To support the increasingly complex circuits needed for plant synthetic biology applications, additional constitutive promoters are essential. Reusing promoter parts can lead to difficulty in cloning, increased heterogeneity between transformants, transgene silencing and trait instability. We have developed a pipeline to identify genes that have stable expression across a wide range of Arabidopsis tissues at different developmental stages and have identified a number of promoters that are well expressed in both transient (Nicotiana benthamiana) and stable (Arabidopsis) transformation assays. We have also introduced two genome-orthogonal gRNA target sites in a subset of the screened promoters, converting them into NOR logic gates. The work here establishes a pipeline to screen for additional constitutive promoters and can form the basis of constructing more complex information processing circuits in the future.

Phenotypic plasticity is a heritable trait that provides sessile organisms a strategy to rapidly mitigate negative effects of environmental change. Yet, we have little understanding of the mode of inheritance and genetic architecture of plasticity in different focal traits relevant to agricultural applications. This study builds on our recent discovery of genes controlling temperature-mediated flower size plasticity in Arabidopsis thaliana and focuses on dissecting the mode of inheritance and combining ability of plasticity in the context of plant breeding. We created a full diallel cross using 12 A. thaliana accessions displaying different temperature-mediated flower size plasticities, scored as the fold change between two temperatures. Griffing’s analysis of variance in flower size plasticity indicated that non-additive genetic action shapes this trait and pointed at challenges and opportunities when breeding for reduced plasticity. Our findings provide an outlook of flower size plasticity that is important for developing resilient crops for future climates.

Plant organ morphogenesis spans several orders of magnitude in time and space. Because of limitations in live-imaging, analysing whole organ growth from initiation to mature stages typically rely on static data sampled from different timepoints and individuals. We introduce a new model-based strategy for dating organs and for reconstructing morphogenetic trajectories over unlimited time windows based on static data. Using this approach, we show that Arabidopsis thaliana leaves are initiated at regular 1-day intervals. Despite contrasted adult morphologies, leaves of different ranks exhibited shared growth dynamics, with linear gradations of growth parameters according to leaf rank. At the sub-organ scale, successive serrations from same or different leaves also followed shared growth dynamics, suggesting that global and local leaf growth patterns are decoupled. Analysing mutants leaves with altered morphology highlighted the decorrelation between adult shapes and morphogenetic trajectories, thus stressing the benefits of our approach in identifying determinants and critical timepoints during organ morphogenesis.

Mobilisation of seed storage reserves is important for seedling establishment in Arabidopsis. In this process, sucrose is synthesised from triacylglycerol via core metabolic processes. Mutants with defects in triacylglycerol-to-sucrose conversion display short etiolated seedlings. We found that whereas sucrose content in the indole-3-butyric acid response 10 (ibr10) mutant was significantly reduced, hypocotyl elongation in the dark was unaffected, questioning the role of IBR10 in this process. To dissect the metabolic complexity behind cell elongation, a quantitative-based phenotypic analysis combined with a multi-platform metabolomics approach was applied. We revealed that triacylglycerol and diacylglycerol breakdown were disrupted in ibr10, resulting in low sugar content and poor photosynthetic ability. Importantly, batch-learning self-organised map clustering revealed that threonine level was correlated with hypocotyl length. Consistently, exogenous threonine supply stimulated hypocotyl elongation, indicating that sucrose levels are not always correlated with etiolated seedling length, suggesting the contribution of amino acids in this process.

Plants acclimate to various types of mechanical stresses through thigmomorphogenesis and alterations in their mechanical properties. Although resemblance between wind- and touch-induced responses provides the foundation for studies where wind influence was mimicked by mechanical perturbations, factorial experiments revealed that it is not always straightforward to extrapolate results induced by one type of perturbation to the other. To investigate whether wind-induced changes in morphological and biomechanical traits can be reproduced, we subjected Arabidopsis thaliana to two vectorial brushing treatments. Both treatments significantly affected the length, mechanical properties and anatomical tissue composition of the primary inflorescence stem. While some of the morphological changes were found to be in line with those induced by wind, changes in the mechanical properties exhibited opposite trends irrespective of the brushing direction. Overall, a careful design of the brushing treatment gives the possibility to obtain a closer match to wind-induced changes, including a positive tropic response.

The zygote is the first cell of a multicellular organism. In most angiosperms, the zygote divides asymmetrically to produce an embryo-precursor apical cell and a supporting basal cell. Zygotic division should properly segregate symbiotic organelles, because they cannot be synthesized de novo. In this study, we revealed the real-time dynamics of the principle source of ATP biogenesis, mitochondria, in Arabidopsis thaliana zygotes using live-cell observations and image quantifications. In the zygote, the mitochondria formed the extended structure associated with the longitudinal array of actin filaments (F-actins) and were polarly distributed along the apical–basal axis. The mitochondria were then temporally fragmented during zygotic division, and the resulting apical cells inherited mitochondria at higher concentration compared to the basal cells. Further observation of postembryonic organs showed that these mitochondrial behaviours are characteristic of the zygote. Overall, our results showed that the zygote has spatiotemporal regulation that unequally distributes the mitochondria.

We examined the combined effects of temperature, carbon dioxide (CO2) and watering regime during seed maturation on subsequent germinability and total phenolics of Arabidopsis thaliana [wild-type (WT) and abi1-1 mutant] seeds. Mature seeds were collected from plants that were grown under lower (22/18°C, 16 h light and 8 h dark) or higher (28/24°C, 16 h light and 8 h dark) temperatures, at ambient (400 μmol mol–1) or elevated (700 μmol mol–1) CO2 concentration, and well-watered or water-stressed. Germinated and non-germinated (viable, rotten and empty) seed percentages, germination rate and total phenolics were determined for both genotypes. Higher maturation temperatures increased seed germination percentage, but decreased germination rate, percentage of rotten and non-germinated viable seeds, and total phenolics. Elevated CO2 increased seed total phenolics. Water stress decreased the percentage of non-germinated viable seeds. Neither of the two latter factors affected other measured parameters. Seeds of the abi1-1 mutant had higher total phenolics. The fate of seeds was mostly affected by higher temperatures and water stress. Also, seeds of the abi1-1 mutant had higher germination rate, empty seed percentage and total phenolics than seeds of the WT genotype. Germination percentage was highest for the WT seeds that matured on the water-stressed plants that were grown under higher temperatures at ambient CO2. It can be concluded that higher temperatures had highest effects on seed germinability and other parameters, and elevated CO2 did not alleviate the negative effects of higher temperatures on seed viability.

Seeds are notoriously high in polyphenols and polysaccharides, which reduce RNA quality and yield, and interfere with downstream applications. We present simple modifications to a rapid RNA extraction protocol for use with seeds. The inclusion of polyethylene glycol in place of polyvinylpyrrolidone reduced polyphenol and polysaccharide contamination. In addition, replacing NaCl with KCl improved the RNA yield from Arabidopsis seeds still bound by mucilage. On extraction of Arabidopsis seed recovered from field soils clean RNA pellets with no accompanying gelatinous matrix (polysaccharide) were seen, with A260/230 ratios greater than 1.8 confirming the lack of polysaccharide carry-over. When Brassica oleracea and Sinapis arvensis seeds were extracted, 260/230 ratios greater than 1.8 were seen. RNA yields in excess of 10 µg per 100 mg seed suitable for RT-QPCR were obtained.

Genetics can be a powerful adjunct to just about any kind of physiological study, including weed physiology or weed/herbicide interactions. Making, mapping, and reverting mutations is simple and straightforward. Making mutants can be as simple as isolating variant individuals from the “wild”, as uncomplicated as doing seed mutagenesis in your laboratory, or as sneaky as recovering mutants as sectors in whole plants. The overall principles for successful development of a protocol for seed mutagenesis of weeds are described and potential problem areas noted. These generalities are illustrated with a specific case history, that of chlorsulfuron. Although chlorsulfuron is accurately described as an inhibitor of the synthesis of branched chain amino acids, careful physiological examination suggests that it kills plant cells, not by starvation for amino acids, but by active toxicity of a metabolite, α-amino butyric acid, produced from a precursor available for diversion in cells with inhibited acetolactate synthase (EC 4.1.3.18, ALS). The story of dominant resistance due to an altered ALS enzyme is well known; analysis using additional mutants fleshes out the story of how chlorsulfuron works. Such analysis has the potential to help unravel other problems in weed physiology.

Isoxaben is an inhibitor of the synthesis of cellulose from glucose. Some dicot weed species are relatively insensitive to isoxaben inhibition. This study investigates mechanisms by which decreased sensitivity may occur in three dicot weed species: catchweed bedstraw, redroot pigweed, and velvetleaf. Dose response curves were generated to determine I50 values for the inhibition of glucose incorporation into cellulose by isoxaben and compared to that of a sensitive species, mouse-ear cress. Metabolic detoxification and uptake rates were measured and the degree of tolerance conferred by these mechanisms was calculated. In all cases, metabolic detoxification was negligible. Lower uptake rates were significant but minor components of tolerance in all species. It is suggested that the principal cause of isoxaben tolerance in these dicot weed species is decreased sensitivity at the target site.

Genome sequences and genome-wide transcript profiles are becoming increasingly available, opening a way to use this information in analyzing how groups of genes are connected in pathways or “regulons” that might explain how organisms accomplish the integration on an organismal level. We have begun to explore the large datasets that are available for transcripts of the best characterized plant model, Arabidopsis thaliana, setting up a gene network using clustering methods. A network, based on the Graphical Gaussian Model (GGM), describes coregulation of genes under a variety of external factors: abiotic, biotic, and chemical treatments. In its present structure, the network reveals coregulation for more than 7,000 genes in the Arabidopsis genome. The network appears to be particularly suited to reveal the regulatory structure of biochemical pathways and environmental stress responses. Examples describe network predictions centered on a trehalose-6-phosphate phosphatase, an Arabidopsis response regulator and EPSPS. Results from the statistical analysis and bioinformatics of large data sets provide hypotheses that must be checked by additional studies. However, networks, which should be expanded from transcripts to also include proteins and metabolites, can be expected to explain not only how the Arabidopsis gene network is structured, but also provide insight in how similar networks in weed species might deviate or show correspondence and overlap.

Aromatic plants and their volatile compounds affect seed germination and plant growth, and therefore hold potential for agriculture uses as plant growth regulators and bioherbicides. In the present study 17 major monoterpenes were selected, and their mechanisms of plant toxicity were elucidated using transgenic Arabidopsis thaliana at various growth stages. Microtubulin and the plant cell membrane were identified as the focal targets through which phytotoxicity and herbicidal activity acted. Variability in monoterpene mechanisms was observed. Limonene and (+)-citronellal had strong antimicrotubule efficacy, whereas citral, geraniol, (−)-menthone, (+)-carvone, and (−)-citronellal demonstrated moderate antimicrotubule efficacy. Pulegone, (−)-carvone, carvacrol, nerol, geranic acid, (+)/(−)-citronellol, and citronellic acid lacked antimicrotubule capacity. An enantioselective disruption of microtubule assembly was recorded for (+)/(−)-citronellal and (+)/(−)-carvone. The (+) enatiomers were more potent than their (−) counterparts. Citral, limonene, carvacrol, and pulegone were also tested for phytotoxicity and herbicidal activity. Pulegone had no detectable effect on microtubules or membranes. Citral disrupted microtubules but did not cause membrane damage. Carvacrol lacked a detectable effect on microtubules but incited membrane leakage, and limonene disrupted microtubules and membrane leakage. Therefore, only limonene was herbicidal at the tested concentrations. In planta quantification of residues revealed that citral was biotransformed into nerol and geraniol, and limonene was converted into carvacrol, which could explain its dual capacity with respect to microtubules and membrane functionality. The results obtained are an important added value to commercial efforts in selecting appropriate aromatic plants to be sources of bioherbicidal compounds for sustainable weed management with a limited potential for herbicide resistance evolution in weed populations.

Terminal-repeat retrotransposon in miniature (TRIM) elements are a miniature form of retrotransposons and play an important role in genome organization. The Cassandra TRIM family has been identified in over 50 plant species, including both monocots and dicots. Cassandra elements carry an independently transcribed 5S RNA sequence in their terminal repeat regions, which is unique compared with other TRIM families. Although the existence of Cassandra elements has been documented in many plants, much work remains to characterize Cassandra family members and elucidate their distribution. In this study, we comparatively analysed the Cassandra family members in the Brassica oleracea, B. rapa and Arabidopsis thaliana genomes. A total of 602, 451 and 173 members, of which 130, 60 and 9 were relatively intact, were identified from the B. oleracea, B. rapa and A. thaliana genomes, respectively. Most of the Cassandra elements (1120/1226) were found in intergenic spaces, but 106 elements were inserted in genic regions such as introns, exons and untranslated regions. Our comparative analysis of the Cassandra family members in A. thaliana, B. rapa and B. oleracea reveals that some Cassandra elements have been commonly retained during the last 20 million years in three species and some elements have been uniquely evolved in Brassica species. This study promotes our understanding of the role and utility of Cassandra elements in the evolution of the Brassicaceae family.

Replication of chloroplast in plant cells is an essential process that requires co-assembly of the tubulin-like plastid division proteins FtsZ1 and FtsZ2 at mid-chloroplast to form a ring structure called the Z-ring. The Z-ring is stabilized via its interaction with the transmembrane protein ARC6 on the inner envelope membrane of chloroplasts. Plants lacking ARC6 are defective in plastid division and contain only one or two enlarged chloroplasts per cell with abnormal localization of FtsZ: instead of a single Z-ring, many short FtsZ filaments are distributed throughout the chloroplast. ARC6 is thought to be the anchoring point for FtsZ assemblies. To investigate the role of ARC6 in FtsZ anchoring, the mobility of green fluorescent protein–tagged FtsZ assemblies was assessed by single particle tracking in mutant plants lacking the ARC6 protein. Mean square displacement analysis showed that the mobility of FtsZ assemblies is to a large extent characterized by anomalous diffusion behavior (indicative of intermittent binding) and restricted diffusion suggesting that besides ARC6-mediated anchoring, an additional FtsZ-anchoring mechanism is present in chloroplasts.

During the last two decades, molecular genetic studies and the completion of thesequencing of the Arabidopsis thaliana genome have increased knowledge ofhormonal regulation in plants. These signal transduction pathways act in concert throughgene regulatory and signalling networks whose main components have begun to be elucidated.Our understanding of the resulting cellular processes is hindered by the complex, andsometimes counter-intuitive, dynamics of the networks, which may be interconnected throughfeedback controls and cross-regulation. Mathematical modelling provides a valuable tool toinvestigate such dynamics and to perform in silico experiments that may not be easilycarried out in a laboratory. In this article, we firstly review general methods formodelling gene and signalling networks and their application in plants. We then describespecific models of hormonal perception and cross-talk in plants. This mathematicalanalysis of sub-cellular molecular mechanisms paves the way for more comprehensivemodelling studies of hormonal transport and signalling in a multi-scale setting.

Dehydrins are late embryogenesis abundant (LEA) proteins that accumulate during seed maturation and in response to abiotic stresses in vegetative tissues. They are thought to protect cellular components from dehydration stress. However, whether they play a role in survival in the dry state is not clear. In this study, an RNA interference (RNAi)-construct against the seed-expressed dehydrin of Arabidopsis thaliana, LEA14 (At2g21490), was introduced to wild-type plants, which led to a strong reduction in transcript abundance of the target gene as well as that of two other seed-expressed dehydrin homologues, XERO1 (At3g50980) and RAB18 (responsive to abscisic acid 18, At5g66400) in the transformants. Mature, dry seeds from the RNAi plants germinated to at least 95% after rehydration, indicating that seed desiccation tolerance was not affected, while they exhibited a twofold reduction in longevity. When stored at 75% relative humidity and 35°C, the seeds of two independent RNAi lines lost 50% of their viability in 10 d and 5 d, respectively, while it took 17 d for wild-type seeds to lose 50% viability. In addition, when seeds were imbibed in the presence of 100 mM NaCl, the seeds of RNAi plants exhibited reduced germination compared to wild-type seeds, suggesting that at least one of the three seed-specific dehydrins plays a role both against deterioration during storage at low moisture content and when imbibed tissues are submitted to salt stress at high moisture.

In Arabidopsis, a variety of mapping populations have been used for the detection of quantitative trait loci (QTLs) responsible for natural variation. In this study, we present an overview of the advantages and disadvantages of the different types of populations used. To do this, we compare the results of both experimental and natural populations for the commonly analysed trait flowering time. It is expected that genome wide association (GWA) mapping will be an increasingly important tool for QTL mapping because of the high allelic richness and mapping resolution in natural populations. In Arabidopsis, GWA mapping becomes ever more facilitated by the increasing availability of re-sequenced genomes of many accessions. However, specifically designed mapping populations such as recombinant inbred lines and near isogenic lines will remain important. The high QTL detection power of such experimental populations can identify spurious GWA associations, and their unique genomic structure is superior for investigating the role of low-frequency alleles. Future QTL studies will therefore benefit from a combined approach of GWA and classical linkage analysis.

A method was developed, which enables substances to be injected into the feeding structure (syncytium) established by Heterodera schachtii in roots of Arabidopsis thaliana. The technique was used to study the uptake of nutrients by the feeding nematode. The fluorescent dye lucifer yellow CH (LYCH) and fluorescence-labelled dextrans of different molecular weights were injected into the thin and translucent roots of A. thaliana. Such roots are a feature of this plant and they provide optimal conditions for microinjection. Injected LYCH was taken up by feeding juveniles and adults, indicated by the staining of the alimentary duct and the digestive system. Fluorescent dextrans of 3, 10 and 20 kDa but not of 40 and 70 kDa were ingested, suggesting that molecules of a maximum Stokes radius of 3·2 to 4·4 nm are taken up. It is likely that the feeding tube, forming the interface between the plant cytosol and the nematode's digestive system, is responsible for this size exclusion effect. The injected fluorescent substances were not detected in plant cells adjacent to the syncytium or in the root vascular elements. Injections into parts of roots which were infested by several nematodes revealed that feeding H. schachtii individuals may share one syncytium.

Several ultrastructural changes occur during dehydration and subsequent rehydration of Arabidopsis thaliana pollen. The cytoplasmic channels, present in the outer part of the intine of the mature, dehydrating pollen grain, degenerate and develop into electron-dense inclusions. At the same time a large quantity of electron-dense material is deposited in the cavities of the exine. A large number of vesicles is produced in the vegetative cell, and they become predominantly located in the peripheral region near the intine. Starch of amyloplasts is consumed and the lipid bodies which originally surround the sperm cells become randomly distributed. In addition, the individual lipid bodies become enveloped by single rough endoplasmic reticulum cisterns.

Homologues of the pea SBP65, a late embryogenesis abundant (LEA) biotinylated protein that behaves as a putative sink for the free vitamin biotin during embryo development, were characterized biochemically in various plant species, including soybean, lentil, peanut, rape, cabbage, carrot and sugarbeet. Based on sequence homologies, the genome of Arabidopsis thaliana contains a gene putatively encoding a homologue of pea SBP65. These proteins exhibit two remarkable features. First, they only accumulate in seeds, particularly during late stages of embryo development. The results strongly suggest that these seed-specific biotinylated proteins belong to the class of plant proteins called seed maturation proteins, which are presumed to play major roles in embryo development. Secondly, covalent attachment of biotin occurs at a lysine residue within a conserved motif of (V/M)GKF, which shows no resemblance to the highly conserved AMKM tetrapeptide that houses the target lysine residue in the well-characterized biotin-dependent carboxylases and decarboxylases. These findings highlight novel structural features for protein biotinylation.