Jinrui Shi

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 (Pi) 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 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 acidlpa2 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)P3 5/6-kinase genes. The purified recombinant ZmIpk protein has kinase activity on several inositol polyphosphates, including Ins(1,3,4)P3, Ins(3,5,6)P3, Ins(3,4,5,6)P4, and Ins(1,2,5,6)P4. 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 F2family. 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 accumulatemyo-inositol and inositol phosphates as in thelpa2 mutant. Allelic tests showed that the ZmIpkMu insertion mutants are allelic to thelpa2. Southern-blot analysis, cloning, and sequencing of the ZmIpk gene from lpa2 revealed that thelpa2-1 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.

ARGOS8 variants generated by CRISPR‐Cas9 improve maize grain yield under field drought stress conditions

Summary Maize 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.

Overexpression of ARGOS Genes Modifies Plant Sensitivity to Ethylene, Leading to Improved Drought Tolerance in Both Arabidopsis and Maize

Reducing ethylene sensitivity by modifying the expression of a negative regulator of ethylene signal transduction improves grain yield in maize under drought stress environments. Lack of sufficient water is a major limiting factor to crop production worldwide, and the development of drought-tolerant germplasm is needed to improve crop productivity. The phytohormone ethylene modulates plant growth and development as well as plant response to abiotic stress. Recent research has shown that modifying ethylene biosynthesis and signaling can enhance plant drought tolerance. Here, we report novel negative regulators of ethylene signal transduction in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). These regulators are encoded by the ARGOS gene family. In Arabidopsis, overexpression of maize ARGOS1 (ZmARGOS1), ZmARGOS8, Arabidopsis ARGOS homolog ORGAN SIZE RELATED1 (AtOSR1), and AtOSR2 reduced plant sensitivity to ethylene, leading to enhanced drought tolerance. RNA profiling and genetic analysis suggested that the ZmARGOS1 transgene acts between an ethylene receptor and CONSTITUTIVE TRIPLE RESPONSE1 in the ethylene signaling pathway, affecting ethylene perception or the early stages of ethylene signaling. Overexpressed ZmARGOS1 is localized to the endoplasmic reticulum and Golgi membrane, where the ethylene receptors and the ethylene signaling protein ETHYLENE-INSENSITIVE2 and REVERSION-TO-ETHYLENE SENSITIVITY1 reside. In transgenic maize plants, overexpression of ARGOS genes also reduces ethylene sensitivity. Moreover, field testing showed that UBIQUITIN1:ZmARGOS8 maize events had a greater grain yield than nontransgenic controls under both drought stress and well-watered conditions.

Maize and Arabidopsis ARGOS proteins interact with ethylene receptor signaling complex, supporting a regulatory role for ARGOS in ethylene signal transduction

ARGOS proteins regulate ethylene signal transduction via protein-protein interactions. The phytohormone ethylene regulates plant growth and development as well as plant response to environmental cues. ARGOS genes reduce plant sensitivity to ethylene when overexpressed in transgenic Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). A previous genetic study suggested that the endoplasmic reticulum and Golgi-localized maize ARGOS1 targets the ethylene signal transduction components at or upstream of CONSTITUTIVE TRIPLE RESPONSE1, but the mechanism of ARGOS modulating ethylene signaling is unknown. Here, we demonstrate in Arabidopsis that ZmARGOS1, as well as the Arabidopsis ARGOS homolog ORGAN SIZE RELATED1, physically interacts with Arabidopsis REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1), an ethylene receptor interacting protein that regulates the activity of ETHYLENE RESPONSE1. The protein-protein interaction was also detected with the yeast split-ubiquitin two-hybrid system. Using the same yeast assay, we found that maize RTE1 homolog REVERSION-TO-ETHYLENE SENSITIVITY1 LIKE4 (ZmRTL4) and ZmRTL2 also interact with maize and Arabidopsis ARGOS proteins. Like AtRTE1 in Arabidopsis, ZmRTL4 and ZmRTL2 reduce ethylene responses when overexpressed in maize, indicating a similar mechanism for ARGOS regulating ethylene signaling in maize. A polypeptide fragment derived from ZmARGOS8, consisting of a Pro-rich motif flanked by two transmembrane helices that are conserved among members of the ARGOS family, can interact with AtRTE1 and maize RTL proteins in Arabidopsis. The conserved domain is necessary and sufficient to reduce ethylene sensitivity in Arabidopsis and maize. Overall, these results suggest a physical association between ARGOS and the ethylene receptor signaling complex via AtRTE1 and maize RTL proteins, supporting a role for ARGOS in regulating ethylene perception and the early steps of signal transduction in Arabidopsis and maize.

Ectopic expression of ARGOS8 reveals a role for ethylene in root lodging resistance in maize

Summary Ethylene plays a critical role in many diverse processes in plant development. Recent studies have demonstrated that overexpression of the maize ARGOS8 gene reduces the plants response to ethylene by decreasing ethylene signaling and enhances grain yield in transgenic maize plants. The objective of this study was to determine the effects of ethylene on the development of nodal roots, which are primarily responsible for root‐lodging resistance in maize. Exogenous application of the ethylene precursor 1‐aminocyclopropane‐1‐carboxylic acid (ACC) was found to promote the emergence of nodal roots. Transcriptome analysis of nodal tissues revealed that the expression of genes involved in metabolic processes and cell wall biogenesis was upregulated in response to ACC treatment, supporting the notion that ethylene is a positive regulator for the outgrowth of young root primordia. In BSV::ARGOS8 transgenic plants with reduced ethylene sensitivity due to constitutive overexpression of ARGOS8, nodal root emergence was delayed and the promotional effect of ACC on nodal root emergence decreased. Field tests showed that the BSV::ARGOS8 plants had higher root lodging relative to non‐transgenic controls. When ARGOS8 expression was controlled by the developmentally regulated promoter FTM1, which conferred ARGOS8 overexpression in adult plants but not in the nodal roots and nodes in juvenile plants, the FTM1::ARGOS8 plants had no significant difference in root lodging compared with the wild type but produced a higher grain yield. These results suggest that ethylene has a role in promoting nodal root emergence and that a delay in nodal root development has a negative effect on root‐lodging resistance in maize.