The Messenger RNA Population in the Embryonic Axes of Phaseolus vulgaris During Development and Following Germination

Misra, S. and Bewley, J. D. 1985. The messenger RNA populationin the embryonic axes of Phaseolus vulgaris during developmentand following germination.—J. exp. Bot. 36: 1644–1652. Messenger RNAs were extracted from young, mid-maturation, late-maturation,mature-dry, and 20-h-germinated embryonic axes of Phaseolusvulgaris cv. Taylor's Horticultural. They were translated invitro in a rabbit reticulocyte lysate protein synthesizing system.Analysis of the translation products using two-dimensional polyacrylamidegel electrophoresis indicated that there were substantial changesin the messenger RNA populations of developing and germinatingaxes. The number of polypeptides synthesized increased sharplyat 20–22 d after pollination and then declined. Therewas a parallel increase and decrease in the Poly(A)⁺ contentof the seed axis. The analysis showed that certain messageswere present throughout development and were stored in maturedry seed. These messages were degraded upon subsequent rehydration.Some messages appeared during mid-maturation but declined duringlater stages of development and were absent from the matureseed. In the germinating seed a set of messages unique to germinationappeared. Key words: —Seed development, germination, mRNA, in vitro translation, Phaseolus vulgaris     

Desiccation of Phaseolus vulgaris Seeds During and Following Germination, and its Effect Upon the Translatable mRNA Population of the Seed Axes

Misra, S. and Bewley, J. D. 1986. Desiccation of Phaseolus vulgansseeds during and following germination, and its effect uponthe translatable mRNA population of the seed axes.—J.exp. BoL 37: 364–374. After imbibition and germination, seeds of P. vulgaris passfrom a stage where they are insensitive to desiccation to astage where they are sensitive. Desiccation of seeds duringthe sensitive stage results in an almost total impairment ofprotein synthesis upon subsequent rehydration. Seeds desiccatedduring the desiccation-tolerant stage, however, resume proteinsynthesis at almost control levels. The protein patterns obtained following in Vitro translationof bulk RNA from fresh imbibed, desiccated, and desiccated-rehydratedseed axes were qualitatively similar at 5 HAI (the desiccation-tolerant stage). The drying treatment resulted in increasedintensity of extant proteins at 5 and 12 HAI. At 12 HAI (thetransition stage between the desiccation-tolerant and desiccation-intolerantphases) desiccation and subsequent rehydration triggered synthesisof a unique set of proteins-the rehydration proteins. At 20HAI (the desiccation-intolerant stage) desiccation resultedin an overall decline in the intensity of proteins synthesizedin vitro. Also the rehydration proteins were not synthesizedin response to a drying and rehydration treatment at this time. Key words: Seed germination, desiccation, mRNA, in vitro translation, Phaseolus vulgaris     

Protein Synthesis in the Axes of Polyethylene Glycol-Treated Pea Seed and during Subsequent Germination

Germination of Alaska pea seeds is inhibited by –0.3 MPapolyethylene glycol but upon subsequent transfer to water, germinationis completed rapidly and radicle emergence occurs more quicklythan in water-imbibed seeds. Protein synthesis is reduced inthe axes of seeds imbibed on PEG but increases upon their returnto water, though not to the level exhibited by axes germinatedon water. Mobilization of proteins in the axes is retarded bytheir failure to complete germination on PEG, although somedoes occur. The quantitative reduction in protein synthesisresulting from incubation in osmoticum is not accompanied bymarked qualitative changes. The block to germination is notobviously associated with a restriction in synthesis of anyparticular protein or set of proteins; conversely, no ‘water-stress’proteins are synthesized in the presence of PEG. The synthesisof growth-specific proteins is prevented by PEG, but these increaseupon relief from the osmoconditioning treatments. These observationsdispute earlier claims for accelerated protein synthesis resultingfrom PEG treatments. Key words: Osmotic priming, Pisum sativum, germination, protein synthesis     

Long-lived Messenger RNA and its Relationship to Protein Synthesis During Germination of Pea (Pisum sativum L.) Seeds

Synthesis of both protein and RNA is initiated very early ingermination in the embryo axes of pea seeds. The early RNA synthesisinvolves all three types, although there is some evidence forpreferential synthesis of mRNA in the first few hours afterthe onset of imbibition. In addition to newly synthesized mRNA,the embryo axis also contains long-lived mRNA. The amount ofthis long-lived mRNA declines markedly during the first 20 hof germination. Synthesis of both protein and RNA is initiated very early ingermination in the embryo axes of pea seeds. The early RNA synthesisinvolves all three types, although there is some evidence forpreferential synthesis of mRNA in the first few hours afterthe onset of imbibition. In addition to newly synthesized mRNA,the embryo axis also contains long-lived mRNA. The amount ofthis long-lived mRNA declines markedly during the first 20 hof germination. Results from in vitro and in vivo protein synthesis experimentsand from studies of polysome formation suggest that much ofthe long-lived mRNA present in the embryo axis does not directprotein synthesis. The increase in the rate of protein synthesisduring germination is thus dependent on recruitment of newlysynthesized mRNA molecules. Pea, Pisum sativum L., germination, mRNA, protein synthesis     

Desiccation of Axes of Phaseolus vulgaris during Development of a Switch from a Development Pattern of Protein Synthesis to a Germination Pattern

Immature seeds of Phaseolus vulgaris removed from the pod at 32 days of development do not germinate unless first subjected to a desiccation treatment. This change from development to germination caused by premature drying is mirrored in the pattern of protein synthesis by the axes. Rehydrated axes from 32-day-developed seeds cease to synthesize proteins that are uniquely associated with development, but instead synthesize some proteins that are similar to those made in the germinating axes from mature dry seeds. Desiccation of 22-day-developed seeds does not lead to their germination, nor does it cause a switch from a developmental to a germination mode of protein synthesis by the axes. It is proposed that desiccation plays a role in permanently suppressing developmental protein synthesis and in inducing germination protein synthesis.     

Messenger RNA movement on the ribosome

Recent X-ray and cryo-EM studies of 70S ribosome complexes containing different types of messenger RNAs (mRNA) and transfer RNA (tRNA) have been reviewed. Changes of the mRNA path on the ribosome at initiation and elongation states have been described. Authors suggested, that the specific region of ribosomal 30S subunit ("platform") is a ribosome binding site of regulatory domains of mRNA which locates on the non-translated 5'-end of the mRNA.     

Messenger RNA movement on the ribosome

The review summarizes the recent structural data obtained for 70S ribosome complexes with various mRNAs and tRNAs by X-ray analysis and cryoelectron microscopy. The mRNA region interacting with the ribosome at translation initiation and elongation is described. A specific part (platform) of the 30S ribosome subunit was assumed to bind the regulatory elements located in the 5′-untranslated region of mRNA.     

Effect of Temperature and Desiccation during Storage on Germination and Keeping Quality of Kochia indica Seeds

Data are given for Kochia indica seeds showing retention ofviability after storage for various periods of time open tothe air under laboratory conditions, open at 30° C., openat 38° C., and sealed over CaCl₂ at 30° C. Seeds have been stored without deterioration at 30° C. sealedover CaC1₂ for over 14 months. Rapid deterioration of seed inopen storage at laboratory temperature and at 30° C. showsthat loss of viability is accelerated by moisture more thanby temperature.     

Messenger RNA changes during differentiation of murine erythroleukemia cells

During differentiation of murine erythroleukemia cells, the levels of certain mRNA were observed to change. To characterize the various patterns of changes that occur during differentiation, cDNA libraries made from RNA isolated from uninduced and differentiating cells were screened with labeled cDNA or RNA labeled in vivo for different periods of time. cDNA clones that corresponded to individual mRNAs whose level remained constant, increased, or decreased during differentiation were identified. These clones were used to analyze Northern blots containing RNA from uninduced and differentiated cells. A number of characteristic changes in individual mRNAs in differentiating murine erythroleukemia cells could be identified, such as no change, increase in concentration, increase in concentration and slight change in size, decrease in concentration, decrease in concentration and change in size, appearance of new band(s) of entirely different size, and change in relative concentrations of two related mRNAs. Measurements of rates of mRNA synthesis and degradation suggest that both parameters change during differentiation and that these changes are instrumental in establishing cellular concentration of specific mRNAs. It seems that the changes in mRNA stability observed in differentiating murine erythroleukemia cells may be associated with changes in the primary structure of the transcribed portion of mRNA. The observation that specific mRNA synthesized before and after induction may have very different stabilities at the same point in differentiation supports this hypothesis.     

Behavior of the Nucleoside Triphosphate Diphosphatase in Pea Cotyledon Chromatin during Germination

The behavior of nucleoside triphosphate diphosphatase (NTDPase)of pea cotyledon chromatin was investigated by a comparisonof the activities of amylase and protease in the cytoplasm.The activities of NTDPase and amylase increased from the 3rdto the 5th day during germination, but the activity of proteaseincreased more rapidly at the stage of imbibition. The inhibitorsactinomycin D and cycloheximide markedly inhibited the increasein the activities of NTDPase and amylase, but their inhibitionof protease was much less. Inhibition of germination by saltstress was accompanied by reductions in the increases in NTDPaseand protease activities, but not by a reduction in amylase activity.Removal of the embryo from dormant seeds had the same effecton these activities as salt stress did. These results suggestthat formation of the NTDPase in the chromatin of the cotyledonis under the control of the embryonic tissue. (Received October 22, 1980; Accepted January 20, 1981)     

Desiccation Resistance in Four Drosophila Species: Sex and Population Effects

Desiccation resistance and body mass were measured in multiple populations of each of four species of Drosophila: two desert endemic species (D. nigrospiracula and D. mojavensis), and two with more widespread distributions (D. melanogaster and D. pseudoobscura). While flies from the desert species were more desiccation tolerant, there was, in certain cases, significant variation in desiccation resistance among populations of the same species. A significant difference in desiccation resistance was observed between the sexes, females were more resistant than males, but this relationship was reversed when taking into account body mass differences between the sexes. The degree of observed within-species variability demonstrates that studies focusing upon differences between species can produce different conclusions if they rely on observations for only single populations of a given species. Our data also suggest the existence of multiple mechanisms for desiccation resistance.     

Biochemical Studies on Development of Mitochondria in Pea Cotyledons during the Early Stage of Germination: Effects of Antibiotics on the Development

l-Leucine-U-¹⁴C was incorporated into mitochondrial protein in pea (Pisum sativum var. Alaska) cotyledons during the imbibing stages. Incorporation was almost completely inhibited by cycloheximide but not by chloramphenicol. Both antibiotics did not affect increases in mitochondrial activities and components of the cotyledons during imbibition. Therefore, mitochondrial development seems to be achieved by a transfer of protein pre-existing in the cytoplasm into the mitochondria rather than by de novo synthesis of mitochondrial protein. Cycloheximide stimulated an increase in bile saltsoluble protein of mitochondria in imbibing pea cotyledons. The recovery of cytochrome oxidase activity after sucrose density gradient centrifugation was enhanced, and the morphological properties of mitochondria were altered by cycloheximide.     

Trichilia dregeana胚轴的脱水敏感性与抗坏血酸的抗氧化作用

以顽拗性Trichilia dregeana Sond.种子为材料,研究其胚轴的脱水敏感性与抗坏血酸的抗氧化作用.T.dregeana胚轴的脱水耐性随着脱水进程逐渐下降,50%的胚轴被脱水致死的含水量(W50)大约为0.16 g H2O/g DW.在脱水过程中,胚轴的电解质渗漏速率逐渐增加,超氧物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)、过氧化氢酶 (CAT)、谷胱苷肽还原酶(GR)和脱氢抗坏血酸还原酶(DHAR)的活性下降,硫代巴比妥酸(TBA)-活性产物的含量增加.2.5～10.0 mmol/L抗坏血酸处理能有效地增加胚轴的脱水耐性和SOD、APX、CAT和GR的活性,降低电解质渗漏速率和TBA活性产物的含量.结果表明,T.dregeana胚轴的脱水耐性与抗氧化酶的活性增加和脂质过氧化作用的降低密切相关.     

Effects of Extreme Acceleration on the Germination, Growth and Cell Wall Composition of Pea Epicotyls

Pea (Pisum sativum) seedlings were germinated and grown in darknessfor 5 d whilst being subjected to continuous accelerations ofbetween 1 ? g and 1054 ? g. Increased acceleration retardedthe elongation of both epicotyls and roots, and inhibited lateralroot growth. Changes in the cell wall carbohydrate and lignincomponents were also recognized. Nevertheless, increasing accelerationfrom 1 ? g to 1054 ? g had no effect on germination and peaswere able to germinate and grow successfully at up to approximately10 000 ? g. Key words: Acceleration, cell walls, Pisum     

Messenger RNA and microRNA profiling during early mouse EB formation

Embryonic stem (ES) cells can be induced to differentiate into embryoid bodies (EBs) in a synchronised manner when plated at a fixed density in hanging drops. This differentiation procedure mimics post-implantation development in mouse embryos and also serves as the starting point of protocols used in differentiation of stem cells into various lineages. Currently, little is known about the potential influence of microRNAs (miRNAs) on mRNA expression patterns during EB formation. We have measured mRNA and miRNA expression in developing EBs plated in hanging drops until day 3, when discrete structural changes occur involving their differentiation into three germ layers. We observe significant alterations in mRNA and miRNA expression profiles during this early developmental time frame, in particular of genes involved in germ layer formation, stem cell pluripotency and nervous system development. Computational target prediction using Pictar, TargetScan and miRBase Targets reveals an enrichment of binding sites corresponding to differentially and highly expressed miRNAs in stem cell pluripotency genes and a neuroectodermal marker, Nes. We also find that members of let-7 family are significantly down-regulated at day 3 and the corresponding up-regulated genes are enriched in let-7 seed sequences. These results depict how miRNA expression changes may affect the expression of mRNAs involved in EB formation on a genome-wide scale. Understanding the regulatory effects of miRNAs during EB formation may enable more efficient derivation of different cell types in culture.     

Development of Mitochondrial Activities in Pea Cotyledons during and following Germination of the Axis

Development of mitochondrial activities in pea cotyledons during early times after the start of imbibition occurred in two phases. In the first phase (0 to 8 hours after the start of imbibition), succinate or NADH oxidation increased rapidly, while malate or α-ketoglutarate oxidation remained low. The latter activities developed only 8 to 12 hours after the start of imbibition (the second phase). Development in the first phase was induced by water uptake, but some development occurred even when the cotyledons were fully imbibed. The presence of the axis was required for the second phase of the development. It is suggested that mitochondrial development in the second phase is brought about by activation of the electron transfer path at a site between the oxidation of endogenous NADH and the reduction of ubiquinone.     

Changes in the Content of Phytochrome I and II Apoproteins in Embryonic Axes of Pea Seeds during Imbibition

The contents of phytochrome I and II in crude extracts fromembryonic axes of Pisum sativum cv. Alaska seeds were immunochemicallydetermined using purified pea phytochrome I and II as standards.We have produced and used three different types of mouse monoclonalanti-pea phytochrome antibodies (mAP) such as one reacting preferentiallywith phytochrome I, one with phytochrome II, and one with bothI and II. Phytochrome II was separated from I in the samplesusing immobilized column chromatography with mAPl. The amountsof two phytochrome species were quantitatively measured withwestern blotting and ELISA. Ca. 0.2 µg /axis of phytochromeI and ca. 0.05 µg /axis of phytochrome II were detectedby ELISA after imbibition for 12 h in the dark, though smallamounts of both were detected in dry axes. Ca. 0.05 µg/axis each of phytochrome I and II were detected by ELISA afterimbibition for 12 h in the light, and the results were confirmedby western blotting. This study showed that phytochrome II isnot green-tissue-specific, being also found in dark-imbibedembryonic axes, and that although light significantly lowersthe content of phytochrome I in the axis, it does not significantlyaffect that of phytochrome II. (Received June 10, 1987; Accepted August 27, 1987)     

Trichiliadregeana胚轴的脱水敏感性与抗坏血酸的抗氧化作用

以顽拗性TrichiliadregeanaSond.种子为材料,研究其胚轴的脱水敏感性与抗坏血酸的抗氧化作用。T.dregeana胚轴的脱水耐性随着脱水进程逐渐下降,50%的胚轴被脱水致死的含水量(W50)大约为0.16gH2O/gDW。在脱水过程中,胚轴的电解质渗漏速率逐渐增加,超氧物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)、过氧化氢酶(CAT)、谷胱苷肽还原酶(GR)和脱氢抗坏血酸还原酶(DHAR)的活性下降,硫代巴比妥酸(TBA)-活性产物的含量增加。2.5~10.0mmol/L抗坏血酸处理能有效地增加胚轴的脱水耐性和SOD、APX、CAT和GR的活性,降低电解质渗漏速率和TBA活性产物的含量。结果表明,T.dregeana胚轴的脱水耐性与抗氧化酶的活性增加和脂质过氧化作用的降低密切相关。