Current challenges and future of agricultural genomes to phenomes in the USA.

Dramatic improvements in measuring genetic variation across agriculturally relevant populations (genomics) must be matched by improvements in identifying and measuring relevant trait variation in such populations across many environments (phenomics). Identifying the most critical opportunities and challenges in genome to phenome (G2P) research is the focus of this paper. Previously (Genome Biol, 23(1):1-11, 2022), we laid out how Agricultural Genome to Phenome Initiative (AG2PI) will coordinate activities with USA federal government agencies expand public-private partnerships, and engage with external stakeholders to achieve a shared vision of future the AG2PI. Acting on this latter step, AG2PI organized the "Thinking Big: Visualizing the Future of AG2PI" two-day workshop held September 9-10, 2022, in Ames, Iowa, co-hosted with the United State Department of Agriculture's National Institute of Food and Agriculture (USDA NIFA). During the meeting, attendees were asked to use their experience and curiosity to review the current status of agricultural genome to phenome (AG2P) work and envision the future of the AG2P field. The topic summaries composing this paper are distilled from two 1.5-h small group discussions. Challenges and solutions identified across multiple topics at the workshop were explored. We end our discussion with a vision for the future of agricultural progress, identifying two areas of innovation needed: (1) innovate in genetic improvement methods development and evaluation and (2) innovate in agricultural research processes to solve societal problems. To address these needs, we then provide six specific goals that we recommend be implemented immediately in support of advancing AG2P research.

Embryo-specific silencing of a transporter reduces phytic acid content of maize and soybean seeds.

Phytic acid in cereal grains and oilseeds is poorly digested by monogastric animals and negatively affects animal nutrition and the environment. However, breeding programs involving mutants with less phytic acid and more inorganic phosphate (P(i)) have been frustrated by undesirable agronomic characteristics associated with the phytic acid-reducing mutations. We show that maize lpa1 mutants are defective in a multidrug resistance-associated protein (MRP) ATP-binding cassette (ABC) transporter that is expressed most highly in embryos, but also in immature endosperm, germinating seed and vegetative tissues. Silencing expression of this transporter in an embryo-specific manner produced low-phytic-acid, high-Pi transgenic maize seeds that germinate normally and do not show any significant reduction in seed dry weight. This dominant transgenic approach obviates the need for incorporating recessive lpa1 mutations to create maize hybrids with reduced phytic acid. Suppressing the homologous soybean MRP gene also generated low-phytic-acid seed, suggesting that the strategy might be feasible for many crops.

The maize low-phytic acid 3 encodes a myo-inositol kinase that plays a role in phytic acid biosynthesis in developing seeds.

Phytic acid, myo-inositol-1,2,3,4,5,6-hexakisphosphate or Ins P6, is the most abundant myo-inositol phosphate in plant cells, but its biosynthesis is poorly understood. Also uncertain is the role of myo-inositol as a precursor of phytic acid biosynthesis. We identified a low-phytic acid mutant, lpa3, in maize. The Mu-insertion mutant has a phenotype of reduced phytic acid, increased myo-inositol and lacks significant amounts of myo-inositol phosphate intermediates in seeds. The gene responsible for the mutation encodes a myo-inositol kinase (MIK). Maize MIK protein contains conserved amino acid residues found in pfkB carbohydrate kinases. The maize lpa3 gene is expressed in developing embryos, where phytic acid is actively synthesized and accumulates to a large amount. Characterization of the lpa3 mutant provides direct evidence for the role of myo-inositol and MIK in phytic acid biosynthesis in developing seeds. Recombinant maize MIK phosphorylates myo-inositol to produce multiple myo-inositol monophosphates, Ins1/3P, Ins4/6P and possibly Ins5P. The characteristics of the lpa3 mutant and MIK suggest that MIK is not a salvage enzyme for myo-inositol recycling and that there are multiple phosphorylation routes to phytic acid in developing seeds. Analysis of the lpa2/lpa3 double mutant implies interactions between the phosphorylation routes.

The maize low-phytic acid mutant lpa2 is caused by mutation in an inositol phosphate kinase gene.

Reduced phytic acid content in seeds is a desired goal for genetic improvement in several crops. Low-phytic acid mutants have been used in genetic breeding, but it is not known what genes are responsible for the low-phytic acid phenotype. Using a reverse genetics approach, we found that the maize (Zea mays) low-phytic acid lpa2 mutant is caused by mutation in an inositol phosphate kinase gene. The maize inositol phosphate kinase (ZmIpk) gene was identified through sequence comparison with human and Arabidopsis Ins(1,3,4)P(3) 5/6-kinase genes. The purified recombinant ZmIpk protein has kinase activity on several inositol polyphosphates, including Ins(1,3,4)P(3), Ins(3,5,6)P(3), Ins(3,4,5,6)P(4), and Ins(1,2,5,6)P(4). The ZmIpk mRNA is expressed in the embryo, the organ where phytic acid accumulates in maize seeds. The ZmIpk Mutator insertion mutants were identified from a Mutator F(2) family. In the ZmIpk Mu insertion mutants, seed phytic acid content is reduced approximately 30%, and inorganic phosphate is increased about 3-fold. The mutants also accumulate myo-inositol and inositol phosphates as in the lpa2 mutant. Allelic tests showed that the ZmIpk Mu insertion mutants are allelic to the lpa2. Southern-blot analysis, cloning, and sequencing of the ZmIpk gene from lpa2 revealed that the lpa2-1 allele is caused by the genomic sequence rearrangement in the ZmIpk locus and the lpa2-2 allele has a nucleotide mutation that generated a stop codon in the N-terminal region of the ZmIpk open reading frame. These results provide evidence that ZmIpk is one of the kinases responsible for phytic acid biosynthesis in developing maize seeds.