Bruce J. Drummond

The myb-homologous P gene controls phlobaphene pigmentation in maize floral organs by directly activating a flavonoid biosynthetic gene subset

The maize P gene, which specifies red pigmentation of the kernel pericarp, cob, and other floral organs, has been an important model since the early days of modern genetics. Here we show that P encodes a Myb homolog that recognizes the sequence CCT/AACC, in sharp contrast with the C/TAACGG bound by vertebrate Myb proteins. P binds to and activates transcription of the A1 gene required for 3-deoxy flavonoid and phlobaphene biosynthesis, but not the Bz1 gene required for anthocyanin biosynthesis. The maize C1 gene, which also encodes a Myb homolog, activates both the A1 and Bz1 genes, but only in the presence of a basic-helix-loop-helix coactivator encoded by the maize genes R or B. These results indicate that Myb homologs can differentially regulate gene expression by binding different DNA sequences, through combinatorial interactions with other factors, or both.

Transgenic tobacco plants and their progeny derived by microprojectile bombardment of tobacco leaves.

Transgenic tobacco plants and progeny carrying coding sequences for neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) were recovered following microprojectile bombardment of tobacco leaves. Transgenic plants were regenerated from bombarded leaf pieces of tobacco cvs. ‘Xanthi’ and ‘Ky 17’ which were cultured in the presence of 100 or 200 μg/ml kanamycin for six to eight weeks. Among 160 putative transgenic plants from at least 16 independent transformation events 76% expressed NPTII, and 50% expressed GUS. Southern analysis of plants expressing either one or both of the enzymes indicated DNA in high molecular weight DNA in 8 of 9 independent transformants analyzed. Two independent transformants and their progeny were analyzed in detail. Analysis of progeny for quantitative enzyme levels of NPTII and GUS, and Southern analysis of parents and progeny clearly demonstrated that the genes were transmitted to progeny. One transformant demonstrated Mendelian ratios for seed germination on kanamycin-containing medium while the other transformant had non-Mendelian ratios. DNA analysis of progeny indicate complex integration of the plasmid DNA, and suggest that rearrangements of this DNA has occurred. These results are consistent with other methods of direct DNA uptake into cells, and verify that the microprojectile bombardment method is capable of DNA delivery into intact plant cells which can give rise to transgenic plants and progeny.

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.

Gene Transfer Mediated by Site-Specific Recombination Systems

Stable genetic transformation depends on DNA recombination, a process defined as a physical exchange of genetic information between interacting DNA molecules. DNA recombination reactions are conveniently divided into three categories: homologous recombination, site-specific recombination, and all others pejoratively described as illegitimate recombination. Current plant transformation techniques primarily rely on the least understood mechanisms, that is, illegitimate recombination. As a result, genetic transformations are unpredictable with respect to the efficiency of transformation and reliability of transgene expression. The use of homologous recombination in DNA integration processes (described as gene targeting) is still a very inefficient process despite substantial ongoing research efforts to develop such techniques both for animal and plant applications [1, 2], Site-specific recombination may bridge the gap between random, unpredictable illegitimate integration and future fully controllable genomic DNA manipulations based on homologous recombination or other mechanisms. There is already available evidence that site-specific recombination can increase the efficiency of genetic transformation, improve its reliability, and reduce the extent of modifications imposed on genomic DNA during the transformation process.

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.