Heterologous Overexpression of Arabidopsis cel1 Enhances Grain Yield, Biomass and Early Maturity in Setaria viridis.

Heterologous overexpression of Arabidopsis cellulase 1 (Atcel1) results in enhanced yield, early maturity, and increased biomass in dicotyledonous species like poplar and eucalyptus but has not been demonstrated in monocots. We produced transgenic Setaria viridis accession A10.1 plants overexpressing a monocotyledonous codon optimized (MCO) Atcel1. Agronomic characterization of the transgenic events showed that heterologous overexpression of MCOAtcel1 caused enhanced grain yield, shoot biomass, and accelerated maturation rate in the model grass species S. viridis under growth chamber conditions. The agronomic trait differences observed were consistent with previous reports in dicots but are here described in a monocot species and associated with increased seed yield. Overexpression of Atcel1 in S. viridis was shown to increase the number of panicles and seeds by 24-30%, enhance overall grain yield by up to 26%, and lead to a shoot dry biomass increase of 16-19%. Overexpression also reduced time to plant maturation and senescence by 12.5%. Our findings in S. viridis suggest that manipulation of Atcel1 has potential for developing early-maturing and higher-yielding monocotyledonous biomass crops suitable for climate-smart agriculture.

Interaction Between Induced and Natural Variation at oil yellow1 Delays Reproductive Maturity in Maize.

We previously demonstrated that maize (Zea mays) locus very oil yellow1 (vey1) encodes a putative cis-regulatory expression polymorphism at the magnesium chelatase subunit I gene (aka oil yellow1) that strongly modifies the chlorophyll content of the semi-dominant Oy1-N1989 mutants. The vey1 allele of Mo17 inbred line reduces chlorophyll content in the mutants leading to reduced photosynthetic output. Oy1-N1989 mutants in B73 reached reproductive maturity four days later than wild-type siblings. Enhancement of Oy1-N1989 by the Mo17 allele at the vey1 QTL delayed maturity further, resulting in detection of a flowering time QTL in two bi-parental mapping populations crossed to Oy1-N1989 The near isogenic lines of B73 harboring the vey1 allele from Mo17 delayed flowering of Oy1-N1989 mutants by twelve days. Just as previously observed for chlorophyll content, vey1 had no effect on reproductive maturity in the absence of the Oy1-N1989 allele. Loss of chlorophyll biosynthesis in Oy1-N1989 mutants and enhancement by vey1 reduced CO2 assimilation. We attempted to separate the effects of photosynthesis on the induction of flowering from a possible impact of chlorophyll metabolites and retrograde signaling by manually reducing leaf area. Removal of leaves, independent of the Oy1-N1989 mutant, delayed flowering but surprisingly reduced chlorophyll contents of emerging leaves. Thus, defoliation did not completely separate the identity of the signal(s) that regulates flowering time from changes in chlorophyll content in the foliage. These findings illustrate the necessity to explore the linkage between metabolism and the mechanisms that connect it to flowering time regulation.

A Very Oil Yellow1 Modifier of the Oil Yellow1-N1989 Allele Uncovers a Cryptic Phenotypic Impact of Cis-regulatory Variation in Maize.

Forward genetics determines the function of genes underlying trait variation by identifying the change in DNA responsible for changes in phenotype. Detecting phenotypically-relevant variation outside protein coding sequences and distinguishing this from neutral variants is not trivial; partly because the mechanisms by which DNA polymorphisms in the intergenic regions affect gene regulation are poorly understood. Here we utilized a dominant genetic reporter to investigate the effect of cis and trans-acting regulatory variation. We performed a forward genetic screen for natural variation that suppressed or enhanced the semi-dominant mutant allele Oy1-N1989, encoding the magnesium chelatase subunit I of maize. This mutant permits rapid phenotyping of leaf color as a reporter for chlorophyll accumulation, and mapping of natural variation in maize affecting chlorophyll metabolism. We identified a single modifier locus segregating between B73 and Mo17 that was linked to the reporter gene itself, which we call very oil yellow1 (vey1). Based on the variation in OY1 transcript abundance and genome-wide association data, vey1 is predicted to consist of multiple cis-acting regulatory sequence polymorphisms encoded at the wild-type oy1 alleles. The vey1 locus appears to be a common polymorphism in the maize germplasm that alters the expression level of a key gene in chlorophyll biosynthesis. These vey1 alleles have no discernable impact on leaf chlorophyll in the absence of the Oy1-N1989 reporter. Thus, the use of a mutant as a reporter for magnesium chelatase activity resulted in the detection of expression-level polymorphisms not readily visible in the laboratory.

A genome-wide association study of the maize hypersensitive defense response identifies genes that cluster in related pathways.

Much remains unknown of molecular events controlling the plant hypersensitive defense response (HR), a rapid localized cell death that limits pathogen spread and is mediated by resistance (R-) genes. Genetic control of the HR is hard to quantify due to its microscopic and rapid nature. Natural modifiers of the ectopic HR phenotype induced by an aberrant auto-active R-gene (Rp1-D21), were mapped in a population of 3,381 recombinant inbred lines from the maize nested association mapping population. Joint linkage analysis was conducted to identify 32 additive but no epistatic quantitative trait loci (QTL) using a linkage map based on more than 7000 single nucleotide polymorphisms (SNPs). Genome-wide association (GWA) analysis of 26.5 million SNPs was conducted after adjusting for background QTL. GWA identified associated SNPs that colocalized with 44 candidate genes. Thirty-six of these genes colocalized within 23 of the 32 QTL identified by joint linkage analysis. The candidate genes included genes predicted to be in involved programmed cell death, defense response, ubiquitination, redox homeostasis, autophagy, calcium signalling, lignin biosynthesis and cell wall modification. Twelve of the candidate genes showed significant differential expression between isogenic lines differing for the presence of Rp1-D21. Low but significant correlations between HR-related traits and several previously-measured disease resistance traits suggested that the genetic control of these traits was substantially, though not entirely, independent. This study provides the first system-wide analysis of natural variation that modulates the HR response in plants.

A connected set of genes associated with programmed cell death implicated in controlling the hypersensitive response in maize.

Rp1-D21 is a maize auto-active resistance gene conferring a spontaneous hypersensitive response (HR) of variable severity depending on genetic background. We report an association mapping strategy based on the Mutant Assisted Gene Identification and Characterization approach to identify naturally occurring allelic variants associated with phenotypic variation in HR. Each member of a collection of 231 diverse inbred lines of maize constituting a high-resolution association mapping panel were crossed to a parental stock heterozygous for Rp1-D21, and the segregating F(1) generation testcrosses were evaluated for phenotypes associated with lesion severity for 2 years at two locations. A genome-wide scan for associations with HR was conducted with 47,445 SNPs using a linear mixed model that controlled for spurious associations due to population structure. Since the ability to identify candidate genes and the resolution of association mapping are highly influenced by linkage disequilibrium (LD), we examined the extent of genome-wide LD. On average, marker pairs separated by >10 kbp had an r(2) value of <0.1. Genomic regions surrounding SNPs significantly associated with HR traits were locally saturated with additional SNP markers to establish local LD structure and precisely identify candidate genes. Six significantly associated SNPs at five loci were detected. At each locus, the associated SNP was located within or immediately adjacent to candidate causative genes predicted to play significant roles in the control of programmed cell death and especially in ubiquitin pathway-related processes.