BackgroundMembers of the cation diffusion facilitator (CDF) family are integral membrane divalent cation transporters that transport metal ions out of the cytoplasm either into the extracellular space or into internal compartments such as the vacuole. The spectrum of cations known to be transported by proteins of the CDF family include Zn, Fe, Co, Cd, and Mn. Members of this family have been identified in prokaryotes, eukaryotes, and archaea, and in sequenced plant genomes. CDF families range in size from nine members in Selaginella moellendorffii to 19 members in Populus trichocarpa. Phylogenetic analysis suggests that the CDF family has expanded within plants, but a definitive plant CDF family phylogeny has not been constructed.ResultsRepresentative CDF members were annotated from diverse genomes across the Viridiplantae and Rhodophyta lineages and used to identify phylogenetic relationships within the CDF family. Bayesian phylogenetic analysis of CDF amino acid sequence data supports organizing land plant CDF family sequences into 7 groups. The origin of the 7 groups predates the emergence of land plants. Among these, 5 of the 7 groups are likely to have originated at the base of the tree of life, and 2 of 7 groups appear to be derived from a duplication event prior to or coincident with land plant evolution. Within land plants, local expansion continues within select groups, while several groups are strictly maintained as one gene copy per genome.ConclusionsDefining the CDF gene family phylogeny contributes to our understanding of this family in several ways. First, when embarking upon functional studies of the members, defining primary groups improves the predictive power of functional assignment of orthologous/paralogous genes and aids in hypothesis generation. Second, defining groups will allow a group-specific sequence motif to be generated that will help define future CDF family sequences and aid in functional motif identification, which currently is lacking for this family in plants. Third, the plant-specific expansion resulting in Groups 8 and 9 evolved coincident to the early primary radiation of plants onto land, suggesting these families may have been important for early land colonization.

SummaryTo avoid metal toxicity, organisms have evolved mechanisms including ef¯ux of metal ions from cells and sequestration into internal cellular compartments. Members of the ubiquitous cation diffusion facilitator (CDF) family are known to play an important role in these processes. Overexpression of the plant CDF family member metal tolerance protein 1 (MTP1) from the Ni/Zn hyperaccumulator Thlaspi goesingense (TgMTP1), in the Saccharomyces cerevisiae D zinc resistance conferring (zrc)1D cobalt transporter (cot)1 double mutant, suppressed the Zn sensitivity of this strain. T. goesingense was found to contain several allelic variants of TgMTP1, all of which confer similar resistance to Zn in Dzrc1Dcot1. Similarly, MTP1 from various hyperaccumulator and non-accumulator species also confer similar resistance to Zn. Dzrc1Dcot1 lacks the ability to accumulate Zn in the vacuole and has lower accumulation of Zn after either long-or short-term Zn exposure. Expression of TgMTP1 in Dzrc1Dcot1 leads to further lowering of Zn accumulation and an increase in Zn ef¯ux from the cells. Expression of TgMTP1 in a V-type ATPase-de®cient S. cerevisiae strain also confers increased Zn resistance. In vivo and in vitro immunological staining of hemagglutinin (HA)-tagged TgMTP1::HA reveals the protein to be localized in both the S. cerevisiae vacuolar and plasma membranes. Taken together, these data are consistent with MTP1 functioning to enhance plasma membrane Zn ef¯ux, acting to confer Zn resistance independent of the vacuole in S. cerevisiae. Transient expression in Arabidopsis thaliana protoplasts also reveals that TgMTP1::green¯uorescent protein (GFP) is localized at the plasma membrane, suggesting that TgMTP1 may also enhance Zn ef¯ux in plants.

SummaryThe integral membrane protein Thlaspi goesingense metal tolerance protein 1 (TgMTP1) has been suggested to play an important role in Zn hyperaccumulation in T. goesingense. Here, we show that the TgMTP1 protein is accumulated to high levels at the vacuolar membrane in shoot tissue of T. goesingense. TgMTP1 is likely to act in the transport of Zn into the vacuole, enhancing both Zn accumulation and tolerance. By specifically expressing TgMTP1 in Arabidopsis thaliana shoots, we show that TgMTP1, localized at the vacuolar membrane, can drive the enhanced shoot accumulation of Zn by initiating a systemic Zn deficiency response. The systematic response includes increased expression of Zn transporters (ZIP3, ZIP4, ZIP5 and ZIP9) in both shoot and root tissue. Furthermore, shoot-specific accumulation of TgMTP1 at the vacuolar membrane also leads to increased resistance to Zn in A. thaliana, probably through enhanced Zn compartmentalization in the vacuole. Such evidence leads to the conclusion that the high levels of TgMTP1 at the vacuolar membrane in shoot tissue of the Zn hyperaccumulator T. goesingense play a role in both Zn tolerance and enhanced Zn uptake and accumulation, via the activation of a systemic Zn deficiency response.

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Maize kernel density affects milling quality of the grain. Kernel density of bulk samples can be predicted by near-infrared reflectance (NIR) spectroscopy, but no accurate method to measure individual kernel density has been reported. This study demonstrates that individual kernel density and volume are accurately measured using X-ray microcomputed tomography (μCT). Kernel density was significantly correlated with kernel volume, air space within the kernel, and protein content. Embryo density and volume did not influence overall kernel density. Partial least-squares (PLS) regression of μCT traits with single-kernel NIR spectra gave stable predictive models for kernel density (R(2) = 0.78, SEP = 0.034 g/cm(3)) and volume (R(2) = 0.86, SEP = 2.88 cm(3)). Density and volume predictions were accurate for data collected over 10 months based on kernel weights calculated from predicted density and volume (R(2) = 0.83, SEP = 24.78 mg). Kernel density was significantly correlated with bulk test weight (r = 0.80), suggesting that selection of dense kernels can translate to improved agronomic performance.

The extreme phenotype of zinc (Zn) hyperaccumulation, which is found in several Brassicaceae species, is determined by mechanisms that promote elevated Zn tolerance and high Zn accumulation in shoots.We used reciprocal grafting between a Zn hyperaccumulator, Thlaspi caerulescens, and a Zn nonaccumulator, Thlaspi perfoliatum, to determine the relative importance of roots and shoots in Zn hyperaccumulation and hypertolerance.Leaves from plants with a T. perfoliatum rootstock and a T. caerulescens shoot scion did not hyperaccumulate Zn, whereas plants with a T. caerulescens rootstock and a T. perfoliatum shoot scion did hyperaccumulate Zn. However, although leaves from plants with a T. caerulescens rootstock and a T. perfoliatum shoot scion hyperaccumulated Zn, at high Zn loads these leaves showed significant symptoms of Zn toxicity, unlike leaves of self grafted T. caerulescens.Hyperaccumulation of Zn in leaves of the hyperaccumulator T. caerulescens is pri-marily dictated by root processes. Further, the mechanisms controlling Zn hypertolerance in the hyperaccumulator T. caerulescens are driven primarily by shoot processes.

Seed size and composition are important traits in food crops and can be affected by nutrient availability in the soil. Phosphorus (P) is a non-renewable, essential macronutrient, and P deficiency limits soybean (Glycine max) yield and quality. To investigate the associations of seed traits in low- and high-P environments, soybean recombinant inbred lines (RILs) from a cross of cultivars Fiskeby III and Mandarin (Ottawa) were grown under contrasting P availability environments. Traits including individual seed weight, seed number, and intact mature pod weight were significantly affected by soil P levels and showed transgressive segregation among the RILs. Surprisingly, P treatments did not affect seed composition or weight, suggesting that soybean maintains sufficient P in seeds even in low-P soil. Quantitative trait loci (QTLs) were detected for seed weight, intact pods, seed volume, and seed protein, with five significant QTLs identified in low-P environments and one significant QTL found in the optimal-P environment. Broad-sense heritability estimates were 0.78 (individual seed weight), 0.90 (seed protein), 0.34 (seed oil), and 0.98 (seed number). The QTLs identified under low P point to genetic regions that may be useful to improve soybean performance under limiting P conditions.

Positional cloning in maize (Zea mays) requires development of markers in the region of interest. We found that primers designed to amplify annotated insertion–deletion polymorphisms of seven base pairs or greater between B73 and Mo17 produce polymorphic markers at a 97% frequency with 49% of the products showing co-dominant fragment length polymorphisms. When the same polymorphisms are used to develop markers for B73 and W22 or Mo17 and W22 mapping populations, 22% and 31% of markers are co-dominant, respectively. There are 38,223 Indel polymorphisms that can be converted to markers providing high-density coverage throughout the maize genome. This strategy significantly increases the efficiency of marker development for fine-mapping in maize.