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

BACKGROUND: The 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. RESULTS: INO 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. CONCLUSIONS: The 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.

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

BACKGROUND: The 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. RESULTS: Significant 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. CONCLUSIONS: At 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.

Developing tools for investigating the multiple roles of ethylene: identification and mapping genes for ethylene biosynthesis and reception in barley.

The plant hormone ethylene is important to many plant processes from germination through senescence, including responses to in vitro growth and plant regeneration. Knowledge of the number and function of genes that are involved in ethylene biosynthesis and reception is necessary to determine the role of specific genes within gene families known to influence ethylene biosynthesis and other aspects of ethylene function in plants. Our objective was built on previous studies that have established the critical role of ethylene in the in vitro response of barley (Hordeum vulgare L.), and that have identified ethylene-related QTL in the barley genome. In this study, we have identified the locations of genes in the barley 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS), ACC oxidase (ACO), and ethylene receptor (ETR) gene families. Specific primers for PCR amplification of each gene were developed and used to map these genes in the Oregon Wolf Barley mapping population. Five ACS, 8 ACO, and 7 ETR genes were identified and mapped to six of the barley chromosomes. Gene locations were syntenous to the orthologs in rice except for two that mapped to chromosome 6H. Gene duplication was evident for ACO genes on chromosomes 5H and 6H. Gene-specific primers will be useful for determining expression of each gene under various environmental conditions, including in vitro environments, to better understand the role of ethylene. Of the six known QTL for green plant regeneration in barley, three were located near the genes mapped in this study.

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

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.

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.

Reaching to multiple targets when standing: the spatial organization of feedforward postural adjustments.

We examined the spatial organization of feedforward postural adjustments produced prior to and during voluntary arm reaching movements executed while standing. We sought to investigate whether the activity of postural muscles before and during reaching was directionally tuned and whether a strategy of horizontal force constraint could be observed. To this end, eight human subjects executed self-paced reach-to-point movements on the random illumination of one of 13 light targets placed within a 180 degrees array centered along the midline of the body. Analysis was divided into two periods: a first corresponding to the 250 ms preceding the onset of the reaching movements (termed pPA period) and a second 250-ms period immediately preceding target attainment (the aPA period). For both periods, electromyographic activity of the lower limb muscles revealed a clear directional tuning, with groups of muscles being activated for similar directions of reach. Analysis of horizontal ground reaction forces supported the existence of a force constraint strategy only for the pPA period, however, with those in the aPA period being more widely dispersed. We suggest that the strategy adopted for feedforward pPAs is one where the tuned muscle synergies constrain the forces diagonally away from the center of mass (CoM) to move it within the support base. However, the need to control for final finger and body position for each target during the aPA phase resulted in a distribution of vectors across reaching directions. Overall, our results would support the idea that endpoint limb force during postural tasks depends on the use of functional muscle synergies, which are used to displace the CoM or decelerate the body at the end of the reach.

Mitochondrial GFA2 is required for synergid cell death in Arabidopsis.

Little is known about the molecular processes that govern female gametophyte (FG) development and function, and few FG-expressed genes have been identified. We report the identification and phenotypic analysis of 31 new FG mutants in Arabidopsis. These mutants have defects throughout development, indicating that FG-expressed genes govern essentially every step of FG development. To identify genes involved in cell death during FG development, we analyzed this mutant collection for lines with cell death defects. From this analysis, we identified one mutant, gfa2, with a defect in synergid cell death. Additionally, the gfa2 mutant has a defect in fusion of the polar nuclei. We isolated the GFA2 gene and show that it encodes a J-domain-containing protein. Of the J-domain-containing proteins in Saccharomyces cerevisiae (budding yeast), GFA2 is most similar to Mdj1p, which functions as a chaperone in the mitochondrial matrix. GFA2 is targeted to mitochondria in Arabidopsis and partially complements a yeast mdj1 mutant, suggesting that GFA2 is the Arabidopsis ortholog of yeast Mdj1p. These data suggest a role for mitochondria in cell death in plants.