Synthetic auxin herbicide 2,4-D and its influence on a model BY-2 suspension.

2,4-D is a broadly used auxin herbicide. The presence of the 2,4-D synthetic auxin in the medium is imperative for long-term BY-2 tobacco suspension viability. The precise mechanism of this symbiosis of the suspension and the synthetic auxin remains unclear. Our goal was to study the hormonal regulation of the growth of the cell suspension; and to describe the experiments clarifying the interaction between the chosen growth regulators and phytohormones on the cellular level, specifically between the 2,4-D synthetic auxin and the native stress phytohormone - ethylene. This study examined the influence of low 2,4-D concentrations stimulating cell growth in vitro as well as the influence of high herbicide concentrations on the model tobacco BY-2 suspension. The culture took 6 days. Different parameters were evaluated, including the influence of different 2,4-D concentrations on the production of the phytohormone ethylene and its precursor 1-Aminocyclopropane-1-carboxylic acid (ACC) in the tobacco cells. The content of 2,4-D in the cells and the medium was established. The observations of the morphological changes showed that a heavy impregnation of the cell walls taking place depending on the concentration of 2,4-D. A dramatic increase in protective polysaccharides and a remodulation of the cell walls by the formation of a pectin shield in artificial conditions were expected and observed. At the same time, massive production of the stress phytohormone ethylene took place, and, because of that, plant mutagenicity, anomalous tumour-type proliferation growth, and the production of supercells were observed. The hypothesis of the protective shield is discussed.

Molecular understanding of anthocyanin biosynthesis activated by PAP1 and regulated by 2, 4-dichlorophenoxyacetic acid in engineered red Artemisia annua cells.

MAIN CONCLUSION: Eight promoters were cloned, from which AC and G-box cis-elements were identified. PAP1 enhanced the promoter activity. 2,4-D reduced the anthocyanin biosynthesis via downregulating the expression of the PAP1 transgene. Artemisia annua is an effective antimalarial medicinal crop. We have established anthocyanin-producing red cell cultures from this plant with the overexpression of Production of Anthocyanin Pigment 1 (PAP1) encoding a R2R3MYB transcription factor. To understand the molecular mechanism by which PAP1 activated the entire anthocyanin pathway, we mined the genomic sequences of A. annua and obtained eight promoters of the anthocyanin pathway genes. Sequence analysis identified four types of AC cis-elements from six promoters, the MYB response elements (MRE) bound by PAP1. In addition, six promoters were determined to have at least one G-box cis-element. Eight promoters were cloned for activity analysis. Dual luciferase assays showed that PAP1 significantly enhanced the promoting activity of seven promoters, indicating that PAP1 turned on the biosynthesis of anthocyanins via the activation of these pathway gene expression. To understand how 2,4-dichlorophenoxyacetic acid (2,4-D), an auxin, regulates the PAP1-activated anthocyanin biosynthesis, five different concentrations (0, 0.05, 0.5, 2.5, and 5 µM) were tested to characterize anthocyanin production and profiles. The resulting data showed that the concentrations tested decreased the fresh weight of callus growth, anthocyanin levels, and the production of anthocyanins per Petri dish. HPLC-qTOF-MS/MS-based profiling showed that these concentrations did not alter anthocyanin profiles. Real-time RT-PCR was completed to characterize the expression PAP1 and four representative pathway genes. The results showed that the five concentrations reduced the expression levels of the constitutive PAP1 transgene and three pathway genes significantly and eliminated the expression of the chalcone synthase gene either significantly or slightly. These data indicate that the constitutive PAP1 expression depends on gradients added in the medium. Based on these findings, the regulation of 2,4-D is discussed for anthocyanin engineering in red cells of A. annua.

Micropropagation of pokeweed (Phytolacca americana L.) and comparison of phenolic, flavonoid content, and antioxidant activity between pokeweed callus and other parts.

BACKGROUND: Pokeweed (Phytolacca americana L.) is regarded as an invasive plant in many parts of the world but possesses therapeutic characteristics used for antitumor and rheumatism treatment. This study investigated the effects of auxins and four explants on pokeweed callus induction. The effects of cytokinins and combinations between cytokinins and NAA on shoot and root induction were also studied. TPC, TFC and antioxidant activity of calli were screened and compared with other pokeweed plant parts. METHODS: Four explants were used to induce callus using 2,4-D and IBA at 1, 2, 3 and 4 mg/l for each auxin. Direct shoot organogenesis from nodal explants was investigated using BAP, kinetin and TDZ (1, 2 and 4 mg/l for each cytokinin). Combined effects between cytokinins and NAA at 0.1, 0.2 and 0.3 mg/l were further simultaneously estimated with root induction. Calli derived from the leaves were compared with other plant parts for TPC, TFC and antioxidant activity using the Folin-Ciocalteu, AlCl3 colorimetric assay and DPPH assays, respectively. RESULTS: Results showed that MS medium containing 2 mg/l 2,4-D induced callus formation on leaf explants that provided highest fresh and dry weights. Three types of synthetic cytokinins as kinetin, TDZ and BAP were used for direct shoot organogenesis from pokeweed nodes. MS medium containing 2 mg/l kinetin was effective in stimulating normal shoots, with the largest number of shoots and leaves and the longest shoots. The combination between cytokinins and NAA showed no positive effect on shoot and root induction from pokeweed nodal explants. For TPC and TFC determination, pokeweed seeds and leaves possessed the highest phenolic and flavonoid contents, respectively. Highest phenolic content of pokeweed seeds led to lowest IC50 by DPPH assay. Phenolic content was higher than flavonoid content. CONCLUSION: Results suggested promising conditions for callus induction. Leaf explants cultured on MS medium with 2 mg/l 2,4-D and nodal explants cultured on MS medium with 2 mg/l kinetin provided the largest number of normal shoots and leaves. NAA did not show positive effects on shoot and root induction when combined with cytokinins. Chemical constituent screening indicated that seeds and leaves provided highest TPC and TFC, respectively, while pokeweed calli contained higher phenolic than flavonoid content. This is the first report describing chemical constituent screening and antioxidant activity of calli and other parts of the pokeweed plant. Results provided significant information to further enhance bioactive compound contents of pokeweed calli using elicitation methods.

Comparing the effects of different exogenous hormone combinations on seed-derived callus induction in Nicotiana tabacum.

Callus from Nicotiana tabacum is used as a model in plant developmental research. We tested several phytohormone (Indoleacetic acid - IAA; 2,4-Dichlorophenoxyacetic acid - 2,4-D; kinetin - KIN; 6-Benzylaminopurine - BAP) combinations to compare different approaches to callus induction directly from the seeds of Nicotiana tabacum. Callus formation was observed up to 4 weeks after sowing and the most effective were 0.5 mg/L of 2,4-D with 0.25 mg/L of BAP and 2 mg/L 2,4-D with 1 mg/L of BAP. The calli were green, photosynthetically active and after 6 weeks of growth, no stress symptoms (estimated on the basis of fluorescence of chlorophyll a in photosystem II) were noticed.

2,4-Dichlorophenoxyacetic Thiosemicarbazides as a New Class of Compounds Against Stomach Cancer Potentially Intercalating with DNA.

hiosemicarbazide is a useful structural moiety that has the biological potential. Optimization of this structure can result in groundbreaking discovery of a new class of therapeutic agents. In the light of this, 1-(2,4-dichlorophenoxy)acetyl-4-(1-naphthyl)thiosemicarbazide (1) and 1,4-bis[(2,4-dichlorophenoxy)acetylthiosemicarbazide]phenyl (2) were synthesized and characterized by spectroscopic method. Cytotoxicity of obtained compounds was evaluated on MKN74 gastric cancer cell line and human skin fibroblast BJ based on methylthiazolyldiphenyl-tetrazolium bromide (MTT) test. The apoptosis/necrosis and cell cycle analysis were conducted using image cytometry. Additionally, in DNA of treated cells, abasic sites (AP) and double strands breaks (DSB) presence were measured. Intercalating properties of active compounds were evaluated using the UV-spectroscopic method. Among newly synthesized derivatives, compound 2 showed toxic effects on gastric cancer cells with simultaneous lack of toxicity to normal fibroblasts. Cell cycle analysis revealed that both compounds influence cell division mainly at the stage of replication. Simultaneously with DNA synthesis disorders, DNA damages like AP-sites and DSBs were observed. Spectroscopic studies revealed possible DNA intercalating properties of tested compounds. Obtained results indicate that the newly synthesized thiosemicarbazide derivatives are a promising group of compounds with potential anticancer activity resulted from interactions with DNA and cell cycle interrupt.

Identification of Novel Inhibitors of Auxin-Induced Ca2+ Signaling via a Plant-Based Chemical Screen.

Many signal perception mechanisms are connected to Ca2+-based second messenger signaling to modulate specific cellular responses. The well-characterized plant hormone auxin elicits a very rapid Ca2+ signal. However, the cellular targets of auxin-induced Ca2+ are largely unknown. Here, we screened a biologically annotated chemical library for inhibitors of auxin-induced Ca2+ entry in plant cell suspensions to better understand the molecular mechanism of auxin-induced Ca2+ and to explore the physiological relevance of Ca2+ in auxin signal transduction. Using this approach, we defined a set of diverse, small molecules that interfere with auxin-induced Ca2+ entry. Based on annotated biological activities of the hit molecules, we found that auxin-induced Ca2+ signaling is, among others, highly sensitive to disruption of membrane proton gradients and the mammalian Ca2+ channel inhibitor bepridil. Whereas protonophores nonselectively inhibited auxin-induced and osmotic stress-induced Ca2+ signals, bepridil specifically inhibited auxin-induced Ca2+ We found evidence that bepridil severely alters vacuolar morphology and antagonized auxin-induced vacuolar remodeling. Further exploration of this plant-tailored collection of inhibitors will lead to a better understanding of auxin-induced Ca2+ entry and its relevance for auxin responses.

Inhibition effects of pesticides on glutathione-S-transferase enzyme activity of Van Lake fish liver.

Glutathione-S-transferases (GSTs) have a function in xenobiotic metabolism. They are a significant multifunctional family with a wide variety of catalytic activities. In the current study, we determined in vitro inhibition effects of 2,4-dichlorophenoxyacetic acid dimethylamine salt (2,4-D DMA), haloxyfop-P-methyl, glyphosate isopropylamine, dichlorvos, and λ-cyhalothrin on purified GST. For this purpose, GST were purified from Van Lake fish (Chalcalburnus tarichii Pallas) liver with 29.25 EU mg-1 specific activity and 10.76% yield using GSH-agarose affinity chromatographic method. The pesticides were tested at various concentrations on in vitro GST activity. Ki constants were calculated as 0.17 ± 0.01, 0.25 ± 0.05, 3.72 ± 0.32, 0.42 ± 0.06, and 0.025 ± 0.004 mM, for 2,4-D DMA, haloxyfop-P-methyl, glyphosate isopropylamine, dichlorvos, and λ-cyhalothrin, respectively. λ-Cyhalothrin showed a better inhibitory effect compared to the other pesticides. The inhibition mechanisms of λ-cyhalothrin were competitive, while the other pesticides were noncompetitive.

Kinetic, spectroscopic and computational docking study of the inhibitory effect of the pesticides 2,4,5-T, 2,4-D and glyphosate on the diphenolase activity of mushroom tyrosinase.

The inhibitory effect of 2,4,5-T, 2,4-D, glyphosate and paraquat on the diphenolase activity of mushroom tyrosinase for oxidation of L-DOPA has been investigated by kinetic measurements, fluorescence spectroscopy and computational docking analysis. 2,4,5-T and 2,4-D inhibit the diphenolase activity of the enzyme following a competitive mechanism, while glyphosate is a mixed inhibitor according to Lineweaver-Burk kinetic analysis. The inhibitory activity follows the order glyphosate >2,4,5-T > 2,4-D with IC50 values of 65, 90 and 106 µM, respectively. Intrinsic tyrosinase fluorescence quenching and computational docking analysis suggest that 2,4,5-T and 2,4-D interact with the active site of the enzyme through hydrophobic interactions, while glyphosate also interacts with external residues of the active site of the enzyme by hydrogen bonding and hydrophilic interactions inducing conformational changes in the protein structure.

Holistic protocol for callus culture optimization using statistical modelling.

Plants endue a key role against illnesses caused by oxidative stress. These attributes are frequently associated with polyphenolic compounds. However, presence and concentration of secondary metabolites are affected by abiotic factors. The in vitro culture techniques can solve these drawbacks. Peppers can be a suitable alternative to obtain polyphenols. Aiming to optimise the callus culture stage from Capsicum baccatum to produce polyphenols, this work evaluated systemically the effects of the explant's origin (root, hypocotyl and cotyledon), growth hormone type (2,4-dichlorophenoxyacetic acid (2,4-D), benzylaminopurine (BAP) and a combination of 2,4-D/BAP at five-to-one ratio) and concentration (0.023-10.000 mg L-1) on callus culture efficiency parameters using a multilevel factorial design. The root explant in combination with BAP at 1.138 mg L-1 ensured the optimal values of the assessed responses; ​callus mass (225.03 mg), antioxidant activity (35.95%), total phenols (11.48 mg of GAE/g DE) and flavonoids (15.92 mg of RU/g DE) production.

2,4-D attenuates salinity-induced toxicity by mediating anatomical changes, antioxidant capacity and cation transporters in the roots of rice cultivars.

Growth regulator herbicides are widely used in paddy fields to control weeds, however their role in conferring environmental stress tolerance in the crop plants are still elusive. In this study, the effects of recommended dose of 2,4-dichlorophenoxyacetic acid (2,4-D)  on growth, oxidative damage, antioxidant defense, regulation of cation transporter genes and anatomical changes in the roots of rice cultivars XS 134 (salt resistant) and ZJ 88 (salt sensitive) were investigated under different levels of saline stress. Individual treatments of saline stress and 2,4-D application induced oxidative damage as evidenced by decreased root growth, enhanced ROS production, more membrane damage and Na+ accumulation in sensitive cultivar compared to the tolerant cultivar. Conversely, combined treatments of 2,4-D and saline stress significantly alleviated the growth inhibition and oxidative stress in roots of rice cultivars by modulating lignin and callose deposition, redox states of AsA, GSH, and related enzyme activities involved in the antioxidant defense system. The expression analysis of nine cation transporter genes showed altered and differential gene expression in salt-stressed roots of sensitive and resistant cultivars. Together, these results suggest that 2,4-D differentially regulates the Na+ and K+ levels, ROS production, antioxidant defense, anatomical changes and cation transporters/genes in roots of rice cultivars.

Production of the dammarene sapogenin (protopanaxadiol) in transgenic tobacco plants and cultured cells by heterologous expression of PgDDS and CYP716A47.

KEY MESSAGE: Protopanaxadiol (PPD) is an aglycone of dammarene-type ginsenoside and has high medicinal values. In this work, we reported the PPD production in transgenic tobacco co-overexpressing PgDDS and CYP716A47. PPD is an aglycone of ginsenosides produced by Panax species and has a wide range of pharmacological activities. PPD is synthesized via the hydroxylation of dammarenediol-II (DD) by CYP716A47 enzyme. Here, we established a PPD production system via cell suspension culture of transgenic tobacco co-overexpressing the genes for PgDDS and CYP716A47. The concentration of PPD in transgenic tobacco leaves was 2.3-5.7 µg/g dry weight (DW), depending on the transgenic line. Leaf segments were cultured on medium with various types of hormones to induce callus. Auxin treatment, particularly 2,4-D, strongly enhanced the production of DD (783.8 µg g(-1) DW) and PPD (125.9 µg g(-1) DW). Treatment with 2,4-D enhanced the transcription of the HMG-Co reductase (HMGR) and squalene epoxidase genes. PPD production reached 166.9 and 980.9 µg g(-1) DW in a 250-ml shake flask culture and in 5-l airlift bioreactor culture, respectively.

An efficient in vitro system for somatic embryogenesis and podophyllotoxin production in Podophyllum hexandrum Royle.

Podophyllum hexandrum Royle known as Indian mayapple is an important medicinal plant found only in higher altitudes (2,700 to 4,200 m) of the Himalayas. The highly valued anticancer drug Podophyllotoxin is obtained from the roots of this plant. Due to over exploitation, this endemic plant species is on the verge of extinction. In vitro culture for efficient regeneration and the production of podophyllotoxin is an important research priority for this plant. Hence, in the present study, an efficient plant regeneration system for mass multiplication through somatic embryogenesis was developed. We have screened P. hexandrum seeds collected from three different regions in the Himalayas to find their regenerative potentials. These variants showed variation in germination percentage as well as somatic embryogenic frequency. The seeds collected from the Milam area of Pithoragarh district showed better germination response (99.3%) on Murashige and Skoog (MS) medium fortified with Gibberellic acid (GA3 [5 mg/l]) and higher direct somatic embryogenic frequency (89.6%). Maximum production of embryogenic callus (1.2 g fresh weight [FW]) was obtained when cotyledons containing the direct somatic embryo clusters were cultured in MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D [1.5 mg/l]) after 4 week of culture in complete darkness. In the present investigation, somatic embryogenesis was accomplished either by direct organogenesis or callus mediated pathways. The latter method resulted in a higher frequency of somatic embryo induction in hormone-free MS medium yielding 47.7 embryos/50 mg of embryogenic callus and subsequent germination in MS medium supplemented with GA3 (5 mg/l). Seventy-nine percent of embryos attained complete maturity and germinated into normal plants with well-developed roots. Systematic histological analysis revealed the origin of somatic embryo and their ontogenesis. The higher level of podophyllotoxin (1.8 mg/g dry weight [DW]) was recorded in germinated somatic embryos when compared to field grown plants. The present system can be widely used for mass propagation, transgenic recovery, and podophyllotoxin production for commercial utilization.

Regulation of anthocyanin biosynthesis in Arabidopsis thaliana red pap1-D cells metabolically programmed by auxins.

Red pap1-D cells of Arabidopsis thaliana have been cloned from production of anthocyanin pigmentation 1-Dominant (pap1-D) plants. The red cells are metabolically programmed to produce high levels of anthocyanins by a WD40-bHLH-MYB complex that is composed of the TTG1, TT8/GL3 and PAP1 transcription factors. Here, we report that indole 3-acetic acid (IAA), naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) regulate anthocyanin biosynthesis in these red cells. Seven concentrations (0, 0.2, 0.4, 2.2, 9, 18 and 27 µM) were tested for the three auxins. IAA and 2,4-D at 2.2-27 µM reduced anthocyanin levels. NAA at 0-0.2 µM or above 9 µM also decreased anthocyanin levels, but from 0.4 to 9 µM, it increased them. HPLC-ESI-MS analysis identified seven cyanin molecules that were produced in red pap1-D cells, and their levels were affected by auxins. The expression levels of ten genes, including six transcription factors (TTG1, EGL3, MYBL2, TT8, GL3 and PAP1) and four pathway genes (PAL1, CHS, DFR and ANS) involved in anthocyanin biosynthesis were analyzed upon various auxin treatments. The resulting data showed that 2,4-D, NAA and IAA control anthocyanin biosynthesis by regulating the expression of TT8, GL3 and PAP1 as well as genes in the anthocyanin biosynthetic pathway, such as DFR and ANS. In addition, the expression of MYBL2, PAL1 and CHS in red pap1-D and wild-type cells differentially respond to the three auxins. Our data demonstrate that the three auxins regulate anthocyanin biosynthesis in metabolically programmed red cells via altering the expression of transcription factor genes and pathway genes.

Callogenesis in leaves of Kalanchoe pinnata Lam. by 2, 4-D and BA action / Calogênese em folhas de Kalanchoe pinnata Lam. pela ação de 2, 4-D e BAP

The Kalanchoe pinnata Lam. is a bush species of the Crassulaceae that is distinguished by its important medicinal properties. Its leaves are used as cataplasm to treat headaches and wounds. There is evidence for a hypotensive and anti-inflammatory effect. Techniques of plant tissue culture have been applied to plant species that produce substances likely to be explored in pharmacology, cell suspension being the main technique. At the industrial level, this method utilizes bioreactors in order to produce secondary metabolites on a large scale. The objective of this study was to evaluate the effects of in vitro combinations of 2,4-dichlorophenoxiacetic acid (2,4-D) and benzylaminopurine (BA) on callus induction in leaf explants of K. pinnata. Leaf fragments were inoculated in MS medium supplemented with 3.0% sucrose, 0.8% agar and factorial combinations of 2,4-D (0.00, 4.52, 9.06, 18.12 µM) and BA (0.00, 4.44, 8.88, 17.76 µM). The cultures were kept in the darkness at 24±2ºC for 50 days. The percentage of callus induction and the area of explants covered by callus cells were evaluated. In the absence of growth regulators, callus induction did not occur, with necrosis of all explants. The highest percentage of callus induction was 100%, obtained with the combination of 9.06 µM 2,4-D and 8.88 µM BA, but the calluses covered only 25% of the leaf area. The most efficient combination was 4.52 µM 2,4-D and 8.88 µM BA, resulting in 91% callus induction with 50 to 100% of the explants being covered by callus cells.

Kalanchoe pinnata Lam. é uma espécie arbustiva da família Crassulaceae que apresenta interessantes propriedades medicinais. Suas folhas são utilizadas em cataplasma para tratar enxaqueca e ferimentos. Há evidência de seu efeito como hipotensiva e anti-inflamatória. Técnicas de cultura de tecidos vegetais têm sido aplicadas para espécies que possuem substâncias passíveis de exploração na farmacologia, sendo a suspensão celular a principal técnica utilizada. A nível industrial, este método utiliza biorreatores para produzir metabólitos secundários em larga escala. Este estudo teve como objetivo avaliar os efeitos in vitro de combinações do ácido 2,4-diclorofenoxiacético (2,4-D) e de benzilaminopurina (BAP) na indução de calos em explantes foliares de K. pinnata. Fragmentos foliares foram inoculados em meio MS contendo 3% de sacarose, 0,8% de ágar e combinações fatoriais de 2,4-D (0,00; 4,52; 9,06 e 18,12 µM) e BAP (0,00; 4,44; 8,88 e 17,76 µM). Os cultivos foram mantidos no escuro, a 24±2ºC por 50 dias. A porcentagem de indução de calos e a área dos explantes coberta por células de calos foram avaliadas. Na ausência de reguladores de crescimento não ocorreu indução de calos, com necrose de todos os explantes. A porcentagem mais alta de indução de calos foi de 100%, obtida com a combinação de 9,06 µM de 2,4-D e 8,88 µM de BAP, mas estes calos cobriram apenas 25% da área foliar. A combinação mais eficiente foi de 9,06 µM de 2,4-D e 8,88 µM de BAP, que resultou em 91% de indução e 50 a 100% da área dos explantes coberta por células de calos.

Stimulative influence of germination and growth of maize seedlings originating from aged seeds by 2,4-D potencies.

BACKGROUND: The 2,4-D (2,4-dichlorophenoxyacetic acid) is using as a growth regulator in tissue culture media. Maize seeds have poor ability to maintain germination rate in the long term. OBJECTIVE: To examine the possible restorative effect of homeopathic 2,4-D potencies on maize seedlings originating from seeds damaged by accelerated aging. METHODS: Seeds of four maize lines were subjected to accelerated aging stress treatment. Seed samples were treated with distilled water (control) and a range of potencies of 2,4-D: 3C, 3.75C, 4.5C, 5.25C and 6C. The germination capacity, fresh substance (FS) and length of root and shoot were determined. Hydrolysis and biosynthesis, GSH/GSSG ratio and redox capacity were calculated. RESULTS: Induced seed aging decreased germination rate and growth of seedlings. 2,4-D potencies did not have a statistically significant effect on germination. However, there were statistically significant effects on FS production, root and shoot length and redox capacity. The 3C potency had the largest effect on the FS accumulation, 4.5C increased root and shoot length, compared to control (statistically significant). The GSH/GSSG ratio and the redox capacity were decreased by aging. The 3C and 4.5C potencies tended to reverse the GSH/GSSG ratio (statistically significant) in the root and shoot, (i.e., shifted the redox balance to the reduced state). CONCLUSION: Homeopathic potencies of 2,4-D appear to have a beneficial effect on artificially aged maize seeds: they stimulate growth through better substance conversion from seed rest, and shift the redox capacity towards a reduced environment. Further work is required to determine if this is an useful means of improving maize seed germination and growth.

In vivo analysis of the calcium signature in the plant Golgi apparatus reveals unique dynamics.

The Golgi apparatus is thought to play a role in calcium homeostasis in plant cells. However, the calcium dynamics in this organelle is unknown in plants. To monitor the [Ca2+]Golgiin vivo, we obtained and analyzed Arabidopsis thaliana plants that express aequorin in the Golgi. Our results show that free [Ca2+] levels in the Golgi are higher than in the cytosol (0.70 µM vs. 0.05 µM, respectively). Stimuli such as cold shock, mechanical stimulation and hyperosmotic stress, led to a transient increase in cytosolic calcium; however, no instant change in the [Ca2+]Golgi concentration was detected. Nevertheless, a delayed increase in the [Ca2+]Golgi up to 2-3 µM was observed. Cyclopiazonic acid and thapsigargin inhibited the stimuli-induced [Ca2+]Golgi increase, suggesting that [Ca2+]Golgi levels are dependent upon the activity of Ca2+-ATPases. Treatment of these plants with the synthetic auxin analog, 2,4-dichlorophenoxy acetic acid (2,4-D), produced a slow decrease of free calcium in the organelle. Our results indicate that the plant Golgi apparatus is not involved in the generation of cytosolic calcium transients and exhibits its own dynamics modulated in part by the activity of Ca2+ pumps and hormones.

Direct and indirect method of plant regeneration from root explants of Caesalpinia bonduc (L.) Roxb.--a threatened medicinal plant of Western Ghats.

An in vitro regeneration protocol has been standardized via direct and indirect methods from excised root explants of C. bonduc, a threatened woody legume used for the treatment of contagious diseases, inflammation, leprosy, antiperiodic, febrifuge, anthelmenthic, urinary disorders, leucorrhoea, piles and to heal wounds. MS medium supplemented with 17.75 micromol BAP and 2.46 micromol IBA, induced a mean of 3.40 +/- 1.07 shoots directly from the surface of excised root explant. Subsequently, the shoots rooted readily on MS half strength medium with out growth regulators. In indirect organogenesis, callogenic frequency was optimized (96.66%) at the concentration of 9.04 micromol 2, 4-D and 0.88 micromol BAP. An average, 15.30 +/- 5.25 shoots were differentiated from the root callus at the concentration of 17.57 micromol BAP and 2.85 micromol IAA. Shoots regenerated through callus were rooted well on MS half strength medium with growth regulators at 2.95 micromol IBA. Rooted plantlets were transferred to the pots containing sterilized soil and were successfully hardened at greenhouse condition for three weeks then exposed to the natural environment. Survival rate was more (95%) in plantlets derived through direct organogenesis than (60%) the plantlets regenerated through root calli.

A peroxisomal carrier delivers NAD⁺ and contributes to optimal fatty acid degradation during storage oil mobilization.

The existence of a transport protein that imports cytosolic NAD(+) into peroxisomes has been controversially discussed for decades. Nevertheless, the biosynthesis of NAD(+) in the cytosol necessitates the import of NAD(+) into peroxisomes for numerous reduction/oxidation (redox) reactions. However, a gene encoding such a transport system has not yet been identified in any eukaryotic organism. Here, we describe the peroxisomal NAD(+) carrier in Arabidopsis. Our candidate gene At2g39970 encodes for a member of the mitochondrial carrier family. We confirmed its peroxisomal localization using fluorescence microscopy. For a long time At2g39970 was assumed to represent the peroxisomal ATP transporter. In this study, we could show that the recombinant protein mediated the transport of NAD(+) . Hence, At2g39970 was named PXN for peroxisomal NAD(+) carrier. The loss of PXN in Arabidopsis causes defects in NAD(+) -dependent ß-oxidation during seedling establishment. The breakdown of fatty acid released from storage oil was delayed, which led to the retention of oil bodies in pxn mutant seedlings. Based on our results, we propose that PXN delivers NAD(+) for optimal fatty acid degradation during storage oil mobilization.

Effects of auxins on the production of steroidal alkaloids in rapidly proliferating tissue and cell cultures of Solanum lyratum.

INTRODUCTION: Solanum lyratum, a rare species, is used to treat cancer, tumours and warts. Plant cell and tissue culture of S. lyratum, producing steroidal alkaloids, could be useful supplements to natural sources. OBJECTIVE: To study the production of solanine, solanidine and solasodine by adding auxin-type phytohormones including indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) to cell and callus cultures of S. lyratum. METHODOLOGY: Methanolic extracts were made from callus and cell cultures of S. lyratumand and analysed using RP C18 HPLC with UV detection. RESULTS: 2,4-D-induced calli from roots led to a significant enhancement in solanine production with a value of 4.13 mg/g dry weight (DW). The maximal solanidine and solasodine levels of 6.26 and 7.69 mg/g DW were respectively obtained with IBA- and IAA-treated S. lyratum cells at concentrations of 1 and 5 mg/L. CONCLUSION: Auxins were found to be useful phytohormones for the production of steroidal alkaloids. The callus and cell culture system developed is simple and can hence be a method of production of steroidal alkaloids in S. lyratum and other Solanaceae species.

Control of root hair development in Arabidopsis thaliana by an endoplasmic reticulum anchored member of the R2R3-MYB transcription factor family.

The evolution of roots and root hairs was a crucial innovation that contributed to the adaptation of plants to a terrestrial environment. Initiation of root hairs involves transcriptional cues that in part determine cell patterning of the root epidermis. Once root hair initiation has occurred, elongation of the root hair takes place. Although many genes have been identified as being involved in root hair development, many contributors remain uncharacterized. In this study we report on the involvement of a member (here dubbed maMYB) of the plant-specific R2R3-MYB family of transcription factors in root hair elongation in Arabidopsis. We show that maMYB is associated with the endoplasmic reticulum membrane with the transcription factor domain exposed to the cytosol, suggesting that it may function as a membrane-tethered transcription factor. We demonstrate that a truncated form of maMYB (maMYB84⁻³°9), which contains the R2R3-MYB transcription factor domain, is localized and retained in the nucleus, where it regulates gene expression. Silencing of maMyb resulted in plants with significantly shorter root hairs but similar root hair density compared with wild type, implying a role of the protein in root hair elongation. 2,4-D (2,4-dichlorophenoxyacetic acid), an exogenous auxin analog that promotes root hair elongation, rescued the short root hair phenotype and maMyb mRNA was induced in the presence of 2,4-D and IAA (indole-3-acetic acid). These results indicate a functional role of maMYB, which is integrated with auxin, in root hair elongation in Arabidopsis.