Onset Of Imbibition Research Articles

In-situ 3D measurements of water films on the natural grain surface of porous rocks

During fluid displacement, e.g. in aquifers or other fluid reservoirs such as CO2 or H2 storage sites or hydrocarbon reservoirs, water films are expected to form along the internal pore surface due to its wetting properties and capillarity. Near connate water saturation, these water films would dominate the macroscopic multiphase flow behaviour when e.g. water penetrates the porous medium. We use Atomic Force Microscopy (AFM) to study the sub-pore scale configuration of microscale water films on the rough pore surface of Ketton and Estaillades rocks, after drainage of the medium with decane. Then, we compare the results with a numerical simulation and use a film flow model to estimate the relative permeability through these films, which can be potentially used to link the water film configuration to pore- and core-scale responses. We find that the surface coverage and connectivity of the water films varies substantially with capillary pressure. This can affect the permeability of the films up to several orders of magnitude and thus potentially dictate the displacement efficiency at the onset of imbibition, when the brine phase re-enters the pore space. The novel experimental workflow proposed in this study may help future digital assessment of multiphase flow behaviour near connate saturation.

Castor (Ricinus communis L.) differential cell cycle and metabolism reactivation, germinability, and seedling performance under NaCl and PEG osmoticum: Stress tolerance related to genotype-preestablished superoxide dismutase activity

Castor (Ricinus communis) is a relevant industrial oilseed feedstock for many industrial applications, being globally mainly cultivated by smallholder farmers in semiarid areas, where abiotic stresses predominate. Therefore, susceptible to generating reactive oxygen species (ROS) and subsequent oxidative stress, compromising cell metabolism upon seed imbibition and germination, seedling and crop establishment, and yield. The present study evaluated the consequences of water restriction by Polyethylene glycol (PEG) and Sodium chloride (NaCl) on cell cycle and metabolism reactivation on germinability, seedling growth, and vigor parameters in 2 commercial castor genotypes (Nordestina and Paraguaçu). PEG water restriction inhibited germination completely at −0.23 MPa or higher, presumably due to reduced oxygen availability. The restrictive effects of NaCl saline stress on germination were observed only from −0.46 MPa onwards, affecting dry mass accumulation and the production of normal seedlings. In general, superoxide dismutase (SOD) activity increased in NaCl −0.23 MPa, whereas its modulation during the onset of imbibition (24h) seemed to depend on its initial levels in dry seeds in a genotype-specific manner, therefore, resulting in the higher stress tolerance of Nordestina compared to Paraguaçu. Overall, results show that Castor germination and seedling development are more sensitive to the restrictive effects of PEG than NaCl at similar osmotic potentials, contributing to a better understanding of the responses to water restriction stresses by different Castor genotypes. Ultimately, SOD may constitute a potential marker for characterizing castor genotypes in stressful situations during germination, early seedling, and crop establishment, and a target for breeding for Castor-improved stress tolerance.

Simulation of Counter-Current Spontaneous Imbibition Based on Momentum Equations with Viscous Coupling, Brinkman Terms and Compressible Fluids

A numerical model is investigated representing counter-current spontaneous imbibition of water to displace oil or gas from a core plug. The model is based on mass and momentum conservation equations in the framework of the theory of mixtures. We extend a previous imbibition model that included fluid–rock friction and fluid–fluid drag interaction (viscous coupling) by including fluid compressibility and Brinkman viscous terms. Gas compressibility accelerated recovery due to gas expansion from high initial non-wetting pressure to ambient pressure at typical lab conditions. Gas compressibility gave a recovery profile with two characteristic linear sections against square root of time which could match tight rock literature experiments. Brinkman terms decelerated recovery and delayed onset of imbibition. Experiments where this was prominent were successfully matched. Both compressibility and Brinkman terms caused recovery deviation from classical linearity with the square root of time. Scaling yielded dimensionless numbers when Brinkman term effects were significant.Article HighlightsSpontaneous imbibition with viscous coupling, compressibility and Brinkman terms.Viscous coupling reduces spontaneous imbibition rate by fluid–fluid friction.Brinkman terms delay early recovery and explain seen delayed onset of imbibition.Gas compressibility accelerates recovery and can be significant at lab conditions.Gas compressibility gives recovery with two root of time lines as seen for shale.

Early Growth and Development of Horseweed (<i>Conyza canadensis</i> (L.) Cronq.)

Horseweed (Conyza canadensis (L.) Cronq.) produces thousands of small elongated seeds which are botanically defined as achenes; yet, relative to the quantity of achenes produced, few seedlings survive to produce mature plants. The developmental progression from achene to 4 mm seedlings was documented, and seedling response to moisture deprivation was described. Radical protrusion through the pericarp occurred between 18 and 30 hours after onset of imbibition in water or when germinated on soil at or greater than field capacity. A ring of root hair initials formed immediately after radical emergence at the interface of what was to become the separation between the root and hypocotyl. By 48 hours post imbibition, radicals differentiated into a distinct root with root cap and a hypocotyl, and root hairs elongated. By 72 hours post imbibition, seedlings had emerged from the pericarp, and had: expanded photosynthetic cotyledons, a clearly defined hypocotyl, a ring of elongated root hairs exceeding 1 mm in length, and a root equal or longer than the hypocotyl. The epicotyl had not yet emerged, and the total seedling length was approximately 3 to 4 mm. Germination was delayed on soil at or below field capacity. More than 95% of two- and four-day-old seedlings that had been desiccated for more than 24 hours died after being rehydrated.

Efficiency of energy conversion and growth of gamma irradiated embryos and young seedlings of Triticum monococcum L. cultivar Einkorn

The study was conducted to determine whether the efficiency of energy conversion into growth can be used as an indicator for the determination of the optimal gamma irradiation dosage for mutation breeding. To meet this objective, embryo growth, shoot growth, root growth, mobilization of food reserves, respiration and energy conversion were studied in gamma-irradiated wheat Triticum monococcum L. cultivar Einkorn kernels. Kernels were exposed to 50, 150, 250 and 350 Gy and germinated. Kernels were collected 12 h after onset of imbibition and then every 12 h until 168 h. Irradiated seed demonstrated retardation in all parameters, which increased as the gamma irradiation dosage increased. For the most, dosage and time, as well as dosage by time interaction were highly significant. Root growth appeared to be the most sensitive to gamma irradiation, followed by shoot growth, mobilization of food reserves and efficiency of energy conversion. Full recovery of the efficiency of energy conversion took place at 50 Gy, with an increase in inefficiency with an increase in dosage. The point where full recovery of efficiency of energy conversion into growth gives way to incomplete recovery (100 Gy) is in line with the suggested dosages for practical mutation breeding in Triticum monococcum L. by the FAO/IAEA and is therefore an ideal indicator for predicting the dosage that will be optimal for plant mutation breeding.

Water content, adenylate kinase, and mitochondria drive adenylate balance in dehydrating and imbibing seeds.

Water and life are inexorably linked, but some organisms are capable of losing almost all cellular water to enter a non-metabolic state of anhydrobiosis. This raises intriguing questions about how energy metabolism is managed during such transitions. Here, we have investigated adenylate metabolism during seed imbibition and drying using intact or fragmented pea (Pisum sativum L.) seeds. AMP was confirmed as the major adenylate stored in dry seeds, and normal adenylate balance was rapidly restored upon rehydration of the tissues. Conversely, re-drying of fully imbibed seeds reversed the balance toward AMP accumulation. The overall analysis, supported by in vitro enzyme mimicking experiments, shows that during tissue dehydration, when oxidative phosphorylation is no longer efficient because of decreasing water content, the ATP metabolic demand is met by adenylate kinase, resulting in accumulation of AMP. During seed imbibition, adenylate balance is rapidly restored from the AMP stock by the concerted action of adenylate kinase and mitochondria. The adenylate balance in orthodox seeds, and probably in other anhydrobiotes, appears to be simply driven by water content throughout the interplay between ATP metabolic demand, adenylate kinase, and oxidative phosphorylation, which requires mitochondria to be energetically efficient from the onset of imbibition.

Changes in low-molecular-weight thiol-disulphide redox couples are part of bread wheat seed germination and early seedling growth

The tripeptide antioxidant glutathione (γ-l-glutamyl-l-cysteinyl-glycine; GSH) essentially contributes to thiol-disulphide conversions, which are involved in the control of seed development, germination, and seedling establishment. However, the relative contribution of GSH metabolism in different seed structures is not fully understood. We studied the GSH/glutathione disulphide (GSSG) redox couple and associated low-molecular-weight (LMW) thiols and disulphides related to GSH metabolism in bread wheat (Triticum aestivum L.) seeds, focussing on redox changes in the embryo and endosperm during germination. In dry seeds, GSH was the predominant LMW thiol and, 15 h after the onset of imbibition, embryos of non-germinated seeds contained 12 times more LMW thiols than the endosperm. In germinated seeds, the embryo contained 17 and 11 times more LMW thiols than the endosperm after 15 and 48 h, respectively. This resulted in the embryo having significantly more reducing half-cell reduction potentials of GSH/GSSG and cysteine (Cys)/cystine (CySS) redox couples (EGSSG/2GSH and ECySS/2Cys, respectively). Upon seed germination and early seedling growth, Cys and CySS concentrations significantly increased in both embryo and endosperm, progressively contributing to the cellular LMW thiol-disulphide redox environment (Ethiol-disulphide). The changes in ECySS/2Cys could be related to the mobilisation of storage proteins in the endosperm during early seedling growth. We suggest that EGSSG/2GSH and ECySS/2Cys can be used as markers of the physiological and developmental stage of embryo and endosperm. We also present a model of interaction between LMW thiols and disulphides with hydrogen peroxide (H2O2) in redox regulation of bread wheat germination and early seedling growth.

Accurate early‐time and late‐time modeling of countercurrent spontaneous imbibition

AbstractSpontaneous countercurrent imbibition into a finite porous medium is an important physical mechanism for many applications, included but not limited to irrigation, CO2 storage, and oil recovery. Symmetry considerations that are often valid in fractured porous media allow us to study the process in a one‐dimensional domain. In 1‐D, for incompressible fluids and homogeneous rocks, the onset of imbibition can be captured by self‐similar solutions and the imbibed volume scales with . At later times, the imbibition rate decreases and the finite size of the medium has to be taken into account. This requires numerical solutions. Here we present a new approach to approximate the whole imbibition process semianalytically. The onset is captured by a semianalytical solution. We also provide an a priori estimate of the time until which the imbibed volume scales with . This time is significantly longer than the time it takes until the imbibition front reaches the model boundary. The remainder of the imbibition process is obtained from a self‐similarity solution. We test our approach against numerical solutions that employ parametrizations relevant for oil recovery and CO2 sequestration. We show that this concept improves common first‐order approaches that heavily underestimate early‐time behavior and note that it can be readily included into dual‐porosity models.

Stored and neosynthesized mRNA in Arabidopsis seeds: effects of cycloheximide and controlled deterioration treatment on the resumption of transcription during imbibition

Dry seeds accumulate translatable mRNAs as well as functional proteins for transcription and translation. They are possibly involved in early physiological responses after imbibition, however, their functions remain poorly understood. The aim of this study is to investigate the function of seed stored transcriptional machinery in resumption of gene expression after the onset of imbibition in Arabidopsis thaliana. First, we examined the characters of stored mRNAs in A. thaliana dry seeds using microarray data from non-dormant Columbia (Col) and dormant Cape Verde Islands (Cvi) accessions. Transcriptomes of Col and Cvi dry seeds resembled one another, suggesting that patterns of stored mRNA do not reflect either the degree of dormancy or germination potential, but rather reflect the developmental context, such as seed maturation. Upon imbibition, changes in mRNA abundance of many genes were initiated between 1- and 2-h after the onset of imbibition. RT-PCR expression analysis of imbibition-responsive genes indicates that early induction was not altered by treatment of cycloheximide. This suggests that de novo protein synthesis is not required for gene expression during early imbibition stages. Moreover, controlled deterioration treatment (CDT), which causes artificial damages on dry seeds, disrupted gene expression specifically during the first 3 h after the start of imbibition, suggesting that seed stored transcription factors play a pivotal role in gene expression during early imbibition periods. Furthermore, the negligible effect of CDT on germination indicates that early imbibition response is dispensable and de novo synthesized proteins compensate for the function of stored proteins for germination.

Temporal Expression Patterns of Hormone Metabolism Genes during Imbibition of Arabidopsis thaliana Seeds: A Comparative Study on Dormant and Non-Dormant Accessions

Seed imbibition is a prerequisite for subsequent dormancy and germination control. Here, we investigated imbibition responses of Arabidopsis seeds by transcriptomic and hormone profile analyses using dormant [Cape Verde Islands (Cvi)] and non-dormant [Columbia (Col)] accessions. Once imbibed, seeds of both accessions swelled most up to 3 h, reflecting water uptake. Microarray analysis showed that in both accessions, seeds imbibed for 15 min, 30 min and 1 h were less active in gene expression than at 3 h. More than 2,000 genes were either up-regulated or down-regulated in seeds imbibed for 3 h. Some genes up-regulated at 3 h were already induced in seeds imbibed for 1 h, suggestive of genome reprogramming early after the onset of imbibition. Imbibition-induced genes in seeds imbibed for 3 h included those up-regulated in both Col and Cvi (common) or unique to either accession (accession specific). Up-regulated genes that were both common and Cvi-specific were over-represented for sugar metabolism and the pentose phosphate pathway, whereas Col-specific genes were over-represented for ribosomal protein genes. Quantification of plant hormones showed that ABA and salicylic acid (SA) contents were higher, but gibberellin A(4) (GA(4)), N(6)-(Delta(2)-isopentenyl)adenine (iP), jasmonic acid (JA), JA-isoleucine (JA-Ile) and IAA were lower in imbibed seeds of Cvi compared with Col. In addition, changes in IAA and JA were initiated before 1 h, whereas ABA and JA-Ile declined 3 h after the onset of imbibition. An increase in GA(4) and iP appeared to be correlated temporally with the initiation of secondary water uptake, which marks the completion of germination.

After-ripening alters the gene expression pattern of oxidases involved in the ethylene and gibberellin pathways during early imbibition of Sisymbrium officinale L. seeds

After-ripening (AR) in Sisymbrium officinale seeds altered SoACS7, SoACO2, SoGA20ox2, SoGA3ox2, and SoGA2ox6 gene expression. Except for SoGA20ox2 expression, which sharply diminished, the expression of the other genes rose during development, particularly that of SoACS7. In contrast, only the SoACO2 and SoGA2ox6 transcripts increased with seed desiccation; the others decreased. AR increased the SoGA3ox2 transcript in dry seed, but dramatically decreased the SoACS7 transcript. At the onset of imbibition, AR inhibited SoACS7 and SoACO2 expression and stimulated that of SoGA20ox2, SoGA3ox2, and SoGA2ox6, demonstrating that the participation of ethylene (ET) and gibberellins (GAs) differs in after-ripened and non-after-ripened seeds. The inhibition of SoACO2 expression in the presence of GA4+7, paclobutrazol (PB), inhibitors of ET synthesis and signalling (IESS), and notably ET+GA4+7 indicated ET–GA cross-talk in non-after-ripened seeds. A positive effect of AR in reversing this inhibition was found. The idea of ET–GA cross-talk is also supported by the effect of ET on SoGA3ox2 expression, notably induced by the AR process. In contrast, SoGA20ox2 expression did not appear to be susceptible to AR. SoGA2ox6 expression, poorly known in seeds, suggests that AR prompted an up-regulation under all treatments studied, whereas in non-after-ripened seeds expression was down-regulated. On the other hand, the β-mannanase (MAN) activity dramatically increased in dry after-ripened seed, being significantly boosted by ET. The absence of MAN inhibition by IESS suggests that although ET seems to be one of the factors controlling MAN, its presence did not appear to be essential. GA4+7 only increased MAN in seeds wich were after-ripened. Here, it is proposed that ET and GAs participate actively in establishing the AR process.

Is seed conditioning essential for Orobanche germination?

Parasitic Orobanchaceae germinate only after receiving a chemical stimulus from roots of potential host plants. A preparatory phase of several days that follows seed imbibition, termed conditioning, is known to be required; thereafter the seeds can respond to germination stimulants. The aim of this study was to examine whether conditioning is essential for stimulant receptivity. Non-conditioned seeds of both Orobanche cumana Wallr. and O. aegyptiaca Pers. [syn. Phelipanche aegyptiaca (Pers.) Pomel] were able to germinate in response to chemical stimulation by GR24 even without prior conditioning. Stimulated seeds reached maximal germination rates about 2 weeks after the onset of imbibition, no matter whether the seeds had or had not been conditioned before stimulation. Whereas the lag time between stimulation and germination response of non-conditioned seeds was longer than for conditioned seeds, the total time between imbibition and germination was shorter for the non-conditioned seeds. Unlike the above two species, O. crenata Forsk. was found to require conditioning prior to stimulation. Seeds of O. cumana and O. aegyptiaca are already receptive before conditioning. Thus, conditioning is not involved in stimulant receptivity. A hypothesis is put forward, suggesting that conditioning includes (a) a parasite-specific early phase that allows the imbibed seeds to overcome the stress caused by failing to receive an immediate germination stimulus, and (b) a non-specific later phase that is identical to the pregermination phase between seed imbibition and actual germination that is typical for all higher plants.

Function and stress tolerance of seed mitochondria

Seeds of higher plant are desiccation tolerant, which suggests that their mitochondria exhibit particular properties. Insight into the function of seed mitochondria, especially in legume and model plants, has been fostered by the development of proteomics. Seed mitochondria are functional at the onset of imbibition, and their integrity and performance systematically improves during germination. This suggests that repair and biogenesis mechanisms exist, and this is supported by morphological and biochemical evidence. Seed mitochondria generate and operate in a hypoxic environment. They accumulate stress proteins, such as a small heat‐shock protein and a late embryogenesis abundant protein. The mitochondria of pea (Pisum sativum L.) seed also display a biased phospholipid composition likely to favour desiccation tolerance. These specific biochemical properties surely contribute to the remarkable tolerance of seed mitochondria to extreme temperatures. Recent progress towards the resolution of the seed mitochondrial proteome is discussed in light of the growing body of genomic data.

Root growth inhibition by aluminum is probably caused by cell death due to peroxidase-mediated hydrogen peroxide production

The effect of aluminum on hydrogen peroxide production and peroxidase-catalyzed NADH oxidation was studied in barley roots germinated and grown between two layers of moistened filter paper. Guaiacol peroxidase activity significantly increased after 48 h and was approximately two times higher after 72 h in Al-treated roots. The oxidation of NADH was also significantly increased and, like guaiacol peroxidase activity, it was two times higher in A1-treated roots than in controls. Elevated H2O2 production was observed both 48 and 72 h after the onset of imbibition in the presence of A1. Separation on a cation exchange column allowed the detection of two peaks with NADH peroxidase and H2O2 production activity. However, a difference between control and Al-treated plants was found only in one fraction, in which four times higher guaiacol peroxidase activity and five times higher NADH peroxidase activity were expressed and about three times more H2O2 was produced. One anionic peroxidase and three cationic peroxidases were detected in this fraction by native polyacrylamide gel electrophoresis. The anionic peroxidase was activated in the Al-treated root tips and also oxidized NADH but was detectable only after a long incubation time. Two of the cationic peroxidases were capable of oxidizing NADH and producing a significant amount of H2O2, but only one of these was activated by A1 stress. The role of these peroxidases during A1 stress in barley root tips is discussed.

Activity of some enzymes in barley caryopses during imbibition in aluminium presence

Peroxidase, superoxide dismutase, acid and alkaline phosphatase, esterase and glucosidase activities were studied during imbibition of barley caryopses in the presence of aluminium. Antioxidative enzymes (peroxidase, superoxid dismutase) showed elevated activity already 2 h after the onset of imbibition in the presence of Al. In contrast hydrolytic enzymes (phosphatases, glucosidase, esterase) were only moderately activated at low Al concentrations (1–2mM), while strong inhibition was observed at higher Al concentrations (4–8mM). In in vitro conditions 8mM Al had no effect on the activity of acid phosphatase, moderately inhibited alkaline phosphatase and glucosidase and strongly esterase activity. During imbibition of caryopses in solution without Al an increase of the pH value of the imbibition solution from 4 to 6 has occurred, while in the presence of Al the shift in pH value was less expressive and dependent on Al concentration. At 8mM Al concentration no change in the pH value of imbibition solution was observed. The SDS-PAGE analysis of polypeptides released to the imbibition solution in the presence of Al revealed the accumulation of two polypeptides with relative molecular mass of 35 and 18 kDa. The release of 96 and 27.5 kDa polypeptides was completely inhibited at 8mM Al concentration. These results confirmed that Al is able to influence different physiological processes already during seed imbibition and early growth phases of barley seedlings.

The ascorbic acid system in seeds: to protect and to serve

The ascorbic acid (ASC) system functions dynamically in seeds, although the strategies for ASC production and utilization may vary according to seed developmental and functional stages. In orthodox seeds, ASC content and ASC peroxidase activity increase during the early stages of development, then decrease during the desiccation stage, so that, at quiescence, seeds have neither ASC nor ASC peroxidase, but retain a small amount of dehydroascorbic acid (DHA) and significant activities of ASC recycling enzymes. ASC and ASC peroxidase activity re-start after a few hours from the onset of imbibition. In contrast, the ASC system is little affected during germination of recalcitrant seeds. Although the presence of the ASC system in seeds has often been considered only within the framework of seed antioxidant defences, ASC function in seeds is also likely to be related to its action as a specific co-substrate required for the activity of dioxygenases (e.g. 1-aminocyclopropane carboxylate oxidase, gibberellic acid hydroxylases and 9-cis-epoxycarotenoid dioxygenases) involved in the synthesis of ethylene, gibberellins and abscisic acid, respectively. The possible role of ASC in coordinating the activities of these key enzymes is discussed.

Membrane integrity and oxidative properties of mitochondria isolated from imbibing pea seeds after priming or accelerated ageing

AbstractGermination is an energy-demanding process that requires the operation of mitochondria, which must survive desiccation in the quiescent seed and become rapidly functional after imbibition to meet the ATP demand. The relationship between germination and mitochondrial performance was addressed by analysing the properties of mitochondria isolated from control, primed and aged pea (Pisum sativum L.) seeds. Mitochondria were isolated and purified at early stages of germination (before radicle protrusion), and their oxidative properties, membrane integrity and ultrastructure were examined. Mitochondria isolated after 12 h of imbibition readily oxidized exogenous NADH and Krebs cycle substrates at high rates. However, their phosphorylation efficiency was restricted by poor membrane integrity. After 22 h from the beginning of imbibition, purified seed mitochondria had intact outer membranes and oxidized the substrates at slightly lower rates, but with higher respiratory control (improved capacity for phosphorylation). Purified seed mitochondria were always found to be deficient in endogenous NAD, although the organelles were capable of importing and retaining the cofactor. While the priming treatment appeared to slightly increase the performance of mitochondria, seed deterioration by accelerated ageing strongly affected the oxidative properties of mitochondria, which were badly impaired in ATP production. Outer and inner membrane integrity was identified as the primary target for desiccation and ageing stress. A link between mitochondrial function and seed quality was also corroborated by respiration measurements of seed fragments at the onset of imbibition.

Temporal and photoregulated expression of five tomato phytochrome genes.

Quantitative measurements of the absolute amounts of mRNAs transcribed from each of five phytochrome genes (PHYA, PHYB1, PHYB2, PHYE, PHYF) throughout the life cycle of a tomato plant and in response to changes in ambient light conditions are reported here. From their lowest level in unimbibed seed, all five transcripts increase by from 10- to 1000-fold during the first 24 h following the onset of imbibition, both in continuous darkness and in a greenhouse. In a greenhouse and on a whole-plant basis, all continue to increase throughout day 6, after which all but PHYE decline over the next week to a plateau at about one-half of the maximal value. PHYE mRNA differs in that in continues to increase in abundance during the first 2-3 weeks and thereafter remains at that maximal level. In adult plants, on a whole-plant basis and in decreasing order of abundance, PHYA, PHYB1, PHYE, PHYB2, and PHYF transcripts were present at approximately 120, 40, 40, 15, and 8 mumol microgram-1 of poly(A)(+)-enriched RNA, respectively. The data are consistent with the demonstrated roles of phytochromes A and B1 during seedling development and lead to the suggestion that phytochrome E might have a more important role in mature plants. Somewhat unexpectedly, PHYA and PHYB2 expression patterns are very similar. In seedlings, PHYA and PHYB2 exhibit the greatest increase in expression following a light-to-dark transition, as well as the greatest decrease following a dark-to-light transition. PHYA and PHYB2 are also similar in that both exhibit comparable variation on a natural diurnal cycle, while PHYB1 also exhibits variation but with a markedly different phase. The diurnal variation in expression of PHYA, PHYB1 and PHYB2 is consistent with the possibility that one or more of the phytochromes they encode is important with respect not only to photoperiodic behavior but also to the regulation of other events whose photosensitivity varies during a diurnal cycle.

A vacuolar cysteine endopeptidase (SH-EP) that digests seed storage globulin: Characterization, regulation of gene expression, and posttranslational processing

Summary A cysteine endopeptidase named SH-EP was isolated as one of the major hydrolytic enzymes involved in the mobilization of storage globulin in the cotyledon of germinated seeds of Vigna mungo . The enzyme, by itself or in combination with a serine endopeptidase, digests the globulin subunits to smaller peptides in vitro . Protein immunoblot analysis with antiserum raised against SH-EP showed that this enzyme is synthesized in the cotyledon after the onset of imbibition and increases until day 4 and decreases thereafter. Application of plant hormones and growth regulators, as well as amino acids as proteolytic end-products, appears to affect the development of SH-EP to a limited extent in the cotyledon of the germinated seeds. SH-EP of 33 kDa is synthesized on membrane-bound polysomes as a large, inactive 45-kDa precursor, which is cotranslationally processed to a 43-kDa intermediate through cleavage of a signal peptide. The amino-terminal propeptide region of the intermediate is cleaved further to form the 33-kDa mature enzyme by a multistep processing. The 43-kDa intermediate is also subjected to the cleavage of the carboxy-terminal decapeptide containing a Lys-Asp-Glu-Leu tail, which is known as a retention signal for the endoplasmic reticulum lumen. An asparaginyl endopeptidase, designated VmPE-1, was isolated as one of the processing enzymes responsible for the cleavage of the amino-terminal propeptide region of the precursor to SH-EP.

Enzymes of the Primary Phosphatidylethanolamine Biosynthetic Pathway in Postgermination Castor Bean Endosperm (Developmental Profiles and Partial Purification of the Mitochondrial CTP:Ethanolaminephosphate Cytidylyltransferase).

Ethanolamine kinase, CTP:ethanolaminephosphate cytidylyltransferase (ECT), and ethanolaminephosphotransferase, which sequentially catalyze the primary pathway for phosphatidylethanolamine synthesis, were measured in castor bean (Ricinus communis L. var Hale) endosperm for 6 d after the onset of imbibition. Ethanolamine kinase (EC 2.7.1.82) activity was cytosolic, increasing slowly during the first 5 d and then declining. Total ECT (EC 2.7.7.14) activity increased until the 4th d, but the endoplasmic reticulum fraction of the activity peaked at d 3, and the mitochondrial activity peaked at d 4. Diacylglycerol:CDPethanolamine ethanolaminephosphotransferase (EC 2.7.8.1) increased during the first 2 d after imbibition began, after which it declined. The lowest activity of ethanolamine kinase during postgermination was more than 5-fold higher than the maximum activity of ECT, and the total activity of diacylglycerol:CDPethanolamine ethanolaminephosphotransferase at d 2 was at least triple that of ECT of the endoplasmic reticulum. We have partially purified ECT from mitochondrial fractions of postgermination castor bean endosperm starting with mitochondria purified by sucrose (Suc) density gradient centrifugation and broken by osmotic shock and homogenization. The membrane-bound ECT was solubilized with 1.5% 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate and purified approximately 118-fold by polyethylene glycol precipitation, chromatography on Sephacryl S-200, and then Suc gradient centrifugation. The continuous presence of both salt (0.5 M NaCl) and detergent (1% [w/v] 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate) was necessary to prevent aggregation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the final activity peak resulted in a prominent protein band at 35 kD, which correlated with bands from peak ECT activity fractions after both Suc gradient centrifugation and gel filtration on Sephacryl S-200. The activity of this enzyme was enhanced by the addition of several phospholipids.