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Carotenoid cleavage oxygenases (CCOs) enzymes play a vital role in plant growth and development through the synthesis of apocarotenoids and their derivative. These chemicals are necessary for flower and fruit coloration, as well as the manufacture of plant hormones such as abscisic acid (ABA) and strigolactones, which control a variety of physiological processes. The CCOs gene family has not been characterized in Arachis hypogaea. Genome mining of A. hypogaea identifies 24 AhCCO gene members. The AhCCO gene family was divided into two subgroups based on the recent study of the Arabidopsis thaliana CCO gene family classification system. Twenty-three AhCCO genes, constituting 95.8% of the total, were regulated by 29 miRNAs, underscoring the significance of microRNAs (miRNAs) in governing gene expression in peanuts. AhCCD19 is the only gene that lacks a miRNA target site. The physicochemical characteristics of CCO genes and their molecular weights and isoelectric points were studied further. The genes were then characterized regarding chromosomal distribution, structure, and promoter cis-elements. Light, stress development, drought stress, and hormone responsiveness were discovered to be associated with AhCCO genes, which can be utilized in developing more resilient crops. The investigation also showed the cellular location of the encoded proteins and discovered that the peanut carotenoid oxygenase gene family's expansion was most likely the result of tandem, segmental, and whole-genome duplication events. The localization expresses the abundance of genes mostly in the cytoplasm and chloroplast. Expression analysis shows that AhCCD7 and AhCCD14 genes show the maximum expression in the apical meristem, lateral leaf, and pentafoliate leaf development, while AhNCED9 and AhNCED13 express in response to Aspergillus flavus resistance. This knowledge throws light on the evolutionary history of the AhCCO gene family and may help researchers better understand the molecular processes behind gene duplication occurrences in plants. An integrated synteny study was used to find orthologous carotenoid oxygenase genes in A. hypogaea, whereas Arabidopsis thaliana and Beta vulgaris were used as references for the functional characterization of peanut CCO genes. These studies provide a foundation for future research on the regulation and functions of this gene family. This information provides valuable insights into the genetic regulation of AhCCO genes. This technology could create molecular markers for breeding programs to develop new peanut lines., (© 2024. The Author(s).)

The peanut plant is one of the most economically important crops around the world. Abiotic stress, such as drought, causes over five hundred million dollars in losses in peanut production per year. Peanuts are known to produce prenylated stilbenoids to counteract biotic stress. However, their role in abiotic stress tolerance has not been elucidated. To address this issue, hairy roots with the capacity to produce prenylated stilbenoids were established. An RNA-interference (RNAi) molecular construct targeting the stilbenoid-specific prenyltransferase AhR4DT-1 was designed and expressed via Agrobacterium rhizogenes-mediated transformation in hairy roots of peanut cultivar Georgia Green. Two transgenic hairy roots with the RNAi molecular construct were established, and the downregulation of AhR4DT-1 was validated using reverse transcriptase quantitative PCR. To determine the efficacy of the RNAi-approach in modifying the levels of prenylated stilbenoids, the hairy roots were co-treated with methyl jasmonate, hydrogen peroxide, cyclodextrin, and magnesium chloride to induce the production of stilbenoids and then the stilbenoids were analyzed in extracts of the culture medium. Highly reduced levels of prenylated stilbenoids were observed in the RNAi hairy roots. Furthermore, the hairy roots were evaluated in a polyethylene glycol (PEG) assay to assess the role of prenylated stilbenoids on water-deficit stress. Upon PEG treatment, stilbenoids were induced and secreted into the culture medium of RNAi and wild-type hairy roots. Additionally, the biomass of the RNAi hairy roots decreased by a higher amount as compared to the wild-type hairy roots suggesting that prenylated stilbenoids might play a role against water-deficit stress., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

The frequency and severity of extreme climatic conditions such as drought, salinity, cold, and heat are increasing due to climate change. Moreover, in the field, plants are affected by multiple abiotic stresses simultaneously or sequentially. Thus, it is imperative to compare the effects of stress combinations on crop plants relative to individual stresses. This study investigated the differential regulation of physio-biochemical and metabolomics parameters in peanut ( Arachis hypogaea L.) under individual (salt, drought, cold, and heat) and combined stress treatments using multivariate correlation analysis. The results showed that combined heat, salt, and drought stress compounds the stress effect of individual stresses. Combined stresses that included heat had the highest electrolyte leakage and lowest relative water content. Lipid peroxidation and chlorophyll contents did not significantly change under combined stresses. Biochemical parameters, such as free amino acids, polyphenol, starch, and sugars, significantly changed under combined stresses compared to individual stresses. Free amino acids increased under combined stresses that included heat; starch, sugars, and polyphenols increased under combined stresses that included drought; proline concentration increased under combined stresses that included salt. Metabolomics data that were obtained under different individual and combined stresses can be used to identify molecular phenotypes that are involved in the acclimation response of plants under changing abiotic stress conditions. Peanut metabolomics identified 160 metabolites, including amino acids, sugars, sugar alcohols, organic acids, fatty acids, sugar acids, and other organic compounds. Pathway enrichment analysis revealed that abiotic stresses significantly affected amino acid, amino sugar, and sugar metabolism. The stress treatments affected the metabolites that were associated with the tricarboxylic acid (TCA) and urea cycles and associated amino acid biosynthesis pathway intermediates. Principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA), and heatmap analysis identified potential marker metabolites (pinitol, malic acid, and xylopyranose) that were associated with abiotic stress combinations, which could be used in breeding efforts to develop peanut cultivars that are resilient to climate change. The study will also facilitate researchers to explore different stress indicators to identify resistant cultivars for future crop improvement programs.

Nitric oxide-mediated S-nitrosation through S-nitrosoglutathione reductase (GSNOR) plays important roles in cellular processes and signaling of plants; however, the regulatory mechanism of programmed cell death (PCD) by S-nitrosation remains unclear. In this study, the S-nitrosated proteomic and functions of GSNOR during Al-induced PCD in peanut were investigated. Al stress induced an increase of S-nitrosothiol (SNO) content and GSNOR activity in Al-induced PCD. There was significant positive correlation between SNO content and hydrogen peroxide content. The S-nitrosated proteomic analysis identified 402 S-nitrosated proteins containing 551 S-nitrosated sites during Al-induced PCD in the root tips of peanut. These S-nitrosated proteins were involved in regulation of various biological processes including energy metabolism, maintenance of cell wall function and organic acid secretion. Among them, 128 S-nitrosated proteins were up-regulated and one was down-regulated after Al stress. Experiments with recombinant AhGSNOR revealed that activity of the enzyme was inhibited by its S-nitrosation, with a moderate decrease of 17.9 % after 100 μM GSNO incubation. These data provide novel insights to understanding the functional mechanism of NO-mediated S-nitrosation during plant PCD., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Stenotrophomonas maltophilia has plant growth-promoting potential, and interaction with Arachis hypogaea changes host-plant physiology, biochemistry, and metabolomics, which provides tolerance under the N 2 starvation conditions. About 226 suppression subtractive hybridization clones were obtained from plant-microbe interaction, of which, about 62% of gene sequences were uncharacterized, whereas 23% of sequences were involved in photosynthesis. An uncharacterized SSH clone, SM409 (full-length sequence showed resemblance with Cytb6), showed about 4-fold upregulation during the interaction was transformed to tobacco for functional validation. Overexpression of the AhCytb6 gene enhanced the seed germination efficiency and plant growth under N 2 deficit and salt stress conditions compared to wild-type and vector control plants. Results confirmed that transgenic lines maintained high photosynthesis and protected plants from reactive oxygen species buildup during stress conditions. Microarray-based whole-transcript expression of host plants showed that out of 272,410 genes, 8704 and 24,409 genes were significantly (p < 0.05) differentially expressed (> 2 up or down-regulated) under N 2 starvation and salt stress conditions, respectively. The differentially expressed genes belonged to different regulatory pathways. Overall, results suggested that overexpression of AhCytb6 regulates the expression of various genes to enhance plant growth under N 2 deficit and abiotic stress conditions by modulating plant physiology.

Background: As an important cash crop, the yield of peanut is influenced by soil acidification and pathogen infection. Receptor-like protein kinases play important roles in plant growth, development and stress responses. However, little is known about the number, location, structure, molecular phylogeny, and expression of RLKs in peanut, and no comprehensive analysis of RLKs in the Al stress response in peanuts have been reported., Results: A total of 1311 AhRLKs were identified from the peanut genome. The AhLRR-RLKs and AhLecRLKs were further divided into 24 and 35 subfamilies, respectively. The AhRLKs were randomly distributed across all 20 chromosomes in the peanut. Among these AhRLKs, 9.53% and 61.78% originated from tandem duplications and segmental duplications, respectively. The ka/ks ratios of 96.97% (96/99) of tandem duplication gene pairs and 98.78% (646/654) of segmental duplication gene pairs were less than 1. Among the tested tandem duplication clusters, there were 28 gene conversion events. Moreover, all total of 90 Al-responsive AhRLKs were identified by mining transcriptome data, and they were divided into 7 groups. Most of the Al-responsive AhRLKs that clustered together had similar motifs and evolutionarily conserved structures. The gene expression patterns of these genes in different tissues were further analysed, and tissue-specifically expressed genes, including 14 root-specific Al-responsive AhRLKs were found. In addition, all 90 Al-responsive AhRLKs which were distributed unevenly in the subfamilies of AhRLKs, showed different expression patterns between the two peanut varieties (Al-sensitive and Al-tolerant) under Al stress., Conclusions: In this study, we analysed the RLK gene family in the peanut genome. Segmental duplication events were the main driving force for AhRLK evolution, and most AhRLKs subject to purifying selection. A total of 90 genes were identified as Al-responsive AhRLKs, and the classification, conserved motifs, structures, tissue expression patterns and predicted functions of Al-responsive AhRLKs were further analysed and discussed, revealing their putative roles. This study provides a better understanding of the structures and functions of AhRLKs and Al-responsive AhRLKs.

Polyamines play an important role in stress response. In the pathway of polyamines synthesis, S-adenosylmethionine decarboxylase (SAMDC) is one of the key enzymes. In this study, a full length cDNA of SAMDC (AhSAMDC) was isolated from peanut (Arachis hypogaea L.). Phylogenetic analysis revealed high sequence similarity between AhSAMDC and SAMDC from other plants. In peanut seedlings exposed to sodium chloride (NaCl), the transcript level of AhSAMDC in roots was the highest at 24 h that decreased sharply at 72 and 96 h after 150 mM NaCl treatment. However, the expression of AhSAMDC in peanut leaves was significantly inhibited, and the transcript levels in leaves were not different compared with control These results implied the tissue-specific and time-specific expression of AhSAMDC. The physiological effects and functional mechanism of AhSAMDC were further evaluated by overexpressing AhSAMDC in tobaccos. The transgenic tobacco lines exhibited higher germination rate and longer root length under salt stress. Reduced membrane damage, higher antioxidant enzyme activity, and higher proline content were also observed in the transgenic tobacco seedlings. What's more, AhSAMDC also led to higher contents of spermidine and spermine, which can help to scavenge reactive oxygen species. Together, this study suggests that AhSAMDC enhances plant resistance to salt stress by improving polyamine content and alleviating membrane damage., (© 2020 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.)

The resveratrol-producing rice (Oryza sativa L.) inbred lines, Iksan 515 (I.515) and Iksan 526 (I.526), developed by the expression of the groundnut (Arachis hypogaea) resveratrol synthase 3 (AhRS3) gene in the japonica rice cultivar Dongjin, accumulated both resveratrol and its glucoside, piceid, in seeds. Here, we investigated the effect of the AhRS3 transgene on the expression of endogenous piceid biosynthesis genes (UGTs) in the developing seeds of the resveratrol-producing rice inbred lines. Ultra-performance liquid chromatography (UPLC) analysis revealed that I.526 accumulates significantly higher resveratrol and piceid in seeds than those in I.515 seeds and, in I.526 seeds, the biosynthesis of resveratrol and piceid reached peak levels at 41 days after heading (DAH) and 20 DAH, respectively. Furthermore, RNA-seq analysis showed that the expression patterns of UGT genes differed significantly between the 20 DAH seeds of I.526 and those of Dongjin. Quantitative real-time PCR (RT-qPCR) analyses confirmed the data from RNA-seq analysis in seeds of Dongjin, I.515 and I.526, respectively, at 9 DAH, and in seeds of Dongjin and I.526, respectively, at 20 DAH. A total of 245 UGTs, classified into 31 UGT families, showed differential expression between Dongjin and I.526 seeds at 20 DAH. Of these, 43 UGTs showed more than 2-fold higher expression in I.526 seeds than in Dongjin seeds. In addition, the expression of resveratrol biosynthesis genes (PAL, C4H and 4CL) was also differentially expressed between Dongjin and I.526 developing seeds. Collectively, these data suggest that AhRS3 altered the expression pattern of UGT genes, and PAL, C4H and 4CL in developing rice seeds., Competing Interests: The authors have declared that no competing interests exist.

Flavonol is an important functional bioactive substance in peanut seeds, and plays important roles responding to abiotic stress. The flavonol content is closely related to the activity and regulation of gene expression patterns of flavonol synthase (FLS). In this study, eight FLS genes, AhFLSs were cloned and their expression characterization in different peanut organ and seedling under different abiotic stress were conducted. The results showed that the expressions levels of AhFLSs were differed in all assayed peanut organs and seedlings under abiotic stress treatments. Expression levels of AhFLS2, AhFLS3, AhFLS4, and AhFLS6 were higher than those of other AhFLSs. The flavonol contents of peanut organs and seedlings under different abiotic stress were also determined using high performance liquid chromatography (HPLC). Dried mature peanut seeds were the organ tissue with the highest flavonol content, and flavonol content increased with seed development. Under abiotic stress treatments, the types of flavonols induced differed among stress treatments. Correlation analysis results suggested that eight AhFLS genes may have different functions in peanut. Moreover, changes in the expression of the eight genes appear to has substrate preference. These results can lay the foundation for the study of improving nutritional value of peanut seed and resistance of peanut plant.

GPAT, the rate-limiting enzyme in triacylglycerol (TAG) synthesis, plays an important role in seed oil accumulation. In this study, two AhGPAT9 genes were individually cloned from the A- and B- genomes of peanut, which shared a similarity of 95.65%, with 165 site differences. The overexpression of AhGPAT9 or the knock-down of its gene expression increased or decreased the seed oil content, respectively. Allelic polymorphism analysis was conducted in 171 peanut germplasm, and 118 polymorphic sites in AhGPAT9A formed 64 haplotypes (a1 to a64), while 94 polymorphic sites in AhGPAT9B formed 75 haplotypes (b1 to b75). The haplotype analysis showed that a5, b57, b30 and b35 were elite haplotypes related to high oil content, whereas a7, a14, a48, b51 and b54 were low oil content types. Additionally, haplotype combinations a62/b10, a38/b31 and a43/b36 were associated with high oil content, but a9/b42 was a low oil content haplotype combination. The results will provide valuable clues for breeding new lines with higher seed oil content using hybrid polymerization of high-oil alleles of AhGPAT9A and AhGPAT9B genes.

Crop plants always encounter multiple stresses in the natural environment. Here, the effects of the fungicide zinc thiazole (ZT) on propagation of Ralstonia solanacearum, a bacterial pathogen, were investigated in peanut seedlings under salt stress. Compared with water control, salt stress markedly reduced pathogen resistance in peanut seedlings. However, impaired pathogen resistance was alleviated by treatment with dimethylthiourea, a specific ROS scavenger, or ZT. Subsequently, salt stress or combined salt and pathogen treatment resulted in inhibition of photosynthesis, loss of chlorophyll and accumulation of thiobarbituric acid reactive substances, which could be reversed by ZT. In addition, ZT treatment suppressed the salt stress up-regulated Na+ content and Na+/K+ ratios in peanut roots. Furthermore, salt stress or combined salt and pathogen treatment impaired the activities of antioxidant (e.g. superoxide dismutase/SOD and catalase/CAT), and defense-related (e.g. phenylalanine ammonia lyase /PAL and polyphenol oxidase/PPO) enzymes, which could be rescued by addition of ZT. In contrast, only slight changes of SOD and CAT activities were observed in pathogen-infected seedlings. Similarly, activities of PAL and PPO were slightly modified by salt stress in peanut seedlings. These results suggest that the ZT-enhanced pathogen resistance can be partly attributed to the improvement of photosynthetic capacity and defense enzyme activities, and also the inhibition of Na+/K+ ratios, in this salt-stressed crop plant., Competing Interests: The authors have declared that no competing interests exist.

The development of male sterile plants is a prerequisite to developing hybrid varieties to harness the benefits of hybrid vigor in crops and enhancing crop productivity for sustainable agriculture. In plants, cysteine proteases have been known for their multifaceted roles during programmed cell death, and in ubiquitin- and proteasome-mediated proteolysis. Here, we showed that Arachis diogoi cysteine protease (AdCP) expressed under the TA-29 promoter induced complete male sterility in Indian mustard, Brassica juncea. The herbicide resistance gene bar was used for the selection of transgenic plants. Mustard transgenic plants exhibited male sterile phenotype and failed to produce functional pollen grains. Irregularly shaped aborted pollen grains with groove-like structures were observed in male sterile plants during scanning electron microscopy analysis. The T 1 progeny plants obtained from the seed of primary transgenic male sterile plants crossed with the wild-type plants exhibited segregation of the progeny into male sterile and fertile plants with normal seed development. Further, male sterile plants exhibited higher transcript levels of AdCP in anther tissues, which is consistent with its expression under the tapetum-specific promoter. Our results clearly suggest that the targeted expression of AdCP provides a potential tool for developing male sterile lines in crop plants by the malfunction of tapetal cells leading to male sterility as shown earlier in tobacco transgenic plants (Shukla et al. 2014, Funct Integr Genomics 14:307-317).

Background: Increasing the content of oleic acid in peanut seeds is one of the major goals in peanut breeding due to consumer and industry benefits, such as anti-oxidation and long shelf-life. Homeologous ahFAD2A and ahFAD2B genes encode fatty acid desaturases, which are the key enzymes for converting oleic acid to linoleic acid that oxidizes readily. To date, all high oleic acid peanut varieties result from natural mutations occurred in both genes. A method to induce mutations in the genes of other elite cultivars could speed introgression of this valuable trait. The gene-editing approach utilizing CRISPR/Cas9 technology was employed to induce de novo mutations in the ahFAD2 genes using peanut protoplasts and hairy root cultures as models., Results: The hot spot of natural mutation in these genes was selected as the target region. Appropriate sgRNAs were designed and cloned into a CRISPR/Cas9 expression plasmid. As a result of CRISPR/Cas9 activity, three mutations were identified - G448A in ahFAD2A, and 441&#95;442insA and G451T in ahFAD2B. The G448A and 441&#95;442insA mutations are the same as those seen in existing high oleate varieties and the G451T is new mutation. Because natural mutations appear more often in the ahFAD2A gene than in the ahFAD2B gene in subspecies A. hypogaea var. hypogaea, the mutations induced in ahFAD2B by gene editing may be useful in developing high oleate lines with many genetic backgrounds after validation of oleic acid content in the transformed lines. The appearance of the G448A mutation in ahFAD2A is a further benefit for high oleic acid oil content., Conclusions: Overall, these results showed that mutations were, for the first time, induced by CRISPR-based gene editing approach in peanut. This research demonstrated the potential application of gene editing for mutagenesis in peanut and suggested that CRISPR/Cas9 technology may be useful in the peanut breeding programs.

The present work describes efficient hydrolysis of native starch by a novel β-amylase from peanut (Arachis hypogaea). The Dextrose Equivalent value, which is a measure of starch hydrolysis, for potato and corn starch increased significantly by 40% and 10%, respectively, releasing maltose. Scanning electron microscopy revealed that enzymatic corrosion occurred mainly at the surface of starch granules, leaving broken granules to smaller particles at later stage of digestion. Further, X-ray analysis and Fourier transform infrared spectroscopy displayed the loss of ordered structure in the enzyme degraded starches. These results described the pattern of hydrolysis. Since the action of already known plant β-amylases (sweet potato and soybean) on native starch granule is not very effective and requires gelatinization for maltose production, β-amylase from peanut could be a useful alternative in the present endeavor. It would potentially save time and money arising from gelatinization and lead to improvements in industrial maltose production., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The delta-12 fatty acid desaturase (Δ¹² FAD or FAD2) is a key enzyme that catalyzes oleic acid to linoleic acid by dehydrogenation at Δ¹² position of fatty acid carbon chain. In peanut, reduction or loss of FAD2 activity could enhance the relative content of oleic acid in kernels, and improve the quality and oxidation stability of peanut kernels and products. RNA interference (RNAi) technology could lead to non-expression or down-regulated expression of AhFAD2 gene. We constructed two RNA interference expression vectors with the inverted repeat sequence of partial AhFAD2 gene, which were driven separately by cauliflower mosaic virus (CaMV) 35S promoter or soybean agglutinin lectin seed-specific promoter. Homozygous transgenic lines carrying the two constructs stably in genetics were developed by peanut genetic transformation. There were no significant differences between the transgenic lines and the control through investigating the main agronomic traits. We analyzed the transcriptional level expression of AhFAD2 gene in transgenic lines and the control by real-time fluorescence quantitative PCR (qRT-PCR). The results suggested that the target genes of transgenic lines were likely suppressed by RNA interference, but showed different transcriptional levels in different peanut transgenic lines. Compared with untransformed lines, the resulting down-regulation of AhFAD2 gene resulted in a 15.09% or 36.40% increase in oleic acid content in the seeds of transformed HY23 and FH1 lines respectively, and the content of linoleic acid decreased by 16.19% or 29.81%, correspondingly, the ratio of oleic acid and linoleic acid (O/L) improved by 38.02%, 98.10%. The oleic acid content had significant differences between the two transformation constructs, and also among different transgenic lines. Moreover, the inhibition effect of RNAi was more obvious in the transgenic lines with FH1 as the receptor, and with transformation structure driven by seed specific promoter. The suppressed expression of AhFAD2 gene enabled the development of peanut fatty acid, which indicated that RNA interference would be a reliable technique for the genetic modification of peanut seed quality and the potential for improvement of other traits as well.

β-Amylase from un-germinated seeds of peanut (Arachis hypogaea) was purified to apparent electrophoretic homogeneity with final purification fold of 205 and specific activity of 361μmol/min/mg protein. The enzyme was purified employing simple purification techniques for biochemical characterization. The purified enzyme was identified as β-amylase with M r of 31kDa. The enzyme displayed its optimum catalytic activity at pH5.0 and 60°C with activation energy of 4.5kcal/mol and Q 10 1.2. The enzyme displayed K m and V max values, for soluble potato starch of 1.28mg/mL and 363.63μmol/min/mg, respectively. Thermal inactivation of β-amylase at 65°C resulted into first-order kinetics with rate constant 0.0126min -1 and t ½ 55min. The enzyme was observed to act on native granular potato starch, which could minimize the high cost occurring from solubilization of starch in industries. Enzyme fractions scavenge 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical, indicating its antioxidative nature. In addition, the purified β-amylase was successfully utilized for the improvement of antioxidant potential of wheat. These findings suggest that β-amylase from peanuts have potential application in food and pharmaceutical industries., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Stability of enzymes is an important parameter for their industrial applicability. Here, we report successful immobilization of β-amylase (bamyl) from peanut (Arachis hypogaea) onto Graphene oxide-carbon nanotube composite (GO-CNT), Graphene oxide nanosheets (GO) and Iron oxide nanoparticles (Fe 3 O 4 ). The Box-Behnken Design of Response Surface Methodology (RSM) was used which optimized parameters affecting immobilization and gave 90%, 88% and 71% immobilization efficiency, respectively, for the above matrices. β-Amylase immobilization onto GO-CNT (bamyl@GO-CNT) and Fe 3 O 4 (bamyl@Fe 3 O 4 ), resulted into approximately 70% retention of activity at 65 °C after 100 min of exposure. We used atomic force microscopy (AFM), scanning and transmission electron microscopy (SEM and TEM), Fourier transformed infrared (FT-IR) spectroscopy and fluorescence microscopy for characterization of free and enzyme bound nanostructures (NS). Due to the non-toxic nature of immobilization matrices and simple but elegant immobilization procedure, these may have potential utility as industrial biocatalysts for production of maltose., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Rhizobia-legume interaction activates the SYM pathway that recruits cytokinin signaling for induction of nodule primordia in the cortex. In Arachis hypogaea, bradyrhizobia invade through natural cracks developed in the lateral root base and are directly endocytosed in the cortical cells to generate the nodule primordia. To unravel the role of cytokinin signaling in A. hypogaea, RNA-interference (RNAi) of cytokinin receptor histidine-kinase1 (AhHK1) was done. AhHK1-RNAi downregulated the expression of type-A response regulators such as AhRR5 and AhRR3 along with several symbiotic genes, indicating that both cytokinin signaling and the SYM pathway were affected. Accordingly, there was a drastic downregulation of nodulation in AhHK1-RNAi roots and the nodules that developed were ineffective. These nodules were densely packed, with infected cells having a higher nucleo-cytoplasmic ratio and distinctively high mitotic index, where the rod-shaped rhizobia failed to differentiate into bacteroids within spherical symbiosomes. In accordance with the proliferating state, expression of a mitotic-cyclin AhCycB2.1 was higher in AhHK1-RNAi nodules, whereas expression of a retinoblastoma-related (AhRBR) nodule that restrains proliferation was lower. Also, higher expression of the meristem maintenance factor WUSCHEL-RELATED HOMEOBOX5 correlated with the undifferentiated state of AhHK1-RNAi nodules. Our results suggest that AhHK1-mediated cytokinin signaling is important for both inception and differentiation during nodule development in A. hypogaea.

Imaging approaches that track biological molecules within cells are essential tools in modern biochemistry. Lipids are particularly challenging to visualize, as they are not directly genetically encoded. Phospholipids, the most abundant subgroup of lipids, are structurally diverse and accomplish many cellular functions, acting as major structural components of membranes and as signaling molecules that regulate cell growth, division, apoptosis, cytoskeletal dynamics, and numerous other physiological processes. Cells regulate the abundance, and therefore bioactivity, of phospholipids by modulating the activities of their biosynthetic enzymes. Thus, techniques that enable monitoring of flux through individual lipid biosynthetic pathways can provide key functional information. For example, the choline analogue propargylcholine (ProCho) can report on de novo biosynthesis of phosphatidylcholine by conversion to an alkynyl lipid that can be imaged following click chemistry tagging with an azido fluorophore. We report that ProCho is also a substrate of phospholipase D enzymes-which normally hydrolyze phosphatidylcholine to generate the lipid second messenger phosphatidic acid-in a transphosphatidylation reaction, generating the identical alkynyl lipid. By controlling the activities of phosphatidylcholine biosynthesis and phospholipase D enzymes, we establish labeling conditions that enable this single probe to selectively report on two different biosynthetic pathways. Just as nature exploits the economy of common metabolic intermediates to efficiently diversify biosynthesis, so can biochemists in interrogating such pathways with careful probe design. We envision that ProCho's ability to report on multiple metabolic pathways will enable studies of membrane dynamics and improve our understanding of the myriad roles that lipids play in cellular homeostasis.

Defense responses of peanut ( Arachis hypogaea ) to biotic and abiotic stresses include the synthesis of prenylated stilbenoids. Members of this compound class show several protective activities in human disease studies, and the list of potential therapeutic targets continues to expand. Despite their medical and biological importance, the biosynthetic pathways of prenylated stilbenoids remain to be elucidated, and the genes encoding stilbenoid-specific prenyltransferases have yet to be identified in any plant species. In this study, we combined targeted transcriptomic and metabolomic analyses to discover prenyltransferase genes in elicitor-treated peanut hairy root cultures. Transcripts encoding five enzymes were identified, and two of these were functionally characterized in a transient expression system consisting of Agrobacterium -infiltrated leaves of Nicotiana benthamiana We observed that one of these prenyltransferases, AhR4DT-1, catalyzes a key reaction in the biosynthesis of prenylated stilbenoids, in which resveratrol is prenylated at its C-4 position to form arachidin-2, whereas another, AhR3'DT-1, added the prenyl group to C-3' of resveratrol. Each of these prenyltransferases was highly specific for stilbenoid substrates, and we confirmed their subcellular location in the plastid by fluorescence microscopy. Structural analysis of the prenylated stilbenoids suggested that these two prenyltransferase activities represent the first committed steps in the biosynthesis of a large number of prenylated stilbenoids and their derivatives in peanut. In summary, we have identified five candidate prenyltransferases in peanut and confirmed that two of them are stilbenoid-specific, advancing our understanding of this specialized enzyme family and shedding critical light onto the biosynthesis of bioactive stilbenoids., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Fatty acid desaturases are enzymes that introduce double bonds into fatty acyl chains. Extensive studies of fatty acid desaturases have been done in many plants. However, less is known about the diversity of this gene family in peanut (Arachis hypogaea L.), an important oilseed crop that is cultivated worldwide., Results: In this study, twelve novel AhFADs genes were identified and isolated from peanut. Quantitative real-time PCR analysis indicated that the transcript abundances of AhFAB2-2 and AhFAD3-1 were higher in seeds than in other tissues examined, whereas the AhADS and AhFAD7-1 transcripts were more abundant in leaves. AhFAB2-3, AhFAD3-2, AhFAD4, AhSLD-4, and AhDES genes were highly expressed in flowers, whereas AhFAD7-2, AhSLD-2, and AhSLD-3 were expressed most strongly in stems. During seed development, the expressions of AhFAB2-2, AhFAD3-1, AhFAD7-1, and AhSLD-3 gradually increased in abundance, reached a maximum expression level, and then decreased. The AhFAB2-3, AhFAD3-2, AhFAD4, AhADS, and AhDES transcript levels remained relatively high at the initial stage of seed development, but decreased thereafter. The AhSLD-4 transcript level remained relatively low at the initial stage of seed development, but showed a dramatic increase in abundance at the final stage. The AhFAD7-2 and AhSLD-2 transcript levels remained relatively high at the initial stage of seed development, but then decreased, and finally increased again. The AhFAD transcripts were differentially expressed following exposure to abiotic stresses or abscisic acid. Moreover, the functions of one AhFAD6 and four AhSLD genes were confirmed by heterologous expression in Synechococcus elongates or Saccharomyces cerevisiae., Conclusions: The present study provides valuable information that improves understanding of the biological roles of FAD genes in fatty acid synthesis, and will help peanut breeders improve the quality of peanut oil via molecular design breeding.

Key Message: The binding site for miR398 in an isoform of Cu/Zn superoxide dismutase (CSD1) is eliminated by alternative splicing to bypass miR398-mediated gene down-regulation under drought stress. MicroRNA (miRNA) binding sites (MBSs) are frequently interrupted by introns and therefore require proper splicing to generate functional MBSs in target transcripts. MBSs can also be excluded during splicing of pre-messenger RNA, leading to different regulation among isoforms. Previous studies have shown that levels of Cu/Zn superoxide dismutase (CSD) are down-regulated by miR398. In this study, sequences and transcript levels of peanut CSD1 isoforms (AhCSD1-1, AhCSD1-2.1, and AhCSD1-2.2) were analyzed under the drought stress. Results demonstrated that a miR398 binding site is eliminated in AhCSD1-2.2 as a consequence of alternative splicing, which bypasses miRNA-mediated down-regulation under drought stress. This alternative isoform was not only identified in peanut but also in soybean and Arabidopsis. In addition, transgenic Arabidopsis plants expressing AhCSD1 were more tolerant to osmotic stress. We hypothesize that the level of AhCSD1 is increased to allow diverse plant responses to overcome environmental challenges even in the presence of increased miR398 levels. These findings suggest that studies on the role of alternatively spliced MBSs affecting transcript levels are important for understanding plant stress responses.

Arsenic (As)-polluted groundwater constitutes a serious problem for peanut plants, as roots can accumulate the metalloid in their edible parts. Characterization of stress responses to As may help to detect potential risks and identify mechanisms of tolerance, being the induction of oxidative stress a key feature. Fifteen-day old peanut plants were treated with arsenate in order to characterize the oxidative stress indexes and antioxidant response of the legume under realistic groundwater doses of the metalloid. Superoxide anion (O 2 - ) and hydrogen peroxide (H 2 O 2 ) histochemical staining along with the activities of NADPH oxidase, superoxide dismutase (SOD), catalase (CAT) and thiol (glutathione and thioredoxins) metabolism were determined in roots. Results showed that at 20 μM H 2 AsO 4 - , peanut growth was reduced and the root architecture was altered. O 2 - and H 2 O 2 accumulated at the root epidermis, while lipid peroxidation, NADPH oxidase, SOD, CAT and glutathione S-transferase (GST) activities augmented. These variables increased with increasing As concentration (100 μM) while glutathione reductase (GR) and glutathione peroxidase/peroxiredoxin (GPX/PRX) were significantly decreased. These findings demonstrated that the metalloid induced physiological and biochemical alterations, being the NADPH oxidase enzyme implicated in the oxidative burst. Additionally, the strong induction of GST activity, even at the lowest H 2 AsO 4 - doses studied, can be exploited as suitable biomarker of As toxicity in peanut plants, which may help to detect risks of As accumulation and select tolerant cultivars., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Lipoxygenase (LOX) genes are widely distributed in plants and play crucial roles in resistance to biotic and abiotic stress. Although they have been characterized in various plants, little is known about the evolution of legume LOX genes. In this study, we identified 122 full-length LOX genes in Arachis duranensis, Arachis ipaënsis, Cajanus cajan, Cicer arietinum, Glycine max, Lotus japonicus and Medicago truncatula. In total, 64 orthologous and 36 paralogous genes were identified. The full-length, polycystin-1, lipoxygenase, alpha-toxin (PLAT) and lipoxygenase domain sequences from orthologous and paralogous genes exhibited a signature of purifying selection. However, purifying selection influenced orthologues more than paralogues, indicating greater functional conservation of orthologues than paralogues. Neutrality and effective number of codons plot results showed that natural selection primarily shapes codon usage, except for C. arietinum, L. japonicas and M. truncatula LOX genes. GCG, ACG, UCG, CGG and CCG codons exhibited low relative synonymous codon usage (RSCU) values, while CCA, GGA, GCU, CUU and GUU had high RSCU values, indicating that the latter codons are strongly preferred. LOX expression patterns differed significantly between wild-type peanut and cultivated peanut infected with Aspergillus flavus, which could explain the divergent disease resistance of wild progenitor and cultivars.

Prenylated stilbenoids synthesized in some legumes exhibit plant pathogen defense properties and pharmacological activities with potential benefits to human health. Despite their importance, the biosynthetic pathways of these compounds remain to be elucidated. Peanut (Arachis hypogaea) hairy root cultures produce a diverse array of prenylated stilbenoids upon treatment with elicitors. Using metabolic inhibitors of the plastidic and cytosolic isoprenoid biosynthetic pathways, we demonstrated that the prenyl moiety on the prenylated stilbenoids derives from a plastidic pathway. We further characterized, to our knowledge for the first time, a membrane-bound stilbenoid-specific prenyltransferase activity from the microsomal fraction of peanut hairy roots. This microsomal fraction-derived resveratrol 4-dimethylallyl transferase utilizes 3,3-dimethylallyl pyrophosphate as a prenyl donor and prenylates resveratrol to form arachidin-2. It also prenylates pinosylvin to chiricanine A and piceatannol to arachidin-5, a prenylated stilbenoid identified, to our knowledge, for the first time in this study. This prenyltransferase exhibits strict substrate specificity for stilbenoids and does not prenylate flavanone, flavone, or isoflavone backbones, even though it shares several common features with flavonoid-specific prenyltransferases., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

A serine protease activity was detected in aqueous peanuts seeds extracts, partially purified and characterized as a thiol-dependent serine protease. The potential role of this proteolytic activity on allergic reaction to peanuts was prospected through complement activation studies in human plasma and serum, and MDCK cells to investigate a possible occludin degradation in tight junctions. The peanut protease activity induced the production of anaphylatoxins C3a and C5a, and of the terminal membrane attack complex SC5b-9 whatever the complement activation pathway. The protease activity was also involved in the partial digestion of occludin within tight junctions, with for result, an increase of the epithelial permeability to antigen absorption., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

To clarify the mechanisms of selenium (Se) tolerance in peanut seedlings, we grew peanut seedlings with sodium selenite (0, 3, and 6 mg/L), and investigated the phenylpropanoids metabolism in seedling roots. The results showed that selenite up-regulated the expression of genes and related enzyme activities involving in the phenylpropanoids biosynthesis cascade, such as phenylalanine ammonia-lyase, trans-cinnamate-4-hydroxylase, chalcone synthase, chalcone isomerase, and cinnamyl-alcohol dehydrogenase. Selenite significantly increased phenolic acids and flavonoids, which contributed to the alleviation of selenite-induced stress. Moreover, selenite enhanced the formation of endodermis in roots, which may be attributed to the up-regulation of lignin biosynthesis mediated by the selenite-induced changes of H2O2 and NO, which probably regulated the selenite uptake from an external medium. Accumulation of polyphenolic compounds via the phenylpropanoid pathway may be one of the mechanisms of the increasing selenite tolerance in plants, by which peanut seedlings survived in seleniferous soil, accompanied by accumulation of Se.

Fertility restoration in male sterile plants is an essential requirement for their utilization in hybrid seed production. In an earlier investigation, we have demonstrated that the targeted expression of a cysteine protease in tapetal cell layer resulted in complete male sterility in tobacco transgenic plants. In the present investigation, we have used a cystatin gene, which encodes for a cysteine protease inhibitor, from a wild peanut, Arachis diogoi and developed a plant gene based restoration system for cysteine protease induced male sterile transgenic tobacco plants. We confirmed the interaction between the cysteine protease and a cystatin of the wild peanut, A. diogoi through in silico modeling and yeast two-hybrid assay. Pollen from primary transgenic tobacco plants expressing cystatin gene under the tapetum specific promoter- TA29 restored fertility on cysteine protease induced male sterile tobacco plants developed earlier. This has confirmed the in vivo interaction of cysteine protease and cystatin in the tapetal cells, and the inactivation of cysteine protease and modulation of its negative effects on pollen fertility. Both the cysteine protease and cystatin genes are of plant origin in contrast to the analogous barnase-barstar system that deploys genes of prokaryotic origin. Because of the deployment of genes of plant origin, this system might not face biosafety problems in developing hybrids in food crops., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Selenite can enhance the selenium nutrition level of crops, but excessive selenite may be toxic to plant growth. To elucidate the mechanisms underlying the role of selenite in production and detoxification of oxidative toxicity, peanut seedlings were developed with sodium selenite (0, 3, and 6 mg/L). The effects of selenite on antioxidant capacity, transcript levels of antioxidant enzyme genes, and enzyme activities in hypocotyl were investigated. The CuZn-SOD, GSH-Px, GST, and APX gene expression levels and their enzyme activities in selenite treatments were 1.0-3.6-fold of the control. Selenite also significantly increased the glutathione and ascorbate concentrations by mediating the ascorbate-glutathione cycle, and the selenite-induced hydrogen peroxide may act as a second messenger in the signaling pathways. This work has revealed a complex antioxidative response to selenite in peanut seedling. Understanding these mechanisms may help future research in increasing selenite tolerance and selenium accumulation in peanut and other crops.

The study was conducted to examine the antioxidant enzymes induced by Trichoderma viride JAU60 as initial defense response during invasion of rot pathogen Aspergillus niger Van Tieghem in five groundnut varieties under pot culture. Seed treatment of T. viride JAU60 reduced 51-58% collar rot disease incidence in different groundnut varieties under pathogen infected soil culture. The activities of the antioxidant enzymes, viz., superoxide dismutase (SOD, EC 1.15.1.1), guaiacol peroxidase (GPX, EC 1.11.1.7) and ascorbate peroxidase (APX, EC 1.11.1.11), elevated in response to pathogen infection, in higher rate by tolerant varieties (J-11 and GG-2) compared with susceptible (GAUG-10, GG-13, GG-20) and further induced by T. viride treatment. Trichoderma treatment remarkably increased the 2.3 fold SOD, 5 fold GPX and 2.5 fold APX activities during disease development in tolerant varieties and the same was found about 1.2, 1.5 and 2.0 folds, respectively, in susceptible varieties. Overall, T. viride JAU60 treated seedlings (T3) witnessed higher activities of SOD (1.5 fold), GPX (3.25 fold) and APX (1.25 fold) than pathogen treatment (T2) possibly suggest the induction of antioxidant defense response by Trichoderma bio-controller to combat oxidative burst produced by invading pathogen., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Through its production of phosphatidic acid (PA), phospholipase D (PLD) is strongly involved in vesicular trafficking and cell signaling, making this enzyme an important therapeutic target. However, most PLD assays developed so far are either discontinuous or based on the indirect determination of choline released during PLD-catalyzed phosphatidylcholine hydrolysis, making its kinetic characterization difficult. We present here the development of a direct, specific, and continuous PLD assay that is based on the chelation-enhanced fluorescence property of 8-hydroxyquinoline (8HQ) following Ca(2+) complexation with PLD-generated PA. The real-time fluorescence intensity from 8HQ/Ca(2+)/PA complexes can be converted to concentrations of product using a calibration curve, with a detection limit of 1.2 μM of PA on a microplate scale, thus allowing measurement of the PLD-catalyzed reaction rate parameters. Hence, this assay is well adapted for studying the substrate specificity of PLD, together with its kinetic parameters, using natural phospholipids with various headgroups. In addition, the assay was found to be effective in monitoring the competitive inhibition of PA formation in the production of phosphatidylalcohols following the addition of primary alcohols, such as ethanol, propan-1-ol, or butan-1-ol. Finally, this assay was validated using the purified recombinant Vigna unguiculata PLD, as well as the PLD from Streptomyces chromofuscus, cabbage, or peanuts, and no PA production could be detected using phospholipase A1, phospholipase A2, or phospholipase C, allowing for a reliable determination of PLD activity in crude protein extract samples. This easy to handle PLD assay constitutes, to our knowledge, the first direct and continuous PA determination method on a microplate scale.

Chitinase is an important pathogenesis-related protein in plants, and it can accumulate when induced by salicylic acid (SA) or other elicitors. Here, we found that chitinase mRNA levels were 4.5-times greater when peanut seedlings were sprayed with 1.5 mM SA, as compared to water. The upstream promoter sequence of the chitinase gene was cloned by TAIL-PCR and the potential cis-regulatory elements in this promoter were predicted by the cis-element databases PLACE and plantCARE. Elements in the promoter related to SA induction and disease resistance response included AS-1, GT1-motif, GRWAAW, TGTCA, W-box, and WB-box. The full-length promoter (P) and a series of 5'-deleted promoters (P1-P5) were cloned and then substituted for the 35S promoter of pCAMBIA1301-xylA, which carries the xylose isomerase gene as the selectable marker and GUS as the reporter gene. Six plant expression vectors (pCAMBIA1301-xylA-P-pCAMBIA1301-xylA-P5) were obtained. The six expression vectors were then transferred into onion epidermal cells and peanut plants by Agrobacterium-mediated transformation. Both the full-length and deleted promoters resulted in GUS staining of the onion epidermis cells when induced by SA. In onion epidermis cells, GUS enzyme activity was greater after SA induction. In transgenic peanut plants, GUS mRNA levels were greater after SA induction. Consideration of the cis-regulatory elements predicted by PLACE and plantCARE suggested that AS-1, GRWAAW, and W-box are positive regulatory elements in P2 and P3 and that GT1-motif and TGTCA are negative regulatory elements between P and P2.

Resveratrol is an important antioxidant that confers several beneficial effects on human health. 4-coumarate coenzyme A ligase (4CL) and resveratrol synthase (RS) are key rate-limiting enzymes in the biosynthetic pathway of resveratrol. Using gene fusion technology, the fusion gene, 4CL::RS, was constructed by the 4CL gene from Arabidopsis thaliana and RS gene from Arachis hypogaea. DNAMAN analysis showed that the fusion gene encoded a 964-amino acid protein with an approximate weight of 104.7 kDa and a pI of 5.63. A prokaryotic expression vector containing Nco-I and EcoR-I restriction sites, pET-30a/4CL::RS, was identified by liquid culture bacterial PCR, enzyme digestion, and sequencing, and then used in the induction of expression. Subsequently, a biosynthetic pathway of resveratrol was constructed in Escherichia coli BL21(DE3) that harbored pET-30a/4CL::RS. The recombinant strains were induced to express the fusion protein at 28 °C for 8 h. After bacterial cells were disrupted by hypothermic ultrasonication, the 4CL::RS fusion protein was thoroughly separated from tags using Ni-NTA affinity chromatography, and then detected by SDS-PAGE analysis. When the recombinant strains expressed the fusion protein, the precursor, p-coumaric acid, was converted to resveratrol. In the present study, the final concentration of resveratrol derived from 1 mM p-coumaric acid was 80.524 mg/L, with a 35.28 % (mol/mol) conversion yield.

The FAD2 gene family is functionally responsible for the conversion of oleic acid to linoleic acid in oilseed plants. Multiple members of the FAD gene are known to occur in several oilseed species. In this study, six novel full-length cDNA sequences (named as AhFAD2-1, -2, -3, -4, -5, and -6) were identified in peanut (Arachis hypogaea L.), an analysis of which revealed open reading frames of 379, 383, 394, or 442 amino acids. Sequence comparisons showed that AhFAD2-1 and AhFAD2-2 shared 76% identity, while AhFAD2-2, -3, and -4 displayed highly significant homology. There was only 27% identity overlap between the microsomal ω-6 fatty acid desaturase and the chloroplast ω-6 fatty acid desaturase encoded by AhFAD2-1, -2, -3, -4, and AhFAD2-5, -6, respectively. The phylogeny tree of FAD2 transcripts showed five major groups, and AhFAD2-1 was clearly separated from other groups. Analysis of AhFAD2-1 and AhFAD2-2 transcript distribution in different peanut tissues showed that the AhFAD2-1 gene showed upward of a 70-fold increase in expression of fatty acid than the AhFAD2-2 gene in peanut developing seeds, while the AhFAD2-2 gene expressed most abundantly in peanut flowers. Because the AhFAD2-1 gene played a major role in the conversion of oleic to linoleic acid during seed development, the identification of this novel member in this study would facilitate the further genetic manipulation of peanut oil quality. The implications of overall results also suggest that there may be more candidate genes controlling levels of oleate acid in developing seeds. Results also may be due to the presence of complex gene networks controlling the fluxes between the endoplasmic reticulum and the chloroplast within the peanut cells.

Key Message: Overexpression of CuZnSOD gene from Arachis hypogaea demonstrating its involvement in abiotic stress tolerance. Abiotic stress is accompanied by the formation of reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, and hydroxyl radicals, causing extensive cellular damage and inhibition of photosynthesis that limit the plant productivity. The level of ROS in cells needs to be tightly regulated and the toxic effects of ROS are countered by enzymatic as well as non-enzymatic antioxidant systems. The superoxide dismutase is the first enzyme involved in the detoxification of ROS and converts superoxide (O2(·-)) radicals to H2O2. A full-length cDNA clone encoding a CuZnSOD, named AhCuZnSOD, was isolated from the salt tolerant cell lines of Arachis hypogaea, stably thriving at 200 mM NaCl. The cell line showed higher transcript accumulation under multiple abiotic stresses, including drought, salinity, cold and oxidative stress treatment. The functional role of AhCuZnSOD in alleviation of abiotic stress was assessed by its overexpression in transgenic tobacco plants. The T1 transgenic plants showed improved tolerance to salinity and dehydration stress as indicated by higher seed germination and better chlorophyll content. The transgenic plants survived under longer periods of water deficiency and salinity stress and displayed improved recovery after rehydration compared to the wild type (WT) plants. The enhanced level of the transgene correlated with higher relative water content, less electrolyte damage, less malondialdehyde, higher antioxidant enzyme activity, H2O2 and O2(·-) accumulation under stress conditions compared to WT plants. Our results substantiate that increased levels of SOD activity brought about by overexpression of AhCuZnSOD gene may play an important role in ameliorating oxidative injury induced by various environmental stresses.

Vacuolar processing enzymes are cysteine proteases responsible for maturation of vacuolar proteins. They have been shown to possess caspase-1-like activity, mediate cell death and display increased activity during pathogen infections. A transcript derived fragment corresponding to VPE was found to be up-regulated in a cDNA-AFLP analysis of host responses of a wild peanut, Arachis diogoi upon challenge from the late leaf spot pathogen Phaeoisariopsis personata, which was subsequently validated by q-PCR in a time course analysis, where susceptible peanut did not show its upregulation. In transient conditional and constitutive expression studies in tobacco leaves using agroinfiltration, we have observed that expression of AdVPE was associated with hypersensitive response (HR) like cell death. AdVPE expression was found to be high at 24 h post estradiol application and this was associated with the enhanced co-expression of molecular markers of HR cell death genes and genes for pathogenesis related proteins indicating that AdVPE positively regulates defense responses and its estradiol induced expression is sufficient for HR-like cell death in tobacco. We found that AdVPE expression was very strongly induced in response to sodium nitroprusside, which indicates its involvement in stress signaling. Induced expression of AdVPE in response to jasmonic acid and ethylene also indicates its involvement in an interconnected network of signaling. Transgenic tobacco plants ectopically expressing AdVPE exhibited enhanced resistance against Phytophthora parasitica var. nicotianae, Alternaria alternata var.  nicotianae and Rhizoctonia solani. To our knowledge, this is the first report on the heterologous expression of a pathogen induced VPE enhancing resistance to fungal pathogens with cell death phenomenon under transient expression.

In this study, we investigated the effects of exogenous calcium nitrate on photoinhibition and thylakoid protein level in peanut plants under heat (40°C) and high irradiance (HI) (1,200 µmol/m(2) per s) stress. Compared with control seedlings (cultivated in 0 mmol/L Ca(NO3 )2 medium), the maximal photochemical efficiency of photosystem II (PSII) in Ca(2+) -treated plants showed a slight decrease after 5 h stress, accompanied by lower degree of PSII closure (1-qP), higher non-photochemical quenching, and lower level of membrane damage. Ca(2+) inhibitors were used to analyze the varieties of antioxidant enzymes activity and PSII proteins. These results indicated that Ca(2+) could protect the subunits of PSII reaction centers from photoinhibition by reducing the generation of reactive oxygen species. In the presence of both ethyleneglycol-bis(2-aminoethylether)-tetraacetic acid and ascorbic acid (AsA), the net degradation of the damaged D1 protein was faster than that only treated with AsA. Our previous study showed that either the transcriptional or the translational level of calmodulin was obviously higher in Ca(2+) -treated plants. These results suggested that, under heat and HI stress, the Ca(2+) signal transduction pathway can alleviate the photoinhibition through regulating the protein repair process besides an enhanced capacity for scavenging reactive oxygen species., (© 2014 The Authors. Journal of Integrative Plant Biology published by Wiley Publishing Asia Pty Ltd on behalf of Institute of Botany, The Chinese Academy of Sciences.)

In this study, the full-length cDNA encoding allene oxide cyclase (AhAOC) was isolated from peanut (Arachis hypogaea L.). The deduced amino acid sequence of AhAOC showed high homology with other plant AOCs. The transcript of AhAOC was found to be abundantly expressed in roots. Expression analysis demonstrated that AhAOC was induced by abscisic acid, methyl-jasmonic acid, salicylic acid, salinity, polyethylene glycol, and cold stresses, particularly by high salinity. Overexpression of AhAOC in rice increased root elongation and plant height compared with expression in control plants and conferred tolerance against salinity. Thus, the AhAOC gene may play an important role in increasing the expression of transcription factors (MYB2 and OsONAC045) and functional genes (DREB1F and LEA3) in transgenic rice under salt stress as well as improve stress tolerance through the accumulation of compatible solutes (proline and soluble sugar). The AhAOC gene is a potential resource for enhancing salt tolerance in crop species.

Aims: We evaluated whether reactive oxygen species (ROS) production and the plant antioxidant system are involved in the symbiotic interaction between bradyrhizobia and legumes infected by crack entry, without intracellular infection threads (IT) formation, such as Arachis hypogaea L. (peanut). The role of bradyrhizobial Nod factors (NF) in modulating the plants' oxidative burst was also analysed., Methods and Results: Histochemical and quantitative procedures were used to detect ROS levels in inoculated and in NF-treated peanut roots. Increase in root H2O2 production was determined at 10 min postinoculation with Bradyrhizobium sp. SEMIA 6144 or after NF addition. ROS production was modulated by NF. From 15 to 30 min postinoculation, the compatibility of Bradyrhizobium sp.-peanut interaction depends mostly on the H2O2 detoxification via catalase., Conclusions: We demonstrated for the first time that the early events of the symbiotic interaction in legumes invaded by crack entry trigger an increase in ROS production (represented exclusively by a higher H2O2 content) in which NADPH-oxidase seems not to be involved. NF modulate this response by enhancing the plant antioxidant machinery, contributing to the creation of adequate conditions for symbiosis development., Significance and Impact of the Study: Our data provide new insights into the mechanism involves in the symbiotic interaction that establish legumes infected by crack entry and suggest that ROS response shows differences compared with legumes invaded by IT formation., (© 2014 The Society for Applied Microbiology.)

Background: Induced resistance to Helicoverpa armigera through exogenous application of jasmonic acid (JA) and salicylic acid (SA) was studied in groundnut genotypes (ICGV 86699, ICGV 86031, ICG 2271 and ICG 1697) with different levels of resistance to insects and the susceptible check JL 24 under greenhouse conditions. Activities of oxidative enzymes and the amounts of secondary metabolites and proteins were quantified at 6 days after JA and SA application/insect infestation. Data were also recorded on plant damage and H. armigera larval weights and survival., Results: Higher levels of enzymatic activities and amounts of secondary metabolites were observed in the insect-resistant genotypes pretreated with JA and then infested with H. armigera than in JL 24. The insect-resistant genotypes suffered lower insect damage and resulted in poor survival and lower weights of H. armigera larvae than JL 24. In some cases, JA and SA showed similar effects., Conclusion: JA and SA induced the activity of antioxidative enzymes in groundnut plants against H. armigera, and reduced its growth and development. However, induced response to application of JA was greater than to SA, and resulted in reduced plant damage, and larval weights and survival, suggesting that induced resistance can be used as a component of pest management in groundnut., (© 2014 Society of Chemical Industry.)

Chalcone isomerase (CHI) plays critical roles in plant secondary metabolism, which is important for the interaction between plants and the environment. CHI genes are widely studied in various higher plants. However, little information about CHI genes is available in peanut. Based on conservation of CHI gene family, we cloned the peanut type II CHI gene (AhCHI II) cDNA and genome sequence. The amino acid sequence of peanut CHI II was highly homologous to type II CHI from other plant species. qRT-PCR results showed that peanut CHI II is mainly expressed in roots; however, peanut CHI I is mainly expressed in tissues with high content of anthocyanin. Gene duplication and gene cluster analysis indicated that CHI II was derived from CHI I 65 million years ago approximately. Our gene structure analysis results are not in agreement with the previous hypothesis that CHI II was derived from CHI I by the insertion of an intron into the first exon. Moreover, no positive selection pressure was found in CHIs, while, 32.1 % of sites were under neutral selection, which may lead to mutation accumulation and fixation during great changes of environment.

Background: Peanut is one of the major source for human consumption worldwide and its seed contain approximately 50% oil. Improvement of oil content and quality traits (high oleic and low linoleic acid) in peanut could be accelerated by exploiting linked markers through molecular breeding. The objective of this study was to identify QTLs associated with oil content, and estimate relative contribution of FAD2 genes (ahFAD2A and ahFAD2B) to oil quality traits in two recombinant inbred line (RIL) populations., Results: Improved genetic linkage maps were developed for S-population (SunOleic 97R × NC94022) with 206 (1780.6 cM) and T-population (Tifrunner × GT-C20) with 378 (2487.4 cM) marker loci. A total of 6 and 9 QTLs controlling oil content were identified in the S- and T-population, respectively. The contribution of each QTL towards oil content variation ranged from 3.07 to 10.23% in the S-population and from 3.93 to 14.07% in the T-population. The mapping positions for ahFAD2A (A sub-genome) and ahFAD2B (B sub-genome) genes were assigned on a09 and b09 linkage groups. The ahFAD2B gene (26.54%, 25.59% and 41.02% PVE) had higher phenotypic effect on oleic acid (C18:1), linoleic acid (C18:2), and oleic/linoleic acid ratio (O/L ratio) than ahFAD2A gene (8.08%, 6.86% and 3.78% PVE). The FAD2 genes had no effect on oil content. This study identified a total of 78 main-effect QTLs (M-QTLs) with up to 42.33% phenotypic variation (PVE) and 10 epistatic QTLs (E-QTLs) up to 3.31% PVE for oil content and quality traits., Conclusions: A total of 78 main-effect QTLs (M-QTLs) and 10 E-QTLs have been detected for oil content and oil quality traits. One major QTL (more than 10% PVE) was identified in both the populations for oil content with source alleles from NC94022 and GT-C20 parental genotypes. FAD2 genes showed high effect for oleic acid (C18:1), linoleic acid (C18:2), and O/L ratio while no effect on total oil content. The information on phenotypic effect of FAD2 genes for oleic acid, linoleic acid and O/L ratio, and oil content will be applied in breeding selection.

Accumulative evidence indicates that the lipoxygenase (LOX) pathway plays a significant role in the Aspergillus-seed interaction, such as interfering with activities of endogenous fungal oxylipins or producing antimicrobial compounds and signaling molecules. In this study, we characterized the LOX pathway in peanut seed during Aspergillus parasiticus colonization in a model of two cultivars distinguished as resistant ('PI337394') and susceptible ('Florman INTA') to Aspergillus spp. infection and aflatoxin contamination. The LOX activity together with the content of LOX substrate and LOX products demonstrated the presence of a differential response mechanism to A. parasiticus infection between cultivars. Our findings suggest that this mechanism is under transcriptional control of previously identified (LOX 2 and LOX 3) and novel (LOX 4 and LOX 5) LOX genes. The results of this study support the role of these enzymes in defense during fungus infection in peanut seed.

Symbiosis Receptor Kinase (SYMRK), a member of the Nod factor signaling pathway, is indispensible for both nodule organogenesis and intracellular colonization of symbionts in rhizobia-legume symbiosis. Here, we show that the intracellular kinase domain of a SYMRK (SYMRK-kd) but not its inactive or full-length version leads to hyperactivation of the nodule organogenic program in Medicago truncatula TR25 (symrk knockout mutant) in the absence of rhizobia. Spontaneous nodulation in TR25/SYMRK-kd was 6-fold higher than rhizobia-induced nodulation in TR25/SYMRK roots. The merged clusters of spontaneous nodules indicated that TR25 roots in the presence of SYMRK-kd have overcome the control over both nodule numbers and their spatial position. In the presence of rhizobia, SYMRK-kd could rescue the epidermal infection processes in TR25, but colonization of symbionts in the nodule interior was significantly compromised. In summary, ligand-independent deregulated activation of SYMRK hyperactivates nodule organogenesis in the absence of rhizobia, but its ectodomain is required for proper symbiont colonization., (© 2014 American Society of Plant Biologists. All Rights Reserved.)

Allopolyploidization in plants entails the merger of two divergent nuclear genomes, typically with only one set (usually maternal) of parental plastidial and mitochondrial genomes and with an altered cytonuclear stoichiometry. Thus, we might expect cytonuclear coevolution to be an important dimension of allopolyploid evolution. Here, we investigate cytonuclear coordination for the key chloroplast protein rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase), which is composed of nuclear-encoded, small subunits (SSUs) and plastid-encoded, large subunits. By studying gene composition and diversity as well as gene expression in four model allopolyploid lineages, Arabidopsis, Arachis, Brassica, and Nicotiana, we demonstrate that paralogous nuclear-encoded rbcS genes within diploids are subject to homogenization via gene conversion and that such concerted evolution via gene conversion characterizes duplicated genes (homoeologs) at the polyploid level. Many gene conversions in the polyploids are intergenomic with respect to the diploid progenitor genomes, occur in functional domains of the homoeologous SSUs, and are directionally biased, such that the maternal amino acid states are favored. This consistent preferential maternal-to-paternal gene conversion is mirrored at the transcriptional level, with a uniform transcriptional bias of the maternal-like rbcS homoeologs. These data, repeated among multiple diverse angiosperm genera for an important photosynthetic enzyme, suggest that cytonuclear coevolution may be mediated by intergenomic gene conversion and altered transcription of duplicated, now homoeologous nuclear genes., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Diacylglycerol acyltransferase (DGAT) catalyzes the final and only committed acylation step in the synthesis of triacylglycerols. In this study, three novel AhDGATs genes were identified and isolated from peanut. Quantitative real-time RT-PCR analysis indicated that the AhDGAT1-2 transcript was more abundant in roots, seeds, and cotyledons, whereas the transcript abundances of AhDGAT1-1 and AhDGAT3-3 were higher in flowers than in the other tissues examined. During seed development, transcript levels of AhDGAT1-1 remained relatively low during the initial developmental stage but increased gradually during later stages, peaking at 50 days after pegging (DAP). Levels of AhDGAT1-2 transcripts were higher at 10 and 60 DAPs and much lower during other stages, whereas AhDGAT3-3 showed higher expression levels at 20 and 50 DAPs. In addition, AhDGAT transcripts were differentially expressed following exposure to abiotic stresses or abscisic acid. The activity of the three AhDGAT genes was confirmed by heterologous expression in a Saccharomyces cerevisiae TAG-deficient quadruple mutant. The recombinant yeasts restored lipid body formation and TAG biosynthesis, and preferentially incorporated unsaturated C18 fatty acids into lipids. The present study provides significant information useful in modifying the oil deposition of peanut through molecular breeding.

Plant receptor-like kinases (RLKs) are distinguished by having a tyrosine in the 'gatekeeper' position. Previously we reported Symbiosis Receptor Kinase from Arachis hypogaea (AhSYMRK) to autophosphorylate on the gatekeeper tyrosine (Y670), though this phosphorylation was not necessary for the kinase activity. Here we report that recombinant catalytic domain of AhSYMRK with a phosphomimic substitution in the gatekeeper position (Y670E) is catalytically almost inactive and is conformationally quite distinct from the corresponding native enzyme. Additionally, we show that gatekeeper-phosphorylated AhSYMRK polypeptides are inactive and depletion of this inactive form leads to activation of intramolecular autophosphorylation of AhSYMRK. Together, our results suggest gatekeeper tyrosine autophosphorylation to be autoinhibitory for AhSYMRK., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Abscisic acid (ABA) catabolism is one of the determinants of endogenous ABA levels affecting numerous aspects of plant growth and abiotic-stress responses. The major ABA catabolic pathway is triggered by ABA 8'-hydroxylation catalysed by ABA 8'-hydroxylase, the cytochrome P450 CYP707A family. In this study, the full-length cDNAs of AhCYP707A1 and AhCYP707A2 were cloned and characterized from peanut. Expression analyses showed that AhCYP707A1 and AhCYP707A2 were expressed ubiquitously in peanut roots, stems, and leaves with different transcript accumulation levels, including the higher expression of AhCYP707A1 in roots. The expression of AhCYP707A2 was significantly up-regulated by 20% PEG6000 or 250 mmol/L NaCl in peanut roots, stems, and leaves, whereas the up-regulation of AhCYP707A1 transcript level by PEG6000 or NaCl was observed only in roots instead of leaves and stems. Due to the osmotic and ionic stresses of high concentration of NaCl to plants simultaneously, low concentration of LiCl (30 mmol/L, at which concentration osmotic status of cells is not seriously affected, the toxicity of Li+ being higher than that of Na+) was used to examine whether the effect of NaCl might be related to osmotic or ionic stress. The results revealed visually the susceptibility to osmotic stress and the resistance to salt ions in peanut seedlings. The significant up-regulation of AhCYP707A1, AhCYP707A2 and AhNCED1 transcripts and endogenous ABA levels by PEG6000 or NaCl instead of LiCl, showed that the osmotic stress instead of ionic stress affected the expression of those genes and the biosynthesis of ABA in peanut. The functional expression of AhCYP707A1 cDNA in yeast showed that the microsomal fractions prepared from yeast cell expressing recombinant AhCYP707A1 protein exhibited the catalytic activity of ABA 8'-hydroxylase. These results demonstrate that the expressions of AhCYP707A1 and AhCYP707A2 play an important role in ABA catabolism in peanut, particularly in response to osmotic stress.

Many plants accumulate hydroxycinnamoyl esters to protect against abiotic and biotic stresses. Caffeoyl esters in particular can be substrates for endogenous polyphenol oxidases (PPOs). Recently, we showed that perennial peanut (Arachis glabrata Benth.) leaves contain PPO and identified one PPO substrate, caftaric acid (trans-caffeoyl-tartaric acid). Additional compounds were believed to be cis- and trans-p-coumaroyl tartaric acid and cis- and trans-feruloyl-tartaric acid, but lack of standards prevented definitive identifications. Here we characterize enzymatic activities in peanut leaves to understand how caftaric acid and related hydroxycinnamoyl esters are made in this species. We show that peanut leaves contain a hydroxycinnamoyl-CoA:tartaric acid hydroxycinnamoyl transferase (HTT) activity capable of transferring p-coumaroyl, caffeoyl, and feruloyl moieties from CoA to tartaric acid (specific activities of 11 ± 2.8, 8 ± 1.8, 4 ± 0.8 pkat mg(-1) crude protein, respectively). The HTT activity was used to make cis- and trans-p-coumaroyl- and -feruloyl-tartaric acid in vitro. These products allowed definitive identification of the corresponding cis- and trans-hydroxycinnamoyl esters extracted from leaves. We tentatively identified sinapoyl-tartaric acid as another major phenolic compound in peanut leaves that likely participates in secondary reactions with PPO-generated quinones. These results suggest hydroxycinnamoyl-tartaric acid esters are made by an acyltransferase, possibly a BAHD family member, in perennial peanut. Identification of a gene encoding HTT and further characterization of the enzyme will aid in identifying determinants of donor and acceptor substrate specificity for this important class of biosynthetic enzymes. An HTT gene could also provide a means by genetic engineering for producing caffeoyl- and other hydroxycinnamoyl-tartaric acid esters in forage crops that lack them.

Plant β-1,3-glucanases are commonly involved in disease resistance. This report describes the cloning and genetic transformation of a β-1,3-glucanase gene from peanut. The gene was isolated from both the genomic DNA and cDNA of peanut variety Huayu20 by polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR), respectively. The DNA sequence contained 1471 bp including two exons and one intron, and the coding sequence contained 1047 bp that coded for a 348-amino acid protein with a calculated molecular weight of 38.8 kDa. The sequence was registered in NCBI (GenBank accession No. JQ801335) and was designated as Ah-Glu. As determined by BLAST analysis, the Ah-Glu protein has 42-90% homology with proteins from Oryza sativa (BAC83070.1), Zea mays (NP&#95;001149308), Arabidopsis thaliana (NP&#95;200470.1), Medicago sativa (ABD91577.1), and Glycine max (XP&#95;003530515.1). The over-expression vector pCAMBIA1301-Glu containing Ah-Glu was constructed, confirmed by PCR and restriction enzyme digestion, and transformed into peanut variety Huayu22 by Agrobacterium EHA105-mediated transformation. The putative transformed plants (T0) were confirmed by PCR amplification. RT-PCR analysis and β-glucuronidase (GUS) staining showed that the transferred Ah-Glu was expressed as mRNA and protein. In a laboratory test, the transgenic plants were found to be more resistant to the fungal pathogen Cercospora personata than the non-transgenic plants were.