Experimental approaches targeting carotenoid biosynthetic enzymes have successfully increased the seed b-carotene content of crops. However, linkage analysis of seed carotenoids in Arabidopsis thaliana recombinant inbred populations showed that only 21% of quantitative trait loci, including those for b-carotene, encode carotenoid biosynthetic enzymes in their intervals. Thus, numerous loci remain uncharacterized and underutilized in biofortification approaches. Linkage mapping and genome-wide association studies of Arabidopsis seed carotenoids identified CAROTENOID CLEAVAGE DIOXYGENASE4 (CCD4) as a major negative regulator of seed carotenoid content, especially b-carotene. Loss of CCD4 function did not affect carotenoid homeostasis during seed development but greatly reduced carotenoid degradation during seed desiccation, increasing b-carotene content 8.4-fold relative to the wild type. Allelic complementation of a ccd4 null mutant demonstrated that single-nucleotide polymorphisms and insertions and deletions at the locus affect dry seed carotenoid content, due at least partly to differences in CCD4 expression. CCD4 also plays a major role in carotenoid turnover during dark-induced leaf senescence, with b-carotene accumulation again most strongly affected in the ccd4 mutant. These results demonstrate that CCD4 plays a major role in b-carotene degradation in drying seeds and senescing leaves and suggest that CCD4 orthologs would be promising targets for stabilizing and increasing the level of provitamin A carotenoids in seeds of major food crops.

Branched-chain amino acids (BCAAs) are three of the nine essential amino acids in human and animal diets and are important for numerous processes in development and growth. However, seed BCAA levels in major crops are insufficient to meet dietary requirements, making genetic improvement for increased and balanced seed BCAAs an important nutritional target. Addressing this issue requires a better understanding of the genetics underlying seed BCAA content and composition. Here, a genome-wide association study and haplotype analysis for seed BCAA traits in Arabidopsis thaliana revealed a strong association with a chromosomal interval containing two BRANCHED-CHAIN AMINO ACID TRANSFERASES, BCAT1 and BCAT2. Linkage analysis, reverse genetic approaches, and molecular complementation analysis demonstrated that allelic variation at BCAT2 is responsible for the natural variation of seed BCAAs in this interval. Complementation analysis of a bcat2 null mutant with two significantly different alleles from accessions Bayreuth-0 and Shahdara is consistent with BCAT2 contributing to natural variation in BCAA levels, glutamate recycling, and free amino acid homeostasis in seeds in an alleledependent manner. The seed-specific phenotype of bcat2 null alleles, its strong transcription induction during late seed development, and its subcellular localization to the mitochondria are consistent with a unique, catabolic role for BCAT2 in BCAA metabolism in seeds.

SUMMARYCellular folates function as co-enzymes in one-carbon metabolism and are predominantly decorated with a polyglutamate tail that enhances co-enzyme affinity, subcellular compartmentation and stability. Polyglutamylation is catalysed by folylpolyglutamate synthetases (FPGSs) that are specified by three genes in Arabidopsis, FPGS1, 2 and 3, which reportedly encode plastidic, mitochondrial and cytosolic isoforms, respectively. A mutational approach was used to probe the functional importance of folate polyglutamylation in one-carbon metabolism and development. Biochemical analysis of single FPGS loss-of-function mutants established that folate polyglutamylation is essential for organellar and whole-plant folate homeostasis. However, polyglutamylated folates were still detectable, albeit at lower levels, in organelles isolated from the corresponding isozyme knockout lines, e.g. in plastids and mitochondria of the fpgs1 (plastidial) and fpgs2 (mitochondrial) mutants. This result is surprising given the purported single-compartment targeting of each FPGS isozyme. These results indicate redundancy in compartmentalised FPGS activity, which in turn explains the lack of anticipated phenotypic defects for the single FPGS mutants. In agreement with this hypothesis, fpgs1 fpgs2 double mutants were embryo-lethal, fpgs2 fpgs3 mutants exhibited seedling lethality, and fpgs1 fpgs3 mutants were dwarfed with reduced fertility. These phenotypic, metabolic and genetic observations are consistent with targeting of one or more FPGS isozymes to multiple organelles. These data confirm the importance of polyglutamylation in folate compartmentation, folate homeostasis and folate-dependent metabolic processes, including photorespiration, methionine and pantothenate biosynthesis.

Amino acids are essential for proper growth and development in plants. Amino acids serve as building blocks for proteins but also are important for responses to stress and the biosynthesis of numerous essential compounds. In seed, the pool of free amino acids (FAAs) also contributes to alternative energy, desiccation, and seed vigor; thus, manipulating FAA levels can significantly impact a seed's nutritional qualities. While genome-wide association studies (GWAS) on branched-chain amino acids have identified some regulatory genes controlling seed FAAs, the genetic regulation of FAA levels, composition, and homeostasis in seeds remains mostly unresolved. Hence, we performed GWAS on 18 FAAs from a 313-ecotype Arabidopsis (Arabidopsis thaliana) association panel. Specifically, GWAS was performed on 98 traits derived from known amino acid metabolic pathways (approach 1) and then on 92 traits generated from an unbiased correlation-based metabolic network analysis (approach 2), and the results were compared. The latter approach facilitated the discovery of additional novel metabolic interactions and singlenucleotide polymorphism-trait associations not identified by the former approach. The most prominent network-guided GWAS signal was for a histidine (His)-related trait in a region containing two genes: a cationic amino acid transporter (CAT4) and a polynucleotide phosphorylase resistant to inhibition with fosmidomycin. A reverse genetics approach confirmed CAT4 to be responsible for the natural variation of His-related traits across the association panel. Given that His is a semiessential amino acid and a potent metal chelator, CAT4 orthologs could be considered as candidate genes for seed quality biofortification in crop plants.Free amino acids (FAAs) play a pivotal role in the central metabolism of plants. FAAs serve as building blocks for protein synthesis and also are precursors for osmolytes, alternative energy, hormones, and key secondary metabolites (Rai, 2002; Araújo et al., 2010;Tzin and Galili, 2010; Angelovici et al., 2011). Studies of both developing and germinating seeds also have implicated FAAs in proper seed development and germination (Angelovici et al., 2010;Galili and Amir, 2013). Still, the genetic control of FAA metabolism in seed remains poorly understood. One reason is that FAA metabolism is tightly intertwined with essential cellular processes in plants, and manipulating FAA levels can have strong deleterious, pleiotropic effects on the entire system (Guyer et al., 1995;Galili, 2011;Ingle, 2011;Pratelli and Pilot, 2014). For example, increasing Lys and Thr inhibits the activity of Asp kinase via a feedback inhibition loop (Clark and Lu, 2015). At high concentrations, this inhibition can lead to starvation of a downstream metabolic product, Met, which inhibits plant growth (Bright et al., 1982;Rognes et al., 1983;Heremans and Jacobs, 1995). In addition, several amino acid catabolic products, such as those emanating from branched-chain amino acids (BCAAs) or Lys degradation pathways, can be chan...