SummaryMaize ARGOS8 is a negative regulator of ethylene responses. A previous study has shown that transgenic plants constitutively overexpressing ARGOS8 have reduced ethylene sensitivity and improved grain yield under drought stress conditions. To explore the targeted use of ARGOS8 native expression variation in drought‐tolerant breeding, a diverse set of over 400 maize inbreds was examined for ARGOS8 mRNA expression, but the expression levels in all lines were less than that created in the original ARGOS8 transgenic events. We then employed a CRISPR‐Cas‐enabled advanced breeding technology to generate novel variants of ARGOS8. The native maize GOS2 promoter, which confers a moderate level of constitutive expression, was inserted into the 5′‐untranslated region of the native ARGOS8 gene or was used to replace the native promoter of ARGOS8. Precise genomic DNA modification at the ARGOS8 locus was verified by PCR and sequencing. The ARGOS8 variants had elevated levels of ARGOS8 transcripts relative to the native allele and these transcripts were detectable in all the tissues tested, which was the expected results using the GOS2 promoter. A field study showed that compared to the WT, the ARGOS8 variants increased grain yield by five bushels per acre under flowering stress conditions and had no yield loss under well‐watered conditions. These results demonstrate the utility of the CRISPR‐Cas9 system in generating novel allelic variation for breeding drought‐tolerant crops.

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.

Members of the Arabidopsis calcineurin B-like Ca 2 ؉ binding protein (AtCBL) family are differentially regulated by stress conditions. One AtCBL plays a role in salt stress; another is implicated in response to other stress signals, including drought, cold, and wounding. In this study, we identified a group of novel protein kinases specifically associated with AtCBL-type Ca 2 ؉ sensors. In addition to a typical protein kinase domain, they all contain a unique C-terminal region that is both required and sufficient for interaction with the AtCBL-type but not calmodulin-type Ca 2 ؉ binding proteins from plants. Interactions between the kinases and AtCBLs require micromolar concentrations of Ca 2 ؉ , suggesting that increases in cellular Ca 2 ؉ concentrations may trigger the formation of AtCBL-kinase complexes in vivo. Unlike most serine/threonine kinases, the AtCBL-interacting kinase efficiently uses Mn 2 ؉ to Mg 2 ؉ as a cofactor and may function as a Mn 2 ؉ binding protein in the cell. These findings link a new type of Ca 2 ؉ sensors to a group of novel protein kinases, providing the molecular basis for a unique Ca 2 ؉ signaling machinery in plant cells. INTRODUCTIONAmong the extracellular signals eliciting changes in Ca 2 ϩ concentration in the cytoplasm of plant cells are plant hormones, light, stress factors, and pathogenic or symbiotic elicitors (Knight et al., 1991(Knight et al., , 1996(Knight et al., , 1997Neuhaus et al., 1993; Trewavas and Knight, 1994; Ehrhardt et al., 1996;McAinsh et al., 1997; Wu et al., 1997). In addition, many intrinsic growth and developmental processes, such as elongation of the root hair and pollen tube, are accompanied by Ca 2 ϩ transients (Franklin-Tong et al., 1996; Felle and Hepler, 1997; Holdaway-Clarke et al., 1997; Wymer et al., 1997). Because different signals often elicit distinct and specific cellular responses, an interesting question is how do cells distinguish between the Ca 2 ϩ signals produced by different stimuli?Studies with both animal and plant cells suggest that a Ca 2 ϩ signal is represented not only by Ca 2 ϩ concentration but also by spatial and temporal information, including Ca 2 ϩ localization and oscillation (Franklin-Tong et al., 1996; Holdaway-Clarke et al., 1997; Dolmetsch et al., 1998;Li et al., 1998). Although such complexity in Ca 2 ϩ parameters may partially explain the specificity of cellular responses triggered by a particular stimulus, the signaling components that "sense" and "interpret" the Ca 2 ϩ signals hold the key to linking the changes in these parameters to specific cellular responses.If Ca 2 ϩ signaling pathways constitute "molecular relays," the first "runner" after Ca 2 ϩ should be a component that serves as the Ca 2 ϩ "sensor" to monitor changes in Ca 2 ϩ parameters. Such sensors often are proteins that bind Ca 2 ϩ and, in so doing, change conformation in a Ca 2 ϩ -dependent manner. Several families of Ca 2 ϩ sensors have been identified in higher plants. Perhaps the best known is the family of calmodulin (CaM) and CaM-related prot...

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.Phytic acid, myo-inositol 1,2,3,4,5,6-hexakisphosphate, is an abundant component of plant seeds and is deposited in protein bodies as a mixed salt of mineral cations, such as K ϩ , Mg 2ϩ , Ca 2ϩ , Zn 2ϩ , and Fe 3ϩ . Typically, 50% to 80% of the phosphorus in seeds is found in this compound. Phytic acid serves as a major storage form for myo-inositol, phosphorus, and mineral cations for use during seedling growth. The other known role of phytic acid is the control of inorganic phosphate (Pi) levels in both developing seeds and seedlings (Strother, 1980). In maize (Zea mays) kernels, nearly 90% of the phytic acid is accumulated in embryo and about 10% in aleurone layers. Maize endosperm contains only trace amount of phytic acid (O'Dell et al., 1972). In rice (Oryza sativa), barley (Hordeum vulgare), and wheat (Triticum aestivum), most of the phytic acid (approximately 90%) is found in the aleurone layers and only about 10% in embryo. Reduced phytic acid content in seeds is a desired goal for genetic improvement in several crops, including maize, rice, barley, wheat, and soybean (Glycine max). Because monogastric animals digest phytic acid poorly, animal feed is supplemented with Pi to meet the phosphorus requirement for animal growth. Undigested phytic acid is eliminated and is a leading phosphorus pollution source (Cromwell and Coffey, 1991). Although phytic acid as an antioxidant is sugges...

SummaryPhytic acid, myo-inositol-1,2,3,4,5,6-hexakis phosphate or Ins P 6 , 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, Ins(1/3)P, Ins(4/6)P and possibly Ins(5)P. 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.