AtDSEL, an Arabidopsis cytosolic DAD1-like acylhydrolase, is involved in negative regulation of storage oil mobilization during seedling establishment

Mobilization of seed storage reserves is essential for seed germination and seedling establishment. Here, we report that AtDSEL, an Arabidopsis thalianaDAD1-like Seedling Establishment-related Lipase, is involved in the mobilization of storage oils for early seedling establishment. AtDSEL is a cytosolic member of the DAD1-like acylhydrolase family encoded by At4g18550. Bacterially expressed AtDSEL preferentially hydrolyzed 1,3-diacylglycerol and 1-monoacylglycerol, suggesting that AtDSEL is an sn-1-specific lipase. AtDSEL-overexpressing transgenic Arabidopsis plants (35S:AtDSEL) were defective in post-germinative seedling growth in medium without an exogenous carbon source. This phenotype was rescued by the addition of sucrose to the growth medium. In contrast, loss-of-function mutant plants (atdsel-1 and atdsel-2) had a mildly fast-growing phenotype regardless of the presence of an exogenous carbon source. Electron microscopy revealed that 5-day-old 35S:AtDSEL cotyledons retained numerous peroxisomes and oil bodies, which were exhausted in wild-type and mutant cotyledons. The impaired seedling establishment of 35S:AtDSEL was not rescued by the addition of an exogenous fatty acid source, and 35S:AtDSEL seedling growth was insensitive to 2,4-dichlorophenoxybutyric acid, indicating that β-oxidation was blocked in AtDSEL-overexpressers. These results suggest that AtDSEL is involved in the negative regulation of seedling establishment by inhibiting the breakdown of storage oils.     

Free amino acids and γ-glutamyl peptides in fagaceae

Amino acids have been investigated in seeds and fresh parts of members of the Fagaceae. Seeds from the genus Fagus contain willardiine, 5-hydroxy-6-methylpipecolic acids, N-[N-(3-amino-3-carboxypropyl)-3-amino-3-carboxypropyl]azetidine-2-carboxylic acid and γ-glutamyl peptides, mainly γ-glutamylphenylalanine. These compounds are nearly or totally absent from leaves of F. silvatica and from seedlings and immature seeds of F. silvatica var. purpurea; instead, the seedlings contain large amounts of γ-l-glutamyl-l-isoleucine and γ-l-glutamyl-l-leucine. γ-l-Glutamyl-l-tryptophan and γ-l-glutamyl-γ-l-glutamyl-l-phenylalanine, not previously known from nature, have been isolated from seeds of F. silvatica var. purpurea. The structures have been confirmed by syntheses. 4-Hydroxypipecolic acid (with trans-configuration) has been identified in seeds of F. japonica Maxim. and F. sieboldii Endl. None of the above compounds was found in Quercus or Castanea species whereas argininosuccinic acid was identified in Castanea sativa.     

Arabidopsis galactinol synthase AtGolS2 improves drought tolerance in the monocot model Brachypodium distachyon

Brachypodium distachyon (purple false brome) is a herbaceous species belonging to the grass subfamily Pooideae, which also includes major crops like wheat, barley, oat and rye. The species has been established as experimental model organism for understanding and improving cereal crops and temperate grasses. The complete genome of Bd21, the community standard line of B. distachyon, has been sequenced and protocols for Agrobacterium-mediated transformation have been published. Further improvements to the experimental platform including better evaluation systems for transgenic plants are still needed. Here we describe the growth conditions for Bd21 plants yielding highly responsive immature embryos that can generate embryogenic calli for transformation. A prolonged 20-h photoperiod produced seeds with superior immature embryos. In addition, osmotic treatment of embryogenic calli enhanced the efficiency of transfection by particle bombardment. We generated transgenic plants expressing Arabidopsis thaliana galactinol synthase 2 (AtGolS2) in these experiments. AtGolS2-expressing transgenics displayed significantly improved drought tolerance, increasing with increased expression of AtGolS2. These results demonstrate that AtGolS2 can confer drought tolerance to monocots and confirm that Brachypodium is a useful model to further explore ways to understand and improve major monocot crop species.     

Mixed function oxidases from germinating castor bean endosperm

Homogenates from germinating castor bean endosperm were fractionated by sucrose density gradient centrifugation and examined for mixed function oxidase activity. Activity of cinnamic acid 4-hydroxylase and p-chloro-N-methylaniline N-demethylase was highest in the endoplasmic reticulum fraction. Activity of both enzymes is dependent on NADPH and on molecular oxygen; both activities are inhibited by carbon monoxide. When challenged with a number of potential inhibitors the enzymes responded in ways fairly typical of mixed function oxidases from other plants and animals. The N-demethylase appears to be specific for N-methylarylamines. In the absence of NADPH, cumene hydroperoxide is able to support N-demethylation. The mechanistic significance of this activity is discussed.     

Avocado roots treated with salicylic acid produce phenol-2,4-bis (1,1-dimethylethyl), a compound with antifungal activity

We demonstrated the ability of salicylic acid (SA) to induce a compound in avocado roots that strengthens their defense against Phytophthora cinnamomi. The SA content of avocado roots, before and after the application of exogenous SA, was determined by High-Performance Liquid Chromatography (HPLC). After 4 h of SA feeding, the endogenous level in the roots increased to 223 μg g⁻¹ FW, which was 15 times the amount found in control roots. The methanolic extract obtained from SA-treated avocado roots inhibited the radial growth of P. cinnamomi. A thin layer chromatographic bioassay with the methanolic extract and spores of Aspergillus showed a distinct inhibition zone. The compound responsible for the inhibition was identified as phenol-2,4-bis (1,1-dimethylethyl) by gas chromatography and mass spectrometry. At a concentration of 100 μg/mL, the substance reduced germinative tube length in Aspergillus and radial growth of P. cinnamomi. A commercial preparation of phenol-2,4-bis (1,1-dimethylethyl) caused the same effects on mycelium morphology and radial growth as our isolate, confirming the presence of this compound in the root extracts. This is the first report of the induction of this compound in plants by SA, and the results suggest that it plays an important role in the defense response of avocado.     

Callus mediated shoot organogenesis and regeneration of cytologically stable plants of Ledebouria revoluta: An ethnomedicinal plant with promising antimicrobial potency

Ledebouria revoluta are important ethnomedicinal plant found in India and South Africa. Micropropagation via indirect shoot organogenesis had been established from three types of explant (i.e. scale leaf, leaf lamina and root) of L. revoluta. Scale leaf was found superior as compared to leaf lamina and root explant with respect to their organogenic callus induction potentiality. Murashige and Skoog (1962) [MS] media supplemented with 3.0?mg?L^?1 2,4-dichlorophenoxyacetic acid, 0.75?mg?L^?1 β-naphthoxyacetic acid were best effective for inducing organogenic callus. Maximum 17.0?±?0.52 bulblets were induced from about 500?mg of callus within 42–46?days sub-culturing on a medium containing 0.75?mg?L^?1 kinetin. The bulblets were matured (86.7% success) after one month culture on the same medium composition. The best result of in vitro root induction with 100% response and 8.4?±?0.31 roots per bulb was achieved after 18?days of implantation on MS medium containing 2.0?mg?L^?1 indole-3-butyric acid. Plantlets were acclimatized with a 96.0% survival rate. Chromosomal studies revealed cytological stability of callus cells and all regenerants containing 2n?=?30 chromosomes, same as parental plants. Antimicrobial activity of L. revoluta was tested against two Gram-positive bacteria, three Gram-negative bacteria and two fungi. The methanol and ethanol extract proved more effective against bacteria, whereas acetone and chloroform extract shows potential anti-fungal activities. Present protocol can be applied reliably to produce uniform planting materials in large scale. In addition, this efficient indirect regeneration pathway via callus culture opens a way for improvement through genetic transformation.     

Karyology and sex determination in Aglaothamnion oosumiense Itono (Ceramiaceae,Rhodophyta)

A cytogenetic investigation on male and female reproductive cells of Aglaothamnion oosumiense Itono indicates that the sexuality of this species might be determined by a sex chromosome. Chromosome counts in female and male gametophytes gave 37 and 36, respectively. Sex ratio of gametophytes was 1:1. Both male-derived and female-derived bisexual plants were observed. Bisexual plants were different in gross morphology and position of carpogonial branches from normal unisexual gametophytes. The chromosome number of female-derived bisexual plants was N=37 and male-derived bisexual plants was N=36. Some male plants developed parasporangia in addition. The paraspore germlings showed the same chromosome number as the male plants. The fertilized carpogonium and gonimoblast cells had 2N = ca. 70 chromosomes.     

N-Glycosylation and N-Glycan Moieties of CTB Expressed in Rice Seeds

Cholera toxin B subunit (CTB) is widely used as a carrier molecule and mucosal adjuvant and for the expression of fusion proteins of interest. CTB-fusion proteins are also expressed in plants, but the N-glycan structures of CTB have not been clarified. To gain insights into the N-glycosylation and N-glycans of CTB expressed in plants, we expressed CTB in rice seeds with an N-terminal glutelin signal and a C-terminal KDEL sequence and analyzed its N-glycosylation and N-glycan structures. CTB was successfully expressed in rice seeds in two forms: a form with N-glycosylation at Asn32 that included both plant-specific N-glycans and small oligomannosidic N-glycans and a non-N-glycosylated form. N-Glycan analysis of CTB showed that approximately 50 % of the N-glycans had plant-specific M3FX structures and that almost none of the N-glycans was of high-mannose-type N-glycan even though the CTB expressed in rice seeds contains a C-terminal KDEL sequence. These results suggest that the CTB expressed in rice was N-glycosylated through the endoplasmic reticulum (ER) and Golgi N-glycosylation machinery without the ER retrieval.     

phlF− mutant of Pseudomonas fluorescens J2 improved 2,4-DAPG biosynthesis and biocontrol efficacy against tomato bacterial wilt

Pseudomonas fluorescens J2 can produce 2,4-diacetylphloroglucinol (2,4-DAPG) as the main antibiotic compound and effectively inhibits the wilt pathogens Ralstonia solanacearum and Fusarium oxysporum. The phlF which negatively regulates the 2,4-DAPG synthesis in strain J2 was disrupted by homologous recombination to construct a mutant strain J2-phlF⁻. The mutant J2-phlF⁻ produced much more 2,4-DAPG and showed higher inhibitory effect on R. solanacearum than the wild type strain J2 in vitro. The mutant J2-phlF⁻ also showed more colonization of tomato roots and higher inhibition to R. solanacearum in soil than wild type strain J2. The biocontrol efficiency of mutant J2-phlF⁻ was higher against tomato bacterial wilt than wild type strain J2, but the differences were not significant. However, the application of both strains with organic fertilizer improved the colonization and biocontrol efficiency against tomato bacterial wilt and mutant strain J2-phlF⁻ showed higher biocontrol efficiency against tomato bacterial wilt than wild type strain J2. Both strains, J2 and J2-phlF⁻, could also promote the growth of tomato plants.     

Synthesis and biological evaluation of thiazolidine-2,4-dione-pyrazole conjugates as antidiabetic,anti-inflammatory and antioxidant agents

A series of fourteen novel thiazolidine-2,4-dione derivatives clubbed with pyrazole moiety were synthesized via four step reaction procedure. Reactions were monitored by thin layer chromatography and were characterized by physicochemical and spectrophotometric (IR, Mass, ¹HNMR and ¹³CNMR) analysis. The spectral data were in good agreement with their structures. The title compounds were docked against peroxisome proliferated activated receptors (PPAR-γ) and alpha-amylase and further evaluated for in vivo and in vitro antidiabetic, in vitro anti-inflammatory and antioxidant activities. Compound GB14 exhibited significant blood glucose lowering activity and was also found to be active inhibitor of alpha-amylase. Compound GB7 was found to be potent anti-inflammatory agent in terms of reducing inflammatory markers (TNF-α, IL-β, MDA) and also showed antioxidant activity to good extent. Therefore, these compounds may be considered as promising candidates for the development of new antidiabetic agents.     

Production of transgenic local rice cultivars (Oryza sativa L.) for improved drought tolerance using Agrobacterium mediated transformation

Rice being the staple food of middle and south India, there is an extensive research undertaken in protecting the species and improving the quality and yield. Several recombinations have been made to the rice genome to impart various qualities which lack in the pure breed. Oryza faces various natural stress, like temperature variance, high salinity, etc., drought is one of the major parameters affecting the growth and yield of the plant. Transgenic rice cultivars can be generated for drought tolerance using the Agrobacterium mediated transformations. The current work aims to impart the gene for drought tolerance in Oryza sativa L. using Agrobacterium mediated transformation. The gene targeted in this context is dehydration response element binding factors (DREB). DREB plays a major role in response to drought mediated stress. Sambha mahsuri (Indica type) and Cotton dora sannalu (Indica type) the two local cultivars have been transformed for the gene AtDREB1A under 35s CaMV promoters (pBIH binary vector) for which the vector used was Agrobacterium. The target plant tissue being used was calli. Optimization of the parameters was performed for a lethal dose of hygromycin, cefotaxime level, and acetosyringone level. PCR amplification was used for the confirmation of the transgenic (T₀) species in which 23% and 18% for Sambha mahsuri and Cotton dora sannalu, respectively. Southern blotting was performed for the genomic DNA. Normal growth was shown by the T₁ transgenic plants whose expression was confirmed by RT-PCR. The T₁ transgenic plants showed good tolerance to drought mediated stress for a total period of one and a half week under greenhouse condition. The study can be concluded by producing a potentially successful drought resistance T₁ species produced using Agrobacterium mediated transformation.     

Anion activation of γ-glutamyltransferase from fruiting bodies of Lentinus edodes

Activation by different anions of γ-glutamyltransferase obtained in a. particulate form from fruiting bodies of Lentinus edodes has been studied using either L-γ-glutamyl-p-nitroanlide or lentinic acid as substrate. The mushroom transferase was activated by SCN^?, NO₃^?, Cl^?, Br^?, ClO₃^?, Bro₃^?, N₃^?, I^? and F^?, but not those alkali and earth cations previously believed to activate the animal transferase, nor by citrate, claimed to be effective for the kidney bean transferase. Among anions proved hardly to activate the transferase were ClO₄^?, NO₂?, HCO₃^?, H₂PO₄^?, SO₃^2? and SO₄^2?. A high concentration of these anions more or less impeded the halide activation. Kinetic studies revealed that halides function as activators of increasing V_max while keeping K_m constant. These observations appeared least compatible with the possibility that the anion activation might involve a non-specific effect of high solute concentration, viz. dissociation of the enzyme from the supporting structure in the particulates. The activating effect of halides described here probably extends also to the animal enzymes.     

Lauroylethanolamide is a potent competitive inhibitor of lipoxygenase activity

The lipoxygenase (LOX) pathway was proposed to compete with hydrolysis and be partly responsible for the metabolism of polyunsaturated N-acylethanolamines (PU-NAEs). Treatment of Arabidopsis seedlings with lauroylethanolamide (NAE 12:0) resulted in elevated levels of PU-NAE species, and this was most pronounced in plants with reduced NAE hydrolase activity. Enzyme activity assays revealed that NAE 12:0 inhibited LOX-mediated oxidation of PU lipid substrates in a dose-dependent and competitive manner. NAE 12:0 was 10-20 times more potent an inhibitor of LOX activities than lauric acid (FFA 12:0). Furthermore, treatment of intact Arabidopsis seedlings with NAE 12:0 (but not FFA 12:0) substantially blocked the wound-induced formation of jasmonic acid (JA), suggesting that NAE 12:0 may be used in planta to manipulate oxylipin metabolism.     

Promotion of photosynthesis in transgenic rice over-expressing of maize C4 phosphoenolpyruvate carboxylase gene by nitric oxide donors

We determined the effects of exogenous nitric oxide on photosynthesis and gene expression in transgenic rice plants (PC) over-expressing the maize C₄ pepc gene, which encodes phosphoenolpyruvate carboxylase (PEPC). Seedlings were subjected to treatments with NO donors, an NO scavenger, phospholipase inhibitors, a Ca²⁺ chelator, a Ca²⁺ channel inhibitor, and a hydrogen peroxide (H₂O₂) inhibitor, individually and in various combinations. The NO donors significantly increased the net photosynthetic rate (P_N) of PC and wild-type (WT), especially that of PC. Treatment with an NO scavenger did inhibit the P_N of rice plants. The treatments with phospholipase inhibitors and a Ca²⁺ chelator decreased the P_N of WT and PC, and photosynthesis was more strongly inhibited in WT than in PC. Further analyses showed that the NO donors increased endogenous levels of NO and PLD activity, but decreased endogenous levels of Ca²⁺ both WT and PC. However, there was a greater increase in NO in WT and a greater increase in PLD activity and Ca²⁺ level in PC. The NO donors also increased both PEPC activity and pepc gene expression in PC. PEPC activity can be increased by SNP alone. But the expression of its encoding gene in PC might be regulated by SNP, together with PA and Ca²⁺.     

Branching, nitrogen distribution, and carbon gain in solitary plants of an annual herb, Xanthium canadense

The theory of optimal nitrogen (N) distribution predicts that the carbon gain of plants will be maximised when leaves of higher irradiance have higher N content per area (N _area). Most previous studies have examined optimal N distribution without explicitly considering the branching status of plants. I investigated light environment, N distribution and photosynthetic traits of individual leaves of an herbaceous species, Xanthium canadense. X. canadense was grown solitary under high (HN) and low nutrients (LN). Light availability, leaf mass per unit area and N _area were measured for all leaves within plants. Daily photosynthesis of the plants of actual N distribution was compared with those of optimal and constant N distribution. Branch production was facilitated in HN but not in LN plants. N _area was correlated more with leaf order than with leaf light environment. Although N was more limited and the light environment was less heterogeneous within crowns in LN than in HN plants, leaf N distribution was closer to optimal in the latter. These results suggest that leaf N distribution was not optimised in solitary plants of X. canadense. Because this species often regenerates in a dense stand, leaf N distribution might be selected to maximise carbon gain only in such a stand. Leaf N distribution might thus be constrained by the regeneration strategy of the species.     

Photosynthate metabolism in the source leaves of n(2)-fixing soybean plants

Soybean plants (Glycine max [L.] Merr. cv Williams), which were symbiotic with Bradyrhizobium japonicum, and which grew well upon reduced nitrogen supplied solely through N₂ fixation processes, often exhibited excess accumulation of starch and sucrose and diminished soluble protein in their source leaves. Nitrate and ammonia, when supplied to the nodulated roots of N₂-fixing plants, mediated a reduction of foliar starch accumulation and a corresponding increase in soluble protein in the source leaves. This provided an opportunity to examine the potential metabolic adjustments by which NO₃⁻ and NH₄⁺ (N) sufficiency or deficiency exerted an influence upon soybean leaf starch synthesis. When compared with soybean plants supplied with N, elevated starch accumulation was focused in leaf palisade parenchyma tissue of N₂-fixing plants. Foliar activities of starch synthesis pathway enzymes including fructose-1,6-bisphosphate phosphatase, phosphohexoisomerase, phosphoglucomutase (PGM), as well as adenosine diphosphate glucose pyrophosphorylase (in some leaves) exhibited highest activities in leaf extracts of N₂-fixing plants when expressed on a leaf protein basis. This was interpreted to mean that there was an adaptation of these enzyme activities in the leaves of N₂-fixing plants, and this contributed to an increase in starch accumulation. Another major causal factor associated with increased starch accumulation was the elevation in foliar levels of fructose-6-phosphate, glucose-6-phosphate, and glucose-1-phosphate (G1P), which had risen to chloroplast concentrations considerably in excess of the K_m values for their respective target enzymes associated with starch synthesis, e.g. elevated G1P with respect to adenosine diphosphate glucose pyrophosphorylase (ADPG-PPiase) binding sites. The cofactor glucose-1,6-bisphosphate (G1,6BP) was found to be obligate for maximal PGM activity in soybean leaf extracts of N₂-fixing as well as N-supplemented plants, and G1,6BP levels in N₂-fixing plant leaves was twice that of levels in N-supplied treatments. However the concentration of chloroplastic G1,6BP in illuminated leaves was computed to be saturating with respect to PGM in both N₂-fixing and N-supplemented plants. This suggested that the higher level of this cofactor in N₂-fixing plant leaves did not confer any higher PGM activation and was not a factor in higher starch synthesis rates. Relative to plants supplied with NO₃⁻ and NH₄⁺, the source leaf glycerate-3-phosphate (3-PGA) and orthophosphate (Pi) concentrations in leaves of N₂-fixing plants were two to four times higher. Although Pi is a physiological competitive inhibitor of leaf chloroplast ADPG-PPiase, and hence, starch synthesis, elevated chloroplast 3-PGA levels in N₂-fixing plant leaves apparently prevented interference of Pi with ADPG-PPiase catalysis and starch synthesis.     

Biosynthesis of tropic acid: Feeding experiments with cinnamoyltropine and littorine

The administration of cinnamoyl-[2-¹⁴C]-tropine-[N-methyl-¹⁴C] to Datura stramonium plants resulted in the formation of labeled atropine and scopolamine. However the atropine was found to have almost all its radioactivity located on the N-methyl group of the alkaloid, indicating that the administered ester had undergone hydrolysis in the plant affording tropine and cinnamic acid, the latter not being utilized for the biosynthesis of tropic acid. Dual labeled RS-littorine (3β-(2-hydroxy-3-phenylpropionyloxy-[1-¹⁴C]-tropane-[3β-³H]) was also fed to D. stramonium and radioactive atropine was obtained. However the drastic change in the ³H:¹⁴C ratio found in the atropine indicated that the littorine was not converted directly to the alkaloid, and it is suggested that the littorine is hydrolysed _{in vivo} to tropine and phenyl-lactic acid, the latter undergoing rearrangement to tropic acid prior to esterification with tropine.     

Selenium bioisosteric replacement of adenosine derivatives promoting adiponectin secretion increases the binding affinity to peroxisome proliferator-activated receptor δ

N⁶-(3-Iodobenzyl)adenosine-5′-N-methyluronamide (1a, IB-MECA) exhibited polypharmacological characteristics targeting A₃ adenosine receptor (AR), peroxisome proliferator-activated receptor (PPAR) γ, and PPARδ, simultaneously. The bioisosteric replacement of oxygen in 4′-oxoadenosines with selenium significantly increased the PPARδ-binding activity. 2-Chloro-N⁶-(3-iodobenzyl)-4′-selenoadenosine-5′-N-methyluronamide (3e) and related 4′-selenoadenosine derivatives significantly enhanced adiponectin biosynthesis during adipogenesis in human bone marrow mesenchymal stem cells (hBM-MSCs). The PPARδ-binding affinity, but not the A₃ AR binding affinity, of 4′-selenoadenosine derivatives correlated with their adiponectin secretion stimulation. Compared with the sugar ring of 4′-oxoadenosine, that of 4′-selenoadenosine was more favorable in forming the South sugar conformation. In the molecular docking simulation, the South sugar conformation of compound 3e formed additional hydrogen bonds inside the PPARδ ligand-binding pocket compared with the North conformation. Therefore, the sugar conformation of 4′-selenoadenosine PPAR modulators affects the ligand binding affinity against PPARδ.