Bruna Bucciarelli

RNA Interference Identifies a Calcium-Dependent Protein Kinase Involved in Medicago truncatula Root Development

Changes in cellular or subcellular Ca2+ concentrations play essential roles in plant development and in the responses of plants to their environment. However, the mechanisms through which Ca2+ acts, the downstream signaling components, as well as the relationships among the various Ca2+-dependent processes remain largely unknown. Using an RNA interference–based screen for gene function in Medicago truncatula, we identified a gene that is involved in root development. Silencing Ca2+-dependent protein kinase1 (CDPK1), which is predicted to encode a Ca2+-dependent protein kinase, resulted in significantly reduced root hair and root cell lengths. Inactivation of CDPK1 is also associated with significant diminution of both rhizobial and mycorrhizal symbiotic colonization. Additionally, microarray analysis revealed that silencing CDPK1 alters cell wall and defense-related gene expression. We propose that M. truncatula CDPK1 is a key component of one or more signaling pathways that directly or indirectly modulates cell expansion or cell wall synthesis, possibly altering defense gene expression and symbiotic interactions.

An RNA-Seq Transcriptome Analysis of Orthophosphate-Deficient White Lupin Reveals Novel Insights into Phosphorus Acclimation in Plants

Summary: Analysis of all the expressed genes in white lupin roots and leaves shows that acclimation to phosphorous deficiency involves changes in root development and modifications in metabolism. Phosphorus, in its orthophosphate form (Pi), 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 Pi deficiency remain largely unknown. White lupin (Lupinus albus) has evolved unique adaptations for growth in Pi-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 Pi 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 Pi deficiency with a 2-fold or greater change and P ≤ 0.05. Twelve sequences were consistently differentially expressed due to Pi deficiency stress in three species, Arabidopsis (Arabidopsis thaliana), potato (Solanum tuberosum), and white lupin, making them ideal candidates to monitor the Pi status of plants. Additionally, classic physiological experiments were coupled with RNA-Seq data to examine the role of cytokinin and gibberellic acid in Pi deficiency-induced cluster root development. This global gene expression analysis provides new insights into the biochemical and molecular mechanisms involved in the acclimation to Pi deficiency.

Localization of superoxide dismutases and hydrogen peroxide in legume root nodules.

Superoxide dismutases (SODs) catalyze the dismutation of superoxide radicals to O2 and H2O2 and thus represent a primary line of antioxidant defense in all aerobic organisms. H2O2 is a signal molecule involved in the plants response to pathogen attack and other stress conditions as well as in nodulation. In this work, we have tested the hypothesis that SODs are a source of H2O2 in indeterminate alfalfa (Medicago sativa) and pea (Pisum sativum) nodules. The transcripts and proteins of the major SODs of nodules were localized by in situ RNA hybridization and immunogold electron microscopy, respectively, whereas H2O2 was localized cytochemically by electron microscopy of cerium-perfused nodule tissue. The transcript and protein of cytosolic CuZnSOD are most abundant in the meristem (I) and invasion (II) zones, interzone II-III, and distal part of the N2-fixing zone (III), and those of MnSOD in zone III, especially in the infected cells. At the subcellular level, CuZnSOD was found in the infection threads, cytosol adjacent to cell walls, and apoplast, whereas MnSOD was in the bacteroids, bacteria within infection threads, and mitochondria. The distinct expression pattern of CuZnSOD and MnSOD suggests specific roles of the enzymes in nodules. Large amounts of H2O2 were found at the same three nodule sites as CuZnSOD but not in association with MnSOD. This colocalization led us to postulate that cytosolic CuZnSOD is a source of H2O2 in nodules. Furthermore, the absence or large reduction of H2O2 in nodule tissue preincubated with enzyme inhibitors (cyanide, azide, diphenyleneiodonium, diethyldithiocarbamate) provides strong support to the hypothesis that at least some of the H2O2 originates by the sequential operation of an NADPH oxidase-like enzyme and CuZnSOD. Results also show that there is abundant H2O2 associated with degrading bacteroids in the senescent zone (IV), which reflects the oxidative stress ensued during nodule senescence.

Malate plays a central role in plant nutrition

Malate occupies a central role in plant metabolism. Its importance in plant mineral nutrition is reflected by the role it plays in symbiotic nitrogen fixation, phosphorus acquisition, and aluminum tolerance. In nitrogen-fixing root nodules, malate is the primary substrate for bacteroid respiration, thus fueling nitrogenase. Malate also provides the carbon skeletons for assimilation of fixed nitrogen into amino acids. During phosphorus deficiency, malate is frequently secreted from roots to release unavailable forms of phosphorus. Malate is also involved with plant adaptation to aluminum toxicity. To define the genetic and biochemical regulation of malate formation in plant nutrition we have isolated and characterized genes involved in malate metabolism from nitrogen-fixing root nodules of alfalfa and those involved in organic acid excretion from phosphorus-deficient proteoid roots of white lupin. Moreover, we have overexpressed malate dehydrogenase in alfalfa in attempts to improve nutrient acquisition. This report is an overview of our efforts to understand and modify malate metabolism, particularly in the legumes alfalfa and white lupin.

An RNA-Seq based gene expression atlas of the common bean

BackgroundCommon bean (Phaseolus vulgaris) is grown throughout the world and comprises roughly 50% of the grain legumes consumed worldwide. Despite this, genetic resources for common beans have been lacking. Next generation sequencing, has facilitated our investigation of the gene expression profiles associated with biologically important traits in common bean. An increased understanding of gene expression in common bean will improve our understanding of gene expression patterns in other legume species.ResultsCombining recently developed genomic resources for Phaseolus vulgaris, including predicted gene calls, with RNA-Seq technology, we measured the gene expression patterns from 24 samples collected from seven tissues at developmentally important stages and from three nitrogen treatments. Gene expression patterns throughout the plant were analyzed to better understand changes due to nodulation, seed development, and nitrogen utilization. We have identified 11,010 genes differentially expressed with a fold change ≥ 2 and a P-value < 0.05 between different tissues at the same time point, 15,752 genes differentially expressed within a tissue due to changes in development, and 2,315 genes expressed only in a single tissue. These analyses identified 2,970 genes with expression patterns that appear to be directly dependent on the source of available nitrogen. Finally, we have assembled this data in a publicly available database, The Phaseolus vulgaris Gene Expression Atlas (Pv GEA), http://plantgrn.noble.org/PvGEA/ . Using the website, researchers can query gene expression profiles of their gene of interest, search for genes expressed in different tissues, or download the dataset in a tabular form.ConclusionsThese data provide the basis for a gene expression atlas, which will facilitate functional genomic studies in common bean. Analysis of this dataset has identified genes important in regulating seed composition and has increased our understanding of nodulation and impact of the nitrogen source on assimilation and distribution throughout the plant.

White Lupin Cluster Root Acclimation to Phosphorus Deficiency and Root Hair Development Involve Unique Glycerophosphodiester Phosphodiesterases

White lupin (Lupinus albus) is a legume that is very efficient in accessing unavailable phosphorus (Pi). It develops short, densely clustered tertiary lateral roots (cluster/proteoid roots) in response to Pi limitation. In this report, we characterize two glycerophosphodiester phosphodiesterase (GPX-PDE) genes (GPX-PDE1 and GPX-PDE2) from white lupin and propose a role for these two GPX-PDEs in root hair growth and development and in a Pi stress-induced phospholipid degradation pathway in cluster roots. Both GPX-PDE1 and GPX-PDE2 are highly expressed in Pi-deficient cluster roots, particularly in root hairs, epidermal cells, and vascular bundles. Expression of both genes is a function of both Pi availability and photosynthate. GPX-PDE1 Pi deficiency-induced expression is attenuated as photosynthate is deprived, while that of GPX-PDE2 is strikingly enhanced. Yeast complementation assays and in vitro enzyme assays revealed that GPX-PDE1 shows catalytic activity with glycerophosphocholine while GPX-PDE2 shows highest activity with glycerophosphoinositol. Cell-free protein extracts from Pi-deficient cluster roots display GPX-PDE enzyme activity for both glycerophosphocholine and glycerophosphoinositol. Knockdown of expression of GPX-PDE through RNA interference resulted in impaired root hair development and density. We propose that white lupin GPX-PDE1 and GPX-PDE2 are involved in the acclimation to Pi limitation by enhancing glycerophosphodiester degradation and mediating root hair development.

Knockdown of CELL DIVISION CYCLE16 Reveals an Inverse Relationship between Lateral Root and Nodule Numbers and a Link to Auxin in Medicago truncatula

The postembryonic development of lateral roots and nodules is a highly regulated process. Recent studies suggest the existence of cross talk and interdependency in the growth of these two organs. Although plant hormones, including auxin and cytokinin, appear to be key players in coordinating this cross talk, very few genes that cross-regulate root and nodule development have been uncovered so far. This study reports that a homolog of CELL DIVISION CYCLE16 (CDC16), a core component of the Anaphase Promoting Complex, is one of the key mediators in controlling the overall number of lateral roots and nodules. A partial suppression of this gene in Medicago truncatula leads to a decrease in number of lateral roots and a 4-fold increase in number of nodules. The roots showing lowered expression of MtCDC16 also show reduced sensitivity to phytohormone auxin, thus providing a potential function of CDC16 in auxin signaling.

Recruitment of Novel Calcium-Binding Proteins for Root Nodule Symbiosis in Medicago truncatula

Legume rhizobia symbiotic nitrogen (N2) fixation plays a critical role in sustainable nitrogen management in agriculture and in the Earths nitrogen cycle. Signaling between rhizobia and legumes initiates development of a unique plant organ, the root nodule, where bacteria undergo endocytosis and become surrounded by a plant membrane to form a symbiosome. Between this membrane and the encased bacteria exists a matrix-filled space (the symbiosome space) that is thought to contain a mixture of plant- and bacteria-derived proteins. Maintenance of the symbiosis state requires continuous communication between the plant and bacterial partners. Here, we show in the model legume Medicago truncatula that a novel family of six calmodulin-like proteins (CaMLs), expressed specifically in root nodules, are localized within the symbiosome space. All six nodule-specific CaML genes are clustered in the M. truncatula genome, along with two other nodule-specific genes, nodulin-22 and nodulin-25. Sequence comparisons and phylogenetic analysis suggest that an unequal recombination event occurred between nodulin-25 and a nearby calmodulin, which gave rise to the first CaML, and the gene family evolved by tandem duplication and divergence. The data provide striking evidence for the recruitment of a ubiquitous Ca2+-binding gene for symbiotic purposes.

A Standardized Method for Analysis of Medicago truncatula Phenotypic Development

Medicago truncatula has become a model system to study legume biology. It is imperative that detailed growth characteristics of the most commonly used cultivar, line A17 cv Jemalong, be documented. Such analysis creates a basis to analyze phenotypic alterations due to genetic lesions or environmental stress and is essential to characterize gene function and its relationship to morphological development. We have documented morphological development of M. truncatula to characterize its temporal developmental growth pattern; developed a numerical nomenclature coding system that identifies stages in morphological development; tested the coding system to identify phenotypic differences under phosphorus (P) and nitrogen (N) deprivation; and created visual models using the L-system formalism. The numerical nomenclature coding system, based on a series of defined growth units, represents incremental steps in morphological development. Included is a decimal component dividing growth units into nine substages. A measurement component helps distinguish alterations that may be missed by the coding system. Growth under N and P deprivation produced morphological alterations that were distinguishable using the coding system and its measurement component. N and P deprivation resulted in delayed leaf development and expansion, delayed axillary shoot emergence and elongation, decreased leaf and shoot size, and altered root growth. Timing and frequency of flower emergence in P-deprived plants was affected. This numerical coding system may be used as a standardized method to analyze phenotypic variation in M. truncatula due to nutrient stress, genetic lesions, or other factors and should allow valid growth comparisons across geographically distant laboratories.

Update on White Lupin Cluster Root Acclimation to Phosphorus Deficiency Update on Lupin Cluster Roots

Phosphorus (P) is one of 17 essential elements (nutrients) required for plant growth (Tiessen, 2008; Cordell et al., 2009). Although bound P is quite abundant in many soils, it is largely unavailable for uptake. As such, P is frequently the most limiting element for plant growth and development. Crop yield on 40% to 60% of the world’s arable land is limited by P availability. Mined rock phosphate is the primary source of P fertilizer. Approximately 90% of all mined rock phosphate is used for agriculture (Tiessen, 2008; Cordell et al., 2009). However, rock phosphate is a nonrenewable resource (Steen, 1998; Cordell et al., 2009), and easily mined, high-quality rock phosphate sources are projected to be depleted within 30 to 50 years (Steen, 1998; Tiessen, 2008; Cordell et al., 2009). Peak P production is projected to occur in 2035 to 2040 (Cordell et al., 2009). In addition, the world’s major reserves of rock phosphate are located in geographical areas where uncertain political issues could limit access to the world’s P resources. Sustainable management of P in agriculture requires that plant biologists discover mechanisms that enhance P acquisition and exploit these adaptations to make plants more efficient at acquiring P, develop P-efficient germplasm, and advance crop management schemes that increase soil P availability. Cluster roots (Fig. 1), extremely specialized tertiary lateral root structures, are an important adaptive strategy of plants to cope with nutrient-poor, P-depleted soils (Dinkelaker et al., 1995; Neumann and Martinoia, 2002; Vance et al., 2003; Lambers et al., 2006). They are produced on plants from a diverse range of families (Dinkelaker et al., 1995; Watt and Evans, 1999; Shane and Lambers, 2005). White lupin (Lupinus albus) forms cluster roots in response to P starvation. Cluster roots are characterized as concentrated zones of tertiary lateral roots emerging in waves from secondary roots. Root hair density appears to be greater in mature cluster root zones than typical lateral roots. Such an adaptation leads to a striking increase in root surface area available for P uptake from the rhizosphere (Keerthisinghe et al., 1998; Neumann et al., 1999). Cluster root development and function involve a highly synchronous series of molecular and biochemical processes, including highly enhanced lateral root initiation, increased root hair formation, root exudation of organic acid chelators (citrate and malate), modified carbon assimilation, release of enzymes (acid phosphatase, ferric chelate reductases) into the rhizosphere, and more efficient uptake of P from the rhizosphere (Dinkelaker et al., 1989; Neumann et al., 1999; Watt and Evans, 1999; Liu et al., 2001, 2005; Miller et al., 2001; Uhde-Stone et al., 2003a, 2005; Wasaki et al., 2003). Advances have recently been made in understanding the molecular and biochemical events surrounding cluster root formation and function. As a crop, white lupin is a practical alternative to evaluate acclimation to P deficiency, particularly as related to cluster-rooted species (Johnson et al., 1996; Keerthisinghe et al., 1998; Watt and Evans, 1999; Neumann and Martinoia, 2002). Figure 1. White lupin P deficiency cluster roots emerge as waves of tertiary lateral roots along the axis of secondary roots.Department of Plant Nutrition, China Agricultural University, Key Laboratory of Plant-Soil Interactions, Beijing 100193, People’s Republic of China (L.C., J.S.); Department of Agronomy and Plant Genetics (L.C., B.B., C.P.V.) and Department of Soil, Water, and Climate (D.A.), University of Minnesota, St. Paul, Minnesota 55108; and United States Department of Agriculture Agricultural Research Service, St. Paul, Minnesota 55108 (B.B., C.P.V.)