Quantitative analyses and models are required to connect a plant’s cellular organisation with its metabolism. However, quantitative data are often scattered over multiple studies, and finding such data and converting them into useful information is time-consuming. Consequently, there is a need to centralise the available data and to highlight the remaining knowledge gaps. Here, we present a step-by-step approach to manually extract quantitative data from various information sources, and to unify the data format. First, data from Arabidopsis leaf were collated, checked for consistency and correctness and curated by cross-checking sources. Second, quantitative data were combined by applying calculation rules. They were then integrated into a unique comprehensive, referenced, modifiable and reusable data compendium representing an Arabidopsis reference leaf. This atlas contains the metrics of the 15 cell types found in leaves at the cellular and subcellular levels.

The 1991 review paper by Coen and Meyerowitz on the control of floral organ development set out the evidence available at that time, which led to the now famous ABC model of floral organ identity control. The authors summarised the genetic and molecular analyses that had been carried out in a relatively short time by several laboratories, mainly in Arabidopsis thaliana and Antirrhinum majus. The work was a successful example of how systematic genetic and molecular analysis can decipher the mechanism that controls a developmental process in plants. The ABC model is a combinatorial model in which each floral whorl acquires its identity through a unique combination of floral homeotic gene activities. The review also highlights the similarities in the regulation of floral organ identity between evolutionarily distant plant species, emphasising the general relevance of the model and paving the way for comprehensive studies of the evolution of floral diversity.

Cell division is a tightly regulated mechanism, notably in tissues where malfunctions can lead to tumour formation or developmental defects. This is particularly true in land plants, where cells cannot relocate and therefore cytokinesis determines tissue topology. In plants, cell division is executed in radically different manners than in animals, with the appearance of new structures and the disappearance of ancestral mechanisms. Whilst F-actin and microtubules closely co-exist, recent studies mainly focused on the involvement of microtubules in this key process. Here, we used a root tracking system to image the spatio-temporal dynamics of both F-actin reporters and cell division markers in dividing cells embedded in their tissues. In addition to the F-actin accumulation at the phragmoplast, we observed and quantified a dynamic apico-basal enrichment of F-actin from the prophase/metaphase transition until the end of the cytokinesis.

By forming lateral roots, plants expand their root systems to improve anchorage and absorb more water and nutrients from the soil. Each phase of this developmental process in Arabidopsis is tightly regulated by dynamic and continuous signalling of the phytohormones cytokinin and auxin. While the roles of auxin in lateral root organogenesis and spatial accommodation by overlying cell layers have been well studied, insights on the importance of cytokinin is still somewhat limited. Cytokinin is a negative regulator of lateral root formation with versatile modes of action being activated at different root developmental zones. Here, we review the latest progress made towards our understanding of these spatially separated mechanisms of cytokinin-mediated signalling that shape lateral root initiation, outgrowth and emergence and highlight some of the enticing open questions.

Comparative transcriptomics can be used to translate an understanding of gene regulatory networks from model systems to less studied species. Here, we use RNA-Seq to determine and compare gene expression dynamics through the floral transition in the model species Arabidopsis thaliana and the closely related crop Brassica rapa. We find that different curve registration functions are required for different genes, indicating that there is no single common ‘developmental time’ between Arabidopsis and B. rapa. A detailed comparison between Arabidopsis and B. rapa and between two B. rapa accessions reveals different modes of regulation of the key floral integrator SOC1, and that the floral transition in the B. rapa accessions is triggered by different pathways. Our study adds to the mechanistic understanding of the regulatory network of flowering time in rapid cycling B. rapa and highlights the importance of registration methods for the comparison of developmental gene expression data.

GLP-1 (abnormal germline proliferation) is a Notch-like receptor protein that plays an essential role in pharyngeal development. In this study, an orthologue of Caenorhabditis elegans glp-1 was identified in Meloidogyne incognita. A computational analysis revealed that the orthologue contained almost all the domains present in the C. elegans gene: specifically, the LIN-12/Notch repeat, the ankyrin repeat, a transmembrane domain and different ligand-binding motifs were present in orthologue, but the epidermal growth factor-like motif was not observed. An expression analysis showed differential expression of glp-1 throughout the life cycle of M. incognita, with relatively higher expression in the egg stage. To evaluate the silencing efficacy of Mi-glp-1, transgenic Arabidopsis plants carrying double-stranded RNA constructs of glp-1 were generated, and infection of these plants with M. incognita resulted in a 47–50% reduction in the numbers of galls, females and egg masses. Females obtained from the transgenic RNAi lines exhibited 40–60% reductions in the transcript levels of the targeted glp-1 gene compared with females isolated from the control plants. Second-generation juveniles (J2s), which were descendants of the infected females from the transgenic lines, showed aberrant phenotypes. These J2s exhibited a significant decrease in the overall distance from the stylet to the metacorpus region, and this effect was accompanied by disruption around the metacorporeal bulb of the pharynx. The present study suggests a role for this gene in organ (pharynx) development during embryogenesis in M. incognita and its potential use as a target in the management of nematode infestations in plants.

Induction of nine-cis-epoxycarotenoid dioxygenase 6 (NCED6), an abscisic acid (ABA) biosynthesis gene, alone is sufficient to suspend germination in testa-ruptured seeds, which are at the final step of germination. Molecular consequences of NCED6 induction in imbibed seeds were investigated by RNA sequencing. The analysis identified many unknown and uncharacterized genes that were up-regulated by NCED6 induction, in addition to the major regulators of ABA signalling. Interestingly, other NCEDs were up-regulated by NCED6 induction, suggesting that the major rate-limiting enzymes in the ABA biosynthesis pathway are subject to positive-feedback regulation. ZEAXANTHIN EPOXIDASE and ABSCISIC ALDEHYDE OXIDASE3, which function upstream and downstream of NCED, were also up-regulated in seeds by NCED6 induction, which suggests that the distinct layers of positive feedback loops are coordinately operating in the NCED6-induced seeds. SOMNUS (SOM), which was also up-regulated by NCED6 induction, was the major mediator of enhanced ABA signalling in NCED6-induced seeds. SOM exerted negative effects on GA biosynthesis, which also contributes to a high ABA–GA ratio and reinforces the suppressive state of germination. Besides these coding genes, long intergenic non-coding RNAs (lincRNAs) were also up-regulated upon NCED6 induction (termed N6LINCRs). Conditional expression of N6LINCR1 altered gene expression profiles in seeds. Twenty-six genes were up-regulated and 66 genes were down-regulated by the induction of N6LINCR1. These results suggest that some of N6LINCRs have a regulatory role in gene expression in seeds, which potentially contributes to the regulation of germination by ABA.

Genetically encoded Förster resonance energy transfer (FRET)-based biosensors have been used to report relative concentrations of ions and small molecules, as well as changes in protein conformation, posttranslational modifications, and protein–protein interactions. Changes in FRET are typically quantified through ratiometric analysis of fluorescence intensities. Here we describe methods to evaluate ratiometric imaging data acquired through confocal microscopy of a FRET-based inorganic phosphate biosensor in different cells and subcellular compartments of Arabidopsis thaliana. Linear regression was applied to donor, acceptor, and FRET-derived acceptor fluorescence intensities obtained from images of multiple plants to estimate FRET ratios and associated location-specific spectral correction factors with high precision. FRET/donor ratios provided a combination of high dynamic range and precision for this biosensor when applied to the cytosol of both root and leaf cells, but lower precision when this ratiometric method was applied to chloroplasts. We attribute this effect to quenching of donor fluorescence because high precision was achieved with FRET/acceptor ratios and thus is the preferred ratiometric method for this organelle. A ligand-insensitive biosensor was also used to distinguish nonspecific changes in FRET ratios. These studies provide a useful guide for conducting quantitative ratiometric studies in live plants that is applicable to any FRET-based biosensor.

Chloroplast division requires filamentation temperature-sensitive Z (FtsZ), a tubulin-like GTPase of cyanobacterial endosymbiotic origin. Plants and algae possess two distinct FtsZ protein families, FtsZ1 and FtsZ2 that co-assemble into a ring (Z-ring) at the division site. Z-ring assembly and disassembly and division site positioning is controlled by both positive and negative factors via their specific interactions with FtsZ1 and FtsZ2. Here we present the in planta analysis of Arabidopsis FtsZ1 and FtsZ2 turnover in the context of a native chloroplast division machinery. Fluorescence recovery after photobleaching analysis was conducted using fluorescently tagged FtsZ at wild-type (WT)-like levels. Rapid photobleaching, low signal-to-noise ratio, and phototropic movements of chloroplasts were overcome by (i) using progressive intervals in time-lapse imaging, (ii) analyzing epidermal rather than stromal chloroplasts, and (iii) employing image stack alignment during postprocessing. In plants of WT background, fluorescence recovery half-times averaged 117 and 325 s for FtsZ1 and FtsZ2, respectively. In plants lacking ARC3, the key negative regulator of FtsZ assembly, the turnover was threefold slower. The findings are discussed in the context of previous results conducted in a heterologous system.

Seeds have evolved to be highly efficient environmental sensors that respond not only to their prevailing environment, but also their environmental history, to regulate dormancy and the initiation of germination. In the present work we investigate the combined impact of a number of environmental signals (temperature, nitrate, light) during seed development on the mother plant, during post-shedding imbibition and during prolonged post-shedding exposure in both dry and imbibed states, simulating time in the soil seed bank. The differing response to these environments was observed in contrasting winter (Cvi, Ler) and summer (Bur) annual Arabidopsis ecotypes. Results presented show that environmental signals both pre- and post-shedding determine the depth of physiological dormancy and therefore the germination response to the ambient environment. The ecotype differences in seed response to ambient germination conditions are greatly enhanced by seed maturation in different environments. Further variation in response develops following shedding when seeds do not receive the full complement of environmental signals required for germination and enter the soil seed bank in either dry or imbibed states. Species seed dormancy characteristics cannot therefore be easily defined, as seed dormancy is a dynamic state subject to within-species adaptation to local environments.

Seeds of Sisymbrium officinale display physiological dormancy and require nitrate to germinate. Rupture of the testa precedes radicle protrusion through the endosperm (germination sensu stricto). While both endosperm rupture and testa rupture (TR) required nitrate, endosperm rupture was fully inhibited by abscisic acid (ABA) but TR was not inhibited. The gibberellic acid (GA)-synthesis inhibitor paclobutrazol prevented TR, which was reverted by exogenous GA4 but not by nitrate. The orientation of TR was transverse, which prompted the question whether seeds elongate prior to radicle protrusion, concurrent with an increase in water content. Between 9 h and 1 d no increase in length or water content was observed. During incubation in ABA the length of imbibed seeds without TR did not increase between 1 and 5 d, whereas nitrate added to ABA induced TR and a 94% increase in length. At the same time the water content of seeds without TR increased by 18%, while the water content of seeds with TR increased by 38%. Length and water content were correlated in a single-seed analysis for seeds with TR, but not for seeds without TR. Increased length was also observed in Arabidopsis seeds with nitrate-induced TR. These results indicate that prior to endosperm rupture dormancy release by nitrate is accompanied by TR, seed elongation and an increase in water content. A new multiphasic model is proposed for the imbibition curve, where the second phase of the classical triphasic curve is split into three sub-phases, of which phases IIB and IIC are associated with TR.

The micropylar region of endosperm (ME) is a physical barrier to radicle emergence in seeds of many different species, including tomato (Solanum lycopersicum) and Arabidopsis thaliana. ME is thought to be weakened through cell wall-modifying proteins, and this is supported by transcriptome data showing enrichment of cell wall-associated genes in ME. Gibberellin and ethylene have been suggested to be involved in induction of these genes in ME. However, mechanisms underlying this critical event for germination still remain elusive. In addition to hormonal regulation of ME weakening, recent data from high-throughput analyses suggested that it might be important for the radicle tip to ‘touch’ ME (or mechanosensing), in terms of ME-specific gene induction. This emerging hypothesis can be integrated with previous hypotheses about hormonal regulation of ME-specific gene expression in seeds.

The seed consists of three distinct regions, the zygotic embryo and endosperm and the maternal seed coat. Development of the seed requires co-ordinated growth and differentiation within each of these regions. Although the major events that occur during seed development have been studied extensively at the anatomical, physiological and molecular level, a comprehensive understanding of gene activity in each seed region has been lacking. DNA microarrays and extensive sequencing of cDNA libraries permit genome-wide analyses of mRNA populations of biological samples. We review recent studies of genome-wide gene activity that illustrate approaches used to provide new insights into the processes that occur during seed development.

The production of hydrophilic mucilages by the seed coat or pericarp, which are released upon seed hydration, is a commonly found adaptation in angiosperms, known as myxodiaspory. These are composed primarily of pectins and hemicelluloses that undergo substantive swelling upon hydration. Synthesized in the Golgi apparatus and secreted to an apoplastic space via secretory vesicles, mucilages can also contain cellulose microfibrils or cellulosic fibres that are synthesized at the plasma membrane in association with microtubules. Investigation of mucilage production in, and differentiation of, the mucilage secretory cells of the genetic model plant Arabidopsis thaliana has identified a number of regulatory genes and enzymes involved in pectin synthesis and secretion, in muro pectin modification and secondary cell wall synthesis. Studies of the role of mucilages in both a number of species and in Arabidopsis mutants affected in its production suggest that they have multiple ecological roles. These include facilitation of seed hydration, mediation of germination under waterlogged conditions, prevention of seed dispersal or predation by adherence to soil, and promotion of seed dispersal by attachment to animals. The precise role of mucilages appears to be dependent on species and their environmental context.

Seed storage proteins (SSPs) are synthesized during development and the expression of their genes is under tight tissue-specific and temporal transcriptional regulation. In this review we summarize the current knowledge concerning the regulatory steps controlling SSP synthesis in Arabidopsis and cereals, which involves the interaction of cis regulatory elements with corresponding trans-acting factors. In some cases, the regulation of SSP genes requires the concerted action of multiple transcription factors (TFs). There is an evolutionary conservation between the prolamins (the main group of SSPs in many cereal grains) and a major group of dicot seed albumins; this relates to both the regulatory elements and the TFs that are functionally exchangeable between the monocot and dicot species.

Germination and early seedling development are critical for successful stand establishment of plants. Following germination, the cotyledons, which are derived from embryonic tissue, emerge from the seed. Arabidopsis seedlings at post-germinative stages are supported mainly by the supply of nutrition from the cotyledons until vegetative leaves emerge and initiate photosynthesis. The switch to autotrophic growth is a significant transition at the post-germinative stage. Here, we provide evidence that down-regulation of SQUAMOSA PROMOTER-BINDING PROTEIN LIKE13 (SPL13) by microRNA156 (miR156) plays an important role in the regulation of the post-germinative switch from the cotyledon stage to the vegetative-leaf stage. Silent mutations created in the SPL13 sequence in the region that is complementary to the miR156 sequence caused the deregulation of the mutant form of SPL13 (mSPL13) mRNA from miR156. Mutant seedlings over-accumulated miRNA-resistant messages and exhibited a delay in the emergence of vegetative leaves compared to wild-type seedlings. The delay was not observed in control transgenic plants expressing non-mutated SPL13, indicating that the phenotype was caused specifically by the silent mutations and deregulation of SPL13 from miR156. Characterization of the SPL13 promoter indicated that this gene is expressed mainly in the hypocotyl and affects leaf primordium development. These results suggest that the repression of SPL13 by miR156 is essential for normal post-germinative growth in Arabidopsis.

MicroRNAs (miRNAs) are involved in developmental programmes of plants, including seed germination and post-germination. Here, we provide evidence that two different miRNA pathways, miR156 and miR172, interact during the post-germination stages in Arabidopsis. Mutant seedlings expressing miR156-resistant SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE13 (mSPL13), which has silent mutations in the miR156 complementary sequence, over-accumulated SPL13 mRNA and exhibited a delay in seedling development. Microarray analysis indicated that SCHNARCHZAPFEN (SNZ), an AP2-like gene targeted by miR172, was down-regulated in these mutants. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and miRNA gel blot analyses showed that the MIR172 genes were up-regulated in mSPL13 mutants. These results suggest that the miRNA regulation cascades (miR156⊣SPL13 → miR172⊣SNZ) play a critical role during the post-germination developmental stages in Arabidopsis.

Flavonoids are secondary metabolites that are present at high levels in most plant seeds and grains. These compounds appear to play vital roles in defence against pathogens and predators and contribute to physiological functions such as seed maturation and dormancy. At the same time, particular subclasses of flavonoids, such as the proanthocyanidins (condensed tannins), negatively impact the use of seeds and grains in animal feed and can add undesirable qualities to food products for human consumption. This article presents an overview of investigations into the physiological and agronomic aspects of seed and grain flavonoids as well as a review of molecular genetic studies, particularly in maize, Arabidopsis and soybean, where mutants deficient in flavonoid biosynthesis provide useful tools for stydying the metabolic machinery underlying the accumulation of these compounds in reproductive structures.

X-radiography is a simple, rapid and non-destructive method for analysing the morphology of embryos in dry, mature seeds of Arabidopsis thaliana. In wild type seeds, the cotyledons, hypocotyl and radicle tip can be readily distinguished. In seeds of the mutant types knolle, keule, and the double mutant keulelgnom, aberrations in embryo morphology can be visualized. X-radiography may therefore be useful in the isolation of embryo mutants from Arabidopsis seed samples.

The ability to initiate radicle elongation, or germination potential, occurs in developing embryos before the completion of seed maturation. Green embryos after the walking-stick stage in developing Arabidopsis thaliana seeds germinate when excised from seeds and incubated in Murashige–Skoog (MS) medium containing 1% sucrose. Germination potential is not observed during early embryogenesis at the globular, heart and torpedo stages. Here, we describe an Arabidopsis mutant with embryos arrested at early stages of development, but still exhibiting germination potential. The mutant, termed embryo ball (eb), produced shrunken seeds containing round or irregularly shaped embryos that did not germinate. The round embryos excised from developing eb seeds were capable of growing a primary root with root hairs when incubated in media. In contrast, cotyledons were absent at the apical region of the eb embryos, although the apical region produced leaf-like structures with trichomes, indicating vegetative leaf identity. These observations suggested that morphological maturation was not essential for the induction of germination potential. The eb embryos exhibited partial desiccation tolerance that is characteristic of mature embryos at later stages of development, suggesting that cell maturation was also independent of morphological maturation. The eb mutant provides novel information on cell and tissue identity in developmental biology, as well as a useful tool to dissect the mechanisms underlying the induction of germination potential in developing seeds.