Is proline the quintessential sentinel of plants? A case study of postharvest flower senescence in Dianthus chinensis L.

The present investigation primarily focussed on evaluating the efficacy of exogenous proline on the flower longevity of Dianthus chinensis L. Floral buds were harvested at the paint brush stage (i.e., a day prior to anthesis) and divided into 6 sets, with one set of buds (i.e., control) held in distilled water and rest of the 5 sets were supplemented with various concentrations of proline, viz., 10 mM, 20 mM, 30 mM, 40 mM and 50 mM. The application of proline at 40 mM concentration proved out to be most effective in improving the longevity of the flowers by about 4 days as compared to the control. The ameliorated longevity coincided with enhanced floral diameter, fresh mass, dry mass and water content. The flowers with delayed senescence also maintained higher soluble proteins, sugars and phenols. The results suggest that exogenous proline effectively alleviates oxidative stress in the petal tissue, as evident by a relatively lower maloendialdehyde content, which is manifested in the form of reduced lipid peroxidation (LPO). Reduced LPO was commensurate with increased membrane stability, quantified by membrane stability index. Moreover, the flowers with improved longevity exhibited a decline in lipoxygenase activity and significant augmentation of antioxidant enzymes superoxide dismutase, catalase and ascorbate peroxidase.     

Changes in the Physical State of Membrane Lipids during Senescence of Rose Petals

Changes in the physical state of microsomal membrane lipids during senescence of rose flower petals (Rosa hyb. L. cv Mercedes) were measured by x-ray diffraction analysis. During senescence of cut flowers held at 22°C, lipid in the ordered, gel phase appeared in the otherwise disordered, liquid-crystalline phase lipids of the membranes. This was due to an increase in the phase transition temperature of the lipids. The proportion of gel phase in the membrane lipids of 2-day-old flowers was estimated as about 20% at 22°C. Ethylene may be responsible, at least in part, for the increase in lipid transition temperature during senescence since aminooxyacetic acid and silver thiosulfate inhibited the rise in transition temperature. When flowers were stored at 3°C for 10 to 17 days and then transferrd to 22°C, gel phase lipid appeared in membranes earlier than in freshly cut flowers. This advanced senescence was the result of aging at 3°C, indicated by increases in membrane lipid transition temperature and ethylene production rate during the time at 3°C. It is concluded that changes in the physical state of membrane lipids are an integral part of senescence of rose petals, that they are caused, at least in part, by ethylene action and that they are responsible, at least in part, for the increase in membrane permeability which precedes flower death.     

Effect of gibberellic acid on carnation flower senescence: evidence that the delay of carnation flower senescence by gibberellic acid depends on the stage of flower development

Gibberellic acid at concentrations of 10^–5 M and 10^–4 M delayed the senescence of cut carnation flowers, when applied continuously via the stem, to flowers between the closed brush and fully open stages of development. Older flowers with reflexed petals were unresponsive. Treatment with paclobutrazol, an inhibitor of GA biosynthesis, prevented tight buds from opening fully, reduced the longevity of partially open flowers, but was ineffective when applied continuously to fully open flowers. Gibberellic acid-treated flowers did not show simultaneous petal inrolling, a known indicator of senescence, and the time to complete petal drying was extended. Gibberellic acid modified the climacteric ethylene rise in a manner consistent with the extension of longevity. These results provide evidence for a correlative role of gibberellins in flower development.Abbreviations GA₃ gibberellin A₃ - GLC gas liquid chromatography     

Efficacy of salicylic acid in modulating physiological andbiochemical mechanisms to improve postharvest longevity in cut spikes of Consolida ajacis (L.) Schur.

Postharvest losses of cut flowers is one of the considerable challenges restricting their efficient marketability. Consequently, such challenges have triggered a constant hunt for developing compatible postharvest treatments to mitigate postharvest losses. Interestingly, recent studies entrench extensive role of salicylic acid (SA) in mitigating postharvest losses in various flower systems. The current investigation focusses on role of SA in augmenting physiological and biochemical responses to mitigate postharvest senescence in cut spikes of Consolida ajacis. The cut spikes of C. ajacis were supplemented with various SA treatments viz, 2 mM, 4 mM, 6 mM. The effects of these treatments were evaluated against control set of spikes placed in distilled water. Our study indicates considerable increment in postharvest longevity of cut spikes, besides an increase in solution uptake, sugar and protein content of tepal tissues.SA augmented antioxidant system via upsurge in phenolic content and antioxidant enzymes viz, superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) to forfend reactive oxygen species (ROS) related oxidative damage. SA profoundly reduced lipoxygenase (LOX) activity to preserve the membrane integrity and thus prevented seepage of solutes from tepal tissues. These results authenticate SA particularly 4 mM concentration as effective postharvest treatment to preserve the postharvest quality of C. ajacis cut spikes.     

Physiological temperature regulation by flowers of the sacred lotus

Flowers of the sacred lotus, Nelumbo nucifera Gaertn. (Nelumbonaceae) are thermogenic and physiologically thermoregulatory. The 42 g flowers remain between 30-36°C during a 2 to 4-day period despite fluctuations in environmental temperatures between about 10-45°C. As the ambient temperature drops, the flowers increase heat production in proportion. Temperature regulation apparently occurs at a cellular level, by a steep, reversible thermal inhibition of respiration at flower temperatures above 30°C. There was a marked time lag between change in flower temperature and compensatory response, suggesting regulation through a biochemical feedback mechanism rather than structural changes in enzymes or membranes. By oxidizing carbohydrate, the flowers produce up to 1 W, with about half of the heat coming from the 8.5 g carpellary receptacle. The period of temperature regulation begins before petal opening and continues through the period of stigma receptivity. Temperature regulation may reward insect pollinators with a warm, equable environment, or it possibly enhances and coordinates flower development.     

The Formation and Distribution of Ice within Forsythia Flower Buds

Differential thermal analysis detected two freezing events when dormant forsythia (Forsythia viridissima Lindl.) flower buds were cooled. The first occurred just below 0°C, and was coincident with the freezing of adjacent woody tissues. The second exotherm appeared as a spike between −10 and −25°C and was correlated with the lethal low temperature. Although this pattern of freezing was similar to that observed in other woody species, differences were noted. Both direct observations of frozen buds and examination of buds freeze-fixed at −5°C demonstrated that ice formed within the developing flowers at temperatures above the second exotherm and lethal temperature. Ice crystals had formed within the peduncle and in the lower portions of the developing flower. Ice also formed within the scales. In forsythia buds, the developing floral organ did not freeze as a unit as noted in other species. Instead the low temperature exotherm appeared to correspond to the lethal freezing of supercooled water within the anthers and portions of the pistil.     

Chemical compositions by using LC–MS/MS and GC–MS and antioxidant activities of methanolic extracts from leaf and flower parts of Scabiosa columbaria subsp. columbaria var. columbaria L.

The members of the Scabiosa genus are one of the traditional medicinal plants used in the treatment of many diseases, in particular the treatment of scabies. In this study, it was aimed to determine antioxidant activities and chemical composition of methanolic extracts of leaves and flowers of Scabiosa columbaria subsp. columbaria var. columbaria. The phenolic contents of both parts of the plant were analyzed by LC–MS/MS. A total of 6 phenolic compounds were determined and chlorogenic acid was the major compound in both flower and leaf parts of the plants, with 5936.052 µg/g and 8021.666 µg/g, respectively. 6 different methods were used to determine the antioxidant activity of the plant parts. Both leaf and flower parts of the plant showed high antioxidant activity in all tested methods and the antioxidant activity values of the leaf part were measured higher than those of the flower part for four tests. The methanol extracts of the plant parts was analyzed with GC–MS and number of the essential oil compounds in the leaf and flower parts were determined as 17 and 13, respectively. Linalool compound was also found to be common in both parts of the plant. The major compounds of the essential oils were identified as 4-Octadecenal (30.01%) in the flower and carvone (35.44%) in the leaf. In addition, terpene derivatives was determined as 90.32% of the highest essential oil group in the leaf, while this value was determined as 1.42% in the flower. For the flower, aromatics were determined as the main component group with 21.31%.     

Effect of ATP on the Calcium Efflux in Dialyzed Squid Giant Axons

Dialysis perfusion technique makes it possible to control the internal composition of squid giant axons. Calcium efflux has been studied in the presence and in the virtual absence (<5 µM) of ATP. The mean calcium efflux from axons dialyzed with 0.3 µM ionized calcium, [ATP]_i > 1,000 µM, and bathed in artificial seawater (ASW) was 0.24 ± 0.02 pmol·cm^-2·s^-1 (P/CS) (n = 8) at 22°C. With [ATP]_i < 5 µM the mean efflux was 0.11 ± 0.01 P/CS (n = 15). The curve relating calcium efflux to [ATP]_i shows a constant residual calcium efflux in the range of 1–100 µM [ATP]_i. An increase of the calcium efflux is observed when [ATP]_i is >100 µM and saturates at [ATP]_i > 1,000 µM. The magnitude of the ATP-dependent fraction of the calcium efflux varies with external concentrations of Na⁺, Ca⁺⁺, and Mg⁺⁺. These results suggest that internal ATP changes the affinity of the calcium transport system for external cations.     

Arsenic-induced oxidative stress in the common bean legume,Phaseolus vulgaris L. seedlings and its amelioration by exogenous nitric oxide

The adverse effects of arsenic (As) toxicity on seedling growth, root and shoot anatomy, chlorophyll and carotenoid contents, root oxidizability (RO), antioxidant enzyme activities, H₂O₂ content, lipid peroxidation and electrolyte leakage (EL%) in common bean (Phaseolus vulgaris L.) were investigated. The role of exogenous nitric oxide (NO) in amelioration of As-induced inhibitory effect was also evaluated using sodium nitroprusside (100 μM SNP) as NO donor and 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (200 μM PTIO) as NO scavenger in different combinations with 50 μM As. As-induced growth inhibition was associated with marked anomalies in anatomical features, reduction in pigment composition, increased RO and severe perturbations in antioxidant enzyme activities. While activity of superoxide dismutase and catalase increased, levels of ascorbate peroxidase, dehydroascorbate reductase and glutathione reductase decreased significantly and guaiacol peroxidase remained normal. The over-accumulation of H₂O₂ content along with high level of lipid peroxidation and electrolyte leakage indicates As-induced oxidative damage in P. vulgaris seedlings with more pronounced effect on the roots than the shoots. Exogenous addition of NO significantly reversed the As-induced oxidative stress, maintaining H₂O₂ in a certain level through balanced alterations of antioxidant enzyme activities. The role of NO in the process of amelioration has ultimately been manifested by significant reduction of membrane damage and improvement of growth performance in plants grown on As + SNP media. Onset of oxidative stress was more severe after addition of PTIO, which confirms the protective role of NO against As-induced oxidative damage in P. vulgaris seedlings.     

Effects of pollinia removal and insertion on flower longevity inChloraea alpina (Orchidaceae)

Summary Although it is known that stigmatic pollen deposition may trigger early flower senescence, the existence of a similar plastic response of flower lifespan to pollen removal has been much less studied. Here we report on a factorial, manipulative experiment in which all 2 × 2 flower combinations of pollinia removal and stigmatic pollinia insertion were performed in inflorescences of the Patagonian ground orchidChloraea alpina. This experiment was conducted in the laboratory, in a population of cut inflorescences and in the field. We hypothesized that if expected fitness gains, through both the male and female functions, were weighed against the costs of flower maintenance, then early flower senescence should be triggered by either pollinia removal or insertion. The shortest flower lifespan would be expected in flowers where both processes occurred. Results showed that flower longevity was very strongly affected by pollinia insertion, reducing the flower lifespan by approximately 60%. The response of pollinia removal was much weaker. A significant reduction in flower longevity caused by pollinia removal was only detected in unpollinated flowers (i.e. no pollinia inserted). Within the racemose inflorescences, flowers in basal positions lived longer than flowers in terminal ones, which might be evidence of the importance of resource availability in determining maximum flower longevity. The observed responses of flower lifespan plasticity to pollinia manipulation only partially supported our expectations based on fitness benefit—cost relationships. Other factors that might explain these discrepancies are the different fitness gains that may indeed accrue to the processes of pollinia removal and insertion as they occur in nature, donor manipulation of the recipient flower lifespan associated with the evolution of pollen clustering into pollinia and physiological constraints in terms of the extent to which flower longevity may respond to pollen removal.     

Timing of self-compatibility, flower longevity, and potential for male outcross success in Leptosiphon jepsonii (Polemoniaceae)

When fertilization triggers flower senescence, early autonomous selfing may cause flowers to senesce before pollen has dispersed, discounting unused pollen. Selfing-induced flower senescence was examined in Leptosiphon jepsonii, a species that varies in the timing of self-compatibility. In field and greenhouse experiments, fertilization had a large effect on flower senescence; most outcrossed flowers senesced after 1 d whereas emasculated flowers lasted 2-5 d. In a comparison of inbred lines from three populations, longevity of autonomously selfed flowers of early self-compatible individuals was significantly less than that of late self-compatible individuals. In field experiments, autonomously selfed flowers were shorter-lived in a predominantly early-selfing population than in a predominantly late-selfing population. Pollen was available and viable beyond the first day of anthesis, suggesting that reductions in flower longevity caused by autonomous selfing could incur a cost to male outcross fitness. We argue that this effect is likely to be most pronounced under intermediate rates of pollinator visitation. Observed pollinator visitation rates ranged from 0.035-0.775 visits per flower per day, indicating a potential for selfing-induced flower senescence to incur pollen discounting in Leptosiphon jepsonii.     

DNA barcoding and genetic fidelity assessment of micropropagated Aenhenrya rotundifolia (Blatt.) C.S. Kumar and F.N. Rasm.: a critically endangered jewel orchid

Aenhenrya rotundifolia is a critically endangered terrestrial jewel orchid. It is monotypic and endemic to evergreen forests of southern western ghats of India. In the present study, identification of this plant species is validated with DNA barcoding using matK and rbcL chloroplast markers. Further, germ-free juvenile axillary bud explants were cultured on Mitra medium supplemented with different kinds of cytokinins like 6-benzyladenine, 6-furfurylaminopurine, N⁶-(Δ²-isopentyl) adenine, thidiazuron, zeatin and meta-topolin as well as auxins such as α-naphthaleneacetic acid, indole-3-acetic acid and indole-3-butyric acid at different concentrations and combinations for successful proliferation and establishment in vitro. After 12 weeks of culture, axillary bud explants produced an average of 30.12 ± 0.71 shoots per explant, 3.87 ± 0.06 cm shoot length, 1671 ± 2.82 mg fresh mass of proliferated shoots with a proliferation frequency of 100% on Mitra medium supplemented with 6.20 µM meta-topolin and 2.25 µM thidiazuron. No root formation was observed in in vitro proliferated microshoots. However, tiny hair like projections were observed in some elongated shoots on Mitra medium pertaining to 5.37 µM NAA. The tiny hair like structure bearing plantlets were hardened and acclimatized with 100% survival rate in the polytunnel chamber. After 8–10 months of establishment ex vitro, flowering was observed. Additionally, the genetic fidelity of in vitro derived plants was tested with ISSR and SCoT marker profiling. The test results revealed that the plants derived from the protocol has 99% genetic similarity to that of the donor mother plant. This study can be applied in forensic interventions of this species, describes the maintenance of germplasm in vitro and establishment of new viable population in its original habitats by restoring existing sites of this critically endangered jewel orchid.     

MiR-101 Induces Senescence and Prevents Apoptosis in the Background of DNA Damage in MCF7 Cells

Moderately increased DNA damage due to the exogenous miR-101 (4 fold) over-expression in MCF7 cells was substantiated by an increase in the number of γ-H2AX foci, correlating with a simple-to-do Halo-assay. miR-101 induced mild/moderate DNA damage favoured senescence rather than apoptosis. An experimental support emanated from the induced mild/moderate DNA damage with 1 µM/5 µM etoposide in MCF7 cells, which resulted in an endogenous miR-101 over-expression (10/4 fold, respectively), followed by senescence. On the other hand, the severe DNA damage induced with 10 µM etoposide, resulted in a low (<1 fold) endogenous expression of miR-101 and an elevated percentage of apoptotic cells. Using bioinformatics tools along with in-vitro and in-vivo validations, miR-101 was found to target and downregulate the mRNA expression of UBE2N and SMARCA4, involved in DNA damage repair (DDR) pathways. Recovery of the expression of the two novel targets in anti-miR-101 transfection validated the results. We conclude that a threshold range of over-expressed miR-101, capable of inducing mild/moderate DNA damage, is sensed by cells to become senescent. The observation derives further support from in-silico protein-protein network analysis where the two novel targets showed their involvement in senescence pathway.     

Red mason bees cannot compete with honey bees for floral resources in a cage experiment

Intensive beekeeping to mitigate crop pollination deficits and habitat loss may cause interspecific competition between bees. Studies show negative correlations between flower visitation of honey bees (Apis mellifera) and wild bees, but effects on the reproduction of wild bees were not proven. Likely reasons are that honey bees can hardly be excluded from controls and wild bee nests are generally difficult to detect in field experiments. The goal of this study was to investigate whether red mason bees (Osmia bicornis) compete with honey bees in cages in order to compare the reproduction of red mason bees under different honey bee densities. Three treatments were applied, each replicated in four cages of 18 m³ with 38 red mason bees in all treatments and 0, 100, and 300 honey bees per treatment with 10–20% being foragers. Within the cages, the flower visitation and interspecific displacements from flowers were observed. Niche breadths and resource overlaps of both bee species were calculated, and the reproduction of red mason bees was measured. Red mason bees visited fewer flowers when honey bees were present. Niche breadth of red mason bees decreased with increasing honey bee density while resource overlaps remained constant. The reproduction of red mason bees decreased in cages with honey bees. In conclusion, our experimental results show that in small and isolated flower patches, wild bees can temporarily suffer from competition with honey bees. Further research should aim to test for competition on small and isolated flower patches in real landscapes.     

Kinetics of Phosphomevalonate Kinase from Saccharomyces cerevisiae

The mevalonate-based isoprenoid biosynthetic pathway is responsible for producing cholesterol in humans and is used commercially to produce drugs, chemicals, and fuels. Heterologous expression of this pathway in Escherichia coli has enabled high-level production of the antimalarial drug artemisinin and the proposed biofuel bisabolane. Understanding the kinetics of the enzymes in the biosynthetic pathway is critical to optimize the pathway for high flux. We have characterized the kinetic parameters of phosphomevalonate kinase (PMK, EC 2.7.4.2) from Saccharomyces cerevisiae, a previously unstudied enzyme. An E. coli codon-optimized version of the S. cerevisiae gene was cloned into pET-52b+, then the C-terminal 6X His-tagged protein was expressed in E. coli BL21(DE3) and purified on a Ni²⁺ column. The K_M of the ATP binding site was determined to be 98.3 µM at 30°C, the optimal growth temperature for S. cerevisiae, and 74.3 µM at 37°C, the optimal growth temperature for E. coli. The K_M of the mevalonate-5-phosphate binding site was determined to be 885 µM at 30°C and 880 µM at 37°C. The V_max was determined to be 4.51 µmol/min/mg enzyme at 30°C and 5.33 µmol/min/mg enzyme at 37°C. PMK is Mg²⁺ dependent, with maximal activity achieved at concentrations of 10 mM or greater. Maximum activity was observed at pH = 7.2. PMK was not found to be substrate inhibited, nor feedback inhibited by FPP at concentrations up to 10 µM FPP.     

Artificial asymmetric warming reduces nectar yield in a Tibetan alpine species of Asteraceae

Background and Aims Asymmetric warming is one of the distinguishing features of global climate change, in which winter and night-time temperatures are predicted to increase more than summer and diurnal temperatures. Winter warming weakens vernalization and hence decreases the potential to flower for some perennial herbs, and night warming can reduce carbohydrate concentrations in storage organs. This study therefore hypothesized that asymmetric warming should act to reduce flower number and nectar production per flower in a perennial herb, Saussurea nigrescens, a key nectar plant for pollinators in Tibetan alpine meadows.Methods A long-term (6 years) warming experiment was conducted using open-top chambers placed in a natural meadow and manipulated to achieve asymmetric increases in temperature, as follows: a mean annual increase of 0·7 and 2·7 °C during the growing and non-growing seasons, respectively, combined with an increase of 1·6 and 2·8 °C in the daytime and night-time, respectively, from June to August. Measurements were taken of nectar volume and concentration (sucrose content), and also of leaf non-structural carbohydrate content and plant morphology.Key Results Six years of experimental warming resulted in reductions in nectar volume per floret (64·7 % of control), floret number per capitulum (8·7 %) and capitulum number per plant (32·5 %), whereas nectar concentration remained unchanged. Depletion of leaf non-structural carbohydrates was significantly higher in the warmed than in the ambient condition. Overall plant density was also reduced by warming, which, when combined with reductions in flower development and nectar volumes, led to a reduction of ∼90 % in nectar production per unit area.Conclusions The negative effect of asymmetric warming on nectar yields in S. nigrescens may be explained by a concomitant depletion of leaf non-structural carbohydrates. The results thus highlight a novel aspect of how climate change might affect plant–pollinator interactions and plant reproduction via induction of allocation shifts for plants growing in communities subject to asymmetric warming.     

DNA bending induced by DNA (cytosine-5) methyltransferases

DNA bending induced by six DNA (cytosine-5) methyltransferases was studied using circular permutation gel mobility shift assay. The following bend angles were obtained: M.BspRI (GG^m5CC), 46–50°; M.HaeIII (GG^m5CC), 40–43°; M.SinI (GGW^m5CC), 34–37°; M.Sau96I (GGN^m5CC), 52–57°; M.HpaII (C^m5CGG), 30°; and M.HhaI (G^m5CGC), 13°. M.HaeIII was also tested with fragments carrying a methylated binding site, and it was found to induce a 32° bend. A phase-sensitive gel mobility shift assay, using a set of DNA fragments with a sequence-directed bend and a single methyltransferase binding site, indicated that M.HaeIII and M.BspRI bend DNA toward the minor groove. The DNA curvature induced by M.HaeIII contrasts with the lack of DNA bend observed for a covalent M.HaeIII–DNA complex in an earlier X-ray study. Our results and data from other laboratories show a correlation between the bending properties and the recognition specificities of (cytosine-5) methyltransferases: enzymes recognizing a cytosine 3′ to the target cytosine tend to induce greater bends than enzymes with guanine in this position. We suggest that the observed differences indicate different mechanisms employed by (cytosine-5) methyltransferases to stabilize the helix after the target base has flipped out.     

Nitric oxide modulates specific steps of auxin-induced adventitious rooting in sunflower

Present work on indole-3-acetic acid (IAA)-induced adventitious rooting in sunflower hypocotyl highlights a clear demarcation of nitric oxide (NO)-dependent and NO-independent roles of auxin in this developmental process. Of the three phases of adventitious rooting, induction is strictly auxin-dependent though initiation and extension are regulated by an interaction of IAA with NO. A vital role of auxin-efflux transporters (PIN) is also evident from 1-napthylphthalamic acid (NPA)-triggered suppression of adventitious roots (AR). Use of actin depolymerizing agent, latrunculin B (Lat B), has demonstrated the necessity of functional actin filaments in auxin-induced AR response, possibly through its effect on actin-mediated recycling of auxin transporter proteins. Thus, evidence for a linkage between IAA, NO and actin during AR formation has been established.Key words: adventitious roots, auxin, sunflowerAdventitious roots (AR) are post-embryonic roots known to originate from stem, leaf petiole and non-pericycle tissue of old roots. In young stem, AR commonly arise from the interfascicular parenchyma while they appear from vascular rays near the cambium in older stem. Formation of AR begins with re-differentiation of predetermined cells which switch from their morphogenetic path to act as mother cells for initiation of root primordia.¹ The process of AR formation consists of three physiologically interdependent phases: induction, initiation and extension.¹ Induction phase comprises of various molecular and biochemical events but no morphologically visible changes appear during this phase. Formation of multilayered cells and conception of root primordia occurs during initiation phase. During expression phase, root primordia exhibit intra-stem growth and their emergence through epidermis. Various environmental and endogenous factors, such as temperature, light, hormones (particularly auxin), sugars and mineral salts, act as cues for promoting redifferentiation of predetermined cells resulting in root induction.The three phases of AR formation are known to be regulated by alterations in the endogenous level of auxin.² A transient increase in auxin concentration has been reported during induction phase, which is followed by a decrease and again an increase during expression phase.³ Auxin transport to and from the responding region is essential for root organogenesis. Acropetal transport of auxin occurs through the vascular cylinder and the basipetal transport takes place through the epidermal and subtending cortical cells.⁴ Polar transport of auxin from the shoot apical meristem to the rooting region is primarily facilitated by auxin-influx (AUX1) and -efflux (PIN) transporters. Asymmetric localization of these transporter proteins in the vascular cambium cells is responsible for differential distribution of auxin in a particular zone of cells.⁵ Polar auxin transport is known to be inhibited by 1-napthylphthalamic acid (NPA, a phytotropin). This inhibition is mediated through a binding of NPA molecule to putative NPA-binding protein (NBP), which is functionally associated with PIN proteins.⁶ Efflux transporters exhibit rapid turnover in plasma membrane.⁷ High affinity of NBP for actin filaments,^8,9 suggests its involvement in the cycling and polar distribution of PIN proteins.¹⁰ Organization of actin filaments is known to be rapidly, reversibly and specifically disrupted by Latrunculin B (Lat B), a macrolide toxin isolated from Latrunculia magnificia, a red sea sponge.¹¹ Lat B associates with actin monomers in 1:1 ratio, thereby preventing their repolymerization into filaments, resulting in a complete shift from F-actin to G-actin.¹² Owing to its well-understood and simple mode of action and low effective dosage, Lat B has supplanted the classic actin-depolymerizing drug cytochalasin D¹³ in pharmacological investigations. In the past few years, significant work has been done on NO as a signaling molecule in a variety of plant developmental processes.¹⁴ Nitric oxide is known to play a crucial role in root development.¹⁵Using sunflower as a model system, present work has been undertaken to investigate the possible role of NO during IAA-induced adventitious rooting in hypocotyl explants. Since auxin action is principally based on PIN-regulated polar transport of IAA molecules, and PIN proteins are known to exhibit actin-asssisted rapid recycling in the target cells, attempts have been made in the present work to find a correlation between auxin transport, actin and NO, using specific pharmacological agents. Additionally, effect of Cyclosporin A (CsA), an inhibitor of nitric oxide synthase (NOS) in animal systems,¹⁶ has also been investigated. These specific agents have been used to monitor root initiation at the target sites in our attempts to decipher a signaling cascade for AR.Seeds of Sunflower (Helianthus annuus L. cv. Morden) were germinated on moist germination sheets at 25 ± 2°C under continuous illumination of 4.3 Wm⁻². Hypocotyls from 4 d old seedlings and with similar growth rate, were excised up to 6 cm below the cotyledonary node. Hypocotyl explants with apical meristem intact but cotyledons excised were selected for the present work with a view to provide a continuity of the endogenous auxin source. Similar explants were also recently used by Huang et al.¹⁷ to investigate indole-3-butyric acid (IBA)-induced AR formation in mung bean (Phaseolus radiatus L.). Using IAA instead of IBA for such investigations was preferred for the present work keeping in view that the two auxins seem to employ different transport proteins for their polar transport.¹⁸ Freshly harvested explants were put upright in glass vials with their proximal cut ends dipped in 1 ml of different concentrations (1, 5, 10 and 15 µM) of IAA, thus bathing the hypocotyls up to 5 mm of their lower ends. Explants were maintained in dark during the course of experiments. The number of AR visible on hypocotyl surface was recorded daily up to 4 days of incubation. Concentration of IAA thus observed optimal for rooting (10 µM) was used for all further experiments. Similarly, various concentrations of other test solutions, namely NPA (auxin efflux blocker; 1 and 10 µM), Lat B (an inducer of actin depolymerization; 25, 50 and 100 nM), CsA (an inhibitor of cyclophilins; 1, 5 and 10 µM), sodium nitroprusside (SNP; NO donor; 1, 5, 10 and 100 µM) and 2-phenyl-4,4,5,5-tetramethyllimidazoline-1-oxyl-3-oxide (PTIO; NO scavenger; 1 and 1.5 mM), were initially used to select their respective optimal concentrations. Based on these preliminary experiments NPA, Lat B, CsA, SNP and PTIO were used at 10 µM, 100 nM, 10 µM, 100 µM and 1.5 mM, respectively, for all subsequent experiments. Some other treatment combinations, namely NPA (10 µM) + IAA (10 µM); LatB (100 nM) + IAA (10 µM); CsA (10 µM) + IAA (10 µM); SNP (100 µM) + NPA (10 µM) and PTIO (1.5 mM) + IAA (10 µM) were also used to investigate their effects on adventitious rooting. Hypocotyl explants incubated in distilled water served as control. Morphological observations of rooting response were imaged after 7 days of incubation, using Nikon digital camera fitted on a stereomicroscope (Stemi 2000, Zeiss, Germany). Detailed evaluation of root initiation was observed after clearing by immersing the explants in a 3:1 solution of ethanol: acetic acid overnight. They were then transferred to 2 N NaOH solution, left overnight, washed once with distilled water and stained with safranin solution for 2–3 min. Excess stain was removed by repeated washing in distilled water. The lower 2 cm region of hypocotyl explants was then cut and mounted on a glass slide to examine endogenous root initials, using a stereomicroscope (Stemi 2000, Zeiss, Germany) fitted with a Nikon camera.Root initiation and extension in the basal region of hypocotyl explants maintained in distilled water indicates the expected basipetal transport of the inducing factor (endogenous IAA) from the intact meristem, as also reported earlier.¹⁹ Treatment with IAA (10 µM) elicited two effects on hypocotyl explants in comparison to those subjected to distilled water treatment: (1) Formation of greater number of root initials, (2) Greater extension of the initiated roots (Figs. 1 and ​and22). A response similar to that evoked by IAA is also evident in hypocotyl explants treated with 100 µM of SNP (Figs. 1 and ​and22). Recently SNP (NO donor) has been reported to evoke dose-dependent response on AR formation in marigold.²⁰ In the present work, treatment with variable concentrations of SNP, ranging from 1–100 µM, lead to a gradual increase in the number and extension growth of AR till 100 µM. Pagnussat et al.²¹ and Liao et al.²⁰ have used 10 µM and 50 µM as effective SNP concentration in cucumber and marigold, respectively. Thus, optimal concentration of SNP for AR formation is species-dependent. In presence of PTIO (1.5 mM; a specific NO scavenger), complete suppression of AR was evident in sunflower, as also reported earlier in mung bean.¹⁷ Combination of PTIO with IAA lead to root initiation only (no extension growth). NPA (10 µM) blocked AR initiation by endogenous (distilled water treatment) and exogenous IAA (Fig. 1). Application of NPA inhibits polar auxin transport, thus reducing the optimal concentration of IAA required for AR formation at the hypocotyl base (zone of AR formation). Thus, no evidence of root initials was evident in presence of NPA, which is also reported in cucumber²² and loblolly pine,²³ respectively. Though NO is expected to act downstream of IAA²⁴ but a treatment of SNP in combination with NPA (present work) lead to complete suppression of AR formation. Our unpublished observations have indicated the expression of NO in the interfascicular cells after induction phase (i.e., during AR iniation and extension). Thus, it can be proposed that IAA is involved in induction phase of adventitious rooting independent of NO, while initiation and extension phases appear to involve IAA-NO interaction. CsA-cyclophilin complex is known to inhibit calcineurin (a protein phosphatase) and NOS activity in animal systems.²⁵ Treatment of hypocotyl explants with CsA (10 µM) lead to formation of fewer number of roots which exhibited extension growth. Oh et al.²⁶ reported a significant reduction in the number of roots in the presence of CsA in hypocotyl explants from tomato. Subjecting hypocotyl explants with a combination of CsA and IAA lead to formation of fewer number of root initials, reaffirming the involvement of NO in auxin action in the developmental process under investigation (AR). However, further investigations on the role of cyclophilins and NOS in auxin-modulated AR formation are required to pinpoint their specific sites of action in this developmental process. Treatment with Lat B (+ and − IAA) lead to complete AR suppression in sunflower hypocotyl explants. Actin-mediated polar localization of PIN proteins is responsible for polar auxin transport and disruption of microfilaments by Lat-B would thus, directly affect IAA transport leading to the observed AR suppression. These observations indicate a convergence of the effect of IAA with that of NO and a role of a well organized actin in the responding cells.Open in a separate windowFigure 1Effect of IAA and various other pharmacological agents on adventitious rooting in hypocotyl explants. Morphological observations of rooting response (A and C). Evaluation of endogenous root initiation and elongation observed in cleared explants stained with safranin (B and D). Scale bar represents 3 mm.Open in a separate windowFigure 2Quantitative analysis of AR initiation in presence of distilled water, IAA and various pharmacological agents in hypocotyl explants of sunflower. Each datum presents a mean and standard error from at least three observations.To sum up, present investigations provide evidence for a linkage between auxin-induced AR response in seedling hypocotyls and NO (Fig. 3). Both endogenous and exogenous IAA-mediated AR induction seem to depend on actin. Significance of actin in this developmental response has become evident via its role in the cycling of auxin efflux proteins (PIN). The three phases of AR formation can be differentiated from each other in terms of their sensitivities to IAA and NO. AR induction phase seems to be governed by auxin alone, independent of NO. NO seems to become operative in this auxin-modulated response (AR) during initiation and extension phase only. Investigations are being undertaken in the author''s laboratory to visualize and quantitate the NO signal in the IAA-responding hypocotyl explants so that the phasing of the role of NO during AR formation can be precisely predicted.Open in a separate windowFigure 3Schematic presentation of the probable events associated with NO-mediated adventitious rooting.