Guanylyl cyclase activity during photoperiodic flower induction in <Emphasis Type="Italic">Pharbitis nil</Emphasis>

It is known that the level of cGMP is modulated in plant cells in response to a number of stimuli but intracellular events dependent on cGMP metabolism are not clear. Guanylyl cyclases (GCs) are enzymes which are responsible for synthesis of cGMP in eukaryotic and prokaryotic cells. To collect evidence for the participation of cGMP in light signal transduction we isolated enzyme with guanylyl cyclase activity from Pharbitis nil and analysed its level and activity during photoperiodic flower induction. Soluble proteins were isolated from seedlings of a model short-day plant P. nil, partly purified and identified by in vivo and in vitro enzyme assay. In green plants enzyme activity amounted to 484 nmol cGMP/min/mg protein, whereas in etiolated plants it was three times lower (158 nmol cGMP/min/mg protein). Analyse cyclase consists of a single polypeptide of Mr 40 kDa. In order to determine if changes in guanylyl cyclase activity occurred in response to a long, inductive night, we measured enzyme activity in 4-h intervals and observed its increase at 4, 8 and 16 h of darkness. This pattern also fits well with changes in the endogenous cGMP level during a 16 h long flower inductive night. Immunocytochemical analysis confirmed these observations and revealed that changes in the GC level during light/dark conditions appeared. During 16 h long inductive night the strongest signal was observed in cotyledons after 4 and 16 h of the darkness. A high level of fluorescence was generally distributed in mesophyll, however, it was also observed in guard cells. Staining was apparently absent in the veins and cotyledon body. Furthermore, the location inside the cell was analysed. The protein was immunolocalized preferentially in the cytosol, chloroplasts and peroxysomes. Taken together, these data demonstrate in Pharbitis nil the presence of an enzyme which is able to convert GTP to cGMP. Because its level and activity are affected by light we believe that GC/cGMP play a substantial role in light/dark dependent process in plants, such as photoperiodic flower induction.     

Expression of Peroxidases During Seedling Growth in <Emphasis Type="Italic">Ebenus Cretica</Emphasis> L. as Affected by Light and Temperature Treatments

Peroxidase (POD) activity and isoform patterns were investigated during seedling growth (up to 20 days) of Ebenus cretica L. Seeds germinated to approx. 100% after a 24-h imbibition. Seedling growth proceeded smoothly, in both light and dark conditions. No seedling growth was noticed at 4°C. A positive effect of light and increasing temperature (4, 10, 16, 22 and 28°C) on seedlings growth, lignin content and POD activity was observed. Lignin content was 2.5 times higher in seedlings grown under light than in seedlings grown under darkness. Seedlingsȁ9 POD activity was higher in acid pH (5.5) in comparison to neutral pH (7.0). These activities were higher in seedlings grown under darkness than in those grown under light; since additional POD isoforms were expressed in dark conditions. The increase in POD activity was accompanied with the appearance of new POD isoforms correlated with the growth of the seedlings. Four soluble anionic POD isoforms (named A₁, A₂, A₃ and A₄) and three soluble cationic POD isoforms (named C₁, C₂ and C₃) were displayed depending on the treatment and the course of growth. POD isoforms were detected in gel after PAGE. The fast migrating (A₄) isoform, which appeared in the dark-grown seedlings as well as on day 20 at 28°C in the temperature treatment, was separated by DEAE–Sepharose column chromatography. A slow migrating C₁ isoform slightly appeared in both 4 and 10°C temperature treatments and could be related to the low temperature treatments, while A₁, A₂, A₃ and C₂ to the growth stage of seedlings. The expression of seven POD isoforms during seedling growth seems to be related to different developmental events of growth and could be used as useful biochemical markers in the analysis of metabolic regulation in seedling growth of Ebenus cretica.     

Light effects on α-amylase activity and carbohydrate content in relation to lipid mobilization during the seedling growth of sunflower

The changes in α-amylase activity and in starch and free sugar content were investigated in correlation with lipid mobilization inHelianthus annuus during the first 15 days of seedling growth in discontinuous light and in darkness. Throughout the seedling development α-amylase activity increased more significantly in light than in darkness. It was always lower in cotyledons than in other tissues of the embryo axis. In both culture conditions, most of the transitory carbohydrates accumulated in germinating cotyledons were very likely synthesized by gluconeogenesis from the stored lipid breakdown. Nevertheless, in light-grown cotyledons, photosynthesis contributes to increase the carbohydrate levels. The study of several soluble sugars indicates that 1) sucrose stored in cotyledons of mature seeds was used at the onset of seedling growth, more rapidly in light than in darkness, 2) galactose and xylose, both involved as precursors of some cell-wall polysaccharides, remained at a very low level throughout the 15 days and 3) glucose, fructose and maltose accumulated in old etiolated cotyledons in contrast to what occurred in the light.     

Regulation of reserve mobilisation in sunflower during late seedling establishment in continuous darkness

• Reserve mobilisation, metabolite partitioning and reserve‐degrading enzyme activity were studied in sunflower seedlings cultivated in vitro under a 12‐h photoperiod or in the dark to investigate the involvement of source–sink relation and carbon starvation in the regulation of reserve mobilisation under continuous darkness.
• Reserves, metabolites and enzyme activity were determined with standard spectrophotometric methods.
• At the first 24 h of treatment (acclimation phase), darkness did not affect growth, but restricted carbon and nitrogen use, as indicated by sugar and amino acid accumulation in the different seedling parts. After 5 days of treatment (survival phase), extended darkness limited growth and retarded storage lipid mobilisation due to carbon starvation, as evidenced by the depletion of carbohydrates in cotyledons and hypocotyl, as well as the consumption of amino acids in hypocotyls and roots.
• Alterations in the source–sink relationship might have been a response to prolonged darkness, instead of a mechanism used to regulate reserve mobilisation, as these alterations cannot be associated with negative feedback mediated by metabolite accumulation. Storage lipid degradation depends, at least in part, on mechanisms that co‐ordinately regulate the activities of lipases and isocitrate lyase. Taking these results together, it is possible that reserve mobilisation in sunflower seedlings cultivated in the dark might be regulated by mechanisms that perceive the absence of light and predict carbon starvation, adjusting reserve use according to future energy demands to allow, at least in the short term, seedling survival.

     

Exogenous Melatonin Modulates Endogenous H2S Homeostasis and L-Cysteine Desulfhydrase Activity in Salt-Stressed Tomato (Solanum lycopersicum L. var. cherry) Seedling Cotyledons

Although melatonin has been reported to function as a stress signaling molecule, not much information is available on the biochemical and molecular events associated with probable melatonin-hydrogen sulfide crosstalk in plants. Present work provides evidence on the role of melatonin in the modulation of H₂S homoeostasis during NaCl stress in dark-grown tomato (Solanum lycopersicum L. var. cherry) seedlings. NaCl stress (120 mM) inhibits hypocotyl elongation, promotes primary root growth and enhances electrolytic leakage from tomato seedlings. Treatment with H₂S donor (100 µM; NaHS) tends to reverse these effects, all the more so (additive effect) in the presence of melatonin. NaCl stress and exogenous melatonin (30 µM) treatments modulate endogenous H₂S accumulation and positively upregulate the activity of L-cysteine desulfhydrase (L-DES; EC 4.4.1.15; cytosolic). Melatonin has been observed to temporally modulate the activity of specific isoforms of H₂S biosynthesizing enzyme, L-DES in seedling cotyledons. Zymographic analysis of L-DES isoforms in tomato seedling cotyledons has provided novel findings in plant system. Melatonin treatment decreases H₂S accumulation in NaCl-stressed seedling cotyledons which is accompanied by a contrasting increase in L-DES activity. Melatonin, therefore, regulates endogenous H₂S concentration in seedling cotyledons (NaCl treated), thus indicating the role of H₂S catabolism pathways in H₂S homoeostasis. Present findings thus reveal that exogenous melatonin modulates early H₂S signaling in cotyledons of tomato seedlings subjected to NaCl stress. Furthermore, exogenous melatonin and H₂S in combination (additive effect) ameliorate NaCl stress-induced growth changes in tomato seedlings.

     

Changes in cotyledon mRNA during floral induction of Pharbitis nil CV. Violet

Floral induction in seedlings of Pharbitis nil Choisy cv. Violet, with one cotyledon removed, was manipulated by applying various photoperiodic treatments to the remaining cotyledon. Populations of polyadenylated RNA from treated cotyledons were examined to identify messages specifically involved in floral induction. The RNA was translated in vitro using a wheat-germ system, and the resulting translation products were analysed by two-dimensional polyacrylamide gel electrophoresis. Substantial qualitative and quantitative differences were found between mRNA from cotyledons of seedlings kept in continuous light (non-induced) and of seedlings given a 16-h dark period (induced). In contrast, inhibition of flowering with a night-break resulted only in one detectable, quantitative difference in mRNA.Abbreviations CL continuous light - kDa kilodalton - NB 16 h darkness+10 min red-light break, 8 h into the dark period - poly(A)⁺ RNA polyadenylated RNA (isolated by binding to a cellulose oligodeoxythymidine affinity column) - SD short day (16 h dark) - SDP short-day plant - SDS sodium dodecyl sulfate     

Raffinose in seedlings of winter vetch (<Emphasis Type="Italic">Vicia villosa</Emphasis> Roth.) under osmotic stress and followed by recovery

During germination of winter vetch (Vicia villosa Roth.) seeds, the degradation of raffinose family oligosaccharides and galactosyl pinitols occurred faster in axis than in cotyledons. After 7 days of germination, all α-d-galactosides disappeared and the soluble carbohydrates in seedling tissues consisted of d-pinitol, sucrose, fructose, glucose and myo-inositol. Osmotic stress caused by incubation of seedlings in PEG 8000 solution (−0.5, −1.0, and −1.5 MPa) for 48 h induced the activity of crucial enzymes of the RFOs pathway, i.e. galactinol synthase and raffinose synthase, in both the root and epicotyl but not in cotyledons. The root and epicotyl accumulated elevated amounts of galactinol and raffinose as the osmotic potential was lowered. This process was transient because when PEG solution was replaced with water, galactinol and raffinose were degraded, thus confirming their direct involvement in the response of tissues to osmotic stress. Among other soluble carbohydrates, only sucrose accumulated in response to stress. The results did not show potential role of d-pinitol in the adjustment of winter vetch seedlings to osmotic stress.     

Independent effects of jasmonates and ethylene on inhibition of <Emphasis Type="Italic">Pharbitis nil</Emphasis> flowering

Interactions between methyl jasmonate (JA-Me) and ethylene in the photoperiodic flower induction of short-day plant Pharbitis nil were investigated. Both JA-Me and gaseous ethylene applied during the inductive long night caused a decrease in the number of flower buds generated by P. nil. Application of ethylene did not affected niether the level of endogenous jasmonates in the cotyledons during the 16 h long inductive night, nor the inhibitory effect of JA-Me on the flowering of P. nil accompanied by variations in ethylene production. The application of acetylsalicylic acid (aspirin)—a jasmonate biosynthesis inhibitor—slightly stimulated flowering. Our results have shown that the mechanisms of P. nil flower inhibition by jasmonates and ethylene are independent.     

Jasmonates Inhibit Flowering in Short-Day Plant Pharbitis nil

The role of jasmonates in the photoperiodic flower induction of short-day plant Pharbitis nil was investigated. The plants were grown in a special cycle: 72 h of darkness, 24 h of white light with lowered intensity, 24-h long inductive night, 14 days of continuous light. At 4 h of inductive night the cotyledons of non-induced plants contained about two times the amount of endogenous jasmonates (JA/JA-Me) compared to those induced. A 15-min long pulse of far red light (FR) applied at the end of a 24-h long white light phase inhibited flowering of P. nil. The concentration of jasmonates at 2 and 4 h of inductive night in the cotyledons of the plants treated with FR was similar. Red light (R) could reverse the effect of FR. R light applied after FR light decreased the content of jasmonates by about 50%. Methyl jasmonate (JA-Me) applied to cotyledons, shoot apices and cotyledon petioles of P. nil inhibited the formation of flower buds during the first half of a 24-h long inductive or 14-h long subinductive night. Application of JA-Me to the cotyledons was the most effective. None of the plants treated with JA-Me on the cotyledons in the middle of the inductive night formed terminal flower buds. The aspirin, ibuprofen and phenidone, jasmonates biosynthesis inhibitors partially reversed the effect of FR, stimulating the formation of axillary and terminal flower buds. Thus, the results obtained suggests that phytochrome system control both the photoperiodic flower induction and jasmonates metabolism. Jasmonates inhibit flowering in P. nil.     

Gibberellic acid and kinetin partially reverse the effect of water stress on germination and seedling growth in chickpea

The percent germination and seedling growth of chickpea (Cicer arietinum L. cv. PBG-1) decreased with increasing concentrations of exogenous polyethylene glycol 6000 (PEG). With 15% PEG in the growth medium germination was only 33% while with 10% PEG it was 58% as compared to 93% in control. Addition of gibberellic acid (GA₃) and kinetin to medium containing 10% PEG increased the germination and seedling growth and the effect was maximum with 6 µM GA₃ which was a better inducer of growth and germination under reduced water potential than kinetin. However, indole acetic acid (IAA) inhibited germination and growth of stressed seedlings. The activity of amylase in cotyledons under stress was significantly increased with GA₃ while kinetin and IAA were less effective. Gibberellic acid also enhanced the mobilization of starch from cotyledons of stressed seedlings resulting in low starch levels in cotyledons compared with stressed seedlings.     

Far-red mediated polyribosome formation in radish cotyledons

The total ribosome content of radish cotyledons increases during the first 2–3 days of germination both in darkness and under far-red light irradiation; ribonuclease activity is not under phytochrome control during this period. Changes in ribonuclease activity interfere with the analysis of the polyribosomal population. A maximal ratio of polysomes to monosomes is observed 12 h after the onset of far-red light and then it decreases. A 12 h far-red irradiation stimulates the in vivo incorporation of amino acids into proteins. This stimulation persists when seedlings are transferred for 4 h to the dark.     

ACC conversion to ethylene by sunflower seeds in relation to maturation,germination and thermodormancy

Conversion of exogenous 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene was studied in sunflower (Helianthus annuus L., cv. Mirasol) seeds in relation to germinability. Ethylene production from ACC decreased during seed maturation, and non-dormant mature seeds were practically unable to synthesize ethylene until germination and growth occurred, indicating that ethylene forming enzyme (EFE) activity developed during tissue imbibition and growth. ACC conversion to ethylene was reduced by the presence of pericarp, and in young seedlings it was less in cotyledons than in growing axes.ACC conversion to ethylene by cotyledons from young seedlings was optimal at c. 30°C, and was strongly inhibited at 45°C. Pretreatment of imbibed seeds at high temperature (45°C) induced a thermodormancy and a progressive decrease in EFE activity.Abscisic acid and methyl-jasmonate, two growth regulators which inhibit seed germination and seedling growth, and cycloheximide were also shown to inhibit ACC conversion to ethylene by cotyledons of 3-day-old seedlings and by inbibed seeds.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - CH cycloheximide - EFE ethylene forming enzyme - IAA indole-3-acetic acid - Me-Ja methyl-jasmonate     

Gibberellin A3 reverses the effect of salt stress in chickpea (Cicer arietinum L.) seedlings by enhancing amylase activity and mobilization of starch in cotyledons

The percentage germination of chickpea seeds (Cicer arietinum L.cv. PBG-1) gradually decreased with increasing concentration of NaCl in the growth medium and was completely inhibited with 200 mM NaCl. In the presence of 75 mM NaCl, only 51% of the seeds germinated. Gibberellic acid (GA3) and kinetin at 6 µM concentration induced the maximum increase in % germination and seedling growth under salt stress. However, IAA further inhibited both the germination and growth of stressed seedlings. The reduction in amylase activity in cotyledons of stressed seedlings was partially reversed with GA3 and kinetin whereas IAA did not show any positive effect. GA3 was more effective than kinetin in enhancing the reduced germination and seedling growth of chickpea seeds along with amylase activity in cotyledons under NaCl induced saline conditions. The reduced uptake of radiolabelled 14C sucrose by cotyledons and its reduced distribution in the shoots and roots of stressed seedlings was increased with addition of GA3 in the medium. Cotyledonary amylase was separated into amylase 1 and amylase 2 by sephadex G 150 column chromatography. The reduced activities of both amylase 1 and amylase 2 in cotyledons under salt stress was returned to near normal levels with GA3 and there was also an increase in starch utilization, resulting in its lower concentration in cotyledons of GA3-supplemented stressed cotyledons.     

Influence of IAA,TDZ, and light quality on asymbiotic germination,protocorm formation,and plantlet development of <Emphasis Type="Italic">Cyrtopodium glutiniferum</Emphasis> Raddi., a medicinal orchid

We investigated the influence of light quality on in vitro germination and protocorm formation, and the effect of indole-3-acetic acid (IAA) and thidiazuron (TDZ) on proliferation of protocorm-like bodies (PLBs) and development of plantlets of Cyrtopodium glutiniferum Raddi. Germination was faster under white and blue light, and highest under green light. The protocorm developed more rapidly under white, blue, and green light. Continuous darkness delayed seed germination and reduced protocorm formation. Among the plant growth regulators (PGRs) tested for multiplying PLBs, shoots, and roots from protocorms, IAA proved to be superior. TDZ was effective in inducing PLB fresh weight accumulation, but not morphogenesis, unlike IAA. This study indicated that C. glutiniferum seedlings can be produced in vitro using asymbiotic seed germination techniques. High germination rate and protocorm yield can be obtained by initially cultivating C. glutiniferum seeds on medium without growth regulators under white light, or under white light supplemented with green or blue light. This culture system complies with commercial and conservation requirements for rapid and low-cost propagation.     

Salt stress differentially regulates mobilisation of carbon and nitrogen reserves during seedling establishment of Pityrocarpa moniliformis

• Seedling establishment is a critical step in environment colonisation by higher plants that frequently occurs under adverse conditions. Thus, we carried out an integrated analysis of seedling growth, water status, ion accumulation, reserve mobilisation, metabolite partitioning and hydrolase activity during seedling establishment of the native Caatinga species Piptadenia moniliformis (Benth.) Luckow & R.W. Jobson under salinity.
• Two‐day‐old seedlings were cultivated in vitro for 4 days in water agar (control) or supplemented with 50 or 100 mm NaCl. Biochemical determinations were performed according to standard spectrophotometric protocols.
• We found that 100 mm NaCl stimulated starch degradation, amylase activity and soluble sugar accumulation, but limited storage protein hydrolysis in the cotyledons of P. moniliformis seedlings. Although Na⁺ accumulation in the seedling affected K⁺ partitioning between different organs, it was not possible to associate the salt‐induced changes in reserve mobilisation with Na⁺ toxicity, or water status, in the cotyledons. Remarkably, we found that starch content increased in the roots of P. moniliformis seedlings under 100 mm NaCl, probably in response to the toxic effects of Na⁺.
• The mobilisation of carbon and nitrogen reserves is independently regulated in P. moniliformis seedlings under salt stress. The salt‐induced delay in seedling establishment and the resulting changes in the source–sink relationship may lead to storage protein retention in the cotyledons. Possibly, the intensification of starch mobilisation in the cotyledons supported starch accumulation in the root as a potential mechanism to mitigate Na⁺ toxicity.