Ectopic expression of Triticum polonicum VRT-A2 underlies elongated glumes and grains in hexaploid wheat in a dosage-dependent manner.

Flower development is an important determinant of grain yield in crops. In wheat (Triticum spp.), natural variation for the size of spikelet and floral organs is particularly evident in Triticum turgidum ssp. polonicum (also termed Triticum polonicum), a tetraploid subspecies of wheat with long glumes, lemmas, and grains. Using map-based cloning, we identified VEGETATIVE TO REPRODUCTIVE TRANSITION 2 (VRT2), which encodes a MADS-box transcription factor belonging to the SHORT VEGETATIVE PHASE family, as the gene underlying the T. polonicum long-glume (P1) locus. The causal P1 mutation is a sequence rearrangement in intron-1 that results in ectopic expression of the T. polonicum VRT-A2 allele. Based on allelic variation studies, we propose that the intron-1 mutation in VRT-A2 is the unique T. polonicum subspecies-defining polymorphism, which was later introduced into hexaploid wheat via natural hybridizations. Near-isogenic lines differing for the P1 locus revealed a gradient effect of P1 across spikelets and within florets. Transgenic lines of hexaploid wheat carrying the T. polonicum VRT-A2 allele show that expression levels of VRT-A2 are highly correlated with spike, glume, grain, and floral organ length. These results highlight how changes in expression profiles, through variation in cis-regulation, can affect agronomic traits in a dosage-dependent manner in polyploid crops.

Meiosis in allopolyploid Arabidopsis suecica.

Polyploidy is a major force shaping eukaryote evolution but poses challenges for meiotic chromosome segregation. As a result, first-generation polyploids often suffer from more meiotic errors and lower fertility than established wild polyploid populations. How established polyploids adapt their meiotic behaviour to ensure genome stability and accurate chromosome segregation remains an active research question. We present here a cytological description of meiosis in the model allopolyploid species Arabidopsis suecica (2n = 4x = 26). In large part meiosis in A. suecica is diploid-like, with normal synaptic progression and no evidence of synaptic partner exchanges. Some abnormalities were seen at low frequency, including univalents at metaphase I, anaphase bridges and aneuploidy at metaphase II; however, we saw no evidence of crossover formation occurring between non-homologous chromosomes. The crossover number in A. suecica is similar to the combined number reported from its diploid parents Arabidopsis thaliana (2n = 2x = 10) and Arabidopsis arenosa (2n = 2x = 16), with an average of approximately 1.75 crossovers per chromosome pair. This contrasts with naturally evolved autotetraploid A. arenosa, where accurate chromosome segregation is achieved by restricting crossovers to approximately 1 per chromosome pair. Although an autotetraploid donor is hypothesized to have contributed the A. arenosa subgenome to A. suecica, A. suecica harbours diploid A. arenosa variants of key meiotic genes. These multiple lines of evidence suggest that meiosis in the recently evolved allopolyploid A. suecica is essentially diploid like, with meiotic adaptation following a very different trajectory to that described for autotetraploid A. arenosa.

CDKG1 Is Required for Meiotic and Somatic Recombination Intermediate Processing in Arabidopsis.

The Arabidopsis (Arabidopsis thaliana) cyclin-dependent kinase G1 (CDKG1) is necessary for recombination and synapsis during male meiosis at high ambient temperature. In the cdkg1-1 mutant, synapsis is impaired and there is a dramatic reduction in the number of class I crossovers, resulting in univalents at metaphase I and pollen sterility. Here, we demonstrate that CDKG1 is necessary for the processing of recombination intermediates in the canonical ZMM recombination pathway and that loss of CDKG1 results in increased class II crossovers. While synapsis and events associated with class I crossovers are severely compromised in a cdkg1-1 mutant, they can be restored by increasing the number of recombination intermediates in the double cdkg1-1 fancm-1 mutant. Despite this, recombination intermediates are not correctly resolved, leading to the formation of chromosome aggregates at metaphase I. Our results show that CDKG1 acts early in the recombination process and is necessary to stabilize recombination intermediates. Finally, we show that the effect on recombination is not restricted to meiosis and that CDKG1 is also required for normal levels of DNA damage-induced homologous recombination in somatic tissues.

Molecular and physiological responses to desiccation indicate the abscisic acid pathway is conserved in the peat moss, Sphagnum.

Mosses of the genus Sphagnum are the main components of peatlands, a major carbon-storing ecosystem. Changes in precipitation patterns are predicted to affect water relations in this ecosystem, but the effect of desiccation on the physiological and molecular processes in Sphagnum is still largely unexplored. Here we show that different Sphagnum species have differential physiological and molecular responses to desiccation but, surprisingly, this is not directly correlated with their position in relation to the water table. In addition, the expression of drought responsive genes is increased upon water withdrawal in all species. This increase in gene expression is accompanied by an increase in abscisic acid (ABA), supporting a role for ABA during desiccation responses in Sphagnum. Not only do ABA levels increase upon desiccation, but Sphagnum plants pre-treated with ABA display increased tolerance to desiccation, suggesting that ABA levels play a functional role in the response. In addition, many of the ABA signalling components are present in Sphagnum and we demonstrate, by complementation in Physcomitrium patens, that Sphagnum ABI3 is functionally conserved. The data presented here, therefore, support a conserved role for ABA in desiccation responses in Sphagnum.

µCT trait analysis reveals morphometric differences between domesticated temperate small grain cereals and their wild relatives.

Wheat and barley are two of the founder crops domesticated in the Fertile Crescent, and currently represent crops of major economic importance in temperate regions. Due to impacts on yield, quality and end-use, grain morphometric traits remain an important goal for modern breeding programmes and are believed to have been selected for by human populations. To directly and accurately assess the three-dimensional (3D) characteristics of grains, we combine X-ray microcomputed tomography (µCT) imaging techniques with bespoke image analysis tools and mathematical modelling to investigate how grain size and shape vary across wild and domesticated wheat and barley. We find that grain depth and, to a lesser extent, width are major drivers of shape change and that these traits are still relatively plastic in modern bread wheat varieties. Significant changes in grain depth are also observed to be associated with differences in ploidy. Finally, we present a model that can accurately predict the wild or domesticated status of a grain from a given taxa based on the relationship between three morphometric parameters (length, width and depth) and suggest its general applicability to both archaeological identification studies and breeding programmes.

In Vitro Tissue Culture in Brachypodium: Applications and Challenges.

Brachypodium distachyon has become an excellent model for plant breeding and bioenergy grasses that permits many fundamental questions in grass biology to be addressed. One of the constraints to performing research in many grasses has been the difficulty with which they can be genetically transformed and the generally low frequency of such transformations. In this review, we discuss the contribution that transformation techniques have made in Brachypodium biology as well as how Brachypodium could be used to determine the factors that might contribute to transformation efficiency. In particular, we highlight the latest research on the mechanisms that govern the gradual loss of embryogenic potential in a tissue culture and propose using B. distachyon as a model for other recalcitrant monocots.

The cyclin-dependent kinase G group defines a thermo-sensitive alternative splicing circuit modulating the expression of Arabidopsis ATU2AF65A.

The ability to adapt growth and development to temperature variations is crucial to generate plant varieties resilient to predicted temperature changes. However, the mechanisms underlying plant response to progressive increases in temperature have just started to be elucidated. Here, we report that the cyclin-dependent kinase G1 (CDKG1) is a central element in a thermo-sensitive mRNA splicing cascade that transduces changes in ambient temperature into differential expression of the fundamental spliceosome component, ATU2AF65A. CDKG1 is alternatively spliced in a temperature-dependent manner. We found that this process is partly dependent on both the cyclin-dependent kinase G2 (CDKG2) and the interacting co-factor CYCLIN L1 (CYCL1), resulting in two distinct messenger RNAs. The relative abundance of both CDKG1 transcripts correlates with ambient temperature and possibly with different expression levels of the associated protein isoforms. Both CDKG1 alternative transcripts are necessary to fully complement the expression of ATU2AF65A across the temperature range. Our data support a previously unidentified temperature-dependent mechanism based on the alternative splicing (AS) of CDKG1 and regulated by CDKG2 and CYCL1. We propose that changes in ambient temperature affect the relative abundance of CDKG1 transcripts, and this in turn translates into differential CDKG1 protein expression coordinating the AS of ATU2AF65A.

eIF4A RNA Helicase Associates with Cyclin-Dependent Protein Kinase A in Proliferating Cells and Is Modulated by Phosphorylation.

Eukaryotic initiation factor 4A (eIF4A) is a highly conserved RNA-stimulated ATPase and helicase involved in the initiation of messenger RNA translation. Previously, we found that eIF4A interacts with cyclin-dependent kinase A (CDKA), the plant ortholog of mammalian CDK1. Here, we show that this interaction occurs only in proliferating cells where the two proteins coassociate with 5'-cap-binding protein complexes, eIF4F or the plant-specific eIFiso4F. CDKA phosphorylates eIF4A on a conserved threonine residue (threonine-164) within the RNA-binding motif 1b TPGR. In vivo, a phospho-null (APGR) variant of the Arabidopsis (Arabidopsis thaliana) eIF4A1 protein retains the ability to functionally complement a mutant (eif4a1) plant line lacking eIF4A1, whereas a phosphomimetic (EPGR) variant fails to complement. The phospho-null variant (APGR) rescues the slow growth rate of roots and rosettes, together with the ovule-abortion and late-flowering phenotypes. In vitro, wild-type recombinant eIF4A1 and its phospho-null variant both support translation in cell-free wheat germ extracts dependent upon eIF4A, but the phosphomimetic variant does not support translation and also was deficient in ATP hydrolysis and helicase activity. These observations suggest a mechanism whereby CDK phosphorylation has the potential to down-regulate eIF4A activity and thereby affect translation.

CDKG1 protein kinase is essential for synapsis and male meiosis at high ambient temperature in Arabidopsis thaliana.

The Arabidopsis cyclin-dependent kinase G (CDKG) gene defines a clade of cyclin-dependent protein kinases related to CDK10 and CDK11, as well as to the enigmatic Ph1-related kinases that are implicated in controlling homeologous chromosome pairing in wheat. Here we demonstrate that the CDKG1/CYCLINL complex is essential for synapsis and recombination during male meiosis. A transfer-DNA insertional mutation in the cdkg1 gene leads to a temperature-sensitive failure of meiosis in late Zygotene/Pachytene that is associated with defective formation of the synaptonemal complex, reduced bivalent formation and crossing over, and aneuploid gametes. An aphenotypic insertion in the cyclin L gene, a cognate cyclin for CDKG, strongly enhances the phenotype of cdkg1-1 mutants, indicating that this cdk-cyclin complex is essential for male meiosis. Since CYCLINL, CDKG, and their mammalian homologs have been previously shown to affect mRNA processing, particularly alternative splicing, our observations also suggest a mechanism to explain the widespread phenomenon of thermal sensitivity in male meiosis.

The effect of temperature on the male and female recombination landscape of barley.

Barley (Hordeum vulgare) is a crop of global significance. However, a third of the genes of barley are largely inaccessible to conventional breeding programmes as crossovers are localised to the ends of the chromosomes. This work examines whether crossovers can be shifted to more proximal regions simply by elevating growth temperature. We utilised a genome-wide marker set for linkage analysis combined with cytological mapping of crossover events to examine the recombination landscape of plants grown at different temperatures. We found that barley shows heterochiasmy, that is, differences between female and male recombination frequencies. In addition, we found that elevated temperature significantly changes patterns of recombination in male meiosis only, with a repositioning of Class I crossovers determined by cytological mapping of HvMLH3 foci. We show that the length of synaptonemal complexes in male meiocytes increases in response to temperature. The results demonstrate that the distribution of crossover events are malleable and can be shifted to proximal regions by altering the growth temperature. The shift in recombination is the result of altering the distribution of Class I crossovers, but the higher recombination at elevated temperatures is potentially not the result of an increase in Class I events.

FERONIA receptor kinase pathway suppresses abscisic acid signaling in Arabidopsis by activating ABI2 phosphatase.

Plant growth and development are controlled by a delicate balance of hormonal cues. Growth-promoting hormones and growth-inhibiting counterparts often antagonize each other in their action, but the molecular mechanisms underlying these events remain largely unknown. Here, we report a cross-talk mechanism that enables a receptor-like kinase, FERONIA (FER), a positive regulator of auxin-promoted growth, to suppress the abscisic acid (ABA) response through activation of ABI2, a negative regulator of ABA signaling. The FER pathway consists of a FER kinase interacting with guanine exchange factors GEF1, GEF4, and GEF10 that, in turn, activate GTPase ROP11/ARAC10. Arabidopsis mutants disrupted in any step of the FER pathway, including fer, gef1gef4gef10, or rop11/arac10, all displayed an ABA-hypersensitive response, implicating the FER pathway in the suppression mechanism. In search of the target for the FER pathway, we found that the ROP11/ARAC10 protein physically interacted with the ABI2 phosphatase and enhanced its activity, thereby linking the FER pathway with the inhibition of ABA signaling.

AtMYB93 is a novel negative regulator of lateral root development in Arabidopsis.

Plant root system plasticity is critical for survival in changing environmental conditions. One important aspect of root architecture is lateral root development, a complex process regulated by hormone, environmental and protein signalling pathways. Here we show, using molecular genetic approaches, that the MYB transcription factor AtMYB93 is a novel negative regulator of lateral root development in Arabidopsis. We identify AtMYB93 as an interaction partner of the lateral-root-promoting ARABIDILLO proteins. Atmyb93 mutants have faster lateral root developmental progression and enhanced lateral root densities, while AtMYB93-overexpressing lines display the opposite phenotype. AtMYB93 is expressed strongly, specifically and transiently in the endodermal cells overlying early lateral root primordia and is additionally induced by auxin in the basal meristem of the primary root. Furthermore, Atmyb93 mutant lateral root development is insensitive to auxin, indicating that AtMYB93 is required for normal auxin responses during lateral root development. We propose that AtMYB93 is part of a novel auxin-induced negative feedback loop stimulated in a select few endodermal cells early during lateral root development, ensuring that lateral roots only develop when absolutely required. Putative AtMYB93 homologues are detected throughout flowering plants and represent promising targets for manipulating root systems in diverse crop species.

Variation in Water-Holding Capacity in Sphagnum Species Depends on Both Plant and Colony Structure.

Peatlands have become a focal point in climate mitigation strategies as these ecosystems have significant carbon sequestration capacities when healthy but release CO2 and other greenhouse gases when damaged. However, as drought episodes become more frequent and prolonged, organisms key to the functioning of some peatlands are increasingly under pressure from desiccation. The Sphagnum mosses, which tend to keep their ecosystem waterlogged and many of whom promote peat formation, are only mildly desiccation-tolerant in comparison to other mosses. The role of Sphagnum anatomy and colony structure is poorly understood in the context of desiccation resilience. Using four different Sphagnum species belonging to four different subgenera and positions along the gradient of the water table, we show that plant morphological traits and colony density are important determinants of water storage capacity. Our results show that, as previously postulated, the majority of the water is stored in an easily exchangeable form, probably extracellularly, and that plant morphological traits, specifically the type and presence of branches, are major contributors to water storage and can explain some of the interspecies variation. We also show that plant density is another important determinant for water storage capacity as higher densities hold larger quantities of water per unit of biomass for all four species, which increases resilience to desiccation. The results presented here suggest that species choice and planting density should receive more attention when considering peatland restoration strategies.

The Arabidopsis small GTPase AtRAC7/ROP9 is a modulator of auxin and abscisic acid signalling.

Rac-like GTPases or Rho-related GTPases from plants (RAC/ROPs) are important components of hormone signalling pathways in plants. Based on phylogeny, several groups can be distinguished, and the underlying premise is that members of different groups perform distinct functions in the plant. AtRAC7/ROP9 is phylogenetically unique among 11 Arabidopsis RAC/ROPs, and here it was shown that it functions as a modulator of auxin and abscisic acid (ABA) signalling, a dual role not previously assigned to these small GTPases. Plants with reduced levels of AtRAC7/ROP9 had increased sensitivity to auxin and were less sensitive to ABA. On the other hand, overexpressing AtRAC7/ROP9 activated ABA-induced gene expression but repressed auxin-induced gene expression. In addition, both hormones regulated the activity of the AtRAC7/ROP9 promoter, suggesting a feedback mechanism to modulate the signalling output from the AtRAC7/ROP9-controlled molecular switch. High levels of AtRAC7/ROP9 were detected specifically in embryos and lateral roots, underscoring the important role of this protein during embryo development and lateral root formation. These results place AtRAC7/ROP9 as an important signal transducer in recently described pathways that integrate auxin and ABA signalling in the plant.

Quantitative high resolution mapping of HvMLH3 foci in barley pachytene nuclei reveals a strong distal bias and weak interference.

In barley (Hordeum vulgare L.), chiasmata (the physical sites of genetic crossovers) are skewed towards the distal ends of chromosomes, effectively consigning a large proportion of genes to recombination coldspots. This has the effect of limiting potential genetic variability, and of reducing the efficiency of map-based cloning and breeding approaches for this crop. Shifting the sites of recombination to more proximal chromosome regions by forward and reverse genetic means may be profitable in terms of realizing the genetic potential of the species, but is predicated upon a better understanding of the mechanisms governing the sites of these events, and upon the ability to recognize real changes in recombination patterns. The barley MutL Homologue (HvMLH3), a marker for class I interfering crossovers, has been isolated and a specific antibody has been raised. Immunolocalization of HvMLH3 along with the synaptonemal complex transverse filament protein ZYP1, used in conjunction with fluorescence in situ hybridization (FISH) tagging of specific barley chromosomes, has enabled access to the physical recombination landscape of the barley cultivars Morex and Bowman. Consistent distal localization of HvMLH3 foci throughout the genome, and similar patterns of HvMLH3 foci within bivalents 2H and 3H have been observed. A difference in total numbers of HvMLH3 foci between these two cultivars has been quantified, which is interpreted as representing genotypic variation in class I crossover frequency. Discrepancies between the frequencies of HvMLH3 foci and crossover frequencies derived from linkage analysis point to the existence of at least two crossover pathways in barley. It is also shown that interference of HvMLH3 foci is relatively weak compared with other plant species.

ARABIDILLO proteins have a novel and conserved domain structure important for the regulation of their stability.

ARABIDILLO proteins are F-box-Armadillo (ARM) proteins that regulate root branching in Arabidopsis. Many F-box proteins in plants, yeast and mammals are unstable. In plants, the mechanism for this instability has not been fully investigated. Here, we show that a conserved family of plant ARABIDILLO-related proteins has a unique domain structure consisting of an F-box and leucine-rich repeats (LRRs) followed by ARM-repeats. The LRRs are similar to those found in other plant and animal F-box proteins, including cell cycle proteins and hormone receptors. We demonstrate that the LRRs are required for ARABIDILLO1 function in vivo. ARABIDILLO1 protein is unstable: we show that ARABIDILLO1 protein is associated with ubiquitin and is turned over by the proteasome. Both the F-box and LRR regions of ARABIDILLO1 appear to enable this turnover to occur. Application of known lateral root-regulating signals has no effect on ARABIDILLO1 stability. In addition, plants that lack or overexpress ARABIDILLO proteins respond normally to known lateral root-regulating signals. Thus, we suggest that the signal(s) regulating ARABIDILLO stability in vivo may be either highly specific or novel. The structural conservation between ARABIDILLOs and other plant and animal F-box proteins suggests that the stability of other F-box proteins may be controlled by similar mechanisms.

Arabidopsis and Tobacco superman regulate hormone signalling and mediate cell proliferation and differentiation.

Arabidopsis thaliana superman (SUP) plays an important role during flower development by maintaining the boundary between stamens and carpels in the inner two whorls. It was proposed that SUP maintains this boundary by regulating cell proliferation in both whorls, as loss-of-function superman mutants produce more stamens at the expense of carpels. However, the cellular mechanism that underlies SUP function remains unknown. Here Arabidopsis or tobacco (Nicotiana tabacum) SUP was overexpressed in tobacco plants to substantiate SUP's role as a regulator of cell proliferation and boundary definition and provide evidence that its biological role may be mediated via hormonal changes. It was found that moderate levels of SUP stimulated cell growth and proliferation, whereas high levels were inhibitory. SUP stimulated auxin- and cytokinin-regulated processes, and cells overexpressing SUP displayed reduced hormone dependency for proliferation and regeneration into plants. SUP also induced proliferation of female traits in the second and third flower whorls and promoted differentiation of petaloid properties in sepals, further supporting a role for SUP as a boundary regulator. Moreover, cytokinin suppressed stamen development and promoted differentiation of carpeloid tissues, suggesting that SUP may regulate male and female development via its effect on cytokinin signalling. Taken together, these observations suggest a model whereby the effect of SUP on cell growth and proliferation involves the modulation of auxin- and cytokinin-regulated processes. Furthermore, differential SUP expression or different sensitivities of different cell types to SUP may determine whether SUP stimulates or suppresses their proliferation.

Development of an Image Analysis Pipeline to Estimate Sphagnum Colony Density in the Field.

Sphagnum peatmosses play an important part in water table management of many peatland ecosystems. Keeping the ecosystem saturated, they slow the breakdown of organic matter and release of greenhouse gases, facilitating peatland's function as a carbon sink rather than a carbon source. Although peatland monitoring and restoration programs have increased recently, there are few tools to quantify traits that Sphagnum species display in their ecosystems. Colony density is often described as an important determinant in the establishment and performance in Sphagnum but detailed evidence for this is limited. In this study, we describe an image analysis pipeline that accurately annotates Sphagnum capitula and estimates plant density using open access computer vision packages. The pipeline was validated using images of different Sphagnum species growing in different habitats, taken on different days and with different smartphones. The developed pipeline achieves high accuracy scores, and we demonstrate its utility by estimating colony densities in the field and detecting intra and inter-specific colony densities and their relationship with habitat. This tool will enable ecologists and conservationists to rapidly acquire accurate estimates of Sphagnum density in the field without the need of specialised equipment.

A Functional Kinase Is Necessary for Cyclin-Dependent Kinase G1 (CDKG1) to Maintain Fertility at High Ambient Temperature in Arabidopsis.

Maintaining fertility in a fluctuating environment is key to the reproductive success of flowering plants. Meiosis and pollen formation are particularly sensitive to changes in growing conditions, especially temperature. We have previously identified cyclin-dependent kinase G1 (CDKG1) as a master regulator of temperature-dependent meiosis and this may involve the regulation of alternative splicing (AS), including of its own transcript. CDKG1 mRNA can undergo several AS events, potentially producing two protein variants: CDKG1L and CDKG1S, differing in their N-terminal domain which may be involved in co-factor interaction. In leaves, both isoforms have distinct temperature-dependent functions on target mRNA processing, but their role in pollen development is unknown. In the present study, we characterize the role of CDKG1L and CDKG1S in maintaining Arabidopsis fertility. We show that the long (L) form is necessary and sufficient to rescue the fertility defects of the cdkg1-1 mutant, while the short (S) form is unable to rescue fertility. On the other hand, an extra copy of CDKG1L reduces fertility. In addition, mutation of the ATP binding pocket of the kinase indicates that kinase activity is necessary for the function of CDKG1. Kinase mutants of CDKG1L and CDKG1S correctly localize to the cell nucleus and nucleus and cytoplasm, respectively, but are unable to rescue either the fertility or the splicing defects of the cdkg1-1 mutant. Furthermore, we show that there is partial functional overlap between CDKG1 and its paralog CDKG2 that could in part be explained by overlapping gene expression.

A CRISPR/Cas9-Based Mutagenesis Protocol for Brachypodium distachyon and Its Allopolyploid Relative, Brachypodium hybridum.

The CRISPR/Cas9 system enables precise genome editing and is a useful tool for functional genomic studies. Here we report a detailed protocol for targeted genome editing in the model grass Brachypodium distachyon and its allotetraploid relative B. hybridum, describing gRNA design, a transient protoplast assay to test gRNA efficiency, Agrobacterium-mediated transformation and the selection and analysis of regenerated plants. In B. distachyon, we targeted the gene encoding phytoene desaturase (PDS), which is a crucial enzyme in the chlorophyll biosynthesis pathway. The albino phenotype of mutants obtained confirmed the effectiveness of the protocol for functional gene analysis. Additionally, we targeted two genes related to cell wall maintenance, encoding a fasciclin-like arabinogalactan protein (FLA) and a pectin methylesterase (PME), also in B. distachyon. Two genes encoding cyclin-dependent kinases (CDKG1 and CDKG2), which may be involved in DNA recombination were targeted in both B. distachyon and B. hybridum. Cas9 activity induces mainly insertions or deletions, resulting in frameshift mutations that, may lead to premature stop codons. Because of the close phylogenetic relationship between Brachypodium species and key temperate cereals and forage grasses, this protocol should be easily adapted to target genes underpinning agronomically important traits.