Molecular analysis of SCARECROW genes expressed in white lupin cluster roots

The Scarecrow (SCR) transcription factor plays a crucial role in root cell radial patterning and is required for maintenance of the quiescent centre and differentiation of the endodermis. In response to phosphorus (P) deficiency, white lupin (Lupinus albus L.) root surface area increases some 50-fold to 70-fold due to the development of cluster (proteoid) roots. Previously it was reported that SCR-like expressed sequence tags (ESTs) were expressed during early cluster root development. Here the cloning of two white lupin SCR genes, LaSCR1 and LaSCR2, is reported. The predicted amino acid sequences of both LaSCR gene products are highly similar to AtSCR and contain C-terminal conserved GRAS family domains. LaSCR1 and LaSCR2 transcript accumulation localized to the endodermis of both normal and cluster roots as shown by in situ hybridization and gene promoter::reporter staining. Transcript analysis as evaluated by quantitative real-time-PCR (qRT-PCR) and RNA gel hybridization indicated that the two LaSCR genes are expressed predominantly in roots. Expression of LaSCR genes was not directly responsive to the P status of the plant but was a function of cluster root development. Suppression of LaSCR1 in transformed roots of lupin and Medicago via RNAi (RNA interference) delivered through Agrobacterium rhizogenes resulted in decreased root numbers, reflecting the potential role of LaSCR1 in maintaining root growth in these species. The results suggest that the functional orthologues of AtSCR have been characterized

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

Identification of markers associated with race-specific resistance to Aphanomyces root rot in alfalfa, Poster at the 2017 APS meeting

Aphanomyces root rot, caused by Aphanomyces euteiches, is one of the most important diseases of alfalfa in the United States. Two races of the pathogen are recognized and although most cultivars are resistant to race 1, fewer have resistance to race 2, the predominant race in North America. Molecular markers are needed to facilitate breeding for resistance and to clarify race/resistance gene structure. Resistant and susceptible seedlings were identified from three resistant cultivars, WAPH1, WAPH5 and 53V52, and used as parents to produce F1 populations. Severity of symptoms corresponded with amount of pathogen DNA and oospore density in roots. Race-specific resistance involves a hypersensitive response of individual epidermal or cortical cells upon pathogen attack followed by suberization of cells surrounding the stele and strong autofluorescence in cortical cells, indicating the presence of phenolic compounds. Segregation ratios of F1 populations suggested that resistance to race 1 in WAPH1 is conditioned by a single gene but resistance to race 1 is multigenic in WAPH5 and 53V52, and resistance to race 2 is multigenic in all three cultivars. Segregation for resistance to seven strains of A. euteiches in 70 F1 full-sib plants derived from 53V52 suggested the presence of clustered resistance genes and multiple race types. Identification of resistance gene loci is in progress using genotyping by sequencing and genetic mapping of F1 populations

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 plant's 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.This work was supported by grant AGL2002-02876 from the Dirección General de Investigación, Ministry of Science and Technology (Spain).Peer reviewe

Management Strategies and Distribution of Aphanomyces Root Rot of Alfalfa (Medicago sativa), a continuing threat to forage production in the United States

Alfalfa (Medicago sativa) is one of several legumes that is affected by Aphanomyces root rot (ARR) caused by Aphanomyces euteiches. Symptoms of ARR on alfalfa seedlings include a yellow-grey discolouration of roots, rotting and loss of lateral roots, stunted growth, chlorotic foliage and reduction of nitrogen-producing nodules on roots. Infection can also occur on adult plants leading to loss of lateral roots and nodules. At the seedling stage, ARR decreases alfalfa stand establishment, and field longevity is reduced when adult plants are infected. A. euteiches is an oomycete pathogen that has motile zoospores and thick-walled oospores that can survive for many years in soil. Two races are currently recognized by pathogenicity on differential alfalfa check cultivars. Most alfalfa cultivars contain race 1 resistance, but there is an increasing development of cultivars with resistance to race 2. Management strategies include planting resistant cultivars, avoiding planting in fields with poor drainage and rotating crops with nonhost plants

Knockdown of CELL DIVISION CYCLE16 Reveals an Inverse Relationship between Lateral Root and Nodule Numbers and a Link to Auxin in Medicago truncatula1[W][OA]

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

Covid-19 Occupational Risk Incidence and Working Sectors Involved During the Pandemic in Italy

Background: Starting from March 2020 until December 2021, different phases of Covid-19 pandemic have been identified in Italy, with several containing/lifting measures progressively enforced by the National government. In the present study, we investigate the change in occupational risk during the subsequent pandemic phases and we propose an estimate of the incidence of the cases by economic sector, based on the analysis of insurance claims for compensation for Covid-19. Methods: Covid-19 epidemiological data available for the general population and injury claims of workers covered by the Italian public insurance system in 2020–2021 were analyzed. Monthly Incidence Rate of Covid-19 compensation claims per 100,000 workers (MIRw) was calculated by the economic sector and compared with the same indicator for general population in different pandemic periods. Results: The distribution of Covid-19 MIRw by sector significantly changed during the pandemic related to both the strength of different waves and the mitigation/lifting strategies enforced. The level of occupational fraction was very high at the beginning phase of the pandemic, decreasing to 5% at the end of 2021. Healthcare and related services were continuously hit but the incidence was significantly decreasing in 2021 in all sectors, except for postal and courier activities in transportation and storage enterprises. Conclusion: The analysis of compensation claim data allowed to identify time trends for infection risk in different working sectors. The claim rates were highest for human health and social work activities but the distribution of risk among sectors was clearly influenced by the different stages of the pandemic

Transcript profiling of common bean (<it>Phaseolus vulgaris </it>L.) using the GeneChip^®Soybean Genome Array: optimizing analysis by masking biased probes

<p>Abstract</p> <p>Background</p> <p>Common bean (<it>Phaseolus vulgaris </it>L.) and soybean (<it>Glycine max</it>) both belong to the <it>Phaseoleae </it>tribe and share significant coding sequence homology. This suggests that the GeneChip^®Soybean Genome Array (soybean GeneChip) may be used for gene expression studies using common bean.</p> <p>Results</p> <p>To evaluate the utility of the soybean GeneChip for transcript profiling of common bean, we hybridized cRNAs purified from nodule, leaf, and root of common bean and soybean in triplicate to the soybean GeneChip. Initial data analysis showed a decreased sensitivity and accuracy of measuring differential gene expression in common bean cross-species hybridization (CSH) GeneChip data compared to that of soybean. We employed a method that masked putative probes targeting inter-species variable (ISV) regions between common bean and soybean. A masking signal intensity threshold was selected that optimized both sensitivity and accuracy of measuring differential gene expression. After masking for ISV regions, the number of differentially-expressed genes identified in common bean was increased by 2.8-fold reflecting increased sensitivity. Quantitative RT-PCR (qRT-PCR) analysis of 20 randomly selected genes and purine-ureide pathway genes demonstrated an increased accuracy of measuring differential gene expression after masking for ISV regions. We also evaluated masked probe frequency per probe set to gain insight into the sequence divergence pattern between common bean and soybean. The sequence divergence pattern analysis suggested that the genes for basic cellular functions and metabolism were highly conserved between soybean and common bean. Additionally, our results show that some classes of genes, particularly those associated with environmental adaptation, are highly divergent.</p> <p>Conclusions</p> <p>The soybean GeneChip is a suitable cross-species platform for transcript profiling in common bean when used in combination with the masking protocol described. In addition to transcript profiling, CSH of the GeneChip in combination with masking probes in the ISV regions can be used for comparative ecological and/or evolutionary genomics studies.</p