A Novel Soybean Diacylglycerol Acyltransferase 1b Variant with Three Amino Acid Substitutions Increases Seed Oil Content.

Improving soybean (Glycine max) seed composition by increasing the protein and oil components will add significant value to the crop and enhance environmental sustainability. Diacylglycerol acyltransferase (DGAT) catalyzes the final rate-limiting step in triacylglycerol (TAG) biosynthesis and has a major impact on seed oil accumulation. We previously identified a soybean DGAT1b variant with 14 amino acid substitutions (GmDGAT1b-MOD) that increases total oil content by 3 percentage points when overexpressed in soybean seeds. In the present study, additional GmDGAT1b variants were generated to further increase oil with a reduced number of substitutions. Variants with one to four amino acid substitutions were screened in the model systems S. cerevisiae and transient N. benthamiana leaf. Promising GmDGAT1b variants resulting in high oil accumulation in the model systems were selected for over-expression in soybeans. One GmDGAT1b variant with three novel amino acid substitutions (GmDGAT1b-3aa) increased total soybean oil to levels near the previously discovered GmDGAT1b-MOD variant. In a multiple location field trial, GmDGAT1b-3aa transgenic events had significantly increased oil and protein by up to 2.3 and 0.6 percentage points, respectively. Modeling of the GmDGAT1b-3aa protein structure provided insights into the potential function of the three substitutions. These findings will guide efforts to improve soybean oil content and overall seed composition by CRISPR editing.

Overexpression of zmm28 increases maize grain yield in the field.

Increasing maize grain yield has been a major focus of both plant breeding and genetic engineering to meet the global demand for food, feed, and industrial uses. We report that increasing and extending expression of a maize MADS-box transcription factor gene, zmm28, under the control of a moderate-constitutive maize promoter, results in maize plants with increased plant growth, photosynthesis capacity, and nitrogen utilization. Molecular and biochemical characterization of zmm28 transgenic plants demonstrated that their enhanced agronomic traits are associated with elevated plant carbon assimilation, nitrogen utilization, and plant growth. Overall, these positive attributes are associated with a significant increase in grain yield relative to wild-type controls that is consistent across years, environments, and elite germplasm backgrounds.

A single point mutation in Ms44 results in dominant male sterility and improves nitrogen use efficiency in maize.

Application of nitrogen fertilizer in the past 50 years has resulted in significant increases in crop yields. However, loss of nitrogen from crop fields has been associated with negative impacts on the environment. Developing maize hybrids with improved nitrogen use efficiency is a cost-effective strategy for increasing yield sustainably. We report that a dominant male-sterile mutant Ms44 encodes a lipid transfer protein which is expressed specifically in the tapetum. A single amino acid change from alanine to threonine at the signal peptide cleavage site of the Ms44 protein abolished protein processing and impeded the secretion of protein from tapetal cells into the locule, resulting in dominant male sterility. While the total nitrogen (N) content in plants was not changed, Ms44 male-sterile plants reduced tassel growth and improved ear growth by partitioning more nitrogen to the ear, resulting in a 9.6% increase in kernel number. Hybrids carrying the Ms44 allele demonstrated a 4%-8.5% yield advantage when N is limiting, 1.7% yield advantage under drought and 0.9% yield advantage under optimal growth conditions relative to the yield of wild type. Furthermore, we have developed an Ms44 maintainer line for fertility restoration, male-sterile inbred seed increase and hybrid seed production. This study reveals that protein secretion from the tapetum into the locule is critical for pollen development and demonstrates that a reduction in competition between tassel and ear by male sterility improves grain yield under low-nitrogen conditions in maize.

Transcriptional silencing of heterologous anther promoters in maize: a genetic method to replace detasseling for seed production.

The promoter of the maize male fertility gene ZmMs45, and other anther-specific maize promoters, was previously shown to be transcriptionally silenced by constitutively expressed promoter-inverted repeat RNAs (pIRs). In addition, ZmMS45pIR-mediated male sterility was reversed by co-expression of Ms45 transcribed by promoters not targeted by pIR RNA silencing. In this report, male fertility was restored to ms45 maize by fusing non-maize inflorescence promoters to the ZmMS45 coding region. This complementation assay also established that these rice or Arabidopsis promoters, when expressed as pIRs, functioned to silence sequence identical promoters. These observations were exploited to develop a genetic method to replace maize detasseling during hybrid seed production. In this system, the ZmMS45 coding region was fused to one of two dissimilar non-maize promoters to generate paired sets of ms45 recessive inbred parents which could be self-pollinated and maintained independently. Linked to each unique Ms45 gene was a non-maize pIR which targeted the promoter transcribing the Ms45 copy contained in the paired inbred parent plant. A cross of these pairs brings the dissimilar pIR cassettes together and resulted in silencing both transformed copies of Ms45. The net result uncovers the ms45 allele carried by the inbreds yielding male sterile progeny. The application of heterologous promoters and transcriptional silencing in plants provides an alternative to post-transcriptional gene silencing as a means to restore and silence gene function in plants.

Transcriptional gene silencing as a tool for uncovering gene function in maize.

Transcriptional gene silencing has broad applications for studying gene function in planta. In maize, a large number of genes have been identified as tassel-preferred in their expression pattern, both by traditional genetic methods and by recent high-throughput expression profiling platforms. Approaches using RNA suppression may provide a rapid alternative means to identify genes directly related to pollen development in maize. The male fertility gene Ms45 and several anther-expressed genes of unknown function were used to evaluate the efficacy of generating male-sterile plants by transcriptional gene silencing. A high frequency of male-sterile plants was obtained by constitutively expressing inverted repeats (IR) of the Ms45 promoter. These sterile plants lacked MS45 mRNA due to transcriptional inactivity of the target promoter. Moreover, fertility was restored to these promoter IR-containing plants by expressing the Ms45 coding region using heterologous promoters. Transcriptional silencing of other anther-expressed genes also significantly affected male fertility phenotypes and led to increased methylation of the target promoter DNA sequences. These studies provide evidence of disruption of gene activity in monocots by RNA interference constructs directed against either native or transformed promoter regions. This approach not only enables the correlation of monocot anther-expressed genes with functions that are important for reproduction in maize, but may also provide a tool for studying gene function and identifying regulatory components unique to transcriptional gene control.