Contents Introduction  424 The phosphorus conundrum 424 Adaptations to low P 424 Uptake of P  424 P deficiency alters root development and function 426 P deficiency modifies carbon metabolism 431 Acid phosphatase 436 Genetic regulation of P responsive genes 437 Improving P acquisition 439 Synopsis  440 Summary Phosphorus (P) is limiting for crop yield on > 30% of the world's arable land and, by some estimates, world resources of inexpensive P may be depleted by 2050. Improvement of P acquisition and use by plants is critical for economic, humanitarian and environmental reasons. Plants have evolved a diverse array of strategies to obtain adequate P under limiting conditions, including modifications to root architecture, carbon metabolism and membrane structure, exudation of low molecular weight organic acids, protons and enzymes, and enhanced expression of the numerous genes involved in low‐P adaptation. These adaptations may be less pronounced in mycorrhizal‐associated plants. The formation of cluster roots under P‐stress by the nonmycorrhizal species white lupin (Lupinus albus), and the accompanying biochemical changes exemplify many of the plant adaptations that enhance P acquisition and use. Physiological, biochemical, and molecular studies of white lupin and other species response to P‐deficiency have identified targets that may be useful for plant improvement. Genomic approaches involving identification of expressed sequence tags (ESTs) found under low‐P stress may also yield target sites for plant improvement. Interdisciplinary studies uniting plant breeding, biochemistry, soil science, and genetics under the large umbrella of genomics are prerequisite for rapid progress in improving nutrient acquisition and use in plants.

Phosphorus, in its orthophosphate form (P i ), is one of the most limiting macronutrients in soils for plant growth and development. However, the whole-genome molecular mechanisms contributing to plant acclimation to P i deficiency remain largely unknown. White lupin (Lupinus albus) has evolved unique adaptations for growth in P i -deficient soils, including the development of cluster roots to increase root surface area. In this study, we utilized RNA-Seq technology to assess global gene expression in white lupin cluster roots, normal roots, and leaves in response to P i supply. We de novo assembled 277,224,180 Illumina reads from 12 complementary DNA libraries to build what is to our knowledge the first white lupin gene index (LAGI 1.0). This index contains 125,821 unique sequences with an average length of 1,155 bp. Of these sequences, 50,734 were transcriptionally active (reads per kilobase per million reads $ 3), representing approximately 7.8% of the white lupin genome, using the predicted genome size of Lupinus angustifolius as a reference. We identified a total of 2,128 sequences differentially expressed in response to P i deficiency with a 2-fold or greater change and P # 0.05. Twelve sequences were consistently differentially expressed due to P i deficiency stress in three species, Arabidopsis (Arabidopsis thaliana), potato (Solanum tuberosum), and white lupin, making them ideal candidates to monitor the P i status of plants. Additionally, classic physiological experiments were coupled with RNA-Seq data to examine the role of cytokinin and gibberellic acid in P i deficiency-induced cluster root development. This global gene expression analysis provides new insights into the biochemical and molecular mechanisms involved in the acclimation to P i deficiency.

White lupin (Lupinus albus) adapts to phosphorus deficiency (ϪP) by the development of short, densely clustered lateral roots called proteoid (or cluster) roots. In an effort to better understand the molecular events mediating these adaptive responses, we have isolated and sequenced 2,102 expressed sequence tags (ESTs) from cDNA libraries prepared with RNA isolated at different stages of proteoid root development. Determination of overlapping regions revealed 322 contigs (redundant copy transcripts) and 1,126 singletons (single-copy transcripts) that compile to a total of 1,448 unique genes (unigenes). Nylon filter arrays with these 2,102 ESTs from proteoid roots were performed to evaluate global aspects of gene expression in response to ϪP stress. ESTs differentially expressed in P-deficient proteoid roots compared with ϩP and ϪP normal roots include genes involved in carbon metabolism, secondary metabolism, P scavenging and remobilization, plant hormone metabolism, and signal transduction.Phosphorus (P) is an essential macronutrient for plant growth and development that plays key roles in many processes, including energy metabolism and synthesis of nucleic acids and membranes (Raghothama, 1999). It is second only to nitrogen as the most limiting nutrient for plant growth (Bieleski, 1973; Raghothama, 1999;. In many soils, low availability of P is a limiting factor in crop production . Due to the low availability of soluble P in many ecosystems, plants have developed adaptive mechanisms that aid in the acquisition of P from soil. Strategies that lead to better uptake or acquisition include expanded root surface area through increased root growth and root hair development (Lynch and Brown, 1998;Gilroy and Jones, 2000; Williamson et al., 2001), organic acid synthesis and exudation (Dinkelaker et al., 1989;Johnson et al., 1996a;Jones, 1998; Aono et al., 2001;Massonneau et al., 2001; Sas et al., 2001), exudation of acid phosphatases (Duff et al., 1991; del Pozo et al., 1999;Gilbert et al., 1999;Miller et al., 2001), enhanced expression of phosphate transporters (Leggewie et al., 1997;Liu et al., 1998aLiu et al., , 1998b Chiou et al., 2001;Liu et al., 2001), and mycorrhizal associations (Marschner and Dell, 1994; Smith et al., 1994). Strategies aimed at conserving P involve internal remobilization of P and use of alternative metabolic pathways (Theodorou et al., 1992; Theodorou and Plaxton, 1993;Plaxton and Carswell, 1999).White lupin (Lupinus albus), a species known for its extreme tolerance for low P availability, has proven an illuminating model system for understanding plant adaptations to low P, despite its lack of mycorrhizal symbiosis. Instead, its adaptation to P deficiency (ϪP) is a highly coordinated modification of root development and biochemistry resulting in proteoid (or cluster) roots-short, densely clustered tertiary roots-that resemble bottlebrushes (Gardner et al., 1982(Gardner et al., , 1983Dinkelaker et al., 1995;Johnson et al., 1996b;Neumann et al., 1999;Massonneau et al., 2001). Unlike typical...

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SummaryWhite lupin (Lupinus albus L.) has become an illuminating model for the study of plant adaptation to phosphorus (P) deficiency. It adapts to )P stress with a highly coordinated modification of root development and biochemistry resulting in short, densely clustered secondary roots called proteoid (or cluster) roots. In order to characterize genes involved in proteoid root formation and function in a homologous system, we have developed an Agrobacterium rhizogenes-based transformation system for white lupin roots that allows rapid analysis of reporter genes as well as RNA interference (RNA i )-based gene silencing. We used this system to characterize a lupin multidrug and toxin efflux (Lupinus albus MULTIDRUG AND TOXIN EFFLUX, LaMATE) gene previously shown to have enhanced expression under )P stress. Here, we show that LaMATE had high expression in proteoid roots not only under )P, but also under )Fe, )N, )Mn and þAl stress. A portion containing the putative LaMATE promoter was fused to GUS and enhanced green fluorescence protein (EGFP) reporter genes, and a translational LaMATE::EGFP fusion was constructed under control of the LaMATE promoter. The LaMATE promoter directed P-dependent GUS and EGFP expression to proteoid roots. Confocal microscopy in white lupin and Arabidopsis point to the plasma membrane as the likely location of the LaMATE protein.LaMATE displayed homology to FRD3 in Arabidopsis, but did not complement an Arabidopsis ferric reductase defective 3 (FRD3) mutant. RNA i -based gene silencing was shown to effectively reduce LaMATE expression in transformed white lupin roots. LaMATE RNAi-silenced plants displayed an about 20% reduction in dry weight.

Significant progress in the functional understanding of microRNAs (miRNAs) has been made in mice, but a need remains to develop efficient tools for bi-allelic knockouts of miRNA in the human genome. Transcription activator-like effector nucleases (TALENs) provide an exciting platform for targeted gene ablation in cultured human cells, but bi-allelic modifications induced by TALENs alone occur at low frequency, making screening for double knockouts a tedious task. Here, we present an approach that is highly efficient in bi-allelic miRNA ablation in the human genome by combining TALENs targeting to the miRNA seed region with a homologous recombination donor vector and a positive selection strategy. A pilot test of this approach demonstrates bi-allelic miR-21 gene disruption at high frequency (∼87%) in cultured HEK293 cells. Analysis of three independent clones showed a total loss of miR-21 expression. Phenotypical analysis revealed increased miR-21 target gene expression, reduced cell proliferation, and alterations of global miRNA expression profiles. Taken together, our study reveals a feasible and efficient approach for biallelic miRNA ablation in cultured human cells and demonstrates its usefulness in elucidating miRNA function in human cells.

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White lupin (Lupinus albus L.) is considered a model system for understanding plant acclimation to nutrient deficiency. It acclimates to phosphorus (P) and iron (Fe) deficiency by the development of short, densely clustered lateral roots called proteoid (or cluster) roots; proteoid-root development is further influenced by nitrogen (N) supply. In an effort to better understand proteoid root function under various nutrient deficiencies, we used nylon filter arrays to analyze 2,102 expressed sequence tags (ESTs) from proteoid roots of P-deficient white lupin. These have been previously analyzed for up-regulation in −P proteoid roots, and were here analyzed for upregulation in proteoid roots of N-deprived plants. We identified a total of 19 genes that displayed upregulation in proteoid roots under both P and N Plant Soil (2010) 334:137-150 deprivation. One of these genes showed homology to putative formamidases. The corresponding open reading frame was cloned, overexpressed in E. coli, and the encoded protein was purified; functional characterization of the recombinant protein confirmed formamidase activity. Though many homologues of bacterial and fungal formamidases have been identified in plants, to our knowledge, this is the first report of a functional characterization of a plant formamidase.