Ryan H. Brown

Positive- and negative-acting regulatory elements contribute to the tissue-specific expression of INNER NO OUTER, a YABBY-type transcription factor gene in Arabidopsis

BackgroundThe INNER NO OUTER (INO) gene, which encodes a YABBY-type transcription factor, specifies and promotes the growth of the outer integument of the ovule in Arabidopsis. INO expression is limited to the abaxial cell layer of the developing outer integument of the ovule and is regulated by multiple regions of the INO promoter, including POS9, a positive element that when present in quadruplicate can produce low-level expression in the normal INO pattern.ResultsSignificant redundancy in activity between different regions of the INO promoter is demonstrated. For specific regulatory elements, multimerization or the addition of the cauliflower mosaic virus 35S general enhancer was able to activate expression of reporter gene constructs that were otherwise incapable of expression on their own. A new promoter element, POS6, is defined and is shown to include sufficient positive regulatory information to reproduce the endogenous pattern of expression in ovules, but other promoter regions are necessary to fully suppress expression outside of ovules. The full-length INO promoter, but not any of the INO promoter deletions tested, is able to act as an enhancer-blocking insulator to prevent the ectopic activation of expression by the 35S enhancer. Sequence conservation between the promoter regions of Arabidopsis thaliana, Brassica oleracea and Brassica rapa aligns closely with the functional definition of the POS6 and POS9 regions, and with a defined INO minimal promoter. The B. oleracea INO promoter is sufficient to promote a similar pattern and level of reporter gene expression in Arabidopsis to that observed for the Arabidopsis promoter.ConclusionsAt least two independent regions of the INO promoter contain sufficient regulatory information to direct the specific pattern but not the level of INO gene expression. These regulatory regions act in a partially redundant manner to promote the expression in a specific pattern in the ovule and suppress expression outside of ovules. Establishment of this pattern requires cooperation and competition between multiple positive and negative regulatory elements.

Quantitative trait loci of barley malting quality trait components in the Stellar/01Ab8219 mapping population

Malting barley is of high economic and scientific importance. Determining barley grains that are suitable for malting involves measuring malting quality, which is an expensive and complex process. In order to decrease the cost of phenotyping and accelerate the process of developing superior malting barley cultivars, markers for marker-assisted breeding are needed. In this study, we identified quantitative trait loci (QTLs) for malting traits in a Stellar/01Ab8219 F6:8 recombinant inbred line population grown at Aberdeen and Tetonia, Idaho, USA in 2009 and 2010. We identified QTLs associated with malt extract (ME), wort protein, soluble/total protein (S/T), diastatic power (DP), alpha-amylase, beta-glucan (BG) and free amino nitrogen (FAN) at a logarithm of odds score ≥2.5 using a high-density genetic map produced by merging Diversity Arrays Technology markers with the current single nucleotide polymorphism map. Novel QTLs were identified for DP and FAN on chromosome 5H, S/T on 6H, and BG and ME on 7H. Dissection of the genetic regions associated with malting traits suggests the involvement of multiple molecular pathways. The resulting molecular markers may prove useful for barley improvement.

Expression of ovule and integument-associated genes in reduced ovules of Santalales.

SUMMARY Santalales comprise mainly parasitic plants including mistletoes and sandalwoods. Bitegmic ovules similar to those found in most other angiosperms are seen in many members of the order, but other members exhibit evolutionary reductions to the unitegmic and ategmic conditions. In some mistletoes, extreme reduction has resulted in the absence of emergent ovules such that embryo sacs appear to remain embedded in placental tissues. Three santalalean representatives (Comandra, Santalum, and Phoradendron), displaying unitegmic, and ategmic ovules, were studied. Observed ovule morphologies were consistent with published reports, including Phoradendron serotinum, which we interpret as having reduced ategmic ovules, consistent with earlier reports on this species. For further understanding of the nature of the ovule reductions we isolated orthologs of the Arabidopsis genes AINTEGUMENTA (ANT) and BELL1 (BEL1), which are associated with ovule development in this species. We observed ovular expression of ANT and BEL1 in patterns largely resembling those seen in the integumented ovules of Arabidopsis. These genes were found to be expressed in the integument of unitegmic ovules and in the surface layers of ategmic ovules, and in some cases, expression of BEL1 was also observed in the surrounding carpel tissue. We hypothesize that ategmic ovules derive from a fusion of the integuments with the nucellus or that the nucellus has taken on some of the characteristics confined to integuments in ancestral species.

Conservation of the role of INNER NO OUTER in development of unitegmic ovules of the Solanaceae despite a divergence in protein function

BackgroundThe INNER NO OUTER (INO) gene is expressed in the outermost cell layer of the outer integument of bitegmic ovules and is essential for this organ’s growth. The role and cross-species functional conservation of INO orthologs were examined in members of the Solanaceae, which have unitegmic ovules. Unitegmy has evolved several times in disparate angiosperm lineages. INO expression has been observed in the outermost cell layers of all examined unitegmic ovules, but the functional role of INO in unitegmic ovules has not previously been evaluated.ResultsINO orthologs were unambiguously identified in tobacco and tomato by sequence homology. Expression of the tomato INO gene was limited to the outer cell layer of the single integument indicating that this single integument has properties of the outer integument. Expression occurred only after integument initiation, later than observed in ovules of other examined angiosperms. Virus-induced knock-down of expression of the INO ortholog in tobacco inhibited growth of the outer cell layer of the integument leading to a decrease in both integument extension and curvature of the ovule. The altered ovules closely resemble those of the aberrant testa shape (ats) ino mutant combination in Arabidopsis where we see the effect of the ino mutation on a single fused integument produced by the ats mutation. Despite significant sequence identity and similar expression patterns, the tomato INO coding region was not able to complement the Arabidopsis ino mutant.ConclusionsThe similarity of effects of ino mutations on the unitegmic ovules of tobacco and the fused integuments of the Arabidopsis ats mutant show that: 1) INO orthologs play the same role in promoting integument growth in ovules of tobacco and Arabidopsis; and 2) the unitegmic ovules of tobacco (and hence other solanaceous species) are most likely the result of a congenital fusion of two ancestral integuments. Our results further indicate that INO has a conserved role in growth of the outermost cell layer of integuments. The curvature of solanaceous ovules is driven by unequal growth of the outer layers of the single integument that likely correspond to an ancestral outer integument.

Long-term assessment of transgene behavior in barley: Ds-mediated delivery of bar results in robust, stable, and heritable expression

The utility of transgenic plants for both experimental and practical agronomic purposes is highly dependent on stable, predictable, and heritable expression of the introduced genes. This requirement is frequently unfulfilled, and transgenes often are completely silenced. Studies of transgenic loci have shown that rearrangements of transgenes occur during the integration process, some of which are potent cues that induce silencing. Conversely, intact, single-copy transgenes produced via transposon-mediated gene delivery have shown relatively stable expression, at least in early-generation progeny. To examine the long-term expression stability of a bar expression cassette delivered via Dissociation (Ds)-mediated transposition, we examined qualitative and quantitative expression in barley (Hordeum vulgare L.) populations developed for transposon tagging. Qualitative assessments of herbicide resistance among 106 lines showed bar expression to be stable for at least five generations of advance via self-pollination. Similarly, qualitatively stable expression was observed among 31 near-isogenic lines derived from at least seven backcrosses to the cultivar Garnet. Quantitative RT-PCR measurements of bar expression were conducted for eight near-isogenic lines and their donor parents. The expression of bar was highly correlated in parent and progeny near-isogenic lines, showing high heritability of bar expression. These data demonstrate stable, predictable transgene expression following Ds-mediated delivery.

Unintended consequences: high phosphinothricin acetyltransferase activity related to reduced fitness in barley

Selectable markers used in plant transformation, such as the bar gene, have been chosen for selection efficacy and for the absence of pleiotropic effects. Recent research has suggested that expression of bar in Arabidopsis affects the transcriptome, and in at least one barley cultivar, high bar expression has been associated with severe fitness reductions. However, the observed sensitivity in barley was thought to be an isolated case associated with a specific genetic stock. In this study, fitness reductions were observed in some near-isogenic lines in the background of the cultivar Garnet when the bar locus was homozygous. The fitness of the near-isogenic lines was correlated with the levels of bar mRNA in the donor parents. Previous studies had shown heritability of bar expression in F1 barley hybrids, so these results suggested that Garnet barley is intolerant of high phosphinothricin acetyltransferase activity. Phosphinothricin acetyltransferase activities were determined for eight near-isogenic lines that showed a range of phenotypes (apparently normal to abnormal and nonviable) when homozygous for bar. Fitness was clearly and negatively correlated with phosphinothricin acetyltransferase activity. These results suggest genetic variability for sensitivity to bar expression in barley, and the possibility of negative but non-obvious effects in apparently tolerant cultivars cannot be ruled out. Similar problems may be associated, but not yet detected, with other selectable markers. The potential for pleiotropic effects of selectable markers can be avoided by transformation strategies that enable the elimination of selectable markers.

Students' perspective on genomics: from sample to sequence using the case study of blueberry.

Advances in genomic sequencing technologies in the past decade have revolutionized the field of genomics, resulting in faster and less expensive sequencing. Holding back the potential for innovation, however, is a widespread lack of understanding of genomics and sequencing by the general public. In an attempt to remedy this problem, this paper presents an introduction to the fields of genomics, bioinformatics, and proteomics using the blueberry genome as a model case study of the plant genomics field. The blueberry (Vaccinium sect. Cyanococcus) is often cited as a “super food” in the media due to its nutritional benefits and global economic importance. There have been a number of related genomic publications in the past 20 years; however, a completed genome and a full analysis into the health-related pathways are still needed. As exemplified by this blueberry case study, there are opportunities for future genomic research into numerous beneficial plant species. The solid background presented in this paper provides future researchers the foundation to explore these uncharted areas.