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Abstract 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. 

Abstract 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. 

Abstract 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 (β4-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 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. 

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. 

The plastochron, the time interval between the formation of two successive leaves, is an important determinant of plant architecture. We genetically and phenotypically investigated many-noded dwarf ( mnd ) mutants in barley. The mnd mutants exhibited a shortened plastochron and a decreased leaf blade length, and resembled previously reported plastochron1 ( pla1 ), pla2 , and pla3 mutants in rice. In addition, the maturation of mnd leaves was accelerated, similar to pla mutants in rice. Several barley mnd alleles were derived from three genes— MND1 , MND4 , and MND8 . Although MND4 coincided with a cytochrome P450 family gene that is a homolog of rice PLA1 , we clarified that MND1 and MND8 encode an N-acetyltransferase-like protein and a MATE transporter-family protein, which are respectively orthologs of rice GW6a and maize BIGE1 and unrelated to PLA2 or PLA3 . Expression analyses of the three MND genes revealed that MND1 and MND4 were expressed in limited regions of the shoot apical meristem and leaf primordia, but MND8 did not exhibit a specific expression pattern around the shoot apex. In addition, the expression levels of the three genes were interdependent among the various mutant backgrounds. Genetic analyses using the double mutants mnd4mnd8 and mnd1mnd8 indicated that MND1 and MND4 regulate the plastochron independently of MND8 , suggesting that the plastochron in barley is controlled by multiple genetic pathways involving MND1 , MND4 , and MND8 . Correlation analysis between leaf number and leaf blade length indicated that both traits exhibited a strong negative association among different genetic backgrounds but not in the same genetic background. We propose that MND genes function in the regulation of the plastochron and leaf growth and revealed conserved and diverse aspects of plastochron regulation via comparative analysis of barley and rice. 

Summary The B vitamins provide essential co‐factors for central metabolism in all organisms. In plants, B vitamins have surprising emerging roles in development, stress tolerance and pathogen resistance. Hence, there is a paramount interest in understanding the regulation of vitamin biosynthesis as well as the consequences of vitamin deficiency in crop species. To facilitate genetic analysis of B vitamin biosynthesis and functions in maize, we have mined the UniformMu transposon resource to identify insertional mutations in vitamin pathway genes. A screen of 190 insertion lines for seed and seedling phenotypes identified mutations in biotin, pyridoxine and niacin biosynthetic pathways. Importantly, isolation of independent insertion alleles enabled genetic confirmation of genotype‐to‐phenotype associations. Because B vitamins are essential for survival, null mutations often have embryo lethal phenotypes that prevent elucidation of subtle, but physiologically important, metabolic consequences of sub‐optimal (functional) vitamin status. To circumvent this barrier, we demonstrate a strategy for refined genetic manipulation of vitamin status based on construction of heterozygotes that combine strong and hypomorphic mutant alleles. Dosage analysis ofpdx2alleles in endosperm revealed that endosperm supplies pyridoxine to the developing embryo. Similarly, a hypomorphicbio1allele enabled analysis of transcriptome and metabolome responses to incipient biotin deficiency in seedling leaves. We show that systemic pipecolic acid accumulation is an early metabolic response to sub‐optimal biotin status highlighting an intriguing connection between biotin, lysine metabolism and systemic disease resistance signaling. Seed‐stocks carrying insertions for vitamin pathway genes are available for free, public distribution via the Maize Genetics Cooperation Stock Center.