Adaption of Roots to Nitrogen Deficiency Revealed by 3D Quantification and Proteomic Analysis.

Rapeseed (Brassica napus) is an important oil crop worldwide. However, severe inhibition of rapeseed production often occurs in the field due to nitrogen (N) deficiency. The root system is the main organ to acquire N for plant growth, but little is known about the mechanisms underlying rapeseed root adaptions to N deficiency. Here, dynamic changes in root architectural traits of N-deficient rapeseed plants were evaluated by 3D in situ quantification. Root proteome responses to N deficiency were analyzed by the tandem mass tag-based proteomics method, and related proteins were characterized further. Under N deficiency, rapeseed roots become longer, with denser cells in the meristematic zone and larger cells in the elongation zone of root tips, and also become softer with reduced solidity. A total of 171 and 755 differentially expressed proteins were identified in short- and long-term N-deficient roots, respectively. The abundance of proteins involved in cell wall organization or biogenesis was highly enhanced, but most identified peroxidases were reduced in the N-deficient roots. Notably, peroxidase activities also were decreased, which might promote root elongation while lowering the solidity of N-deficient roots. These results were consistent with the cell wall components measured in the N-deficient roots. Further functional analysis using transgenic Arabidopsis (Arabidopsis thaliana) plants demonstrated that the two root-related differentially expressed proteins contribute to the enhanced root growth under N deficiency conditions. These results provide insights into the global changes of rapeseed root responses to N deficiency and may facilitate the development of rapeseed cultivars with high N use efficiency through root-based genetic improvements.

Exploiting sorghum genetic diversity for enhanced aluminum tolerance: Allele mining based on the AltSB locus.

Root damage due to aluminum (Al) toxicity restricts crop production on acidic soils, which are extensive in the tropics. The sorghum root Al-activated citrate transporter, SbMATE, underlies the Al tolerance locus, AltSB, and increases grain yield under Al toxicity. Here, AltSB loci associated with Al tolerance were converted into Amplification Refractory Mutation System (ARMS) markers, which are cost effective and easy to use. A DNA pooling strategy allowed us to identify accessions harboring rare favorable AltSB alleles in three germplasm sets while greatly reducing genotyping needs. Population structure analysis revealed that favorable AltSB alleles are predominantly found in subpopulations enriched with guinea sorghums, supporting a possible Western African origin of AltSB. The efficiency of allele mining in recovering Al tolerance accessions was the highest in the largest and highly diverse germplasm set, with a 10-fold reduction in the number of accessions that would need to be phenotyped in the absence of marker information. Finally, Al tolerant accessions were found to rely on SbMATE to exclude Al3+ from sensitive sites in the root apex. This study emphasizes gene-specific markers as important tools for efficiently mining useful rare alleles in diverse germplasm, bridging genetic resource conservation efforts and pre-breeding for Al tolerance.

Genomic regions responsible for seminal and crown root lengths identified by 2D & 3D root system image analysis.

BACKGROUND: Genetic improvement of root system architecture is a promising approach for improved uptake of water and mineral nutrients distributed unevenly in the soil. To identify genomic regions associated with the length of different root types in rice, we quantified root system architecture in a set of 26 chromosome segment substitution lines derived from a cross between lowland indica rice, IR64, and upland tropical japonica rice, Kinandang Patong, (IK-CSSLs), using 2D & 3D root phenotyping platforms. RESULTS: Lengths of seminal and crown roots in the IK-CSSLs grown under hydroponic conditions were measured by 2D image analysis (RootReader2D). Twelve CSSLs showed significantly longer seminal root length than the recurrent parent IR64. Of these, 8 CSSLs also exhibited longer total length of the three longest crown roots compared to IR64. Three-dimensional image analysis (RootReader3D) for these CSSLs grown in gellan gum revealed that only one CSSL, SL1003, showed significantly longer total root length than IR64. To characterize the root morphology of SL1003 under soil conditions, SL1003 was grown in Turface, a soil-like growth media, and roots were quantified using RootReader3D. SL1003 had larger total root length and increased total crown root length than did IR64, although its seminal root length was similar to that of IR64. The larger TRL in SL1003 may be due to increased crown root length. CONCLUSIONS: SL1003 carries an introgression from Kinandang Patong on the long arm of chromosome 1 in the genetic background of IR64. We conclude that this region harbors a QTL controlling crown root elongation.

Evolving technologies for growing, imaging and analyzing 3D root system architecture of crop plants.

A plant's ability to maintain or improve its yield under limiting conditions, such as nutrient deficiency or drought, can be strongly influenced by root system architecture (RSA), the three-dimensional distribution of the different root types in the soil. The ability to image, track and quantify these root system attributes in a dynamic fashion is a useful tool in assessing desirable genetic and physiological root traits. Recent advances in imaging technology and phenotyping software have resulted in substantive progress in describing and quantifying RSA. We have designed a hydroponic growth system which retains the three-dimensional RSA of the plant root system, while allowing for aeration, solution replenishment and the imposition of nutrient treatments, as well as high-quality imaging of the root system. The simplicity and flexibility of the system allows for modifications tailored to the RSA of different crop species and improved throughput. This paper details the recent improvements and innovations in our root growth and imaging system which allows for greater image sensitivity (detection of fine roots and other root details), higher efficiency, and a broad array of growing conditions for plants that more closely mimic those found under field conditions.

Iron Absorption from an Intrinsically Labeled Lentil Meal Is Low but Upregulated in Women with Poor Iron Status.

BACKGROUND: Low iron absorption from important staple foods may contribute to iron deficiency in developing countries. To date, few studies have examined the iron bioavailability of pulse crops as commonly prepared and consumed by humans. OBJECTIVE: The objectives were to characterize the iron absorption from a test meal of intrinsically labeled (57)Fe lentils prepared as dal, to compare the bioavailability of iron from (57)Fe in dal with that observed for a reference dose of (58)Fe as ferrous sulfate, and to assess associations between iron absorption and iron status indicators. METHODS: This crossover study included 19 nonpregnant women (n = 6 anemic; hemoglobin: <12.0 g/dL) who consumed 2 test meals on consecutive days in a counter-balanced order, ferrous sulfate (7 mg FeSO4 plus 1 mg (58)Fe) and 330 g dal (lentils enriched to 85.1% with (57)Fe, 8 mg native (57)Fe). Iron absorption was determined by analyzing blood samples taken 14 d after dosing with the use of magnetic sector thermal ionization mass spectrometry. RESULTS: We found that the mean iron absorption from the dal was 2.20% ± 3.40% and was significantly lower than the 23.6% ± 13.2% observed from the same iron load given as ferrous sulfate (P < 0.001). Absorption of non-heme iron from dal and from ferrous sulfate was inversely associated with serum ferritin (SF; r = -0.50, P = 0.05 and r = -0.81, P < 0.001, respectively) and serum hepcidin (r = -0.45, P = 0.05 and r = -0.60, P = 0.007, respectively). Anemic women absorbed more iron from either source (1.20% from dal, P = 0.10; 18.3% from ferrous sulfate, P = 0.001) compared with women who were iron replete. CONCLUSIONS: Iron absorption from the dal was low overall but upregulated in anemic women. Both SF and hepcidin were inversely associated with iron absorption from both a supplemental and a food-based non-heme iron source in nonanemic and anemic women.

Duplicate and conquer: multiple homologs of PHOSPHORUS-STARVATION TOLERANCE1 enhance phosphorus acquisition and sorghum performance on low-phosphorus soils.

Low soil phosphorus (P) availability is a major constraint for crop production in tropical regions. The rice (Oryza sativa) protein kinase, PHOSPHORUS-STARVATION TOLERANCE1 (OsPSTOL1), was previously shown to enhance P acquisition and grain yield in rice under P deficiency. We investigated the role of homologs of OsPSTOL1 in sorghum (Sorghum bicolor) performance under low P. Association mapping was undertaken in two sorghum association panels phenotyped for P uptake, root system morphology and architecture in hydroponics and grain yield and biomass accumulation under low-P conditions, in Brazil and/or in Mali. Root length and root surface area were positively correlated with grain yield under low P in the soil, emphasizing the importance of P acquisition efficiency in sorghum adaptation to low-P availability. SbPSTOL1 alleles reducing root diameter were associated with enhanced P uptake under low P in hydroponics, whereas Sb03g006765 and Sb03g0031680 alleles increasing root surface area also increased grain yield in a low-P soil. SbPSTOL1 genes colocalized with quantitative trait loci for traits underlying root morphology and dry weight accumulation under low P via linkage mapping. Consistent allelic effects for enhanced sorghum performance under low P between association panels, including enhanced grain yield under low P in the soil in Brazil, point toward a relatively stable role for Sb03g006765 across genetic backgrounds and environmental conditions. This study indicates that multiple SbPSTOL1 genes have a more general role in the root system, not only enhancing root morphology traits but also changing root system architecture, which leads to grain yield gain under low-P availability in the soil.

Molecular and physiological analysis of Al³âº and H⁺ rhizotoxicities at moderately acidic conditions.

Al³âº and H⁺ toxicities predicted to occur at moderately acidic conditions (pH [water] = 5-5.5) in low-Ca soils were characterized by the combined approaches of computational modeling of electrostatic interactions of ions at the root plasma membrane (PM) surface and molecular/physiological analyses in Arabidopsis (Arabidopsis thaliana). Root growth inhibition in known hypersensitive mutants was correlated with computed {Al³âº} at the PM surface ({Al³âº}(PM)); inhibition was alleviated by increased Ca, which also reduced {Al³âº}(PM) and correlated with cellular Al responses based on expression analysis of genes that are markers for Al stress. The Al-inducible Al tolerance genes ALUMINUM-ACTIVATED MALATE TRANSPORTER1 and ALUMINUM SENSITIVE3 were induced by levels of {Al³âº}(PM) too low to inhibit root growth in tolerant genotypes, indicating that protective responses are triggered when {Al³âº}(PM) was below levels that can initiate injury. Modeling of the H⁺ sensitivity of the SENSITIVE TO PROTON RHIZOTOXICITY1 knockout mutant identified a Ca alleviation mechanism of H⁺ rhizotoxicity, possibly involving stabilization of the cell wall. The phosphatidate phosphohydrolase1 (pah1) pah2 double mutant showed enhanced Al susceptibility under low-P conditions, where greater levels of negatively charged phospholipids in the PM occur, which increases {Al³âº}(PM) through increased PM surface negativity compared with wild-type plants. Finally, we found that the nonalkalinizing Ca fertilizer gypsum improved the tolerance of the sensitive genotypes in moderately acidic soils. These findings fit our modeling predictions that root toxicity to Al³âº and H⁺ in moderately acidic soils involves interactions between both toxic ions in relation to Ca alleviation.

High-throughput two-dimensional root system phenotyping platform facilitates genetic analysis of root growth and development.

High-throughput phenotyping of root systems requires a combination of specialized techniques and adaptable plant growth, root imaging and software tools. A custom phenotyping platform was designed to capture images of whole root systems, and novel software tools were developed to process and analyse these images. The platform and its components are adaptable to a wide range root phenotyping studies using diverse growth systems (hydroponics, paper pouches, gel and soil) involving several plant species, including, but not limited to, rice, maize, sorghum, tomato and Arabidopsis. The RootReader2D software tool is free and publicly available and was designed with both user-guided and automated features that increase flexibility and enhance efficiency when measuring root growth traits from specific roots or entire root systems during large-scale phenotyping studies. To demonstrate the unique capabilities and high-throughput capacity of this phenotyping platform for studying root systems, genome-wide association studies on rice (Oryza sativa) and maize (Zea mays) root growth were performed and root traits related to aluminium (Al) tolerance were analysed on the parents of the maize nested association mapping (NAM) population.

Three-dimensional root phenotyping with a novel imaging and software platform.

A novel imaging and software platform was developed for the high-throughput phenotyping of three-dimensional root traits during seedling development. To demonstrate the platform's capacity, plants of two rice (Oryza sativa) genotypes, Azucena and IR64, were grown in a transparent gellan gum system and imaged daily for 10 d. Rotational image sequences consisting of 40 two-dimensional images were captured using an optically corrected digital imaging system. Three-dimensional root reconstructions were generated and analyzed using a custom-designed software, RootReader3D. Using the automated and interactive capabilities of RootReader3D, five rice root types were classified and 27 phenotypic root traits were measured to characterize these two genotypes. Where possible, measurements from the three-dimensional platform were validated and were highly correlated with conventional two-dimensional measurements. When comparing gellan gum-grown plants with those grown under hydroponic and sand culture, significant differences were detected in morphological root traits (P < 0.05). This highly flexible platform provides the capacity to measure root traits with a high degree of spatial and temporal resolution and will facilitate novel investigations into the development of entire root systems or selected components of root systems. In combination with the extensive genetic resources that are now available, this platform will be a powerful resource to further explore the molecular and genetic determinants of root system architecture.

Development of a novel aluminum tolerance phenotyping platform used for comparisons of cereal aluminum tolerance and investigations into rice aluminum tolerance mechanisms.

The genetic and physiological mechanisms of aluminum (Al) tolerance have been well studied in certain cereal crops, and Al tolerance genes have been identified in sorghum (Sorghum bicolor) and wheat (Triticum aestivum). Rice (Oryza sativa) has been reported to be highly Al tolerant; however, a direct comparison of rice and other cereals has not been reported, and the mechanisms of rice Al tolerance are poorly understood. To facilitate Al tolerance phenotyping in rice, a high-throughput imaging system and root quantification computer program was developed, permitting quantification of the entire root system, rather than just the longest root. Additionally, a novel hydroponic solution was developed and optimized for Al tolerance screening in rice and compared with the Yoshida's rice solution commonly used for rice Al tolerance studies. To gain a better understanding of Al tolerance in cereals, comparisons of Al tolerance across cereal species were conducted at four Al concentrations using seven to nine genetically diverse genotypes of wheat, maize (Zea mays), sorghum, and rice. Rice was significantly more tolerant than maize, wheat, and sorghum at all Al concentrations, with the mean Al tolerance level for rice found to be 2- to 6-fold greater than that in maize, wheat, and sorghum. Physiological experiments were conducted on a genetically diverse panel of more than 20 rice genotypes spanning the range of rice Al tolerance and compared with two maize genotypes to determine if rice utilizes the well-described Al tolerance mechanism of root tip Al exclusion mediated by organic acid exudation. These results clearly demonstrate that the extremely high levels of rice Al tolerance are mediated by a novel mechanism, which is independent of root tip Al exclusion.

Characterization of AtALMT1 expression in aluminum-inducible malate release and its role for rhizotoxic stress tolerance in Arabidopsis.

Malate transporters play a critical role in aluminum (Al) tolerance responses for some plant species, such as Arabidopsis (Arabidopsis thaliana). Here, we further characterize AtALMT1, an Arabidopsis aluminum-activated malate transporter, to clarify its specific role in malate release and Al stress responses. Malate excretion from the roots of accession Columbia was sharply induced by Al, which is concomitant with the induction of AtALMT1 gene expression. The malate release was specific for Al among rhizotoxic stressors, namely cadmium, copper, erbium, lanthanum, sodium, and low pH, which accounts for the specific sensitivity of a null mutant to Al stress. Al-specific malate excretion can be explained by a combined regulation of AtALMT1 expression and activation of AtALMT1 protein, which is specific for Al. Although low pH treatment slightly induced gene expression, other treatments did not. In addition, malate excretion in Al-activated seedlings was rapidly stopped by removing Al from the solution. Other rhizotoxic stressors were not effective in maintaining malate release. Protein kinase and phosphatase inhibitor studies indicated that reversible phosphorylation was important for the transcriptional and posttranslational regulation of AtALMT1. AtALMT1 promoter-beta-glucuronidase fusion lines revealed that AtALMT1 has restricted expression within the root, such that unnecessary carbon loss is likely minimized. Lastly, a natural nonsense mutation allele of AtALMT1 was identified from the Al-hypersensitive natural accession Warschau-1.

A gene in the multidrug and toxic compound extrusion (MATE) family confers aluminum tolerance in sorghum.

Crop yields are significantly reduced by aluminum toxicity on highly acidic soils, which comprise up to 50% of the world's arable land. Candidate aluminum tolerance proteins include organic acid efflux transporters, with the organic acids forming non-toxic complexes with rhizosphere aluminum. In this study, we used positional cloning to identify the gene encoding a member of the multidrug and toxic compound extrusion (MATE) family, an aluminum-activated citrate transporter, as responsible for the major sorghum (Sorghum bicolor) aluminum tolerance locus, Alt(SB). Polymorphisms in regulatory regions of Alt(SB) are likely to contribute to large allelic effects, acting to increase Alt(SB) expression in the root apex of tolerant genotypes. Furthermore, aluminum-inducible Alt(SB) expression is associated with induction of aluminum tolerance via enhanced root citrate exudation. These findings will allow us to identify superior Alt(SB) haplotypes that can be incorporated via molecular breeding and biotechnology into acid soil breeding programs, thus helping to increase crop yields in developing countries where acidic soils predominate.

Aluminum resistance in maize cannot be solely explained by root organic acid exudation. A comparative physiological study.

Root apical aluminum (Al) exclusion via Al-activated root citrate exudation is widely accepted as the main Al-resistance mechanism operating in maize (Zea mays) roots. Nonetheless, the correlation between Al resistance and this Al-exclusion mechanism has not been tested beyond a very small number of Al-resistant and Al-sensitive maize lines. In this study, we conducted a comparative study of the physiology of Al resistance using six different maize genotypes that capture the range of maize Al resistance and differ significantly in their genetic background (three Brazilian and three North American genotypes). In these maize lines, we were able to establish a clear correlation between root tip Al exclusion (based on root Al content) and Al resistance. Both Al-resistant genotypes and three of the four Al-sensitive lines exhibited a significant Al-activated citrate exudation, with no evidence for Al activation of root malate or phosphate release. There was a lack of correlation between differential Al resistance and root citrate exudation for the six maize genotypes; in fact, one of the Al-sensitive lines, Mo17, had the largest Al-activated citrate exudation of all of the maize lines. Our results indicate that although root organic acid release may play a role in maize Al resistance, it is clearly not the only or the main resistance mechanism operating in these maize roots. A number of other potential Al-resistance mechanisms were investigated, including release of other Al-chelating ligands, Al-induced alkalinization of rhizosphere pH, changes in internal levels of Al-chelating compounds in the root, and Al translocation to the shoot. However, we were unsuccessful in identifying additional Al-resistance mechanisms in maize. It is likely that a purely physiological approach may not be sufficient to identify these novel Al-resistance mechanisms in maize and this will require an interdisciplinary approach integrating genetic, molecular, and physiological investigations.

Identification and characterization of aluminum tolerance loci in Arabidopsis (Landsberg erecta x Columbia) by quantitative trait locus mapping. A physiologically simple but genetically complex trait.

Aluminum (Al) toxicity, which is caused by the solubilization of Al3+ in acid soils resulting in inhibition of root growth and nutrient/water acquisition, is a serious limitation to crop production, because up to one-half of the world's potentially arable land is acidic. To date, however, no Al tolerance genes have yet been cloned. The physiological mechanisms of tolerance are somewhat better understood; the major documented mechanism involves the Al-activated release of Al-binding organic acids from the root tip, preventing uptake into the primary site of toxicity. In this study, a quantitative trait loci analysis of Al tolerance in Arabidopsis was conducted, which also correlated Al tolerance quantitative trait locus (QTL) with physiological mechanisms of tolerance. The analysis identified two major loci, which explain approximately 40% of the variance in Al tolerance observed among recombinant inbred lines derived from Landsberg erecta (sensitive) and Columbia (tolerant). We characterized the mechanism by which tolerance is achieved, and we found that the two QTL cosegregate with an Al-activated release of malate from Arabidopsis roots. Although only two of the QTL have been identified, malate release explains nearly all (95%) of the variation in Al tolerance in this population. Al tolerance in Landsberg erecta x Columbia is more complex genetically than physiologically, in that a number of genes underlie a single physiological mechanism involving root malate release. These findings have set the stage for the subsequent cloning of the genes responsible for the Al tolerance QTL, and a genomics-based cloning strategy and initial progress on this are also discussed.