The nucellus: between cell elimination and sugar transport.

The architecture of the seed is shaped by the processes of tissue partitioning, which determines the volume ratio of maternal and zygotic tissues, and nutrient partitioning, which regulates nutrient distribution among tissues. In angiosperms, early seed development is characterized by antagonistic development of the nucellus maternal tissue and the endosperm fertilization product to become the main sugar sink. This process marked the evolution of angiosperms and outlines the most ancient seed architectures. In Arabidopsis, the endosperm partially eliminates the nucellus and imports sugars from the seed coat. Here, we show that the nucellus is symplasmically connected to the chalaza, the seed nutrient unloading zone, and works as both a sugar sink and source alongside the seed coat. After fertilization, the transient nucellus accumulates starch early on and releases it in the apoplasmic space during its elimination. By contrast, the persistent nucellus exports sugars toward the endosperm through the SWEET4 hexose facilitator. Finally, we analyzed sugar metabolism and transport in the transparent testa 16 mutant, which fails to undergo nucellus cell elimination, which shed light on the coordination between tissue and nutrient partitioning. Overall, this study identifies a path of sugar transport in the Arabidopsis seed and describes a link between sugar redistribution and the nucellus cell-elimination program.

Developmental patterning of the sub-epidermal integument cell layer in Arabidopsis seeds.

Angiosperm seed development is a paradigm of tissue cross-talk. Proper seed formation requires spatial and temporal coordination of the fertilization products - embryo and endosperm - and the surrounding seed coat maternal tissue. In early Arabidopsis seed development, all seed integuments were thought to respond homogenously to endosperm growth. Here, we show that the sub-epidermal integument cell layer has a unique developmental program. We characterized the cell patterning of the sub-epidermal integument cell layer, which initiates a previously uncharacterized extra cell layer, and identified TRANSPARENT TESTA 16 and SEEDSTICK MADS box transcription factors as master regulators of its polar development and cell architecture. Our data indicate that the differentiation of the sub-epidermal integument cell layer is insensitive to endosperm growth alone and to the repressive mechanism established by FERTILIZATION INDEPENDENT ENDOSPERM and MULTICOPY SUPPRESSOR OF IRA1 Polycomb group proteins. This work demonstrates the different responses of epidermal and sub-epidermal integument cell layers to fertilization.

Deposition of a cutin apoplastic barrier separating seed maternal and zygotic tissues.

BACKGROUND: In flowering plants, proper seed development is achieved through the constant interplay of fertilization products, embryo and endosperm, and maternal tissues. Communication between these compartments is supposed to be tightly regulated at their interfaces. Here, we characterize the deposition pattern of an apoplastic lipid barrier between the maternal inner integument and fertilization products in Arabidopsis thaliana seeds. RESULTS: We demonstrate that an apoplastic lipid barrier is first deposited by the ovule inner integument and undergoes de novo cutin deposition following central cell fertilization and relief of the FERTILIZATION INDEPENDENT SEED Polycomb group repressive mechanism. In addition, we show that the WIP zinc-finger TRANSPARENT TESTA 1 and the MADS-Box TRANSPARENT TESTA 16 transcription factors act maternally to promote its deposition by regulating cuticle biosynthetic pathways. Finally, mutant analyses indicate that this apoplastic barrier allows correct embryo sliding along the seed coat. CONCLUSIONS: Our results revealed that the deposition of a cutin apoplastic barrier between seed maternal and zygotic tissues is part of the seed coat developmental program.

Endosperm and Nucellus Develop Antagonistically in Arabidopsis Seeds.

In angiosperms, seed architecture is shaped by the coordinated development of three genetically different components: embryo, endosperm, and maternal tissues. The relative contribution of these tissues to seed mass and nutrient storage varies considerably among species. The development of embryo, endosperm, or nucellus maternal tissue as primary storage compartments defines three main typologies of seed architecture. It is still debated whether the ancestral angiosperm seed accumulated nutrients in the endosperm or the nucellus. During evolution, plants shifted repeatedly between these two storage strategies through molecular mechanisms that are largely unknown. Here, we characterize the regulatory pathway underlying nucellus and endosperm tissue partitioning in Arabidopsis thaliana We show that Polycomb-group proteins repress nucellus degeneration before fertilization. A signal initiated in the endosperm by the AGAMOUS-LIKE62 MADS box transcription factor relieves this Polycomb-mediated repression and therefore allows nucellus degeneration. Further downstream in the pathway, the TRANSPARENT TESTA16 (TT16) and GORDITA MADS box transcription factors promote nucellus degeneration. Moreover, we demonstrate that TT16 mediates the crosstalk between nucellus and seed coat maternal tissues. Finally, we characterize the nucellus cell death program and its feedback role in timing endosperm development. Altogether, our data reveal the antagonistic development of nucellus and endosperm, in coordination with seed coat differentiation.

Seed coat thickness in the evolution of angiosperms.

The seed habit represents a remarkable evolutionary advance in plant sexual reproduction. Since the Paleozoic, seeds carry a seed coat that protects, nourishes and facilitates the dispersal of the fertilization product(s). The seed coat architecture evolved to adapt to different environments and reproductive strategies in part by modifying its thickness. Here, we review the great natural diversity observed in seed coat thickness among angiosperms and its molecular regulation in Arabidopsis.

TWS1, a Novel Small Protein, Regulates Various Aspects of Seed and Plant Development.

Small proteins have long been overlooked due to their poor annotation and the experimental challenges they pose. However, in recent years, their role in various processes has started to emerge, opening new research avenues. Here, we present the isolation and characterization of two allelic mutants, twisted seed1-1 (tws1-1) and tws1-2, which exhibit an array of developmental and biochemical phenotypes in Arabidopsis (Arabidopsis thaliana) seeds. We have identified AT5G01075 as the subtending gene encoding a small protein of 81 amino acids localized in the endoplasmic reticulum. TWS1 is strongly expressed in seeds, where it regulates both embryo development and accumulation of storage compounds. TWS1 loss-of-function seeds exhibit increased starch, sucrose, and protein accumulation at the detriment of fatty acids. TWS1 is also expressed in vegetative and reproductive tissues, where it is responsible for proper epidermal cell morphology and overall plant growth. At the cellular level, TWS1 is responsible for cuticle deposition on epidermal cells and organization of the endomembrane system. Finally, we show that TWS1 is a single-copy gene in Arabidopsis, and it is specifically conserved among angiosperms.

Establishing a framework for the Ad/abaxial regulatory network of Arabidopsis: ascertaining targets of class III homeodomain leucine zipper and KANADI regulation.

The broadly conserved Class III homeodomain leucine zipper (HD-ZIPIII) and KANADI transcription factors have opposing and transformational effects on polarity and growth in all tissues and stages of the plant's life. To obtain a comprehensive understanding of how these factors work, we have identified transcripts that change in response to induced HD-ZIPIII or KANADI function. Additional criteria used to identify high-confidence targets among this set were presence of an adjacent HD-ZIPIII binding site, expression enriched within a subdomain of the shoot apical meristem, mutant phenotype showing defect in polar leaf and/or meristem development, physical interaction between target gene product and HD-ZIPIII protein, opposite regulation by HD-ZIPIII and KANADI, and evolutionary conservation of the regulator-target relationship. We find that HD-ZIPIII and KANADI regulate tissue-specific transcription factors involved in subsidiary developmental decisions, nearly all major hormone pathways, and new actors (such as indeterminate domain4) in the ad/abaxial regulatory network. Multiple feedback loops regulating HD-ZIPIII and KANADI are identified, as are mechanisms through which HD-ZIPIII and KANADI oppose each other. This work lays the foundation needed to understand the components, structure, and workings of the ad/abaxial regulatory network directing basic plant growth and development.

A comprehensive analysis of microProteins reveals their potentially widespread mechanism of transcriptional regulation.

Truncated transcription factor-like proteins called microProteins (miPs) can modulate transcription factor activities, thereby increasing transcriptional regulatory complexity. To understand their prevalence, evolution, and function, we predicted over 400 genes that encode putative miPs from Arabidopsis (Arabidopsis thaliana) using a bioinformatics pipeline and validated two novel miPs involved in flowering time and response to abiotic and biotic stress. We provide an evolutionary perspective for a class of miPs targeting homeodomain transcription factors in plants and metazoans. We identify domain loss as one mechanism of miP evolution and suggest the possible roles of miPs on the evolution of their target transcription factors. Overall, we reveal a prominent layer of transcriptional regulation by miPs, show pervasiveness of such proteins both within and across genomes, and provide a framework for studying their function and evolution.

A per-ARNT-sim-like sensor domain uniquely regulates the activity of the homeodomain leucine zipper transcription factor REVOLUTA in Arabidopsis.

Class III homeodomain leucine zipper (HD-ZIP III) transcription factors regulate critical developmental programs in plants; these include leaf polarity, polarity along the shoot-root axis, and stem cell specification and proliferation. One of the defining features of HD-ZIP III proteins is the presence of a Per-ARNT-Sim-like (PAS-like) MEKHLA domain at the C terminus. PAS-like domains are known to respond to a variety of chemical and physical stimuli. Here, we provide evidence that the MEKHLA domain acts as a negative regulator of Arabidopsis thaliana HD-ZIP III REVOLUTA activity. Based on experiments in yeast and plants, we propose a model in which the MEKHLA domain inhibits dimerization through a sequence-independent steric masking mechanism. This inhibition is relieved in response to a cellular signal that requires the C terminus of the MEKHLA domain for its perception. Overexpression experiments suggest that this signal is unequally distributed and/or sensed in the plant. Our data show that the function of the REVOLUTA MEKHLA domain differs among other HD-ZIP III family members; this difference may explain the genetic differences that have been observed among family members. This finding, combined with our phylogenetic analysis, suggests that REVOLUTA is the latest type of HD-ZIP III protein to have evolved in land plants.

Confocal Imaging of Seeds.

In flowering plants, proper seed development is achieved through the constant interplay of fertilization products, embryo and endosperm, and maternal tissues. Understanding such a complex biological process requires microscopy techniques able to unveil the seed internal morphological structure. Seed thickness and relatively low permeability make conventional tissue staining techniques impractical unless combined with time-consuming dissecting methods. Here, we describe two techniques to imaging the three-dimensional structure of Arabidopsis seeds by confocal laser scanning microscopy. Both procedures, while differing in their time of execution and resolution, are based on cell wall staining of seed tissues with fluorescent dyes.

Interaction of tau protein with the dynactin complex.

Tau is an axonal microtubule-associated protein involved in microtubule assembly and stabilization. Mutations in Tau cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), and tau aggregates are present in Alzheimer's disease and other tauopathies. The mechanisms leading from tau dysfunction to neurodegeneration are still debated. The dynein-activator complex dynactin has an essential role in axonal transport and mutations in its gene are associated with lower motor neuron disease. We show here for the first time that the N-terminal projection domain of tau binds to the C-terminus of the p150 subunit of the dynactin complex. Tau and dynactin show extensive colocalization, and the attachment of the dynactin complex to microtubules is enhanced by tau. Mutations of a conserved arginine residue in the N-terminus of tau, found in patients with FTDP-17, affect its binding to dynactin, which is abnormally distributed in the retinal ganglion cell axons of transgenic mice expressing human tau with a mutation in the microtubule-binding domain. These findings, which suggest a direct involvement of tau in axonal transport, have implications for understanding the pathogenesis of tauopathies.

A TRANSPARENT TESTA Transcriptional Module Regulates Endothelium Polarity.

Seeds have greatly contributed to the successful colonization of land by plants. Compared to spores, seeds carry nutrients, rely less on water for germination, provide a higher degree of protection against biotic and abiotic stresses, and can disperse in different ways. Such advantages are, to a great extent, provided by the seed coat. The evolution of a multi-function seed-coat is inheritably linked to the evolution of tissue polarity, which allows the development of morphologically and functionally distinct domains. Here, we show that the endothelium, the innermost cell layer of the seed coat, displays distinct morphological features along the proximal-distal axis. Furthermore, we identified a TRANSPARENT TESTA transcriptional module that contributes to establishing endothelium polarity and responsiveness to fertilization. Finally, we characterized its downstream gene pathway by whole-genome transcriptional analyses. We speculate that such a regulatory module might have been responsible for the evolution of morphological diversity in seed shape, micropylar pore formation, and cuticle deposition.

Seed Evolution, A 'Simpler' Story.

Seed evolution is often presented as the evolution of morphological complexity. Following the steps of Wilhelm Hofmeister, I argue that changes in the development of one tissue, the megasporangium/nucellus, can explain the origin of the seed habit. Here, I lay down a 'simpler' story that correlates seed evolution to nucellus cell fate.

Seed tissue and nutrient partitioning, a case for the nucellus.

Flowering plants display a large spectrum of seed architectures. The volume ratio of maternal versus zygotic seed tissues changes considerably among species and underlies different nutrient-storing strategies. Such diversity arose through the evolution of cell elimination programs that regulate the relative growth of one tissue over another to become the major storage compartment. The elimination of the nucellus maternal tissue is regulated by developmental programs that marked the origin of angiosperms and outlined the most ancient seed architectures. This review focuses on such a defining mechanism for seed evolution and discusses the role of nucellus development in seed tissues and nutrient partitioning at the light of novel discoveries on its molecular regulation.

Developmental patterning of sub-epidermal cells in the outer integument of Arabidopsis seeds.

The seed, the reproductive unit of angiosperms, is generally protected by the seed coat. The seed coat is made of one or two integuments, each comprising two epidermal cells layers and, in some cases, extra sub-epidermal cell layers. The thickness of the seed-coat affects several aspects of seed biology such as dormancy, germination and mortality. In Arabidopsis, the inner integument displays one or two sub-epidermal cell layers that originate from periclinal cell divisions of the innermost epidermal cell layer. By contrast, the outer integument was considered to be two-cell layered. Here, we show that sub-epidermal chalazal cells grow in between the epidermal outer integument cell layers to create an incomplete three-cell layered outer integument. We found that the MADS box transcription factor TRANSPARENT TESTA 16 represses growth of the chalaza and formation of sub-epidermal outer integument cells. Finally, we demonstrate that sub-epidermal cells of the outer and inner integument respond differently to the repressive mechanism mediated by FERTILIZATION INDEPENDENT SEED Polycomb group proteins and to fertilization signals. Our data suggest that integument cell origin rather than sub-epidermal cell position underlies different responses to fertilization.

Growth of the Arabidopsis sub-epidermal integument cell layers might require an endosperm signal.

The seed, the reproductive unit of angiosperms, is physically protected by the seed coat. The seed coat develops from the ovule integuments after fertilization. The Arabidopsis ovule integuments are made of 5-6 cell layers of epidermal and sub-epidermal origin. The growth of the epidermal integument cell layers responds to an endosperm signal mediated by the AGAMOUS-LIKE 62 MADS box transcription factor with limited embryo contribution. By contrast, the sub-epidermal integument cell layers require the embryo to expand whereas the role of the endosperm is still unclear. Here, we analyzed the development of the sub-epidermal integument cell layers in agl62 mutant seeds, which undergo premature endosperm cellularization and arrest. Our data suggest that embryo and endosperm are both necessary to trigger the expansion of the sub-epidermal integument cell layers.

From Gateway to MultiSite Gateway in one recombination event.

BACKGROUND: Invitrogen Gateway technology exploits the integrase/att site-specific recombination system for directional cloning of PCR products and the subsequent subcloning into destination vectors. One or three DNA segments can be cloned using Gateway or MultiSite Gateway respectively. A vast number of single-site Gateway destination vectors have been created while MultiSite Gateway is limited to few destination vectors and therefore to few applications. The aim of this work was to make the MultiSite Gateway technology available for multiple biological purposes. RESULTS: We created a construct, pDONR-R4-R3, to easily convert any available Gateway destination vector to a MultiSite Gateway vector in a single recombination reaction. In addition, we designed pDONR-R4-R3 so that DNA fragments already cloned upstream or downstream of the Gateway cassette in the original destination vectors can still be utilized for promoter-gene or translational fusions after the conversion. CONCLUSION: Our tool makes MultiSite Gateway a more widely accessible technology and expands its applications by exploiting all the features of the Gateway vectors already available.

A Framework for Discovering, Designing, and Testing MicroProteins to Regulate Synthetic Transcriptional Modules.

Transcription factors often form protein complexes and give rise to intricate transcriptional networks. The regulation of transcription factor multimerization plays a key role in the fine-tuning of the underlying transcriptional pathways and can be exploited to modulate synthetic transcriptional modules. A novel regulation of protein complex formation is emerging: microProteins-truncated transcription factors-engage in protein-protein interactions with transcriptional complexes and modulate their transcriptional activity. Here, we outline a strategy for the discovery, design, and test of putative miPs to fine-tune the activity of transcription factors regulating synthetic or natural transcriptional circuits.

microProtein Prediction Program (miP3): A Software for Predicting microProteins and Their Target Transcription Factors.

An emerging concept in transcriptional regulation is that a class of truncated transcription factors (TFs), called microProteins (miPs), engages in protein-protein interactions with TF complexes and provides feedback controls. A handful of miP examples have been described in the literature but the extent of their prevalence is unclear. Here we present an algorithm that predicts miPs and their target TFs from a sequenced genome. The algorithm is called miP prediction program (miP3), which is implemented in Python. The software will help shed light on the prevalence, biological roles, and evolution of miPs. Moreover, miP3 can be used to predict other types of miP-like proteins that may have evolved from other functional classes such as kinases and receptors. The program is freely available and can be applied to any sequenced genome.