The UniformMu National Public Resource is a widely used, functional genomics tool for maize, constructed by backcross introgression of active Robertson's Mutator (Mu) transposons into the W22 inbred line, creating a large, searchable collection of lines that together carry transposon insertions in thousands of maize genes. This resource provides (1) a ready supply of freely available mutant seed stocks, each linked to mapped gene sequences; (2) uniform controls in an inbred background, for precision analysis of mutant phenotypes; (3) a reliable source of heritable mutants that are consistently recovered in stated lines; and (4) stable mutant lines with no Mu activity. This low-cost resource provides a consummate experimental system for linking gene sequences with their function in a species that has long served humanity, not only as a preeminent genetic model, but also as one of the world's most productive grain crops. Here, we describe how to perform an initial, online search of insertions in the UniformMu population, request seeds, generate segregating families, PCR-genotype seedlings for Mu insertions of interest, and associate genotypes with phenotypes. Resulting analyses provide definitive, in planta evidence for genotype-phenotype relationships that either support or refute hypotheses regarding gene function.

Inositol phosphates (InsP) play diverse signaling roles in regulating development, phosphate sensing, and energy metabolism. Here, we identify 4 maize (Zea mays) mutants, big embryo 2 (bige2), big embryo 3 (bige3), big embryo 4 (bige4), and low phytic acid 1 (lpa1), that show enlargement of the embryo at the expense of endosperm. Bige2 (identical to Lpa2), Bige3 (identical to Lpa3), and Bige4 genes encode inositol phosphate triphosphokinase (ITPK) and mono-inositol phosphate kinase (MIK), both of which catalyze lipid-independent InsP biosynthesis, and inositol polyphosphate kinase (IPK2) in the lipid-dependent InsP pathway, respectively. Lpa1 encodes a tonoplast InsP6 transporter. InsP pathway mutants primarily affect scutellum growth, with each mutant exhibiting a distinct spatial pattern of cell enlargement and/or cell number. Genetic epistasis and transcriptome analyses reveal overlapping and nonredundant roles of lipid-independent and -dependent pathways in regulation of embryo development. Strikingly, ectopic expression of endosperm-specific genes in lpa2-bige2 and bige4 embryos reveals a shift toward endosperm organ identity. We identify a network of NAC transcription factors implicated in shaping lpa2-bige2 and bige4 transcriptomes. Disruption of lipid-independent InsP biosynthesis in lpa2-bige2 is associated with upregulation of a subnetwork of SOG1-related NAC proteins linked to DNA damage repair and endoreduplication. The lpa2-bige2 phenotype is fully suppressed by lpa1, suggesting that a block in InsP6 uptake into the vacuole restores signaling by cytosolic InsP intermediates. Together, these results establish a genetic framework for dissecting complex roles of InsP signaling in seed development.

Geneticists frequently use loss-of-function (knockout) mutations to reveal the effects of a gene's dysfunction at the organismal level, observed as the mutant phenotype. This strategy is facilitated by creation of large, searchable collections of knockout mutants in an organism of interest. Paramount among such resources in maize is the UniformMu National Resource, a large collection of genetic stocks carrying mutations generated by insertions of Robertson's Mutator ( Mu ) transposons. The name UniformMu refers to the phenotypic uniformity of the W22 inbred genetic background in which Mu insertion mutants were created. This community resource continues its pivotal role in providing seeds containing beneficial knockout and knockdown mutations in targeted genes, which can be used to elucidate gene function. The resource offers an invaluable complement to other functional genomics approaches aimed at bridging the gap between genome sequences and plant performance in the field. Several key features are central to the success of the UniformMu National Public Resource. First, mapped insertions are linked to seed stocks that are readily available through the Maize Genetics and Genomics Database (MaizeGDB) and the Maize Genetics Cooperation Stock Center. Second, a uniform inbred background facilitates analysis of mutant phenotypes, by providing uniform wild-type controls. Third, mutant alleles are reliably heritable and consistently recovered in stated lines. Finally, lines are stable, with no continuing transposition of Mu insertions. The collective effort of the maize community allows UniformMu to provide readily accessible knockout and knockdown mutant seeds, as well as, ultimately, highly sought evidence for gene function in planta.

In plants, embryo size is determined via interactions between metabolic and developmental signals. Maize (Zea mays) big embryo 6 (bige6) enhances embryo size while sharply reducing plant growth. Here, we show that BigE6 encodes a plastidial prephenate aminotransferase (PPA-AT), a key enzyme in the arogenate pathway for L-phenylalanine (Phe) and L-tyrosine (Tyr) biosynthesis. The maize BigE6 paralog, BigE6Like, encodes a cytosol-localized PPA-AT, revealing Phe and Tyr biosynthesis via cytosolic arogenate as a potential alternative to the known cytosolic phenylpyruvate pathway. Moreover, the single PPA-AT gene of Arabidopsis (Arabidopsis thaliana) encodes plastidial and cytosolic enzymes by alternative splicing. Transgenic rescue of a ppa-at mutant in Arabidopsis demonstrates that the plastidial PPA-AT is indispensable for seed formation due, in part, to its essential role in the female gametophyte. Leaves of bige6 maize maintained overall homeostasis for aromatic amino acids and downstream metabolites, revealing a resilience of mechanisms that scale growth to a limiting supply of Phe and Tyr. In bige6 seeds, broad perturbation of amino acid homeostasis is associated with transcriptomic upregulation of growth processes in the embryo and endosperm, implicating amino acid signaling in the regulation of embryo size. Our findings reveal the complexity and developmental dependence of growth responses to limiting amino acid biosynthesis.

A transformation in plant cell wall evolution marked the emergence of grasses, grains and related species that now cover much of the globe. Their tough, less digestible cell walls arose from a new pattern of cross-linking between arabinoxylan polymers with distinctive ferulic acid residues. Despite extensive study, the biochemical mechanism of ferulic acid incorporation into cell walls remains unknown. Here we show that ferulic acid is transferred to arabinoxylans via an unexpected sucrose derivative, 3,6-O-diferuloyl sucrose (2-feruloyl-O-α-D-glucopyranosyl-(1'→2)-3,6-O-feruloyl-β-D-fructofuranoside), formed by a sucrose ferulate cycle. Sucrose gains ferulate units through sequential transfers from feruloyl-CoA, initially at the O-3 position of sucrose catalysed by a family of BAHD-type sucrose ferulic acid transferases (SFT1 to SFT4 in maize), then at the O-6 position by a feruloyl sucrose feruloyl transferase (FSFT), which creates 3,6-O-diferuloyl sucrose. An FSFT-deficient mutant of maize, disorganized wall 1 (dow1), sharply decreases cell wall arabinoxylan ferulic acid content, causes accumulation of 3-O-feruloyl sucrose (α-D-glucopyranosyl-(1'→2)-3-O-feruloyl-β-D-fructofuranoside) and leads to the abortion of embryos with defective cell walls. In vivo, isotope-labelled ferulic acid residues are transferred from 3,6-O-diferuloyl sucrose onto cell wall arabinoxylans. This previously unrecognized sucrose ferulate cycle resolves a long-standing mystery surrounding the evolution of the distinctive cell wall characteristics of cereal grains, biofuel crops and related commelinid species; identifies an unexpected role for sucrose as a ferulate group carrier in cell wall biosynthesis; and reveals a new paradigm for modifying cell wall polymers through ferulic acid incorporation.

Abstract Plants release back to the atmosphere about half of the CO2 they capture by photosynthesis. Decreasing the rate of crop respiration could therefore potentially increase yields, store more carbon in the soil and draw down atmospheric CO2. However, decreasing respiration rate has had very little research effort compared to increasing photosynthesis, the historically dominant metabolic paradigm for crop improvement. Conceptual and technical advances, particularly in protein turnover and directed enzyme evolution, have now opened ways to trim the large fraction of respiration that fuels proteome maintenance by lowering the breakdown and resynthesis rates of enzymes and other proteins. In addition to being theoretically possible and practicable, exploring the reduction of respiration is prudential, given that it (i) has barely yet been tried and (ii) could help meet the challenges of sustaining crop productivity and managing atmospheric carbon.

Nearly all eukaryotes carry DNA transposons of the Robertson's Mutator (Mu) superfamily, a widespread source of genome instability and genetic variation. Despite their pervasive impact on host genomes, much remains unknown about the evolution of these transposons. Transposase recognition of terminal inverted repeats (TIRs) is thought to drive and constrain coevolution of MuDR transposase genes and TIRs. To address the extent of this relationship and its impact, we compared separate phylogenies of TIRs and MuDR gene sequences from Mu elements in the maize genome. Five major clades were identified. As expected, most Mu elements were bound by highly similar TIRs from the same clade (homomorphic type). However, a subset of elements contained dissimilar TIRs derived from divergent clades. These "heteromorphs" typically occurred in multiple copies indicating active transposition in the genome. In addition, analysis of internal sequences showed that exchanges between elements having divergent TIRs produced new mudra and mudrb gene combinations. In several instances, TIR homomorphs had been regenerated within a heteromorph clade with retention of distinctive internal MuDR sequence combinations. Results reveal that recombination between divergent clades facilitates independent evolution of transposase (mudra), transposase-binding targets (TIRs), and capacity for insertion (mudrb) of active Mu elements. This mechanism would be enhanced by the preference of Mu insertions for recombination-rich regions near the 5' ends of genes. We suggest that cycles of recombination give rise to alternating homo- and heteromorph forms that enhance the diversity on which selection for Mu fitness can operate.

The maize (Zea mays) ear represents one of the most striking domestication phenotypes in any crop species, with the cob conferring an exceptional yield advantage over the ancestral form of teosinte. Remodeling of the grain-bearing surface required profound developmental changes. However, the underlying mechanisms remain unclear and can only be partly attributed to the known domestication gene Teosinte glume architecture 1 (Tga1). Here we show that a more complete conversion involves strigolactones (SLs), and that these are prominent players not only in the Tga1 phenotype but also other domestication features of the ear and kernel. Genetic combinations of a teosinte tga1 allele with three SL-related mutants progressively enhanced ancestral morphologies. The SL mutants, in addition to modulating the tga1 phenotype, also reshaped kernel-bearing pedicels and cupules in a teosinte-like manner. Genetic and molecular evidence are consistent with SL regulation of TGA1, including direct interaction of TGA1 with components of the SL-signaling system shown here to mediate TGA1 availability by sequestration. Roles of the SL network extend to enhancing maize seed size and, importantly, coordinating increased kernel growth with remodeling of protective maternal tissues. Collectively, our data show that SLs have central roles in releasing kernels from restrictive maternal encasement and coordinating other factors that increase kernel size, physical support, and their exposure on the grain-bearing surface.

Maternal-to-filial nutrition transfer is central to grain development and yield. nitrate transporter 1/peptide transporter (NRT1-PTR)-type transporters typically transport nitrate, peptides, and ions. Here, we report the identification of a maize (Zea mays) NRT1-PTR-type transporter that transports sucrose and glucose. The activity of this sugar transporter, named Sucrose and Glucose Carrier 1 (SUGCAR1), was systematically verified by tracer-labeled sugar uptake and serial electrophysiological studies including two-electrode voltage-clamp, non-invasive microelectrode ion flux estimation assays in Xenopus laevis oocytes and patch clamping in HEK293T cells. ZmSUGCAR1 is specifically expressed in the basal endosperm transfer layer and loss-of-function mutation of ZmSUGCAR1 caused significantly decreased sucrose and glucose contents and subsequent shrinkage of maize kernels. Notably, the ZmSUGCAR1 orthologs SbSUGCAR1 (from Sorghum bicolor) and TaSUGCAR1 (from Triticum aestivum) displayed similar sugar transport activities in oocytes, supporting the functional conservation of SUGCAR1 in closely related cereal species. Thus, the discovery of ZmSUGCAR1 uncovers a type of sugar transporter essential for grain development and opens potential avenues for genetic improvement of seed-filling and yield in maize and other grain crops.

Metabolic engineering uses enzymes as parts to build biosystems for specified tasks. Although a part's working life and failure modes are key engineering performance indicators, this is not yet so in metabolic engineering because it is not known how long enzymes remain functional in vivo or whether cumulative deterioration (wear-out), sudden random failure, or other causes drive replacement. Consequently, enzymes cannot be engineered to extend life and cut the high energy costs of replacement. Guided by catalyst engineering, we adopted catalytic cycles until replacement (CCR) as a metric for enzyme functional life span in vivo. CCR is the number of catalytic cycles that an enzyme mediates in vivo before failure or replacement, i.e., metabolic flux rate/protein turnover rate. We used estimated fluxes and measured protein turnover rates to calculate CCRs for ∼100-200 enzymes each from Lactococcus lactis, yeast, and Arabidopsis CCRs in these organisms had similar ranges (<103 to >107) but different median values (3-4 × 104 in L. lactis and yeast versus 4 × 105 in Arabidopsis). In all organisms, enzymes whose substrates, products, or mechanisms can attack reactive amino acid residues had significantly lower median CCR values than other enzymes. Taken with literature on mechanism-based inactivation, the latter finding supports the proposal that 1) random active-site damage by reaction chemistry is an important cause of enzyme failure, and 2) reactive noncatalytic residues in the active-site region are likely contributors to damage susceptibility. Enzyme engineering to raise CCRs and lower replacement costs may thus be both beneficial and feasible.

BACKGROUND:The maize inbred line A188 is an attractive model for elucidation of gene function and improvement due to its high embryogenic capacity and many contrasting traits to the first maize reference genome, B73, and other elite lines. The lack of a genome assembly of A188 limits its use as a model for functional studies.RESULTS:Here, we present a chromosome-level genome assembly of A188 using long reads and optical maps. Comparison of A188 with B73 using both whole-genome alignments and read depths from sequencing reads identify approximately 1.1 Gb of syntenic sequences as well as extensive structural variation, including a 1.8-Mb duplication containing the Gametophyte factor1 locus for unilateral cross-incompatibility, and six inversions of 0.7 Mb or greater. Increased copy number of carotenoid cleavage dioxygenase 1 (ccd1) in A188 is associated with elevated expression during seed development. High ccd1 expression in seeds together with low expression of yellow endosperm 1 (y1) reduces carotenoid accumulation, accounting for the white seed phenotype of A188. Furthermore, transcriptome and epigenome analyses reveal enhanced expression of defense pathways and altered DNA methylation patterns of the embryonic callus.CONCLUSIONS:The A188 genome assembly provides a high-resolution sequence for a complex genome species and a foundational resource for analyses of genome variation and gene function in maize. The genome, in comparison to B73, contains extensive intra-species structural variations and other genetic differences. Expression and network analyses identify discrete profiles for embryonic callus and other tissues.

Ribosome assembly factors guide the complex process by which ribosomal proteins and the ribosomal RNAs form a functional ribosome. However, the assembly of plant plastid ribosomes is poorly understood. In the present study, we discovered a maize (Zea mays) plastid ribosome assembly factor based on our characterization of the embryo defective 15 (emb15) mutant. Loss of function of Emb15 retards embryo development at an early stage, but does not substantially affect the endosperm, and causes an albino phenotype in other genetic backgrounds. EMB15 localizes to plastids and possesses a ribosome maturation factor M (RimM) domain in the N-terminus and a predicted UDP-GlcNAc pyrophosphorylase domain in the C-terminus. The EMB15 RimM domain originated in bacteria and the UDP-GlcNAc pyrophosphorylase domain originated in fungi; these two domains came together in the ancestor of land plants during evolution. The N-terminus of EMB15 complemented the growth defect of an Escherichia coli strain with a RimM deletion and rescued the albino phenotype of emb15 homozygous mutants. The RimM domain mediates the interaction between EMB15 and the plastid ribosomal protein PRPS19. Plastid 16S rRNA maturation is also significantly impaired in emb15. These observations suggest that EMB15 functions in maize seed development as a plastid ribosome assembly factor, and the C-terminal domain is not important under normal conditions.

Functional and architectural diversification of transcription factor families has played a central role in the independent evolution of complex development in plants and animals. Here, we investigate the role of architectural constraints on evolution of B3 DNA binding domains that regulate plant embryogenesis. B3 domains of ABI3, FUS3, LEC2 and VAL1 proteins recognize the same cis-element. Complex architectures of ABI3 and VAL1 integrate cis-element recognition with other signals, whereas LEC2 and FUS3 have reduced architectures conducive to roles as pioneer activators. In yeast and plant in vivo assays, B3 domain functions correlate with architectural complexity of the parent transcription factor rather than phylogenetic relatedness. In a complex architecture, attenuated ABI3-B3 and VAL1-B3 activities enable integration of cis-element recognition with hormone signaling, whereas hyper-active LEC2-B3 and FUS3-B3 over-ride hormonal control. Three clade-specific amino acid substitutions (beta 4-triad) implicated in interactions with the DNA backbone account for divergence of LEC2-B3 and ABI3-B3. We find a striking correlation between differences in in vitro DNA binding affinity and in vivo activities of B3 domains in plants and yeast. Our results highlight the role of DNA backbone interactions that preserve DNA sequence specificity in adaptation of B3 domains to functional constraints associated with domain architecture.

The thiamin-requiring mutants of Arabidopsis have a storied history as a foundational model for biochemical genetics in plants and have illuminated the central role of thiamin in metabolism. Recent integrative genetic and biochemical analyses of thiamin biosynthesis and utilization imply that leaf metabolism normally operates close to thiamin-limiting conditions. Thus, the mechanisms that allocate thiamin-diphosphate (ThDP) cofactor among the diverse thiamin-dependent enzymes localized in plastids, mitochondria, peroxisomes, and the cytosol comprise an intricate thiamin economy. Here, we show that the classical thiamin-requiring 3 ( th3 ) mutant is a point mutation in plastid localized 5-deoxyxylulose synthase 1 ( DXS1 ), a key regulated enzyme in the methylerythritol 4-phosphate (MEP) isoprene biosynthesis pathway. Substitution of a lysine for a highly conserved glutamate residue (E323) located at the subunit interface of the homodimeric enzyme conditions a hypomorphic phenotype that can be rescued by supplying low concentrations of thiamin in the medium. Analysis of leaf thiamin vitamers showed that supplementing the medium with thiamin increased total ThDP content in both wild type and th3 mutant plants, supporting a hypothesis that the mutant DXS1 enzyme has a reduced affinity for the ThDP cofactor. An unexpected upregulation of a suite of biotic-stress-response genes associated with accumulation of downstream MEP intermediate MEcPP suggests that th3 causes mis-regulation of DXS1 activity in thiamin-supplemented plants. Overall, these results highlight that the central role of ThDP availability in regulation of DXS1 activity and flux through the MEP pathway.

Enzymes have in vivo life spans. Analysis of life spans, i.e., lifetime totals of catalytic turnovers, suggests that nonsurvivable collateral chemical damage from the very reactions that enzymes catalyze is a common but underdiagnosed cause of enzyme death. Analysis also implies that many enzymes are moderately deficient in that their active-site regions are not naturally as hardened against such collateral damage as they could be, leaving room for improvement by rational design or directed evolution. Enzyme life span might also be improved by engineering systems that repair otherwise fatal active-site damage, of which a handful are known and more are inferred to exist. Unfortunately, the data needed to design and execute such improvements are lacking: there are too few measurements of in vivo life span, and existing information about the extent, nature, and mechanisms of active-site damage and repair during normal enzyme operation is too scarce, anecdotal, and speculative to act on. Fortunately, advances in proteomics, metabolomics, cheminformatics, comparative genomics, and structural biochemistry now empower a systematic, data-driven approach for identifying, predicting, and validating instances of active-site damage and its repair. These capabilities would be practically useful in enzyme redesign and improvement of in-use stability and could change our thinking about which enzymes die young in vivo, and why.

Additional file 7. B73 genes overlapped with B73-specific sequences.

Additional file 4. CGRD output with B73Ref4 as the reference.

Abiotic stresses reduce crop growth and yield in part by disrupting metabolic homeostasis and triggering responses that change the metabolome. Experiments designed to understand the mechanisms underlying these metabolomic responses have usually not used agriculturally relevant stress regimes. We therefore subjected maize plants to drought, salt, or heat stresses that mimic field conditions and analyzed leaf responses at metabolome and transcriptome levels. Shared features of stress metabolomes included synthesis of raffinose, a compatible solute implicated in tolerance to dehydration. In addition, a marked accumulation of amino acids including proline, arginine, and γ-aminobutyrate combined with depletion of key glycolysis and tricarboxylic acid cycle intermediates indicated a shift in balance of carbon and nitrogen metabolism in stressed leaves. Involvement of the γ-aminobutyrate shunt in this process is consistent with its previously proposed role as a workaround for stress-induced thiamin-deficiency. Although convergent metabolome shifts were correlated with gene expression changes in affected pathways, patterns of differential gene regulation induced by the three stresses indicated distinct signaling mechanisms highlighting the plasticity of plant metabolic responses to abiotic stress.

The post-translational addition of SUMO plays essential roles in numerous eukaryotic processes including cell division, transcription, chromatin organization, DNA repair, and stress defense through its selective conjugation to numerous targets. One prominent plant SUMO ligase is METHYL METHANESULFONATE-SENSITIVE (MMS)-21/HIGH-PLOIDY (HPY)-2/NON-SMC-ELEMENT (NSE)-2, which has been connected genetically to development and endoreduplication. Here, we describe the potential functions of MMS21 through a collection of UniformMu and CRISPR/Cas9 mutants in maize (Zea mays) that display either seed lethality or substantially compromised pollen germination and seed/vegetative development. RNA-seq analyses of leaves, embryos, and endosperm from mms21 plants revealed a substantial dysregulation of the maize transcriptome, including the ectopic expression of seed storage protein mRNAs in leaves and altered accumulation of mRNAs associated with DNA repair and chromatin dynamics. Interaction studies demonstrated that MMS21 associates in the nucleus with the NSE4 and STRUCTURAL MAINTENANCE OF CHROMOSOMES (SMC)-5 components of the chromatin organizer SMC5/6 complex, with in vitro assays confirming that MMS21 will SUMOylate SMC5. Comet assays measuring genome integrity, sensitivity to DNA-damaging agents, and protein versus mRNA abundance comparisons implicated MMS21 in chromatin stability and transcriptional controls on proteome balance. Taken together, we propose that MMS21-directed SUMOylation of the SMC5/6 complex and other targets enables proper gene expression by influencing chromatin structure.

ABSTRACTFunctional diversification of transcription factor families through variation in modular domain architectures has played a central role in the independent evolution of gene regulatory networks underlying complex development in plants and animals. Here we show that architecture has in turn constrained evolution of B3 DNA binding domains in the B3 network regulating embryo formation in plants. B3 domains of ABI3, FUS3, LEC2 and VAL1 proteins recognize the same cis-element. ABI3 and VAL1 have complex architectures that physically integrate cis-element recognition with other signals, whereas LEC2 and FUS3 have reduced architectures conducive to their roles as pioneer activators. Qualitatively different activities of LEC2 and ABI3 B3 domains measured in vivo and in vitro are attributed in part to clade-specific substitutions in three amino acids that interact with the DNA backbone. Activities of FUS3 and VAL1 B3 domains show a similar correlation with architectural complexity. Domain-swap analyses in planta show that in a complex architecture setting the attenuated activities of ABI3 and VAL1 B3 domains are required for proper integration of ciselement recognition with hormone signalling. These results highlight modes of structural variation affecting non-specific, electrostatic interactions with the DNA backbone as a general mechanism allowing adaptation of DNA binding affinity to architectural constraints while preserving DNA sequence specificity.