Winter warming post floral initiation delays flowering via bud dormancy activation and affects yield in a winter annual crop.

Winter annual life history is conferred by the requirement for vernalization to promote the floral transition and control the timing of flowering. Here we show using winter oilseed rape that flowering time is controlled by inflorescence bud dormancy in addition to vernalization. Winter warming treatments given to plants in the laboratory and field increase flower bud abscisic acid levels and delay flowering in spring. We show that the promotive effect of chilling reproductive tissues on flowering time is associated with the activity of two FLC genes specifically silenced in response to winter temperatures in developing inflorescences, coupled with activation of a BRANCHED1-dependent bud dormancy transcriptional module. We show that adequate winter chilling is required for normal inflorescence development and high yields in addition to the control of flowering time. Because warming during winter flower development is associated with yield losses at the landscape scale, our work suggests that bud dormancy activation may be important for effects of climate change on winter arable crop yields.

Beyond floral initiation: the role of flower bud dormancy in flowering time control of annual plants.

The phenology of temperate perennials, including the timing of vegetative growth and flowering, is well known to be controlled by seasonal dormancy cycles. Dormant structures are known as buds and have specialised covering structures, symplastic isolation from the plant and often autonomous stores of carbon and nitrogen reserves. In contrast, in annual plants our current understanding of the control of the timing of flowering focuses on the mechanisms affecting floral initiation, the transition from a vegetative apical meristem to a inflorescence meristem producing flower primordia in place of leaves. Recently we revealed that annual crops in Brassicaceae exhibit chilling-responsive growth control in a manner closely resembling bud dormancy breakage in perennial species. Here I discuss evidence that vernalisation in autumn is widespread and discuss its role in inducing flower bud set prior to winter, and review evidence that flower bud dormancy has a more wide-spread role in annual plant flowering time control than previously appreciated.

Temperature during seed maturation controls seed vigour through ABA breakdown in the endosperm and causes a passive effect on DOG1 mRNA levels during entry into quiescence.

Temperature variation during seed set is an important modulator of seed dormancy and impacts the performance of crop seeds through effects on establishment rate. It remains unclear how changing temperature during maturation leads to dormancy and growth vigour differences in nondormant seedlings. Here we take advantage of the large seed size in Brassica oleracea to analyse effects of temperature on individual seed tissues. We show that warm temperature during seed maturation promotes seed germination, while removal of the endosperm from imbibed seeds abolishes temperature-driven effects on germination. We demonstrate that cool temperatures during early seed maturation lead to abscisic acid (ABA) retention specifically in the endosperm at desiccation. During this time temperature affects ABA dynamics in individual seed tissues and regulates ABA catabolism. We also show that warm-matured seeds preinduce a subset of germination-related programmes in the endosperm, whereas cold-matured seeds continue to store maturation-associated transcripts including DOG1 because of effects on mRNA degradation before quiescence, rather than because of the effect of temperature on transcription. We propose that effects of temperature on seed vigour are explained by endospermic ABA breakdown and the divergent relationships between temperature and mRNA breakdown and between temperature, seed moisture and the glass transition.

Molecular responses to chilling in a warming climate and their impacts on plant reproductive development and yield.

Responses to prolonged winter chilling are universal in temperate plants which use seasonal temperature cues in the seed, vegetative and reproductive phases to align development with the earth's orbit. Climate change is driving a decline in reliable winter chill and affecting the sub-tropical extent of cultivation for temperate over-wintering crops. Here we explore molecular aspects of plant responses to winter chill including seasonal bud break and flowering, and how variation in the intensity of winter chilling or de-vernalisation can lead to effects on post-chilling plant development, including that of structures necessary for crop yields.

ICE1 and ZOU determine the depth of primary seed dormancy in Arabidopsis independently of their role in endosperm development.

Seed dormancy is a widespread and key adaptive trait that is essential for the establishment of soil seed banks and prevention of pre-harvest sprouting. Herein we demonstrate that the endosperm-expressed transcription factors ZHOUPI (ZOU) and INDUCER OF CBF EXPRESSION1 (ICE1) play a role in determining the depth of primary dormancy in Arabidopsis. We show that ice1 or zou increases seed dormancy and the double mutant has an additive phenotype. This increased dormancy is associated with increased ABA levels, and can be separated genetically from any role in endosperm maturation because loss of ABA biosynthesis or DELAY OF GERMINATION 1 reverses the dormancy phenotype without affecting the aberrant seed morphology. Consistent with these results, ice1 endosperms had an increased capacity for preventing embryo greening, a phenotype previously associated with an increase in endospermic ABA levels. Although ice1 changes the expression of many genes, including some in ABA biosynthesis, catabolism and/or signalling, only ABA INSENSITIVE 3 is significantly misregulated in ice1 mutants. We also demonstrate that ICE1 binds to and inhibits expression of ABA INSENSITIVE 3. Our data demonstrate that Arabidopsis ICE1 and ZOU determine the depth of primary dormancy during maturation independently of their effect on endosperm development.

Trait analysis reveals DOG1 determines initial depth of seed dormancy, but not changes during dormancy cycling that result in seedling emergence timing.

Seedling emergence timing is crucial in competitive plant communities and so contributes to species fitness. To understand the mechanistic basis of variation in seedling emergence timing, we exploited the contrasting behaviour of two Arabidopsis thaliana ecotypes: Cape Verde Islands (Cvi) and Burren (Bur-0). We used RNA-Seq analysis of RNA from exhumed seeds and quantitative trait loci (QTL) analyses on a mapping population from crossing the Cvi and Bur-0 ecotypes. We determined genome-wide expression patterns over an annual dormancy cycle in both ecotypes, identifying nine major clusters based on the seasonal timing of gene expression, and variation in behaviour between them. QTL were identified for depth of seed dormancy and seedling emergence timing (SET). Both analyses showed a key role for DOG1 in determining depth of dormancy, but did not support a direct role for DOG1 in generating altered seasonal patterns of seedling emergence. The principle QTL determining SET (SET1: dormancy cycling) is physically close on chromosome 5, but is distinct from DOG1. We show that SET1 and two other SET QTLs each contain a candidate gene (AHG1, ANAC060, PDF1 respectively) closely associated with DOG1 and abscisic acid signalling and suggest a model for the control of SET in the field.

SeedGerm: a cost-effective phenotyping platform for automated seed imaging and machine-learning based phenotypic analysis of crop seed germination.

Efficient seed germination and establishment are important traits for field and glasshouse crops. Large-scale germination experiments are laborious and prone to observer errors, leading to the necessity for automated methods. We experimented with five crop species, including tomato, pepper, Brassica, barley, and maize, and concluded an approach for large-scale germination scoring. Here, we present the SeedGerm system, which combines cost-effective hardware and open-source software for seed germination experiments, automated seed imaging, and machine-learning based phenotypic analysis. The software can process multiple image series simultaneously and produce reliable analysis of germination- and establishment-related traits, in both comma-separated values (CSV) and processed images (PNG) formats. In this article, we describe the hardware and software design in detail. We also demonstrate that SeedGerm could match specialists' scoring of radicle emergence. Germination curves were produced based on seed-level germination timing and rates rather than a fitted curve. In particular, by scoring germination across a diverse panel of Brassica napus varieties, SeedGerm implicates a gene important in abscisic acid (ABA) signalling in seeds. We compared SeedGerm with existing methods and concluded that it could have wide utilities in large-scale seed phenotyping and testing, for both research and routine seed technology applications.

The potential of the solitary parasitoid Microctonus brassicae for the biological control of the adult cabbage stem flea beetle, Psylliodes chrysocephala.

The cabbage stem flea beetle (CSFB), Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae), is a major pest of oilseed rape, Brassica napus L. (Brassicaceae), within the UK and continental Europe. Following the withdrawal of many broad-spectrum pesticides, most importantly neonicotinoids, and with increased incidence of pyrethroid resistance, few chemical control options remain, resulting in the need for alternative pest management strategies. We identified the parasitoid wasp Microctonus brassicae (Haeselbarth) (Hymenoptera: Braconidae) within CSFB collected from three independent sites in Norfolk, UK. Parasitism of adult CSFB was confirmed, and wasp oviposition behaviour was described. Moreover, we show that within captive colonies parasitism rates are sufficient to generate significant biological control of CSFB populations. A sequence of the M. brassicae mitochondrial cytochrome oxidase 1 (MT-CO1) gene was generated for rapid future identification. Moroccan specimens of Microctonus aethiopoides (Loan), possessing 90% sequence similarity, were the closest identified sequenced species. This study represents the first description published in English of this parasitoid of the adult cabbage stem flea beetle.

Pleiotropy in developmental regulation by flowering-pathway genes: is it an evolutionary constraint?

Pleiotropy occurs when one gene influences more than one trait, contributing to genetic correlations among traits. Consequently, it is considered a constraint on the evolution of adaptive phenotypes because of potential antagonistic selection on correlated traits, or, alternatively, preservation of functional trait combinations. Such evolutionary constraints may be mitigated by the evolution of different functions of pleiotropic genes in their regulation of different traits. Arabidopsis thaliana flowering-time genes, and the pathways in which they operate, are among the most thoroughly studied regarding molecular functions, phenotypic effects, and adaptive significance. Many of them show strong pleiotropic effects. Here, we review examples of pleiotropy of flowering-time genes and highlight those that also influence seed germination. Some genes appear to operate in the same genetic pathways when regulating both traits, whereas others show diversity of function in their regulation, either interacting with the same genetic partners but in different ways or potentially interacting with different partners. We discuss how functional diversification of pleiotropic genes in the regulation of different traits across the life cycle may mitigate evolutionary constraints of pleiotropy, permitting traits to respond more independently to environmental cues, and how it may even contribute to the evolutionary divergence of gene function across taxa.

Awake1, an ABC-Type Transporter, Reveals an Essential Role for Suberin in the Control of Seed Dormancy.

The mother plant plays an important dynamic role in the control of dormancy of her progeny seed in response to environmental signals. In order to further understand the mechanisms by which this dormancy control takes place in Arabidopsis (Arabidopsis thaliana), we conducted a forward genetic screen to isolate mutants that fail to enter dormancy in response to variation in temperature during seed set. We show that, for the first of these mutants, designated awake1, the maternal allele is required for entry into strongly dormant states and that awake1 mutants show seed phenotypes shown previously to be associated with the loss of suberin in the seed. We identify awake1 as an allele of ABCG20, an ATP-binding cassette transporter-encoding gene required for the transport of fatty acids during suberin deposition, and show that further suberin-deficient mutants have seed dormancy defects. Seed coat suberin composition is affected by temperature during seed maturation, but this response appears to be independent of ABCG20. We conclude that seed coat suberin is essential for seed dormancy imposition by low temperature and that the exclusion of oxygen and water from the seed by the suberin and tannin layers is important for dormancy imposition.

Effects of environmental variation during seed production on seed dormancy and germination.

The environment during seed production has major impacts on the behaviour of progeny seeds. It can be shown that for annual plants temperature perception over the whole life history of the mother can affect the germination rate of progeny, and instances have been documented where these affects cross whole generations. Here we discuss the current state of knowledge of signal transduction pathways controlling environmental responses during seed production, focusing both on events that take place in the mother plant and those that occur directly as a result of environmental responses in the developing zygote. We show that seed production environment effects are complex, involving overlapping gene networks active independently in fruit, seed coat, and zygotic tissues that can be deconstructed using careful physiology alongside molecular and genetic experiments.

Maternal temperature history activates Flowering Locus T in fruits to control progeny dormancy according to time of year.

Seasonal behavior is important for fitness in temperate environments but it is unclear how progeny gain their initial seasonal entrainment. Plants use temperature signals to measure time of year, and changes to life histories are therefore an important consequence of climate change. Here we show that in Arabidopsis the current and prior temperature experience of the mother plant is used to control germination of progeny seeds, via the activation of the florigen Flowering Locus T (FT) in fruit tissues. We demonstrate that maternal past and current temperature experience are transduced to the FT locus in silique phloem. In turn, FT controls seed dormancy through inhibition of proanthocyanidin synthesis in fruits, resulting in altered seed coat tannin content. Our data reveal that maternal temperature history is integrated through FT in the fruit to generate a metabolic signal that entrains the behavior of progeny seeds according to time of year.

Linked circadian outputs control elongation growth and flowering in response to photoperiod and temperature.

Clock-regulated pathways coordinate the response of many developmental processes to changes in photoperiod and temperature. We model two of the best-understood clock output pathways in Arabidopsis, which control key regulators of flowering and elongation growth. In flowering, the model predicted regulatory links from the clock to cycling DOF factor 1 (CDF1) and flavin-binding, KELCH repeat, F-box 1 (FKF1) transcription. Physical interaction data support these links, which create threefold feed-forward motifs from two clock components to the floral regulator FT. In hypocotyl growth, the model described clock-regulated transcription of phytochrome-interacting factor 4 and 5 (PIF4, PIF5), interacting with post-translational regulation of PIF proteins by phytochrome B (phyB) and other light-activated pathways. The model predicted bimodal and end-of-day PIF activity profiles that are observed across hundreds of PIF-regulated target genes. In the response to temperature, warmth-enhanced PIF4 activity explained the observed hypocotyl growth dynamics but additional, temperature-dependent regulators were implicated in the flowering response. Integrating these two pathways with the clock model highlights the molecular mechanisms that coordinate plant development across changing conditions.

Regulation of Arabidopsis thaliana seed dormancy and germination by 12-oxo-phytodienoic acid.

We previously demonstrated that the oxylipin 12-oxo-phytodienoic acid (OPDA) acts along with abscisic acid to regulate seed germination in Arabidopsis thaliana, but the mechanistic details of this synergistic interaction remain to be elucidated. Here, we show that OPDA acts through the germination inhibition effects of abscisic acid, the abscisic acid-sensing ABI5 protein, and the gibberellin-sensing RGL2 DELLA protein. We further demonstrate that OPDA also acts through another dormancy-promoting factor, MOTHER-OF-FT-AND-TFL1 (MFT). Both abscisic acid and MFT positively feed back into the OPDA pathway by promoting its accumulation. These results confirm the central role of OPDA in regulating seed dormancy and germination in A. thaliana and underline the complexity of interactions between OPDA and other dormancy-promoting factors such as abscisic acid, RGL2, and MFT.

HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 is required for circadian periodicity through the promotion of nucleo-cytoplasmic mRNA export in Arabidopsis.

Cold acclimation has been shown to be attenuated by the degradation of the INDUCER OF CBF EXPRESSION1 protein by the E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 (HOS1). However, recent work has suggested that HOS1 may have a wider range of roles in plants than previously appreciated. Here, we show that hos1 mutants are affected in circadian clock function, exhibiting a long-period phenotype in a wide range of temperature and light environments. We demonstrate that hos1 mutants accumulate polyadenylated mRNA in the nucleus and that the circadian defect in hos1 is shared by multiple mutants with aberrant mRNA export, but not in a mutant attenuated in nucleo-cytoplasmic transport of microRNAs. As revealed by RNA sequencing, hos1 exhibits gross changes to the transcriptome with genes in multiple functional categories being affected. In addition, we show that hos1 and other previously described mutants with altered mRNA export affect cold signaling in a similar manner. Our data support a model in which altered mRNA export is important for the manifestation of hos1 circadian clock defects and suggest that HOS1 may indirectly affect cold signaling through disruption of the circadian clock.

Differential control of seed primary dormancy in Arabidopsis ecotypes by the transcription factor SPATULA.

Freshly matured seeds exhibit primary dormancy, which prevents germination until environmental conditions are favorable. The establishment of dormancy occurs during seed development and involves both genetic and environmental factors that impact on the ratio of two antagonistic phytohormones: abscisic acid (ABA), which promotes dormancy, and gibberellic acid, which promotes germination. Although our understanding of dormancy breakage in mature seeds is well advanced, relatively little is known about the mechanisms involved in establishing dormancy during seed maturation. We previously showed that the SPATULA (SPT) transcription factor plays a key role in regulating seed germination. Here we investigate its role during seed development and find that, surprisingly, it has opposite roles in setting dormancy in Landsberg erecta and Columbia Arabidopsis ecotypes. We also find that SPT regulates expression of five transcription factor encoding genes: ABA-INSENSITIVE4 (ABI4) and ABI5, which mediate ABA signaling; REPRESSOR-OF-GA (RGA) and RGA-LIKE3 involved in gibberellic acid signaling; and MOTHER-OF-FT-AND-TFL1 (MFT) that we show here promotes Arabidopsis seed dormancy. Although ABI4, RGA, and MFT are repressed by SPT, ABI5 and RGL3 are induced. Furthermore, we show that RGA, MFT, and ABI5 are direct targets of SPT in vivo. We present a model in which SPT drives two antagonistic "dormancy-repressing" and "dormancy-promoting" routes that operate simultaneously in freshly matured seeds. Each of these routes has different impacts and this in turn explains the opposite effect of SPT on seed dormancy of the two ecotypes analyzed here.

Seed production temperature regulation of primary dormancy occurs through control of seed coat phenylpropanoid metabolism.

Environmental changes during seed production are important drivers of lot-to-lot variation in seed behaviour and enable wild species to time their life history with seasonal cues. Temperature during seed set is the dominant environmental signal determining the depth of primary dormancy, although the mechanisms though which temperature changes impart changes in dormancy state are still only partly understood. We used molecular, genetic and biochemical techniques to examine the mechanism through which temperature variation affects Arabidopsis thaliana seed dormancy. Here we show that, in Arabidopsis, low temperatures during seed maturation result in an increase in phenylpropanoid gene expression in seeds and that this correlates with higher concentrations of seed coat procyanidins. Lower maturation temperatures cause differences in coat permeability to tetrazolium, and mutants with increased seed coat permeability and/or low procyanidin concentrations are less able to enter strongly dormant states after exposure to low temperatures during seed maturation. Our data show that maternal temperature signalling regulates seed coat properties, and this is an important pathway through which the environmental signals control primary dormancy depth.

Exploring the pleiotropy of hos1.

Understanding of the roles that HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 1 (HOS1) plays in the plant's ability to sense and respond to environmental signals has grown dramatically. Mechanisms through which HOS1 affects plant development have been uncovered, and the broader consequences of hos1 on the plant's ability to perceive and respond to its environment have been investigated. As such, it has been possible to place HOS1 as a key integrator of temperature information in response to both acute signals and cues that indicate time of year into developmental processes that are essential for plant survival. This review summarizes knowledge of HOS1's form and function, and contextualizes this information so that it is relevant for better understanding the processes of cold signalling, flowering time, and nuclear pore complex function more broadly.

Model selection reveals control of cold signalling by evening-phased components of the plant circadian clock.

Circadian clocks confer advantages by restricting biological processes to certain times of day through the control of specific phased outputs. Control of temperature signalling is an important function of the plant oscillator, but the architecture of the gene network controlling cold signalling by the clock is not well understood. Here we use a model ensemble fitted to time-series data and a corrected Akaike Information Criterion (AICc) analysis to extend a dynamic model to include the control of the key cold-regulated transcription factors C-REPEAT BINDING FACTORs 1-3 (CBF1, CBF2, CBF3). AICc was combined with in silico analysis of genetic perturbations in the model ensemble, and selected a model that predicted mutant phenotypes and connections between evening-phased circadian clock components and CBF3 transcriptional control, but these connections were not shared by CBF1 and CBF2. In addition, our model predicted the correct gating of CBF transcription by cold only when the cold signal originated from the clock mechanism itself, suggesting that the clock has an important role in temperature signal transduction. Our data shows that model selection could be a useful method for the expansion of gene network models.

Network balance via CRY signalling controls the Arabidopsis circadian clock over ambient temperatures.

Circadian clocks exhibit 'temperature compensation', meaning that they show only small changes in period over a broad temperature range. Several clock genes have been implicated in the temperature-dependent control of period in Arabidopsis. We show that blue light is essential for this, suggesting that the effects of light and temperature interact or converge upon common targets in the circadian clock. Our data demonstrate that two cryptochrome photoreceptors differentially control circadian period and sustain rhythmicity across the physiological temperature range. In order to test the hypothesis that the targets of light regulation are sufficient to mediate temperature compensation, we constructed a temperature-compensated clock model by adding passive temperature effects into only the light-sensitive processes in the model. Remarkably, this model was not only capable of full temperature compensation and consistent with mRNA profiles across a temperature range, but also predicted the temperature-dependent change in the level of LATE ELONGATED HYPOCOTYL, a key clock protein. Our analysis provides a systems-level understanding of period control in the plant circadian oscillator.