Soybean Leaf Tissue Research Articles

Exogenous Application of Selenium Nanoparticles (Se-NPs) to Mitigate Salt Stress in Soybean-Evaluation of Physiological, Molecular and Biochemical Processes

AbstractSalt stress is identified as a significant abiotic stress that hampers agricultural sustainability globally. The study was carried out to investigate the potential mitigating effects of selenium nanoparticles (Se-NPs) on salt stress in soybean. Two weeks old grown soybean seedlings were subjected to salt stress conditions (4000 mg L− 1 of sea salts). The plants were foliar sprayed with Se-NPs at concentrations of 0.0, 0.5, 1.0 and 1.5 mg L− 1 twice. The first application was applied at four weeks from sowing and the second application was added after two weeks from the first application. Compared to control, Se-NPs application mitigates the negative effect of salinity on plant growth to a variable extent. This improvement may be attributed to several factors such as increased the concentrations of photosynthetic pigments, total soluble sugars and total protein. In addition, Se-NPs alleviated the adversely effect of oxidative stress by increasing the antioxidant activities and potassium contents without markedly increase in the sodium content of the soybean leaf tissues. Also, Se-NPs enhanced the biosynthesis of secondary metabolites such as total phenolic content under salinity. Moreover, Se-NPs spray significantly reinforced the development of conducting secondary tissues in the leaves and roots of the treated plants. GmHKT1 gene transcription was markedly up-regulated in salinized soybean and foliar sprayed with Se-NPs as a molecular strategy to cope with the salinity. Based on the obtained results, among the different doses of Se-NPs, soybean plants sprayed with 1.0 mg L− 1 Se-NPs showed better salt tolerance. The foliar spray of Se-NPs may be considered as a promising approach to enhance salt tolerance in soybean plants, which could have significant implications for improving agricultural sustainability in salt-affected regions.

A global survey of bicarbonate stress-induced pre-mRNA alternative splicing in soybean via integrative analysis of Iso-seq and RNA-seq

Alternative splicing (AS) plays important roles in modulating environmental stress responses in plants. However, little is known about the functions of bicarbonate-induced AS in cultivated soybean (Glycine max L. Merr.). In this study, we combined PacBio isoform sequencing (Iso-seq) and Illumina RNA sequencing (RNA-seq) to elucidate the bicarbonate-induced AS events in soybean root and leaf tissues. Compared to RNA-seq, Iso-seq identified more novel genes and transcripts, as well as more AS events, indicating that Iso-seq is more efficient in AS detection. Combining these two technologies, we found that intron retention (IR) is the most frequent AS event type. We identified a total of 913 and 1974 bicarbonate stress-responsive differentially alternative spliced genes (DAGs) in soybean leaves and roots respectively, from our RNA-seq results. Additionally, we determined a transcription factor (GmNTL9) and a splicing factor (GmRSZ22), and validated their roles in bicarbonate stress response by AS. Overall, our study opens an avenue for evaluating plant AS regulatory networks, and the obtained global landscape of alternative splicing provides valuable insights into the AS-mediated bicarbonate-responsive mechanisms in plant species.

Quantitative Phosphoproteomic Analysis Provides Insights into the Sodium Bicarbonate Responsiveness of Glycine max.

Sodium bicarbonate stress caused by NaHCO3 is one of the most severe abiotic stresses affecting agricultural production worldwide. However, little attention has been given to the molecular mechanisms underlying plant responses to sodium bicarbonate stress. To understand phosphorylation events in signaling pathways triggered by sodium bicarbonate stress, TMT-labeling-based quantitative phosphoproteomic analyses were performed on soybean leaf and root tissues under 50 mM NaHCO3 treatment. In the present study, a total of 7856 phosphopeptides were identified from cultivated soybeans (Glycine max L. Merr.), representing 3468 phosphoprotein groups, in which 2427 phosphoprotein groups were newly identified. These phosphoprotein groups contained 6326 unique high-probability phosphosites (UHPs), of which 77.2% were newly identified, increasing the current soybean phosphosite database size by 43.4%. Among the phosphopeptides found in this study, we determined 67 phosphopeptides (representing 63 phosphoprotein groups) from leaf tissue and 554 phosphopeptides (representing 487 phosphoprotein groups) from root tissue that showed significant changes in phosphorylation levels under sodium bicarbonate stress (fold change >1.2 or <0.83, respectively; p < 0.05). Localization prediction showed that most phosphoproteins localized in the nucleus for both leaf and root tissues. GO and KEGG enrichment analyses showed quite different enriched functional terms between leaf and root tissues, and more pathways were enriched in the root tissue than in the leaf tissue. Moreover, a total of 53 different protein kinases and 7 protein phosphatases were identified from the differentially expressed phosphoproteins (DEPs). A protein kinase/phosphatase interactor analysis showed that the interacting proteins were mainly involved in/with transporters/membrane trafficking, transcriptional level regulation, protein level regulation, signaling/stress response, and miscellaneous functions. The results presented in this study reveal insights into the function of post-translational modification in plant responses to sodium bicarbonate stress.

Reduction of the severity of Asian soybean rust with foliar application of silicon dioxide

Asian soybean rust (Phakopsora pachyrhizi; ASR) is one of the main diseases that affect soybeans, and it is controlled mainly with chemical fungicides. Induction of plant defense with silicon dioxide (SiO2) application could also be considered to control the disease. However, there is no information on the use of SiO2 as a source of silicon to control ASR. Silicon is an element related to the induction of plant defense mechanisms. Thus, the aim of this study was to verify the in vitro effect of SiO2 on the germination of uredospores of P. pachyrhizi and whether foliar application reduces ASR severity. The in vitro experiment was conducted with six treatments (0; 0.1; 1; 10; 100; 1000 μg.L−1 of SiO2) while the in vivo experiment was carried out with four treatments (0; 1; 2.5; 5% SiO2) under controlled conditions. There was no effect of SiO2 on the germination of uredospores of P. pachyrhizi, yet there was a reduction of up to 85% in the number of uredia with application of 4% SiO2. Plants that received 5% of SiO2 accumulated about 60% more silicon in their leaf tissues compared to the control treatment, increasing 22.99 mg kg−1 of silicon for each percentage point of SiO2 applied. Thus, our study demonstrates that foliar application of SiO2 reduces the severity of ASR and provides greater accumulation of silicon in soybean leaf tissues.

Evaluation of cytosine conversion methods for whole-genome DNA methylation profiling

Background: DNA methylation, the most common epigenetic modification, is defined as the removal or addition of methyl groups to cytosine bases. Studying DNA methylation provides insight into the regulation of gene expression, transposon mobility, genomic stability, and genomic imprinting. Whole-genome DNA methylation profiling (WGDM) is a powerful tool to find DNA methylation. This technique combines standard whole-genome sequencing methodology (e.g., Illumina high-throughput sequencing) with additional steps where unmethylated cytosine is converted to uracil. However, factors such as low cytosine conversion efficiency and inadequate DNA recovery during sample preparation oftentimes render poor-quality data. It is therefore imperative to benchmark sample preparation protocols to increase sequencing data quality and reduce false positives in methylation detection. Methods: A survey analysis was performed to investigate the efficiency of the following commercially available cytosine conversion kits when coupled with the NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB): Zymo Research EZ DNA Methylation™ kit (hereafter known as Zymo Conversion kit), QIAGEN EpiTect Bisulfite kit (hereafter known as QIAGEN Conversion kit), and NEBNext® Enzymatic Methyl-seq Conversion Module (hereafter known as NEB EM-seq kit). Input DNA was derived from soybean (Glycine max [L.] Merrill) leaf tissue. Results: Of those tested, the QIAGEN Conversion kit provided the best sample recovery and the highest number of sequencing reads, whereas the Zymo Conversion kit had the best cytosine conversion efficiency and the least duplication. The sequence library obtained with the NEB EM-seq kit had the highest mapping efficiency (percentage of reads mapped to the genome). The data quality (defined by Phred score) and methylated cytosine call were similar between kits. Conclusions: This study offers the groundwork for selecting an effective DNA methylation detection kit for crop genome research.

Membrane Proteomic Profiling of Soybean Leaf and Root Tissues Uncovers Salt-Stress-Responsive Membrane Proteins.

Cultivated soybean (Glycine max (L.)), the world's most important legume crop, has high-to-moderate salt sensitivity. Being the frontier for sensing and controlling solute transport, membrane proteins could be involved in cell signaling, osmoregulation, and stress-sensing mechanisms, but their roles in abiotic stresses are still largely unknown. By analyzing salt-induced membrane proteomic changes in the roots and leaves of salt-sensitive soybean cultivar (C08) seedlings germinated under NaCl, we detected 972 membrane proteins, with those present in both leaves and roots annotated as receptor kinases, calcium-sensing proteins, abscisic acid receptors, cation and anion channel proteins, proton pumps, amide and peptide transporters, and vesicle transport-related proteins etc. Endocytosis, linoleic acid metabolism, and fatty acid biosynthesis pathway-related proteins were enriched in roots whereas phagosome, spliceosome and soluble NSF attachment protein receptor (SNARE) interaction-related proteins were enriched in leaves. Using label-free quantitation, 129 differentially expressed membrane proteins were found in both tissues upon NaCl treatment. Additionally, the 140 NaCl-induced proteins identified in roots and 57 in leaves are vesicle-, mitochondrial-, and chloroplast-associated membrane proteins and those with functions related to ion transport, protein transport, ATP hydrolysis, protein folding, and receptor kinases, etc. Our proteomic results were verified against corresponding gene expression patterns from published C08 RNA-seq data, demonstrating the importance of solute transport and sensing in salt stress responses.

Glycine max (L.) Merr. (Soybean) metabolome responses to potassium availability

Potassium (K+) has vital physiological and metabolic functions in plants and its availability can impact tolerance to biotic and abiotic stress conditions. Limited studies have investigated the effect of K+ fertilization on soybean metabolism. Using integrated omics, ionomics and metabolomics, we investigated the field-grown Glycine max (soybean) response, after four K+ soil fertilization rates. Soybean leaf and pod tissue (valves and immature seeds) extracts were analysed by ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (UPLC-HRMS) and inductively coupled plasma optical emission spectroscopy (ICP-OES). Multivariate analyses (PCA-X&Y e O2PLS-DA) showed that 51 compounds of 19 metabolic pathways were regulated in response to K+ availability. Under very low potassium availability, soybean plants accumulated of Ca2+, Mg2+, Fe2+, Cu2+, and B in young and old leaves. Potassium fertilization upregulated carbohydrate, galactolipid, and flavonol glycoside biosynthesis in leaves and pod valves, while K+ deficient pod tissues showed increasing amino acids, oligosaccharides, benzoic acid derivatives, and isoflavones contents. Severely K+ deficient soils elicited isoflavones, coumestans, pterocarpans, and soyasaponins in trifoliate leaves, likely associated to oxidative and photodynamic stress status. Additionally, results demonstrate that L-asparagine content is higher in potassium deficient tissues, suggesting this compound as a biomarker of K+ deficiency in soybean plants. These results demonstrate that potassium soil fertilization did not linearly contribute to changes in specialised constitutive metabolites of soybean. Altogether, this work provides a reference for improving the understanding of soybean metabolism as dependent on K+ availability.

ФОТОХІМІЧНА АКТИВНІСТЬ ЛИСТКІВ СОЇ ЗА ДІЇ ФІТОПАТОГЕННИХ БАКТЕРІЙ РІЗНОЇ ВІРУЛЕНТНОСТІ

Soybean plants were grown in a greenhouse. In the phase of 4-6 leaves soybeans were artificially infected with strains of pathogens: Xanthomonas axonopodis pv. glycines 9284 and Xanthomonas axonopodis pv. phaseoli 8843. These strains were obtained from the Ukrainian collection of microorganisms of the Zabolotny institute of Microbiology and Virology of National Academy of Sciences of Ukraine. The photochemical activity of soybean leaves was determined by the biophysical method of chlorophyll fluorescence induction in 13 days after plant inoculation with bacterial strains. A portable Florotest fluorometer was use for measurements. A degree of chlorophyll degradation and suppression of photosynthetic activity in soybean leaves at artificial infection with bacterial strains isolated from different plant species of the Fabaceaeae family dependent from its virulence have been shown. The strain X. axonopodis pv. glycines 9284, isolated from soybean plants, have been highly virulent and acted more negatively than the low virulent to soybean strain X. axonopodis pv. phaseoli 8843. In particular, the value of the minimum fluorescence F0 decreased by 37.0 and 15.4 % for inoculation by strains with different virulence degree: X. axonopodis pv. glycines 9284 (from soybeans) and X. axonopodis pv. phaseoli 8843 (from beans) respectively. There was also a significant decrease in the value of Fm in these variants – by 54.9 % (X. axonopodis pv. glycines 9284) and 19.3 % (X. axonopodis pv. phaseoli 8843). At the same time, the value of maximum quantum yield of PSII (Fv / Fm) was decreased by 11.8 % in the variant with inoculation of soybean plants with the highly virulent strain X. axonopodis pv. glycines 9284, that indicates photoinhibition or damage of antenna complexes of the PSII photosynthetic apparatus of soybean plants. At the same time, only the tendency to reduce the maximum quantum yield of PSII was determined under action infection plants with X. axonopodis pv. phaseoli 8843. A parameter chlorophyll fluorescence decrease ratio Rfd, which shows the activity of CO2 assimilation in the Calvin cycle, was inhibition more at the action of artificial infection with a highly virulent strain – X. axonopodis pv. glycines 9284 – 54.5 and less – at infection with a low virulence strain – X. axonopodis pv. phaseoli 8843 – by 10.8 %. With prolonged action of the pathogenic bacterial strains X.axonopodis pv. glycines 9284 and the X. axonopodis pv. phaseoli 8843 (fortnight) showed significant inhibition of the catalase and peroxidase activity of leaf tissues, which is evidence of an increase in the formation of ROS. It was demonstrated that the highly virulent strain X.axonopodis pv. glycines 9284 most of all suppressed the catalase activity of soybean leaf tissues.

Soybean Leaf Proteomic Profile Influenced by Rhizobacteria Under Optimal and Salt Stress Conditions.

Soil salinity is a major abiotic stressor inhibiting plant growth and development by affecting a range of physiological processes. Plant growth promoting rhizobacteria (PGPR) are considered a sustainable option for alleviation of stress and enhancement of plant growth, yet their mode of action is complex and largely unexplored. In this study, an untargeted proteomic approach provided insights into growth and stress response mechanisms elicited in soybean plants by Rhizobium sp. SL42 and Hydrogenophaga sp. SL48 and co-inoculated with Bradyrhizobium japonicum 532C. The plants were grown under optimal and salt-stressed conditions up to their mid-vegetative stage; shoot growth variables were increased in the bacteria-treated plants. Shotgun proteomics of soybean leaf tissue revealed that a number of proteins related to plant growth and stress tolerance were modulated in the bacterial inoculation treatments. Several key proteins involved in major metabolic pathways of photosynthesis, respiration, and photorespiration were upregulated. These include photosystem I psaK, Rubisco subunits, glyceraldehyde-3-phosphate dehydrogenase, succinate dehydrogenase, and glycine decarboxylase. Similarly, stress response proteins such as catalase and glutathione S-transferase (antioxidants), proline-rich precursor protein (osmolyte), and NADP-dependent malic enzyme (linked to ABA signaling) were increased under salt stress. The functions of proteins related to plant growth and stress adaptation led to an expanded understanding of plant-microbe interactions. These findings suggest that the PGPR strains regulated proteome expression in soybean leaves through multiple signaling pathways, thereby inducing salinity tolerance, and improving plant growth in the presence of this abiotic stress challenge. Data are available via ProteomeXchange with identifier PXD025596.

First report of Mycoleptodiscus terrestris causing root rot of soybean in Indiana.

During the summers in 2019 and 2020, 137 soybean (Glycine max (L.) Merr) seedlings (V1-V3 stage) showing stunting, delayed emergence, and/or crown lesions were collected at Purdue's Agronomy Center for Research and Education in West Lafayette, Indiana. Four seedlings were stunted with reddish-brown girdled lesions along the hypocotyl and crown, rotted tap and lateral roots, and brown discoloration of the cortex and vascular tissues. Four fungal isolates (AC4, AC58, AC96, and AC127) were recovered by plating surface-sterilized symptomatic root tissue onto water agar plates and incubating on the benchtop until mycelia emerged. The growing hyphal tips were transferred to the semi-selective medium DCPA (Andrews and Pitt 1986). On potato dextrose agar, the fungal colonies developed olivaceous green mycelia which melanized into a mat of black microsclerotia with time and no conidia were observed. On 1.5% water agar plates amended with twice autoclaved soybean leaf and root tissue collected from flowering soybean plants, conidia were formed in sporodochia in darkness at 28 οC within one week. Conidia were 1-2 septate, cylindrical with two setae on either end, and measured 20.8 to 26.4 x 4 to 5.6 μm (average 23.9 x 4.7 μm, n=20). The morphological characters matched with the description of Mycoleptodiscus terrestris (Gerd.) Ostaz (Gerdemann 1953). Species identification was further confirmed by sequencing the internal transcribed spacers (ITS) region of rDNA amplified by ITS1 and ITS4 primers (White et al. 1990) and the translation elongation factor 1 alpha (TEF1-α) gene using 983F and 1567R primers with annealing temperature at 53 ○C (Rehner and Buckley 2005). The sequences were deposited in GenBank under the following accession numbers: ITS: MW002684, MT998441, MW010258, and MW010260; and TEF1-α: MW015941-MW015944. The GenBank BLAST searches revealed 100% identity in the ITS region (accession NR_145373.1) and 99.75% identity in the TEF1-α region (MK495977.1) to M. terrestris. Pathogenicity test was conducted on soybean seedlings (cv. Williams) at V1 growth stage using a root dip assay. Isolate AC58 was grown in a modified cotton seed meal broth (CSMB) to produce microsclerotia as inoculum (Gray 1978; Shearer and Jackson 2006). Microsclerotia concentration was measured using a hemocytometer and adjusted to 1.5 x 104 per ml. Five soybean seedlings each were dipped into inoculum or sterile CSMB for 30 minutes then planted individually in vermiculite-filled Styrofoam cups placed on flooded trays in 16-hr photoperiod light racks at room temperature. Seven days after inoculation, all inoculated plants were visibly stunted with root and crown symptoms identical to field symptoms while all controls were healthy. M. terrestris was successfully re-isolated from inoculated plants, but not from the controls, and identified by morphology and sequencing as above. M. terrestris has been previously reported causing root rot of soybean in Illinois (Gray 1978) and Wisconsin (Smith et al. 1998). To our knowledge, this is the first report of M. terrestris infecting soybean in Indiana. Increased geographic distribution of this pathogen warrants more attention for its control. M. terrestris has been proposed as a biological control agent against multiple aquatic weeds (Verma and Charudattan 1993; Shearer and Jackson 2006). Introduction of this fungus into soybean production regions should be avoided.

Analysis of differentially expressed genes in soybean leaf tissue of tolerant and susceptible cultivars under flooding stress revealed by RNA sequencing

Flooding stress causes severe yield reduction in soybean worldwide. The development of stress-tolerant cultivars could be an effective measure to reduce the negative effects of flooding stress. Molecular information on the gene expression pattern of tolerant and susceptible genotypes under flooding stress could be valuable to improve the flooding tolerance in soybean. The objective of this study was to analyze the differentially expressed genes (DEGs) revealed by RNA sequencing in the soybean leaf tissues of tolerant (‘Paldalkong’ and ‘Danbaekkong’) and susceptible (‘NTS1116’) cultivars under flooding stress. Seedlings were grown in a well-watered condition up to the V1–V2 stage and flood-stressed by inundating ~ 10-cm water for 14 days. A total of 22,468 genes were differentially expressed in flood-stressed condition compared to the well-watered control condition, out of which 13,729, 13,405, and 13,160 were differentially expressed in ‘Paldalkong’, ‘Danbaekkong’, and ‘NTS1116’, respectively. A higher number of some of the flooding tolerance-related genes such as lipoxygenase, expansin, glutathione S-transferase, and sugar efflux transporter were up-regulated in the tolerant cultivars than in the susceptible cultivar. The number of some abscisic acid-related transcription factors of basic leucine zipper domain and myeloblastosis families was also higher in the tolerant cultivars than in the susceptible cultivar. The molecular information about the DEGs of tolerant and susceptible cultivars obtained in the present study could be valuable to improve the flooding tolerance in soybeans.

Influence of a Neonicotinoid Seed Treatment on a Nontarget Herbivore of Soybean (Twospotted Spider Mite) and Diet Switching by a Co-occurring Omnivore (Western Flower Thrips).

Insecticidal neonicotinoid seed treatments are a common agricultural insect pest management strategy; however, effects on nontarget pests and omnivorous arthropods are understudied. We used a series of experiments to evaluate impacts of the neonicotinoid seed treatment thiamethoxam on densities of herbivorous twospotted spider mites (Tetranychus urticae Koch [Acari: Tetranychidae]) and feeding behavior of western flower thrips (Frankliniella occidentalis Pergande [Thysanoptera: Thripidae]), an omnivore that feeds on spider mite eggs but is also a significant plant pest. Spider mite densities were higher on neonicotinoid-treated soybeans, but only when mites were not spatially confined. We then examined how availability of thiamethoxam-treated food items (i.e., eggs from spider mites reared on treated soybeans, soybean leaf discs, or a combination of the two), and previous exposure to thiamethoxam-treated soybean impacted thrips feeding. Regardless of the presence of leaf tissue, thrips consumed fewer spider mite eggs laid by females reared on treated soybeans, suggesting spider mite eggs can serve as poisoned prey. Overall, thrips consumed less treated soybean leaf tissue, and thrips on treated leaf discs had a lower percentage of herbivorous feeding events and consumed more nontreated spider mite eggs, indicating a dietary shift from herbivory to predation. The neonicotinoid status of spider mite eggs and prior exposure of thrips also caused shifts in the number and size of leaf scars, likely as a result of altered foraging behavior and/or movement. Shifts between herbivory and predation have implications for thrips damage, virus transmission, and pest management, especially in systems with mixtures of nontreated and neonicotinoid-treated plants.

GmLEA2-1, a Late Embryogenesis Abundant Protein Gene Isolated from Soybean (Glycine max (L.) Merr.), Confers Tolerance to Abiotic Stress

Late embryonic proteins (LEA) gene family was abundant mainly in higher plant embryos, which could protect the embryos from the damage caused by abiotic stress, especially drought and salt stresses. In the present study, GmLEA2-1 was cloned from soybean leaf tissue treated by 10% polyethylene glycol 6000 (PEG6000). The results of quantitative real-time PCR (qRT-PCR) revealed a variety of expression patterns of GmLEA2-1 in various tissues of soybean (root, stem, leaf, flower, pod, early embryo and late embryo). GmLEA2-1 gene shared a lower sequence similarity with other typical LEA genes of same group from different species, but similar functions. Overexpression of GmLEA2-1 in transgenic Arabidopsis thaliana conferred tolerance to drought and salt stresses. The fresh weight and dry weight of seedling, the primary root length and the lateral root density of transgenic Arabidopsis plants were higher than those of wild type Arabidopsis (WT) under drought and salt stresses. Cis-acting regulatory elements in the GmLEA2-1 promoter were also predicted. These data demonstrate that GmLEA2-1 protein play an important role in improving drought and salt tolerance in plants.

High Susceptibility to Cry1Ac and Low Resistance Allele Frequency Reduce the Risk of Resistance of Helicoverpa armigers to Bt Soybean in Brazil.

The Old World bollworm, Helicoverpa armigera (Hübner), was recently introduced into Brazil, where it has caused extensive damage to cotton and soybean crops. MON 87701 × MON 89788 soybean, which expresses the Bt protein Cry1Ac, was recently deployed in Brazil, providing high levels of control against H. armigera. To assess the risk of resistance to the Cry1Ac protein expressed by MON 87701 × MON 89788 soybean in Brazil, we conducted studies to evaluate the baseline susceptibility of H. armigera to Cry1Ac, in planta efficacy including the assessment of the high-dose criterion, and the initial resistance allele frequency based on an F2 screen. The mean Cry1Ac lethal concentration (LC50) ranged from 0.11 to 1.82 μg·mL−1 of diet among all H. armigera field populations collected from crop seasons 2013/14 to 2014/15, which indicated about 16.5-fold variation. MON 87701 × MON 89788 soybean exhibited a high level of efficacy against H. armigera and most likely met the high dose criterion against this target species in leaf tissue dilution bioassays up to 50 times. A total of 212 F2 family lines of H. armigera were established from field collections sampled from seven locations across Brazil and were screened for the presence of MON 87701 × MON 89788 soybean resistance alleles. None of the 212 families survived on MON 87701 × MON 89788 soybean leaf tissue (estimated allele frequency = 0.0011). The responses of H. armigera to Cry1Ac protein, high susceptibility to MON 87701 × MON 89788 soybean, and low frequency of resistance alleles across the main soybean-producing regions support the assumptions of a high-dose/refuge strategy. However, maintenance of reasonable compliance with the refuge recommendation will be essential to delay the evolution of resistance in H. armigera to MON 87701 × MON 89788 soybean in Brazil.

Identification and Comparative Analysis of Differential Gene Expression in Soybean Leaf Tissue under Drought and Flooding Stress Revealed by RNA-Seq.

Drought and flooding are two major causes of severe yield loss in soybean worldwide. A lack of knowledge of the molecular mechanisms involved in drought and flood stress has been a limiting factor for the effective management of soybeans; therefore, it is imperative to assess the expression of genes involved in response to flood and drought stress. In this study, differentially expressed genes (DEGs) under drought and flooding conditions were investigated using Illumina RNA-Seq transcriptome profiling. A total of 2724 and 3498 DEGs were identified under drought and flooding treatments, respectively. These genes comprise 289 Transcription Factors (TFs) representing Basic Helix-loop Helix (bHLH), Ethylene Response Factors (ERFs), myeloblastosis (MYB), No apical meristem (NAC), and WRKY amino acid motif (WRKY) type major families known to be involved in the mechanism of stress tolerance. The expression of photosynthesis and chlorophyll synthesis related genes were significantly reduced under both types of stresses, which limit the metabolic processes and thus help prolong survival under extreme conditions. However, cell wall synthesis related genes were up-regulated under drought stress and down-regulated under flooding stress. Transcript profiles involved in the starch and sugar metabolism pathways were also affected under both stress conditions. The changes in expression of genes involved in regulating the flux of cell wall precursors and starch/sugar content can serve as an adaptive mechanism for soybean survival under stress conditions. This study has revealed the involvement of TFs, transporters, and photosynthetic genes, and has also given a glimpse of hormonal cross talk under the extreme water regimes, which will aid as an important resource for soybean crop improvement.

Soybean Salt Tolerance 1 (GmST1) Reduces ROS Production, Enhances ABA Sensitivity, and Abiotic Stress Tolerance in Arabidopsis thaliana.

Abiotic stresses, including high soil salinity, significantly reduce crop production worldwide. Salt tolerance in plants is a complex trait and is regulated by multiple mechanisms. Understanding the mechanisms and dissecting the components on their regulatory pathways will provide new insights, leading to novel strategies for the improvement of salt tolerance in agricultural and economic crops of importance. Here we report that soybean salt tolerance 1, named GmST1, exhibited strong tolerance to salt stress in the Arabidopsis transgenic lines. The GmST1-overexpressed Arabidopsis also increased sensitivity to ABA and decreased production of reactive oxygen species under salt stress. In addition, GmST1 significantly improved drought tolerance in Arabidopsis transgenic lines. GmST1 belongs to a 3-prime part of Glyma.03g171600 gene in the current version of soybean genome sequence annotation. However, comparative reverse transcription-polymerase chain reaction analysis around Glyma.03g171600 genomic region confirmed that GmST1 might serve as an intact gene in soybean leaf tissues. Unlike Glyma.03g171600 which was not expressed in leaves, GmST1 was strongly induced by salt treatment in the leaf tissues. By promoter analysis, a TATA box was detected to be positioned close to GmST1 start codon and a putative ABRE and a DRE cis-acting elements were identified at about 1 kb upstream of GmST1 gene. The data also indicated that GmST1-transgenic lines survived under drought stress and showed a significantly lower water loss than non-transgenic lines. In summary, our results suggest that overexpression of GmST1 significantly improves Arabidopsis tolerance to both salt and drought stresses and the gene may be a potential candidate for genetic engineering of salt- and drought-tolerant crops.

High susceptibility and low resistance allele frequency of Chrysodeixis includens (Lepidoptera: Noctuidae) field populations to Cry1Ac in Brazil.

The soybean looper (SBL), Chrysodeixis includens (Walker), is one of the most important soybean pests in Brazil. MON 87701 × MON 89788 soybean expressing Cry1Ac has been recently deployed in Brazil, providing high levels of control against the primary lepidopteran pests. To support insect resistance management (IRM) programmes, the baseline susceptibility of SBL to Cry1Ac was assessed, and the resistance allele frequency was estimated on the basis of an F2 screen. The toxicity (LC50 ) of Cry1Ac ranged from 0.39 to 2.01 µg mL(-1) diet among all SBL field populations collected from crop seasons 2008/09 to 2012/13, which indicated approximately fivefold variation. Cry1Ac diagnostic concentrations of 5.6 and 18 µg mL(-1) diet were established for monitoring purposes, and no shift in mortality was observed. A total of 626 F2 family lines derived from SBL collected from locations across Brazil during crop season 2014/15 were screened for the presence of Cry1Ac resistance alleles. None of the 626 families survived on MON 87701 × MON 89788 soybean leaf tissue (joint frequency 0.0004). SBL showed high susceptibility and low resistance allele frequency to Cry1Ac across the main soybean-producing regions in Brazil. These findings meet important criteria for effective IRM strategy. © 2015 Society of Chemical Industry.

Developmental profiling of gene expression in soybean trifoliate leaves and cotyledons.

BackgroundImmediately following germination, the developing soybean seedling relies on the nutrient reserves stored in the cotyledons to sustain heterotrophic growth. During the seed filling period, developing seeds rely on the transport of nutrients from the trifoliate leaves. In soybean, both cotyledons and leaves develop the capacity for photosynthesis, and subsequently senesce and abscise once their function has ended. Before this occurs, the nutrients they contain are mobilized and transported to other parts of the plant. These processes are carefully orchestrated by genetic regulation throughout the development of the leaf or cotyledon.ResultsTo identify genes involved in the processes of leaf or cotyledon development and senescence in soybean, we used RNA-seq to profile multiple stages of cotyledon and leaf tissues. Differentially expressed genes between stages of leaf or cotyledon development were determined, major patterns of gene expression were defined, and shared genes were identified. Over 38,000 transcripts were expressed during the course of leaf and cotyledon development. Of those transcripts, 5,000 were expressed in a tissue specific pattern. Of the genes that were differentially expressed between both later stage tissues, 90 % had the same direction of change, suggesting that the mechanisms of senescence are conserved between tissues. Analysis of the enrichment of biological functions within genes sharing common expression profiles highlights the main processes occurring within these defined temporal windows of leaf and cotyledon development. Over 1,000 genes were identified with predicted regulatory functions that may have a role in control of leaf or cotyledon senescence.ConclusionsThe process of leaf and cotyledon development can be divided into distinct stages characterized by the expression of specific gene sets. The importance of the WRKY, NAC, and GRAS family transcription factors as major regulators of plant senescence is confirmed for both soybean leaf and cotyledon tissues. These results help validate functional annotation for soybean genes and promoters.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0553-y) contains supplementary material, which is available to authorized users.

First Report of Phakopsora pachyrhizi Causing Rust on Soybean in Malawi.

Soybean rust (SBR), caused by Phakopsora pachyrhizi, has become established in Africa since the first report in Uganda in 1996 (2). The urediniospores, as windborne propagules, have infested new regions of Africa, initiating SBR in many countries, including Ghana and Democratic Republic of the Congo in 2007 (4) and Tanzania in 2014 (3). No refereed reports have been published about rust in Malawi, but some people have indicated that soybean rust may have been observed as early as 2008. Typical symptoms and signs of SBR, including leaf yellowing and tan, sporulating uredinia, were observed on soybean in May 2014 during field surveys in the major soybean-growing areas of Malawi, including the central (Dowa, Mchinji, and Kasungu) and southern (Thyolo) regions in nine out of 12 sites surveyed. When microscopically examined, urediniospores were elliptical, echinulate, and hyaline to pale yellowish brown. Leaves exhibiting sporuliferous uredinia were sent by APHIS permit to the University of Illinois. To confirm the pathogen, symptomatic soybean leaf tissue of approximately 1 cm2 was excised from each of the samples, and DNA was extracted using the FastDNA Spin Kit (MP Biomedicals, Solon, OH), with further purification using the MicroElute DNA Clean-up Kit (Omega Bio-Tek, Norcross, GA). The resulting DNA was analyzed by quantitative PCR using published Taqman assays for P. pachyrhizi and P. meibomiae, with a multiplexed exogenous internal control reaction to validate negative results (1). P. pachyrhizi DNA was detected in excess of 180,000 genome equivalents/cm2 in all samples, indicating a substantial infection. P. meibomiae DNA was determined to be absent from all samples, within the limit of quantification of ~2 pg DNA/cm2. Urediniospores dislodged from three leaves and inoculated onto susceptible soybean cultivar Williams 82 produced tan lesions after 2 weeks of incubation in a detached-leaf assay. This is the first confirmed report of P. pachyrhizi causing rust on soybean in Malawi, putting at risk 14,000 ha currently under soybean production. The reports of soybean rust in Malawi and adjoining countries will alter soybean production practices and research interests. In some cases, foliar application of fungicides has increased and planting dates have been changed to avoid conditions that are most conducive for rust development. Efforts to understand the virulence and genetic diversity of the pathogen in the region are needed in order to develop and deploy resistant cultivars.

First Report of Phakopsora pachyrhizi on Soybean Causing Rust in Tanzania.

Phakopsora pachyrhizi Syd. was reported on legume hosts other than soybean in Tanzania as early as 1979 (1). Soybean rust (SBR), caused by P. pachyrhizi, was first reported on soybean in Africa in Uganda in 1996 (3), and its introduction into Africa was proposed to occur through urediniospores blowing from western India to the African east coastal areas by moist northeast monsoon winds (4). The fungus rapidly spread and was reported on soybean in South Africa in 2001, in western Cameroon in 2003, and in Ghana and the Democratic Republic of the Congo in 2007 (5). A second species causing SBR on soybean, P. meibomiae, has not been reported in Africa or elsewhere, outside of the Americas. From 2012 to 2014, symptomatic leaf samples were collected in the major soybean growing areas of the Tanzanian Southern Highlands (Iringa, Mbeya, and Ruvuma regions). Symptoms of SBR included yellowing of leaves and tan sporulating lesions. These symptoms were observed at flowering through seed maturity. From fields surveyed in 2012, 2013, and 2014, SBR was observed in 5 of 14, 7 of 11, and 14 of 31 fields, respectively. Some of the leaves sampled had up to 80% of the leaf area affected. When microscopically examined, urediniospores were elliptical, echinulate, and hyaline to pale yellowish brown. In 2014, sporuliferous uredinia were observed on leaf material collected from the Iringa and Ruvuma regions of Tanzania, and a subset of these samples was sent by APHIS permit to the University of Illinois. To confirm the pathogen, symptomatic soybean leaf tissue of approximately 1 cm2 was excised from each of the samples, and DNA was extracted using the FastDNA Spin Kit (MP Biomedicals, Solon, OH), with further purification using the MicroElute DNA Clean-up Kit (Omega Bio-Tek, Norcross, GA). The DNA was subjected to quantitative PCR using published Taqman assays for P. pachyrhizi, P. meibomiae, and a multiplexed exogenous internal control reaction to validate negative results (2). P. pachyrhizi DNA was detected in excess of 66,000 genome equivalents/cm2 in all samples, and P. meibomiae DNA was determined to be absent from all samples (limit of quantification ~2 pg DNA/cm2). Free surviving urediniospores were dislodged from 12 samples and inoculated onto susceptible soybean cultivar Williams 82, which produced sporulating SBR lesions after 2 weeks of incubation in a detached-leaf assay. Thus, Koch's postulates were completed. This is the first report of P. pachyrhizi causing rust on soybean in Tanzania. In vivo cultures have been established from most of these samples, and ongoing research includes an evaluation of the P. pachyrizi virulence on a differential set, and characterization of the genetic diversity.