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Tucker, M. R.

Publications and source records attributed to Tucker, M. R..

11 recordsLinked to original sources

Barley (Hordeum vulgare L.) HvDEP1 alleles and their effect on agronomic and physical grain traits

The Dense and erect particle 1 (HvDEP1) gene, located on chromosome 5H in barley (Hordeum vulgare L.), encodes a heterotrimeric G-protein {gamma}-subunit that regulates grain size and stem elongation. Multiple alleles of HvDEP1 have been identified, including the widely utilized semi-dwarf allele HvDEP1.GP, caused by an insertion mutation, and a recently discovered variant, HvDEP1.V, characterized by two deletions in the putative cis-regulatory region. In this study, we evaluated the phenotypic effects of HvDEP1.V relative to HvDEP1.GP and the wild-type allele (HvDEP1.WT) using two BC{square}F{square} populations across multi-environment field trials spanning two locations and three years. HvDEP1.V was associated with plants that were 5-14.6 cm taller, had 3-6.7 higher lodging score, and increased head loss compared to HvDEP1.GP. HvDEP1.V showed comparable agronomic attributes to HvDEP1.WT. Substituting HvDEP1.V for HvDEP1.GP significantly increased all physical grain attributes, including grain width (1.44-4.24% in three out of five environments), grain length (4.88-8.69 %), grain area (6.45-11.06%) and thousand-grain weight (6.75-13.8%). Out of five environments, compared to HvDEP1.WT, HvDEP1.V was associated with wider grain in three environments, shorter grain in four environments, and increased grain roundness in four environments. These findings link allelic variation of the HvDEP1 gene to key agronomic and physical grain traits and demonstrate the functional consequences of HvDEP1.V in diverse genetic backgrounds and field conditions, providing valuable insights for barley improvement. Key messageBy evaluating agronomic performance and physical grain traits in two genetically distinct barley populations across multiple environments, we reveal strong environment- and background-dependent effects of HvDEP1 alleles.

genetics↗

AGP-Ca2+ binding is essential for pollen development and pollen tube growth in Arabidopsis thaliana

Arabinogalactan-proteins (AGPs) are highly glycosylated cell wall proteins essential for plant reproduction, although their mode of action remains unclear. AGPs have been proposed to bind and store calcium (Ca2+) in the apoplast via glucuronic acid (GlcA) residues added by glucuronosyltransferases (GLCATs), potentially acting as Ca2+ capacitors. Here we report that Ca2+ binding by AGPs is required for successful double fertilisation in Arabidopsis. Analysis of glcat14a glcat14b glcat14d triple mutants revealed reduced seed set due to the abortion of pollen grains, which lacked cytoplasmic content and an intine layer, as confirmed by the absence of cellulose, resembling the phenotype of AGP-deficient mutants. When grown under Ca2+-deficient conditions, GLCAT mutants showed exacerbated and conditional reproductive defects that were attenuated under standard Ca2+ conditions. Our findings establish a functional requirement for GlcA-mediated Ca2+ binding by AGPs and support their role as apoplastic Ca2+ stores essential for male fertility and reproductive success in plants.

plant biology↗

Control of grain dimensions via ALLENE OXIDE CYCLASE

Cereal grain is a global food staple. Grain formation starts with fertilization of the embryo sac which generates filial tissues, such as the embryo and endosperm, that grow embedded in the maternal ovule and surrounding ovary wall. Maternal tissues provide nutrition and protection but are progressively reduced and degraded, such that filial tissues dominate the mature grain. While we know some enzymatic players, regulation of this process in temperate cereals like wheat and barley, remains less understood. However, previous work suggested a role for the phytohormone Jasmonate (JA). To learn more, we edited the barley ALLENE OXIDASE CYCLASE (HvAOC) gene encoding a JA biosynthetic enzyme. Defective HvAOC alleles caused enhanced vegetative growth, faster flowering and larger, heavier grain - phenotypes rescued by MeJA treatment. HvAOC control of final grain parameters is parentally-derived, consistent with enriched HvAOC expression in maternal compared to filial tissues. Defective HvAOC function enlarged maternal tissues involved in nutrient transfer, correlating with larger endosperm and higher expression of genes linked to sucrose transport. This work provides the first genetic dissection of JA in barley and highlights a key role for HvAOC in the coordination between growth and death of maternal tissues with grain size and weight.

plant biology↗

ARABIDOPSIS Bsister and SEEDSTICK MADS-box transcription factors modulate maternal nutrient flow for seed development in Arabidopsis

Successful seed development in angiosperms depends on the coordinated transport and allocation of sugars from maternal tissues to the developing embryo and endosperm. In Arabidopsis thaliana, ovules function as carbohydrate sink organs, accumulating starch in both gametophytic and sporophytic domains prior to fertilization. This stored starch is later mobilized to support early embryogenesis. Despite extensive knowledge of starch metabolism in photosynthetic tissues, the regulatory mechanisms governing sugar transport in reproductive organs remain poorly understood. Recent studies have identified fertilization-dependent changes in nutrient flow, including callose-mediated modulation of symplastic transport at the phloem unloading site. However, the molecular players orchestrating these transitions are largely unknown. Here, we show that the MIKC MADS domain transcription factors ABS/TT16 and STK play critical roles in regulating maternal nutrient flow during ovule maturation and seed development. We dissect their functional redundancy using omics and genetic approaches, underscoring the importance of different ovule tissues in coordinating sugar transport pathways for post-fertilization development. Our findings reveal a previously underappreciated layer of genetic control over nutrient allocation in reproductive tissues and provide new insights into the metabolic reprogramming required for successful seed formation.

plant biology↗

The p97/VCP adapter UBXD1 drives AAA+ remodeling and ring opening through multi-domain tethered interactions

p97/VCP is an essential cytosolic AAA+ ATPase hexamer that extracts and unfolds substrate polypeptides during protein homeostasis and degradation. Distinct sets of p97 adapters guide cellular functions but their roles in direct control of the hexamer are unclear. The UBXD1 adapter localizes with p97 in critical mitochondria and lysosome clearance pathways and contains multiple p97-interacting domains. We identify UBXD1 as a potent p97 ATPase inhibitor and report structures of intact p97:UBXD1 complexes that reveal extensive UBXD1 contacts across p97 and an asymmetric remodeling of the hexamer. Conserved VIM, UBX, and PUB domains tether adjacent protomers while a connecting strand forms an N-terminal domain lariat with a helix wedged at the interprotomer interface. An additional VIM-connecting helix binds along the second AAA+ domain. Together these contacts split the hexamer into a ring-open conformation. Structures, mutagenesis, and comparisons to other adapters further reveal how adapters containing conserved p97-remodeling motifs regulate p97 ATPase activity and structure.

biochemistry↗

MADS31 supports female germline development by repressing the post-fertilization program in cereal ovules

The female germline of flowering plants develops within a niche of somatic ovule cells, also referred to as the nucellus. How niche cells maintain their own somatic developmental program, yet support the development of adjoining germline cells, remains largely unknown. Here we report that MADS31, a conserved MADS-box transcription factor from the B-sister subclass, is a potent regulator of niche cell identity in barley. MADS31 is preferentially expressed in nucellar cells directly adjoining the germline, and loss-of-function mads31 mutants exhibit deformed and disorganized nucellar cells, leading to impaired germline development and partial female sterility. Molecular assays indicate that MADS31 encodes a potent transcriptional repressor, repressing genes in the ovule that are normally active in the seed. One prominent target of MADS31 is NRPD4b, a seed-expressed component of RNA polymerase IV/V that is involved in gene silencing via RNA directed DNA methylation. NRPD4b is directly repressed by MADS31 in vivo and is de-repressed in mads31 ovules, while overexpression of NRPD4b recapitulates the mads31 ovule phenotype. This coincides with specific changes in histone methylation and is consistent with NRPD4b being directly repressed by MADS31 to maintain ovule niche functionality. Our findings reveal a new mechanism by which somatic ovule tissues maintain their own identity before transitioning to the post-fertilization program.

plant biology↗

Ovule cell wall composition is a maternal determinant of grain size in barley

SummaryO_LIIn cereal species, seed and grain size is influenced by growth of the ovule integuments (seed coat), the spikelet hull (lemma and palea) and the filial endosperm. It has remained unclear whether a highly conserved ovule tissue, the nucellus, has any impact on grain size. C_LIO_LIImmunolabelling revealed that the barley nucellus comprises two distinct cell types that differ in terms of cell wall homogalacturonan (HG) accumulation. Transcriptional profiling of the nucellus identified two pectin methylesterase genes, OVULE PECTIN MODIFIER 1 (OPM1) and OPM2, which are expressed in the ovule but absent from the seed. C_LIO_LIOvules from an opm1 opm2 mutant, and plants expressing an ovule-specific pectin methylesterase inhibitor (PMEI), exhibit reduced HG accumulation. This results in changes to ovule cell size and shape, and ovules that are longer than wild-type controls. At grain maturity, this is manifested as significantly longer grain. C_LIO_LIThese findings indicate that cell wall composition during ovule development acts to limit ovule and seed growth. The investigation of ovule PME and PMEI activity reveals an unexpected role of maternal tissues in controlling grain growth prior to fertilisation, one that has been lacking from models exploring improvements in grain size. C_LI

plant biology↗

HvSL1 and HvMADS16 promote stamen identity to restrict multiple ovary formation in barley

Correct floral development is a consequence of a sophisticated balance between environmental and molecular cues. Floral mutants provide insight into the main genetic determinants that integrate these cues, as well as providing opportunities to assess functional conservation across species. In this study, we characterize the barley (Hordeum vulgare) multiovary mutants mov2.g and mov1 and propose causative gene sequences: a C2H2 zinc-finger HvSL1 and a B-class gene HvMADS16, respectively. In the absence of HvSL1, flowers lack stamens but exhibit functional supernumerary carpels resulting in multiple seeds per floret when artificially pollinated. Deletion of HvMADS16 in mov1 causes homeotic conversion of lodicules and stamens into bract-like organs and carpels that contain non-functional ovules. Based on developmental, genetic, and molecular data we propose a model by which stamen specification in barley is defined by HvSL1 acting upstream of barley B-class genes, specifically the transcriptional up-regulation of HvMADS16. The present work identifies strong conservation of stamen formation pathways with rice, but also reveals intriguing species-specific differences. The findings lay the foundation for a better understanding of floral architecture in Triticeae, a key target for crop improvement. HighlightAnalysis of the barley multiovary mov1 and mov2 loci indicates that HvSL1 and HvMADS16 exhibit both unique and conserved roles in the specification and development of cereal flowers.

developmental biology↗

A structural model of a Ras-Raf signalosome

The protein K-Ras functions as a molecular switch in signaling pathways regulating cell growth. In the MAPK pathway, which is implicated in many cancers, multiple K-Ras proteins are thought to assemble at the cell membrane with Ras-effector proteins from the Raf family. Here we propose an atomistic structural model for such an assembly. Our starting point was an asymmetric, GTP-mediated K-Ras dimer model, which we generated using unbiased molecular dynamics simulations and verified with mutagenesis experiments. Adding further K-Ras monomers in a head-to-tail fashion led to a compact helical assembly, a model we validated using electron microscopy and cell-based experiments. This assembly stabilizes K-Ras in its active state and presents composite interfaces to facilitate Raf binding. Guided by existing experimental data, we then positioned C-Raf, the downstream kinase MEK1, and accessory proteins (Galectin-3 and 14-3-3{sigma}) on the helical assembly. The resulting Ras-Raf signalosome model offers an explanation for a large body of data on MAPK signaling.

molecular biology↗

GWAS reveals the genetic complexity of fructan accumulation patterns in barley grain

We profiled the grain oligosaccharide content of 154 two-row spring barley genotypes and quantified 27 compounds, mainly fructans, that exhibited differential abundance. Clustering revealed two major profile groups where the ‘high’ set contained greater amounts of sugar monomers, sucrose and overall fructans, but lower fructosylraffinose. GWAS identified a significant association for the variability of two fructan types; neoseries-DP7 and inulin-DP9 which showed increased strength when a compound-ratio GWAS was applied. Gene models within this region included five fructan biosynthesis genes, of which three (fructan:fructan 1-fructosyltransferase, sucrose:sucrose 1-fructosyltransferase, and sucrose:fructan 6-fructosyltransferase) have already been described. The remaining two, 6(G)-fructosyltransferase and vacuolar invertase1 have not previously been linked to fructan biosynthesis in barley and showed expression patterns distinct from those of the other three genes, including exclusive expression of 6(G)-fructosyltransferase in outer grain tissues at the storage phase. From exome capture data several SNPs related to inulin- and neoseries-type fructan variability were identified in fructan:fructan 1-fructosyltransferase and 6(G)-fructosyltransferase genes Co-expression analyses uncovered potential regulators of fructan biosynthesis including transcription factors. Our results provide evidence for the distinct biosynthesis of neoseries-type fructans during barley grain maturation plus new gene candidates likely involved in the differential biosynthesis of the various fructan types.Highlight Grain fructan profiles in barley are more complex than previously expected and variations in a diversity panel relate to a genomic region where fructan biosynthesis genes cluster.Abbreviations1-FFTfructan:fructan 1-fructosyltransferase1-SSTsucrose:sucrose 1-fructosyltransferase6-SFTsucrose:fructan 6-fructosyltransferase6G-FFT6(G)-fructosyltransferaseDAPdays after pollinationDPdegree of polymerisationDMdry matterELSDevaporative light scattering detectionFDRfalse discovery rateFOSfructooligosaccharidesFPKMfragments per kilobase, per million mapped readsGWAgenome wide associationGWASgenome wide association studyHAIhours after imbibitionHPAEC–PADhigh pH anion exchange chromatography with pulsed amperometric detectionHPLChigh performance liquid chromatographyKPkestopentaoseKTkestotetraoseLCliquid chromatographyLDlinkage disequilibriumLODlogarithm of oddsMAFminimum allele frequencyMSmass spectrometryNGNeural GasNSneoseries-type fructanPprobability valuePEGpolyethylene glycolQTLquantitative trait lociRFOraffinose family oligosaccharidesRTretention timeSNPsingle nucleotide polymorphismsSPEsolid phase extractionTFAtrifluoroacetic acidTPMtranscripts per millionVI-1vacuolar invertase1View Full Text

plant biology↗

Targeted mutation of barley (1,3;1,4)-β-glucan synthases reveals complex relationships between the storage and cell wall polysaccharide content

Barley (Hordeum vulgare L) grain is comparatively rich in (1,3;1,4)-{beta}-glucan, a source of fermentable dietary fibre that protects against various human health conditions. However, low grain (1,3;1,4)-{beta}-glucan content is preferred for brewing and distilling. We took a reverse genetics approach, using CRISPR/Cas9 to generate mutations in members of the Cellulose synthase-like (Csl) gene superfamily that encode known (HvCslF6 and HvCslH1) and putative (HvCslF3 and HvCslF9) (1,3;1,4)-{beta}-glucan synthases. Resultant mutations ranged from single amino acid (aa) substitutions to frameshift mutations causing premature stop codons, and led to specific differences in grain morphology, composition and (1,3;1,4)-{beta}-glucan content. (1,3;1,4)-{beta}-Glucan was absent in the grain of cslf6 knock-out lines whereas cslf9 knock-out lines had similar (1,3;1,4)-{beta}-glucan content to WT. However, cslf9 mutants showed changes in the abundance of other cell wall-related monosaccharides compared to WT. Thousand grain weight (TGW), grain length, width and surface area were altered in cslf6 knock-outs and to a lesser extent TGW in cslf9 knock-outs. cslf3 and cslh1 mutants had no effect on grain (1,3;1,4)-{beta}-glucan content. Our data indicate that multiple members of the CslF/H family fulfil important functions during grain development but, with the exception of HvCslF6, do not impact the abundance of (1,3;1,4)-{beta}-glucan in mature grain.

plant biology↗