors subsequently upregulate INDEHISCENT (IND) and SPATULA and are indispensable for the binding of IND to some of its targets to allow stigma differentiation. Our findings support a nonhierarchical regulatory scenario in which the combinatorial action of different transcription factors provides exquisite temporal and spatial specificity of their developmental outputs.

New roles of NO TRANSMITTING TRACT and SEEDSTICK during medial domain development in Arabidopsis fruits.

The gynoecium, the female reproductive part of the flower, is key for plant sexual reproduction. During its development, inner tissues such as the septum and the transmitting tract tissue, important for pollen germination and guidance, are formed. In Arabidopsis, several transcription factors are known to be involved in the development of these tissues. One of them is NO TRANSMITTING TRACT (NTT), essential for transmitting tract formation. We found that the NTT protein can interact with several gynoecium-related transcription factors, including several MADS-box proteins, such as SEEDSTICK (STK), known to specify ovule identity. Evidence suggests that NTT and STK control enzyme and transporter-encoding genes involved in cell wall polysaccharide and lipid distribution in gynoecial medial domain cells. The results indicate that the simultaneous loss of NTT and STK activity affects polysaccharide and lipid deposition and septum fusion, and delays entry of septum cells to their normal degradation program. Furthermore, we identified KAWAK, a direct target of NTT and STK, which is required for the correct formation of fruits in Arabidopsis These findings position NTT and STK as important factors in determining reproductive competence.

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.

Arabidopsis COGWHEEL1 links light perception and gibberellins with seed tolerance to deterioration.

Light is a major regulator of plant growth and development by antagonizing gibberellins (GA), and we provide evidence for a role of light perception and GA in seed coat formation and seed tolerance to deterioration. We have identified two activation-tagging mutants of Arabidopsis thaliana, cog1-2D and cdf4-1D, with improved seed tolerance to deterioration linked to increased expression of COG1/DOF1.5 and CDF4/DOF2.3, respectively. These encode two homologous DOF transcription factors, with COG1 most highly expressed in seeds. Improved tolerance to seed deterioration was reproduced in transgenic plants overexpressing these genes, and loss of function from RNA interference resulted in opposite phenotypes. Overexpressions of COG1 and CDF4 have been described to attenuate various light responses mediated by phytochromes. Accordingly, we found that phyA and phyB mutants exhibit increased seed tolerance to deterioration. The phenotype of tolerance to deterioration conferred by gain of function of COG1 and by loss of function of phytochromes is of maternal origin, is also observed under natural aging conditions and correlates with a seed coat with increased suberin and reduced permeability. In developing siliques of the cog1-2D mutant the expression of the GA biosynthetic gene GA3OX3 and levels of GA1 are higher than in the wild type. These results explain the antagonism between phytochromes and COG1 in terms of the inhibition and the activation, respectively, of GA action.

The NTT transcription factor promotes replum development in Arabidopsis fruits.

Fruits are complex plant structures that nurture seeds and facilitate their dispersal. The Arabidopsis fruit is termed silique. It develops from the gynoecium, which has a stigma, a style, an ovary containing the ovules, and a gynophore. Externally, the ovary consists of two valves, and their margins lay adjacent to the replum, which is connected to the septum that internally divides the ovary. In this work we describe the role for the zinc-finger transcription factor NO TRANSMITTING TRACT (NTT) in replum development. NTT loss of function leads to reduced replum width and cell number, whereas increased expression promotes replum enlargement. NTT activates the homeobox gene BP, which, together with RPL, is important for replum development. In addition, the NTT protein is able to bind the BP promoter in yeast, and when this binding region is not present, NTT fails to activate BP in the replum. Furthermore, NTT interacts with itself and different proteins involved in fruit development: RPL, STM, FUL, SHP1 and SHP2 in yeast and in planta. Moreover, its genetic interactions provide further evidence about its biological relevance in replum development.

ARABIDOPSIS THALIANA HOMEOBOX25 uncovers a role for Gibberellins in seed longevity.

Seed longevity is crucial for agriculture and plant genetic diversity, but it is limited by cellular damage during storage. Seeds are protected against aging by cellular defenses and by structures such as the seed coat. We have screened an activation-tagging mutant collection of Arabidopsis (Arabidopsis thaliana) and selected four dominant mutants with improved seed longevity (isl1-1D to isl4-1D) under both natural and accelerated aging conditions. In the isl1-1D mutant, characterized in this work, overexpression of the transcription factor ARABIDOPSIS THALIANA HOMEOBOX25 (ATHB25; At5g65410) increases the expression of GIBBERELLIC ACID3-OXIDASE2, encoding a gibberellin (GA) biosynthetic enzyme, and the levels of GA1 and GA4 are higher (3.2- and 1.4-fold, respectively) in the mutant than in the wild type. The morphological and seed longevity phenotypes of the athb25-1D mutant were recapitulated in transgenic plants with moderate (4- to 6-fold) overexpression of ATHB25. Simultaneous knockdown of ATHB25, ATHB22, and ATHB31 expression decreases seed longevity, as does loss of ATHB25 and ATHB22 function in a double mutant line. Seeds from wild-type plants treated with GA and from a quintuple DELLA mutant (with constitutive GA signaling) are more tolerant to aging, providing additional evidence for a role of GA in seed longevity. A correlation was observed in several genotypes between seed longevity and mucilage formation at the seed surface, suggesting that GA may act by reinforcing the seed coat. This mechanism was supported by the observation of a maternal effect in reciprocal crosses between the wild type and the athb25-1D mutant.

Leaf expansion in Arabidopsis is controlled by a TCP-NGA regulatory module likely conserved in distantly related species.

The NGATHA (NGA) clade of transcription factors (TFs) forms a small subfamily of four members in Arabidopsis thaliana. NGA genes act redundantly to direct the development of apical tissues in the gynoecium, where they have been shown to be essential for style and stigma specification. In addition, NGA genes have a more general role in controlling lateral organ growth. The four NGA genes in Arabidopsis are expressed in very similar domains, although little is known about the nature of their putative regulators. Here, we have identified a conserved region within the four NGA promoters that we have used as a bait to screen a yeast library, aiming to identify such NGA regulators. Three members of the TCP family of TFs, named after the founding factors TEOSINTE BRANCHED 1, CYCLOIDEA and PROLIFERATING CELL FACTOR 1 AND 2), were recovered from this screening, of which two [TCP2 and TCP3, members of the CINCINNATA (CIN) family of TCP genes (CIN-TCP) subclade] were shown to activate the NGA3 promoter in planta. We provide evidence that support that CIN-TCP genes are true regulators of NGA gene expression, and that part of the CIN-TCP role in leaf development is mediated by NGA upregulation. Moreover, we have found that this TCP-NGA regulatory interaction is likely conserved in angiosperms, including important crop species, for which the regulation of leaf development is a target for biotechnological improvement.

A Model for the Gene Regulatory Network Along the Arabidopsis Fruit Medio-Lateral Axis: Rewiring the Pod Shatter Process.

Different convergent evolutionary strategies adopted by angiosperm fruits lead to diverse functional seed dispersal units. Dry dehiscent fruits are a common type of fruit, characterized by their lack of fleshy pericarp and the release of seeds at maturity through openings (dehiscence zones, DZs) in their structure. In previous decades, a set of core players in DZ formation have been intensively characterized in Arabidopsis and integrated in a gene regulatory network (GRN) that explains the morphogenesis of these tissues. In this work, we compile all the experimental data available to date to build a discrete Boolean model as a mechanistic approach to validate the network and, if needed, to identify missing components of the GRN and/or propose new hypothetical regulatory interactions, but also to provide a new formal framework to feed further work in Brassicaceae fruit development and the evolution of seed dispersal mechanisms. Hence, by means of exhaustive in-silico validations and experimental evidence, we are able to incorporate both the NO TRANSMITTING TRACT (NTT) transcription factor as a new additional node, and a new set of regulatory hypothetical rules to uncover the dynamics of Arabidopsis DZ specification.

HDACs MADS-domain protein interaction: a case study of HDA15 and XAL1 in Arabidopsis thaliana.

Cellular behavior, cell differentiation and ontogenetic development in eukaryotes result from complex interactions between epigenetic and classic molecular genetic mechanisms, with many of these interactions still to be elucidated. Histone deacetylase enzymes (HDACs) promote the interaction of histones with DNA by compacting the nucleosome, thus causing transcriptional repression. MADS-domain transcription factors are highly conserved in eukaryotes and participate in controlling diverse developmental processes in animals and plants, as well as regulating stress responses in plants. In this work, we focused on finding out putative interactions of Arabidopsis thaliana HDACs and MADS-domain proteins using an evolutionary perspective combined with bioinformatics analyses and testing the more promising predicted interactions through classic molecular biology tools. Through bioinformatic analyses, we found similarities between HDACs proteins from different organisms, which allowed us to predict a putative protein-protein interaction between the Arabidopsis thaliana deacetylase HDA15 and the MADS-domain protein XAANTAL1 (XAL1). The results of two-hybrid and Bimolecular Fluorescence Complementation analysis demonstrated in vitro and in vivo HDA15-XAL1 interaction in the nucleus. Likely, this interaction might regulate developmental processes in plants as is the case for this type of interaction in animals.

Genetic and Phenotypic Analyses of Carpel Development in Arabidopsis.

Carpels are the female reproductive organs of the flower, organized in a gynoecium, which is likely the most complex organ of the plant. The gynoecium provides protection for the ovules, helps to discriminate between male gametophytes, and facilitates successful pollination. After fertilization, it develops into a fruit, a specialized organ for seed protection and dispersal. To carry out all these functions, coordinated patterning and tissue specification within the developing gynoecium has to be achieved. In this chapter, we provide different methods to characterize defects in carpel morphogenesis and patterning associated with developmental mutations, as well as a list of reporter lines that can be used to facilitate genetic analyses.

Shattering fruits: variations on a dehiscent theme.

Fruits are seed dispersal units, and for that they have evolved different strategies to facilitate separation and dispersal of the progeny from the mother plant. A great proportion of fruits from different clades are dry and dehiscent, opening upon maturity to disperse the seeds. In the last two decades, intense research mainly in Arabidopsis has uncovered the basic network that controls the differentiation of the Arabidopsis fruit dehiscence zone. This review focuses on recent discoveries that have helped to complete the picture, as well as the insights from evo-devo and crop domestication studies that show how the conservation/variation of the elements of this network across species accounts for its evolutionary plasticity and the origin of evolutionary innovations.

Genetic and phenotypic analyses of carpel development in Arabidopsis.

Carpels are the female reproductive organs of the flower, organized in a gynoecium, which is arguably the most complex organ of a plant. The gynoecium provides protection for the ovules, helps to discriminate between male gametophytes, and facilitates successful pollination. After fertilization, it develops into a fruit, a specialized organ for seed protection and dispersal. To carry out all these functions, coordinated patterning and tissue specification within the developing gynoecium have to be achieved. In this chapter, we describe different methods to characterize defects in carpel patterning and morphogenesis associated with developmental mutations as well as a list of reporter lines that can be used to facilitate genetic analyses.

The effect of NGATHA altered activity on auxin signaling pathways within the Arabidopsis gynoecium.

The four NGATHA genes (NGA) form a small subfamily within the large family of B3-domain transcription factors of Arabidopsis thaliana. NGA genes act redundantly to direct the development of the apical tissues of the gynoecium, the style, and the stigma. Previous studies indicate that NGA genes could exert this function at least partially by directing the synthesis of auxin at the distal end of the developing gynoecium through the upregulation of two different YUCCA genes, which encode flavin monooxygenases involved in auxin biosynthesis. We have compared three developing pistil transcriptome data sets from wildtype, nga quadruple mutants, and a 35S::NGA3 line. The differentially expressed genes showed a significant enrichment for auxin-related genes, supporting the idea of NGA genes as major regulators of auxin accumulation and distribution within the developing gynoecium. We have introduced reporter lines for several of these differentially expressed genes involved in synthesis, transport and response to auxin in NGA gain- and loss-of-function backgrounds. We present here a detailed map of the response of these reporters to NGA misregulation that could help to clarify the role of NGA in auxin-mediated gynoecium morphogenesis. Our data point to a very reduced auxin synthesis in the developing apical gynoecium of nga mutants, likely responsible for the lack of DR5rev::GFP reporter activity observed in these mutants. In addition, NGA altered activity affects the expression of protein kinases that regulate the cellular localization of auxin efflux regulators, and thus likely impact auxin transport. Finally, protein accumulation in pistils of several ARFs was differentially affected by nga mutations or NGA overexpression, suggesting that these accumulation patterns depend not only on auxin distribution but could be also regulated by transcriptional networks involving NGA factors.