Arabidopsis DELLA and two HD-ZIP transcription factors regulate GA signalling in the epidermis through the L1 cis-element

Gibberellins (GAs) are plant hormones that affect plant growth and regulate gene expression differentially across tissues. To study the molecular mechanisms underlying GA signaling in Arabidopsis thaliana, we focused on a GDSL lipase gene (LIP1) induced by GA and repressed by DELLA proteins. LIP1 contains an L1 box promoter sequence, conserved in the promoters of epidermis-specific genes, that is bound by ATML1, an HD-ZIP transcription factor required for epidermis specification. In this study, we demonstrate that LIP1 is specifically expressed in the epidermis and that its L1 box sequence mediates GA-induced transcription. We show that this sequence is overrepresented in the upstream regulatory regions of GA-induced and DELLA-repressed transcriptomes and that blocking GA signaling in the epidermis represses the expression of L1 box–containing genes and negatively affects seed germination. We show that DELLA proteins interact directly with ATML1 and its paralogue PDF2 and that silencing of both HD-ZIP transcription factors inhibits epidermal gene expression and delays germination. Our results indicate that, upon seed imbibition, increased GA levels reduce DELLA protein abundance and release ATML1/PDF2 to activate L1 box gene expression, thus enhancing germination potential.

Estrogenic responses in estrogen receptor-α deficient mice reveal a distinct estrogen signaling pathway

Estrogens are thought to regulate female reproductive functions by altering gene transcription in target organs primarily via the nuclear estrogen receptor-α (ER-α). By using ER-α “knock-out” (ERKO) mice, we demonstrate herein that a catecholestrogen, 4-hydroxyestradiol-17β (4-OH-E2), and an environmental estrogen, chlordecone (kepone), up-regulate the uterine expression of an estrogen-responsive gene, lactoferrin (LF), independent of ER-α. A primary estrogen, estradiol-17β (E2), did not induce this LF response. An estrogen receptor antagonist, ICI-182,780, or E2 failed to inhibit uterine LF gene expression induced by 4-OH-E2 or kepone in ERKO mice, which suggests that this estrogen signaling pathway is independent of both ER-α and the recently cloned ER-β. 4-OH-E2, but not E2, also stimulated increases in uterine water imbibition and macromolecule uptake in ovariectomized ERKO mice. The results strongly imply the presence of a distinct estrogen-signaling pathway in the mouse uterus that mediates the effects of both physiological and environmental estrogens. This estrogen response pathway will have profound implications for our understanding of the physiology and pathophysiology of female sex steroid hormone actions in target organs.

Evidence for a Slow-Turnover Form of the Ca2+-Independent Phosphoenolpyruvate Carboxylase Kinase in the Aleurone-Endosperm Tissue of Germinating Barley Seeds1

Phosphoenolpyruvate carboxylase (PEPC) activity was detected in aleurone-endosperm extracts of barley (Hordeum vulgare) seeds during germination, and specific anti-sorghum (Sorghum bicolor) C4 PEPC polyclonal antibodies immunodecorated constitutive 103-kD and inducible 108-kD PEPC polypeptides in western analysis. The 103- and 108-kD polypeptides were radiolabeled in situ after imbibition for up to 1.5 d in 32P-labeled inorganic phosphate. In vitro phosphorylation by a Ca2+-independent PEPC protein kinase (PK) in crude extracts enhanced the enzyme's velocity and decreased its sensitivity to l-malate at suboptimal pH and [PEP]. Isolated aleurone cell protoplasts contained both phosphorylated PEPC and a Ca2+-independent PEPC-PK that was partially purified by affinity chromatography on blue dextran-agarose. This PK activity was present in dry seeds, and PEPC phosphorylation in situ during imbibition was not affected by the cytosolic protein-synthesis inhibitor cycloheximide, by weak acids, or by various pharmacological reagents that had proven to be effective blockers of the light signal transduction chain and PEPC phosphorylation in C4 mesophyll protoplasts. These collective data support the hypothesis that this Ca2+-independent PEPC-PK was formed during maturation of barley seeds and that its presumed underlying signaling elements were no longer operative during germination.

Patterns of Starchy Endosperm Acidification and Protease Gene Expression in Wheat Grains following Germination1

Cereal aleurone responses to gibberellic acid (GA3) include activation of synthesis of hydrolytic enzymes and acidification of the external medium. We have studied the effect of the pH of the incubation medium on the response of wheat (Triticum aestivum) aleurone cells to GA3. De-embryonated half grains show the capacity for GA3-activated medium acidification when incubation is carried out at pH 6.0 to 7.0 but not at lower pHs. In addition, the activating effect of GA3 on the expression of carboxypeptidase III and thiol protease genes is more efficient when the hormone treatment is carried out at neutral pH. In situ pH staining showed that starchy endosperm acidification takes place upon imbibition and advances from the embryo to the distal part of the grain. In situ hybridization experiments showed a similar pattern of expression of a carboxypeptidase III gene, which is up-regulated by GA3 in aleurone cells. However, aleurone gene expression precedes starchy endosperm acidification. These findings imply that in vivo GA perception by the aleurone layer takes place at neutral pH and suggest that the acidification of the starchy endosperm is regulated by GA3 in germinated wheat grains.

Phytochrome Regulates Gibberellin Biosynthesis during Germination of Photoblastic Lettuce Seeds1

Germination of lettuce (Lactuca sativa L.) seed is regulated by phytochrome. The requirement for red light is circumvented by the application of gibberellin (GA). We have previously shown that the endogenous content of GA1, the main bioactive GA in lettuce seeds, increases after red-light treatment. To clarify which step of GA1 synthesis is regulated by phytochrome, cDNAs encoding GA 20-oxidases (Ls20ox1 and Ls20ox2, for L. sativa GA 20-oxidase) and 3β-hydroxylases (Ls3h1 and Ls3h2 for L. sativa GA 3β-hydroxylase) were isolated from lettuce seeds by reverse-transcription polymerase chain reaction. Functional analysis of recombinant proteins expressed in Escherichia coli confirmed that the Ls20ox and Ls3h encode GA 20-oxidases and 3β-hydroxylases, respectively. Northern-blot analysis showed that Ls3h1 expression was dramatically induced by red-light treatment within 2 h, and that this effect was canceled by a subsequent far-red-light treatment. Ls3h2 mRNA was not detected in seeds that had been allowed to imbibe under any light conditions. Expression of the two Ls20ox genes was induced by initial imbibition alone in the dark. The level of Ls20ox2 mRNA decreased after the red-light treatment, whereas that of Ls20ox1 was unaffected by light. These results suggest that red light promotes GA1 synthesis in lettuce seeds by inducing Ls3h1 expression via phytochrome action.

Metabolic Bypass of the Tricarboxylic Acid Cycle during Lipid Mobilization in Germinating Oilseeds1 : Regulation of NAD+-Dependent Isocitrate Dehydrogenase Versus Fumarase

Biosynthesis of sucrose from triacylglycerol requires the bypass of the CO2-evolving reactions of the tricarboxylic acid (TCA) cycle. The regulation of the TCA cycle bypass during lipid mobilization was examined. Lipid mobilization in Brassica napus was initiated shortly after imbibition of the seed and proceeded until 2 d postimbibition, as measured by in vivo [1-14C]acetate feeding to whole seedlings. The activity of NAD+-isocitrate dehydrogenase (a decarboxylative enzyme) was not detected until 2 d postimbibition. RNA-blot analysis of B. napus seedlings demonstrated that the mRNA for NAD+-isocitrate dehydrogenase was present in dry seeds and that its level increased through the 4 d of the experiment. This suggested that NAD+-isocitrate dehydrogenase activity was regulated by posttranscriptional mechanisms during early seedling development but was controlled by mRNA level after the 2nd or 3rd d. The activity of fumarase (a component of the nonbypassed section of the TCA cycle) was low but detectable in B. napus seedlings at 12 h postimbibition, coincident with germination, and increased for the next 4 d. RNA-blot analysis suggested that fumarase activity was regulated primarily by the level of its mRNA during germination and early seedling development. It is concluded that posttranscriptional regulation of NAD+-isocitrate dehydrogenase activity is one mechanism of restricting carbon flux through the decarboxylative section of the TCA cycle during lipid mobilization in germinating oilseeds.

Expression and Localization of Phosphoenolpyruvate Carboxylase in Developing and Germinating Wheat Grains1

Phosphoenolpyruvate carboxylase (PEPC) activity and corresponding mRNA levels were investigated in developing and germinating wheat (Triticum aestivum) grains. During grain development PEPC activity increased to reach a maximum 15 d postanthesis. Western-blot experiments detected two main PEPC polypeptides with apparent molecular masses of 108 and 103 kD. The most abundant 103-kD PEPC subunit remained almost constant throughout the process of grain development and in the scutellum and aleurone layer of germinating grains. The less-abundant 108-kD polypeptide progressively disappeared during the second half of grain development and was newly synthesized in the scutellum and aleurone layer of germinating grains. PEPC mRNA was detected throughout the process of grain development; however, in germinating grains PEPC mRNA accumulated transiently in the scutellum and aleurone layer, showing a sharp maximum 24 h after imbibition. Immunolocalization studies revealed the presence of the enzyme in tissues with a high metabolic activity, as well as in the vascular tissue of the crease area of developing grains. A clear increase in PEPC was observed in the scutellar epithelium of grains 24 h after imbibition. The data suggest that the transiently formed PEPC mRNA in the scutellar epithelium encodes the 108-kD PEPC subunit.

Identification of auxin-responsive elements of parB and their expression in apices of shoot and root.

Detailed analysis of transgenic tobaccos containing a series of chimeric parB promoter/beta-glucuronidase (GUS) gene constructs allowed us to define two auxin-responsive elements (AREs) of 48 bp and 95 bp (positions -210 to -163 and -374 to -280) in the parB promoter. The two AREs responded independently to physiological concentrations of auxin. Gel retardation assays revealed binding of nuclear protein(s) to the sequence conserved between ARE I and ARE II. The auxin responsiveness of the parB promoter did not mediate the pathway through the as-1 element and transcription factor ASF-1. AREs I and II were responsive to auxin at physiological concentrations, whereas as-1 responded only to higher concentrations of auxin which may be interpreted as stress, though as-1 had been reported to be a minimal ARE [Liu, X. & Lam, E. (1994) J. Biol. Chem. 269, 668-675]. Histochemical staining of transgenic tobacco that contained a parB promoter/GUS construct demonstrated the expression of GUS activity in the shoot apex as well as in the root tips, suggesting the involvement of parB expression in meristematic activity or differentiation. The drastic change in auxin responsiveness in the transgenic plants between the 6th and 10th day after imbibition of seeds implies the development or the activation of auxin signal transduction systems during plant development.

Qualidade fisiológica de sementes de Moringa oleifera lam. por meio da análise de imagens

Several tests that evaluate the quality of seeds are destructive and require time, which is considered long and expensive in the processes that involves the production and marketing of seed. Thus, techniques that allow reducing the time related to assess the quality of seed lots is very favorable, considering the technical, economic and scientific point of view. The techniques images of seed analyzed both by X-ray such as digital images, represent alternative for this sector, and are considered reproducible and fast, giving greater flexibility and autonomy to the activities of production systems. Summarily, the objective was to analyze the internal morphology of seeds of this species through x-rayed images and the efficiency of weed seed area increased during soaking through image analysis and compare them with the results of germination tests and force the evaluation of physiological seed quality. For X-ray tests, the seeds were exposed for 0.14 seconds at radiation 40kV and 2.0 mAs. Were analyzed images using the ImageJ program and subsequently put to germinate in B.O.D chamber at 27 ° C, in which there was the comparison of results for germination. To determine the test area increase (% IA), seeds were used with and without seed coat, maintained the B.O.D chamber at 15 ° to 20 ° C, the seeds were photographed before and after the soaking period, the results were compared to the germination rates. For the X-ray test, it was observed that seeds with empty area greater than 20%, showed a higher percentage of abnormal seedlings. And the area increment analysis showed that it is possible to rank the batch after 8 hours of imbibition at 15 ° C according to the germination and vigor tests

Laboratory Investigation of Diluted Bitumen Trapping and Dissolution in Gravel

The Canadian economy is largely dependent on the distribution of large volumes of oil to domestic and international markets by a long network of pipelines. Unfortunately, accidents occur, and oil can leak or spill from these pipelines before it reaches its destination. Of particular concern are the long-term consequences of oil spills in freshwater, which include sinking of oil in water and the contamination of sensitive areas, such as where fish (e.g., salmon) deposit their eggs in gravel-dominated river sediments. There is a knowledge gap regarding the fate and behaviour of oil in river sediment. To this end, this study aimed at finding the potential for diluted bitumen (dilbit) oil to become trapped in gravel and to transfer hydrocarbons into water by dissolution, which are harmful to aquatic life. Two sets of laboratory experiments were conducted to simulate conditions of an oil spill on an exposed bank or in shallow water. In the first set, by conducting capillary pressure-saturation (Pc-Sw) experiments it was found that dilbit can enter gravel pores without much resistance and approximately 14% of the pore volume can be occupied by discontinuous single or multipore blobs of dilbit following imbibition of water. Air-water Pc-Sw experiments done in laboratory 1-D columns required gravity correction and could be successfully scaled to predict dilbit-water Pc-Sw curves, except for the trapped amount of dilbit. Trapped dilbit constituents can be dissolved into the water flowing through gravel pores (hyporheic flow) at different velocities. In the second set, dissolution experiments suggested that for the duration of the test, hydrocarbons that cause acute toxicity dissolve rapidly, likely resulting in a decrease in their effective solubility. However, dilbit saturation changed only <2% within that time. Chronically toxic PAH compounds were also detected in the effluent water. The total concentration of all detected PAHs and alkylPAHs exceeded the threshold literature value to protect early-life stage fish. Observations of decreased concentrations with increased aqueous velocities as well as less than equilibrium concentrations indicated that the mass transfer was rate-limited. A correlation was developed for the mass transfer rate coefficient to understand the mass transfer behaviour beyond the conditions used in the experiments, which had a Reynolds number exponent similar to the studies of NAPL dissolution in groundwater.

Uso de indicadores bioquímicos na qualidade fisiológica de sementes florestais

The scientific research in seed technology is based on techniques that aim the reduction of costs and time, standardization, improvement and establishment of analytical methods while maintaining a high level of reliability of the results. This study sought to elucidate the reliability of electrical conductivity and pH of the exudate compared to the classic germination test, which was developed in two separate studies, however interrelated with each other, as to their final goals. The experimental material of this study consisted of seeds of the species Aspidosperma parvifolium (guatambu), Aspidosperma polyneuron (peroba-rosa), Cabralea canjerana (canjerana), Cariniana legalis (jequitibá), Gallesia integrifolia (pau-d'alho), Handroanthus chrysotrichus (ipê-amarelo), Lonchocarpus campestris (rabo-de-bugio) and Pterogyne nitens (amendoim-do-campo). The physiological quality of the studied seed species was evaluated through the electrical conductivity and pH test of the exudate by mass and individual methods being compared and correlated with the results obtained in the germination test. In addition to the tested methods, imbibition periods of the seeds were evaluated for conductivity and pH, which corresponded to 2, 4, 6, 8, 24 and 48 hours. The electrical conductivity test was efficient in both of the used methods to evaluate the physiological quality of the studied seed species when compared to the standard germination test. The pH test of the exudate applied by the individual method was more efficient and thorough to evaluate the physiological quality of the studied seed species, than the mass method. For the species Gallesia integrifolia, Cariniana legalis and Lonchocarpus campestris the pH tests of the exudate tests were not efficient due to poor or absent correlation between germination and pH.

Desempenho de sementes de soja revestidas com biopolímeros

The use of biopolymers that help to fix pesticides efficiently and degrade easily without harming the environment, and still improve the physiological performance of field soybean seed may bring contributions to the soybean yield. This study aimed to evaluate the effect of cassava starch polymers (AM), sodium alginate (ALG) and polyvinyl alcohol (PVOH), in the concentrations 2, 4 and 6 g / 100 ml of solution, in the physiological attributes of seeds soy, seed speed soaking and performance of soybean seeds after three months of storage. The soybean variety used was the NK 7059 RR. The experimental design used for the three studies was a factorial with 48 experimental units: 3 polymers (AM, ALG and PVOH), 4 different concentrations (0%, 2%, 4% and 6%), with four replications, in a completely randomized design. It was observed the level of significance of the factors and their interactions, applying the test F. The polymers were evaluated by the Tukey test at 5% probability, and the concentrations were evaluated by polynomial regression. The witness obtained better results for most variables studied. Among the polymers, the best coating was observed PVOH because it was the less viscous polymer and visually not served as a substrate for microorganisms. However, also, satisfactory results were obtained for the AM and ALG polymers at a concentration of 2%. There was not interference of the polymers studied with regard to reduction of imbibition rate of soybean seeds. The hydrophilicity of polymers, mainly the AM and ALG accelerated soaking seeds harming germination at concentrations 4% and 6%. In general, the higher the concentration of polymers tended to worse results.

Regulação coordenada da mobilização de lipídios e proteínas por hormônios, fontes de carbono e nitrogênio durante o crescimento pós-germinativo em Girassol

The mobilization of food reserves in storage tissues and allocation of their hydrolysis products in the growing axis are critical processes for the establishment of seedlings after germination. Therefore, it is crucial for mobilization of reserves to be synchronized with the growing axis, so that photosynthetic activity can be started before depletion of reserves. For this, integrative approaches involving different reserves, different hydrolysis products and interaction between storage and growing axis tissues, either through hormones or metabolites with signaling role, can contribute greatly to the elucidation of the regulation mechanisms for reserve mobilization. In this study, was hypothesized that hormones and metabolites have different actions on reserve mobilization, and there must be a crossed effect of sugars on the mobilization of proteins and amino acids on lipids and starch mobilization in sunflower seedlings. This study was conducted with seeds of sunflower (Helianthus annuus L.) hybrid Helio 253 using in vitro culture system. Seeds were germinated on Germitest® paper and grown on agar-water 4 g/L without addition of nutrients during 9 days after imbibition (DAI) for growth curve. To verify the effect of metabolites and hormones, seedlings were transferred in the 2nd DAI to agar-water 4 g/L supplemented with increasing concentrations of sucrose or L-glutamine, abscisic acid, gibberellic acid or indolebutyric acid. The results of this study confirm that the mobilization of lipids and storage proteins occurs in a coordinated manner during post-germination growth in sunflower, corroborating the hypothesis that the application of external carbon (sucrose) and nitrogen (L-glutamine) sources can delay the mobilization of these reserves in a crossed way. Moreover, considering the changes in the patterns of reserve mobilization and partition of their products in seedlings treated with different growth regulators, it is evident that the effects of metabolites and hormones must involve, at least in part, distinct mechanisms of action