Nitric oxide suppresses aluminum-induced programmed cell death in peanut (Arachis hypoganea L.) root tips by improving mitochondrial physiological properties.

Aluminum (Al) stress alters nitric oxide (NO) and induces programmed cell death (PCD) in plants. Recent study has shown that NO inhibits Al-induced PCD. However, the mechanism of NO inhibiting Al-induced PCD has not been revealed yet. Here, we investigated the behavior of mitochondria during Al-induced PCD suppressed by NO in peanut. Seedlings of peanut was grown hydroponically in a controllable growth room. The mitochondrial physiological parameters were determined spectrophotometrically. The expression of AhANT and AhHsp70 was determined by quantitative RT-PCR. Al-induced cell death rapidly in peanut root tips is mitochondria-dependent PCD. There was a significantly negative relationship between PCD and mitochondrial NO/H2O2 level. Compared with Al treatment alone, the addition of NO donor sodium nitroprusside (SNP) increased the ratio of NO/H2O2, down-regulated AhANT expression and inhibited the opening of mitochondrial permeability transition pore (MPTP), up-regulated AhHsp70 expression and increased mitochondrial inner membrane potential (ΔΨm), reduced cytochrome c (Cyt c) release from mitochondria and caspase 3-like protease activity, while the effect of NO specific scavenger cPTIO supplement was opposite. NO suppresses Al-induce PCD in peanut root tips by improving mitochondrial physiological properties.

Cytogenetic evidences on the evolutionary relationships between the tetraploids of the section Rhizomatosae and related diploid species (Arachis, Leguminosae).

Rhizomatosae is a taxonomic section of the South American genus Arachis, whose diagnostic character is the presence of rhizomes in all its species. This section is of particular evolutionary interest because it has three polyploid (A. pseudovillosa, A. nitida and A. glabrata, 2n = 4x = 40) and only one diploid (A. burkartii, 2n = 2x = 20) species. The phylogenetic relationships of these species as well as the polyploidy nature and the origin of the tetraploids are still controversial. The present study provides an exhaustive analysis of the karyotypes of all rhizomatous species and six closely related diploid species of the sections Erectoides and Procumbentes by cytogenetic mapping of DAPI/CMA heterochromatin bands and 5S and 18-26S rDNA loci. Chromosome banding showed variation in the DAPI heterochromatin distribution pattern, which, together with the number and distribution of rDNA loci, allowed the characterization of all species studied here. The bulk of chromosomal markers suggest that the three rhizomatous tetraploid species constitute a natural group and may have at least one common diploid ancestor. The cytogenetic data of the diploid species analyzed evidenced that the only rhizomatous diploid species-A. burkartii-has a karyotype pattern different from those of the rhizomatous tetraploids, showing that it is not likely the genome donor of the tetraploids and the non-monophyletic nature of the section Rhizomatosae. Thus, the tetraploid species should be excluded from the R genome, which should remain exclusively for A. burkartii. Instead, the karyotype features of these tetraploids are compatible with those of different species of the sections Erectoides and Procumbentes (E genome species), suggesting the hypothesis of multiple origins of these tetraploids. In addition, the polyploid nature and the group of diploid species closer to the tetraploids are discussed.

Identification of low Ca(2+) stress-induced embryo apoptosis response genes in Arachis hypogaea by SSH-associated library lift (SSHaLL).

Calcium is a universal signal in the regulation of wide aspects in biology, but few are known about the function of calcium in the control of early embryo development. Ca(2+) deficiency in soil induces early embryo abortion in peanut, producing empty pods, which is a general problem; however, the underlying mechanism remains unclear. In this study, embryo abortion was characterized to be caused by apoptosis marked with cell wall degradation. Using a method of SSH cDNA libraries associated with library lift (SSHaLL), 62 differentially expressed genes were isolated from young peanut embryos. These genes were classified to be stress responses, catabolic process, carbohydrate and lipid metabolism, embryo morphogenesis, regulation, etc. The cell retardation with cell wall degradation was caused by up-regulated cell wall hydrolases and down-regulated cellular synthases genes. HsfA4a, which was characterized to be important to embryo development, was significantly down-regulated under Ca(2+) -deficient conditions from 15 days after pegging (DAP) to 30 DAP. Two AhCYP707A4 genes, encoding abscisic acid (ABA) 8'-hydroxylases, key enzymes for ABA catabolism, were up-regulated by 21-fold under Ca(2+) -deficient conditions upstream of HsfA4a, reducing the ABA level in early embryos. Over-expression of AhCYP707A4 in Nicotiana benthamiana showed a phenotype of low ABA content with high numbers of aborted embryos, small pods and less seeds, which confirms that AhCYP707A4 is a key player in regulation of Ca(2+) deficiency-induced embryo abortion via ABA-mediated apoptosis. The results elucidated the mechanism of low Ca(2+) -induced embryo abortion and described the method for other fields of study.

Pathogen-induced SGT1 of Arachis diogoi induces cell death and enhanced disease resistance in tobacco and peanut.

We have identified a transcript derived fragment (TDF) corresponding to SGT1 in a study of differential gene expression on the resistant wild peanut, Arachis diogoi, upon challenge from the late leaf spot pathogen, Phaeoisariopsis personata, and cloned its full-length cDNA followed by subsequent validation through q-PCR. Sodium nitroprusside, salicylic acid, ethephon and methyl jasmonate induced the expression of AdSGT1, while the treatment with abscisic acid did not elicit its up-regulation. AdSGT1 is localized to both nucleus and cytoplasm. Its overexpression induced hypersensitive-like cell death in tobacco under transient conditional expression using the estradiol system, and this conditional expression of AdSGT1 was also associated with the up-regulation of NtHSR203J, HMGR and HIN1, which have been shown to be associated with hypersensitive response in tobacco in earlier studies. Expression of the cDNA in a susceptible cultivated peanut variety enhanced its resistance against the late leaf spot pathogen, Phaeoisariopsis personata, while the heterologous expression in tobacco enhanced its resistance against Phytophthora parasitica var. nicotianae, Alternaria alternata var. nicotianae and Rhizoctonia solani. Constitutive expression in peanut was associated with the co-expression of resistance-related genes, CC-NB-LRR and some protein kinases.

Reactive oxygen species burst induced by aluminum stress triggers mitochondria-dependent programmed cell death in peanut root tip cells.

Recent studies had certified that aluminum (Al) induced ROS production and programmed cell death (PCD) in higher plants. The relationship between ROS production and PCD occurrence under Al stress is uncovered. The results showed that root elongation inhibition and PCD occurrence was induced by 100 µM AlCl3. Al stress induced ROS burst, up-regulated Rboh and COX gene expression, increased mitochondrial permeability transition pore (MPTP) opening, decreased inner mitochondrial membrane potential (ΔΨm), released cytochrome c from mitochondria to cytoplasm, activated caspase 3-like protease activity. Exogenous H2O2 aggravated the changes caused by Al and accelerated PCD occurrence, but ROS scavenger CAT and AsA reversed the changes caused by Al and inhibited PCD production. A potential cascade of cellular events during Al induced PCD via mitochondria dependent pathway and the mechanism of ROS on regulating PCD induced by Al is proposed.

Phytosynthesis of intracellular and extracellular gold nanoparticles by living peanut plant (Arachis hypogaea L.).

Inorganic nanomaterials of different chemical compositions are conventionally synthesized under harsh environments such as extremes of temperature, pressure, and pH. Moreover, these methods are eco-unfriendly and cumbersome, yield bigger particles, and agglomerate because of not being capped by capping agents. In contrast, biological synthesis of inorganic nanomaterials occurs under ambient conditions, namely room temperature, atmospheric pressure, and physiological pH. These methods are reliable, eco-friendly, and cheap. In this paper, we report for the first time the extracellular and intracellular synthesis of gold nanoparticles (GNPs) using living peanut seedlings. The formed GNPs were highly stable in solution and inside the plant tissue. Transmission electron microscopy revealed that extracellular GNPs distributions were in the form of monodispersed nanoparticles. The nanoparticles ranged from 4 to 6 nm in size. The intercellular nanoparticles were of oval shape and size ranged from 5 to 50 nm. Both extracellular and intracellular nanoparticles were further characterized by standard techniques. The formed GNPs inside the plant tissue were estimated by inductively coupled plasma spectrometry. This opens up an exciting possibility of a plant-based nanoparticle synthesis strategy, wherein the nanoparticles may be entrapped in the biomass in the form of a film or produced in the solution, both of which have interesting applications.

Rhizobial Nod factors are required for cortical cell division in the nodule morphogenetic programme of the Aeschynomeneae legume Arachis.

Nod factors are among the best-studied molecules implicated in the signal exchange that leads to legume-rhizobia symbiosis. The role of these molecules in symbiosis development has been primarily studied in legumes invaded through infection threads. In these plants, Nod factors generate several responses required for nodulation, including the induction of cortical cell division to form the nodule primordium. Arachis hypogaea L. (peanut) exhibits a specific mode of rhizobial infection and nodule morphogenetic programme in which infection threads are never formed. The role of Nod factors in this particular mechanism is unknown. In this work, a peanut symbiont mutant strain unable to produce Nod factors was obtained and characterised. The strain Bradyrhizobium (Arachis) sp. SEMIA 6144 V2 is altered in the nodC gene, which encodes an N-acetylglucosaminyl transferase involved in the first step of the Nod factor biosynthetic pathway. Further research revealed that, although its ability to colonise peanut roots was unaffected, it is not capable of inducing the division of cortical cells. The results obtained indicate that rhizobial Nod factors are essential for the induction of cortical cell division that leads to nodule primordium formation.

Origin of triploid Arachis pintoi (Leguminosae) by autopolyploidy evidenced by FISH and meiotic behaviour.

BACKGROUND AND AIMS: Polyploidy is a dominant feature of flowering-plant genomes, including those of many important crop species. Arachis is a largely diploid genus with just four polyploid species. Two of them are economically important: the cultivated peanut and A. glabrata, a tropical forage crop. Even though it is usually accepted that polyploids within papilionoid legumes have arisen via hybridization and further chromosome doubling, it has been recently suggested that peanut arose through bilateral sexual polyploidization. In this paper, the polyploid nature of the recent, spontaneously originated triploid cytotype of the tropical lucerne, A. pintoi, was analysed, and thereby the mechanism by which polyploids may arise in the genus. METHODS: Chromosome morphology of 2x and 3x A. pintoi was determined by the Feulgens technique and the rDNA sites were mapped by FISH. To investigate whether polyploidization occurred by means of unreduced gametes, a detailed analysis of the microsporogenesis and pollen grains was made. KEY RESULTS: The 2x and 3x plants presented 9m + 1sm and a satellited chromosome type 2 in each haploid genome. Physical mapping revealed a cluster of 18S-26S rDNA, proximally located on chromosome 6, and two 5S rDNA loci on chromosomes 3 and 5. Diploid plants presented 10II in meiosis while trivalents were observed in all triploids, with a maximum of 10III by cell. Diploid A. pintoi produced normal tetrads, but also triads, dyads and monads. Two types of pollen grains were detected: (1) normal-sized with a prolate shape and (2) large ones with a tetrahedral morphology. CONCLUSIONS: Karyotype and meiotic analysis demonstrate that the 3x clone of A. pintoi arose by autopolyploidy. The occurrence of unreduced gametes strongly supports unilateral sexual polyploidization as the most probable mechanism that could have led to the origin of the triploid cytotype. This mechanism of polyploidization would probably be one of the most important mechanisms involved in the origin of economically important species of Arachis, either by triploid bridge or bilateral sexual polyploidization.

Bioprocess and bioreactor: next generation technology for production of potential plant-based antidiabetic and antioxidant molecules.

Globally, diabetes and obesity are two of the most common metabolic diseases of the 21(st) century. Increasingly, not only adults but children and adolescents are being affected. New approaches are needed to prevent and treat these disorders and to reduce the impact of associated disease-related complications. Industrial-scale production using plant-root cultures can produce quantities and quality of inexpensive bioactive small molecules with nutraceutical and pharmaceutical properties. Using this approach, and targeting these diseases, a next generation approach to tackling this emerging global health crisis may be developed. Adventitious roots cultured in bioreactors under controlled and reproducible conditions have been shown effective for production of natural products. The liquid-phase airlift bioreactor in particular has been used successfully for culturing roots on an industrial-scale and thus may provide an economical production platform for expressing promising plant-based antidiabetic and antioxidant molecules. This review focuses on a next-generation, scalable, bioprocessing approach for adventitious and hairy root cultures that are a pesticide-free, seasonally-independent, plant-based source of three molecules that have shown promise for the therapeutic management of diabetes and obesity: corosolic acid, resveratrol and ginsenosides.

Microscopic studies on the Aspergillus flavus infected kernels of commercial peanuts in Georgia.

This article describes the use of microscopy to prove the presence of the aflatoxin producing pathogen, Aspergillus flavus Link ex Fries in commercially available edible peanuts in Georgia. Light microscopy in combination with electron microscopy has been used to describe the infection course established by the fungus. The alkali maceration technique used in the study was successful and sufficient to detect the kernel infection of A. flavus and monitor the infection percentage in edible peanuts. Percentage of infected kernel varied from one commercial outlet to another in the region. Briefly, peanut seeds from Cartersville had the highest percentage of A. flavus infection. Electron microscopy confirmed the seed-borne infection of this mold. Mycelium established inside the host tissues both intercellularly and intracellularly aided by active, continuous branching of young hyphae. Establishment of mycelium was also detected in the xylem vessels of roots indicative of systemic infection. Thus, edible peanuts can form an important source of inoculum and facilitate the spread of the fungus from one peanut to another in commercial outlets and elsewhere. Present study provides strong evidence that A. flavus can escape detection at selling points and lands in commercial outlets via edible peanuts. That these contaminated peanuts could pose public health hazards is discussed.

Transformed hairy roots of Arachis hypogea: a tool for studying root nodule symbiosis in a non-infection thread legume of the Aeschynomeneae tribe.

Arachis hypogea is a non-"infection thread" (IT) legume where rhizobial entry or dissemination in the nodules never involves IT. Rhizobia invade through epidermal "cracks" and directly access the cortical cells to develop the characteristic aeschynomenoid nodules. For investigating these nonclassical nodulation features in Arachis spp., we developed an efficient procedure for Agrobacterium rhizogenes R1000-mediated transformation of this plant. In this study, we optimized the induction of hairy roots and nodulation of composite Arachis hypogea plants in the presence of Bradyrhizobium sp. (Arachis) strain NC92. 35S promoter-driven green fluorescent protein and beta-glucuronidase expression indicated transformation frequency to be above 80%. The transformed roots had the characteristic rosette-type root hairs and had normal level of expression of symbiosis-related genes SymRK and CCaMK. The transgenic nodules resembled the wild-type nodules with an exception of 2 to 3%, where they structurally deviated from the wild-type nodules to form nodular roots. A 16S rRNA profile of an infected-zone metagenome indicated that identical populations of bradyrhizobia invaded both composite wild-type plants grown in natural soil. Our results demonstrate that Arachis hairy root is an attractive system for undertaking investigations of the nonclassical features associated with its nitrogen-fixing symbiotic interactions.

Hemoglobin promotes somatic embryogenesis in peanut cultures.

Critical parameters influencing somatic embryogenesis include growth regulators and oxygen supply. Consequently, the present investigation has focused on optimization of a somatic embryogenic system for peanut (Arachis hypogaea L.) through media supplementation with the auxin, picloram. The latter at 30 mg L(-1) was optimal for inducing regeneration of somatic embryos from cultured explants of zygotic embryos. In contrast, somatic embryogenesis did not occur in the absence of this growth regulator. An assessment has also been made of the beneficial effect on somatic embryogenesis and plant regeneration of the commercial hemoglobin (Hb) solution, Erythrogen. Hemoglobin at 1:50 and 1:100 (v:v) stimulated increases in mean fresh weight (up to a maximum of 57% over control), mean number of explants producing somatic embryos (15%) and mean number of somatic embryos per explant (29%).

[Activity of acid and alkaline phosphatases (intracellular and extracellular) in rhyzosphere fungi from Arachis hypogaea (Papiloneaceae)]. / Actividad de las fosfatasas áicidas y alcalinas (extracelulares e intracelulares) en hongos de la rizosfera de Arachis hypogaea (Papiloneaceae).

The potential role of the fungi, isolated from the peanut rhizosphere, in the production of extracellular and intracellular acid and alkaline phosphatase, was evaluated in vitro. Acid and alkaline extracellular phosphatases showed the highest activities, and the Penicillium species were the most efficient in their production. The correlation analysis showed that extracellular acid and intracellular acid phosphatase produced by Aspergillus niger A. terreus, Penicillium sp. y P. brevicompactum were negatively correlated; while the extracellular and intracellular phosphatase activities, were positively correlated. The extracellular acid phosphatase activities produced in vitro by majority of fungi assayed, were not correlated with the acid phosphatase activity present in the peanut soil rhizosphere. Nevertheless, the extracellular alkaline phosphatase activities produced in vitro, were negatively correlated with the extracellular alkaline phosphatase activities present in the rhizosphere. The ability of phosphatase production by fungi isolated from peanut rhizosphere suggests they have great potential to contribute to the P mineralization in this zone.

Genotypic differences in the presence of hairs on roots and gynophores of peanuts (Arachis hypogaea L.) and their significance for phosphorus uptake.

Root hairs substantially increase the surface area of plant roots with positive effects for phosphorus (P) uptake, but the ability of peanuts to form root hairs has been questioned. The aim was to examine hair development on roots and gynophores of a variety of peanut genotypes and to relate genotypic differences in hair formation to differences in P uptake. Five out of eighteen genotypes completely lacked hairs on both organs whereas others consistently developed hairs on roots and gynophores, although with considerable variation in hair density. The ability to form root hairs as well as root hair density concurred with the presence and density of hairs on gynophores, suggesting a possible connection between both developmental processes. The contribution of root hairs to P uptake was studied in three genotypes differing in hair density. The final amount of P taken up by roots did not differ between genotypes but two distinct P uptake strategies could be identified. The genotype lacking root hairs maintained P uptake due to the development of a large root system whereas densely covered roots of genotype 'Wasedairyu' were three times as efficient in extracting P from a P-deficient soil. Furthermore P uptake through gynophores contributed about 20% to the total P uptake of Wasedairyu but only insignificant amounts to other genotypes. The ability to form hairs on roots and gynophores can therefore be seen as an adaptation to low P availability and if combined with a large root system, could substantially increase the tolerance of peanuts to P deficiency.

[A comparative study on cytochemistry between spring and fall peanut seeds].

A comparative study was conducted on cytochemistry of spring and fall crop seeds in peanut cultivars Quanhua No. 10 and Shanyou 71 respectively. Lipids, protein, and polysaccharides in cells of axis and coteledon were simultaneously shown in the Epon812 buried section by means of cytochemistry, and their morphology, quantity and distribution were compared. Embryo cells of spring crop seed develop fully with big cell more vivid contrasting texture and more regularly disposed organelle, but the counterpart cells in fall crop seeds were not as much mature and their organelle arrangement appeared somewhat irregular. In cotyledon storage cells, there were also some difference between spring crop seed and fall crop seed. Cells of spring crop seeds were full of reserves, with more lipid and protein bodies that were closely ranged and extruded with each other. However, the cell structure in fall crop seeds was more loosely arranged, vacuoles had not been filled with protein, but starch grains accumulated more. Therefore, it was shown clearly that spring crop seeds have some advantages over fall crop seeds on production application. Moreover, some cytochemical techniques for demonstration of lipid, polysaccharide and protein in thick resin section and the stain protection were discussed in the paper.

Factors enhancing Agrobacterium tumefaciens-mediated gene transfer in peanut (Arachis hypogaea L.).

Parameters enhancing Agrobacterium-mediated transfer of foreign genes to peanut (Arachis hypogaea L.) cells were investigated. An intron-containing beta-glucuronidase uidA (gusA) gene under the transcriptional control of CaMV 35S promoter served as a reporter. Transformation frequency was evaluated by scoring the number of sectors expressing GUS activity on leaf and epicotyl explants. The 'Valencia Select' market type cv. New Mexico was more amenable to Agrobacterium transformation than the 'runner' market type cultivars tested (Florunner, Georgia Runner, Sunrunner, or South Runner). The disarmed Agrobacterium tumefaciens strain EHA101 was superior in facilitating the transfer of uidA gene to peanut cells compared to the disarmed strain C58. Rinsing of explants in half-strength Murashige-Skoog (MS) media prior to infection by Agrobacterium significantly increased the transformation efficiency. The use of cocultivation media containing high auxin [1.0 or 2.5 mg/l (4.53 micromolar or 11.31 micromolar) 2,4-D] and low cytokinin [0.25 or 0.5 mg/l (1.0 micromolar or 2.0 micromolar) BA] promoted higher transformation than either hormone-free or thidiazuron-containing medium. The polarity of the epicotyl during cocultivation was important; explants incubated in an inverted (vertically) manner followed by a vertically upright position resulted in improved transformation and shoot regeneration frequencies. Preculture of explants in MS basal medium or with 2.5 mg thidiazuron per l prior to infection drastically decreased the number of transformed zones. The optimized protocol was used to obtain transient transformation frequencies ranging from 12% to 36% for leaf explants, 15% to 42% for epicotyls. Initial evidence of transformation was obtained by polymerase chain reaction and subsequently confirmed by Southern analysis of regenerated plants.

Characterization of two forms of cationic peroxidase from cultured peanut cells.

Two forms of cationic peroxidase from peanut cells were differentiated by concanavalin A affinity chromatography. They differed in molecular mass as well as concanavalin A binding, leading to the initial suggestion that they represented two isozymes of peroxidase. However, similar values for the specific activity, Soret absorption, calcium content, and peptide molecular mass were observed for each of the forms. Therefore, the binding and nonbinding fractions most likely represent two molecular forms of cationic peanut peroxidase, rather than two distinct cationic isozymes. The difference between these two forms is discussed in terms of glycosylation. Through the amino acid sequence analysis of the formic acid treated peptide, the cationic isozyme has been shown to be identical in amino acid sequence to the cDNA clone PNC1.