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Proc. Natl. Acad. Sci. USA Vol. 95, pp. 9009–9013, July 1998 Ferredoxin-1 mRNA is destabilized by changes in photosynthetic
electron transport

MARIE E. PETRACEK*†, LYNN F. DICKEY*, TUYEN T. NGUYEN‡, CHRISTIANE GATZ§, DOLORES A. SOWINSKI*, GEORGE C. ALLEN*, AND WILLIAM F. THOMPSON*Departments of *Botany and ‡Crop Science, North Carolina State University, Raleigh, NC 27695; and §Albrecht von haller Institute for Plant Sciences, University of Gottingen, Untere Karspule 2, 37073 Gottingen, Germany Edited by Elisabeth Gantt, University of Maryland at College Park, College Park, MD, and approved May 29, 1998 (received for review March 12, 1998) In transgenic tobacco, pea Ferredoxin-1
tional initiation context also compromises light regulation of (Fed-1) mRNA accumulates rapidly in response to photosyn-
the Fed-1 mRNA (7). Finally, the Fed-1 mRNA is polyribo- thesis even when the transgene is driven by a constitutive
some-associated in the light but not in the dark (8), thus promoter. To investigate the role of photosynthesis on Fed-1
decreased Fed-1 mRNA abundance is correlated with a de- mRNA stability, we used the tetracycline repressible Top10
crease in Fed-1 translation rates.
promoter system to specifically shut off transcription of the
Fed-1 mRNA levels also decrease when light-grown plants Fed-1 transgene. The Fed-1 mRNA has a half-life of approx-
are treated with the photosynthetic electron transport inhibitor imately 2.4 hr in the light and a half-life of only 1.2 hr in the
dark or in the presence of the photosynthetic electron trans-
reillumination of DCMU-treated plants after a 40-hr dark treatment, Fed-1 mRNA fails to reaccumulate and remains (DCMU). These data indicate that cessation of photosynthe-
dissociated from polyribosomes (8). These results suggest light sis, either by darkness or DCMU results in a destabilization
regulation of Fed-1 mRNA accumulation and translation of the Fed-1 mRNA. Furthermore, the Fed-1 mRNA half-life is
requires photosynthetic electron transport.
reduced immediately upon transfer to darkness, suggesting
It is of interest to know whether the Fed-1 mRNA half-life that Fed-1 mRNA destabilization is a primary response to
is altered by changes in photosynthetic activity. One method of photosynthesis rather than a secondary response to long-term
mRNA half-life determination is to globally inhibit transcrip- dark adaptation. Finally, the two different methods for effi-
tion and monitor the decay of the mRNA of interest over time.
cient tetracycline delivery reported here generally should be
This approach has been used successfully in plants to give useful for half-life measurements of other mRNAs in whole
half-lives that correspond to changes in mRNA steady-state abundance (9, 10). However, apparent secondary effects from global transcriptional inhibition confounded half-life studies in The pea Ferredoxin-1 (Fed-1) gene encodes plastid-localized other cases (11, 12).
photosystem I ferredoxin. Its mRNA is perhaps the best- Attempts to use actinomycin D (ActD) to determine characterized nuclear-encoded transcript in plants that exhib- whether Fed-1 mRNA half-life is altered in the light have been its promoter-independent regulation of mRNA abundance unsuccessful (13). In these experiments, ActD treatment re- (e.g., refs. 1 and 2). Fed-1 initially was identified in a cDNA sulted in an increase rather than the expected decrease in screen for light-regulated mRNAs in pea (3) and was chosen Fed-1 mRNA in the dark, preventing a determination of Fed-1 for further study because Fed-1 mRNA accumulated faster mRNA half-life. There are at least two ways in which these than several other light-regulated mRNAs in etiolated pea results can be reconciled with our observation that the steady- seedlings exposed to light (4). Analysis of transgenic tobacco state abundance of Fed-1 mRNA declines in darkness in a plants containing the transcribed sequence of the pea Fed-1 promoter-independent manner. First, the Fed-1 mRNA may gene under the transcriptional control of the constitutive indeed be more stable in darkness than in light, but this effect cauliflower mosaic virus 35S promoter (P could be overridden if transcriptional elongation is inhibited in 35S) revealed that the light-regulated accumulation of Fed-1 mRNA was conferred the dark. This transcriptional effect on abundance may not be by the mRNA sequence itself rather than by the promoter (1).
detectable by transcriptional run-on assays (reviewed in ref.
Furthermore, the light effect on accumulation of Fed-1 mRNA 14). Second, global inhibition of transcription might lead to transcribed from this chimeric gene was greater in green plants improper Fed-1 light regulation—for example, by causing the than in etiolated seedlings exposed to light for the first time disappearance of a labile factor required for Fed-1 degradation (5). Deletion analysis determined that 95 nt of the 59 UTR plus in the dark.
the first one-third of the Fed-1 coding region ('143 nt) are The secondary effects caused by global transcriptional in- sufficient for light regulation in such constructs. This region is hibitors can be circumvented by making mRNA half-life referred to as the internal light regulatory element (iLRE) (6).
measurements under conditions in which transcription is sup- Further deletion of the Fed-1 iLRE results in a gradual loss of pressed only for the gene of interest, while all other genes are light regulation (7).
transcribed normally. The tetracycline (tet)yVP16 system used Translation appears to be involved in the Fed-1 mRNA light in animals has been adapted for use in plants (15). The Top10 response. Mutation of the Fed-1 initiation codon to either a tetracycline-repressible promoter (P-Top10) is based on the missense or nonsense codon abolishes light regulation of the constitutive P35S, but contains seven tetracycline operator sites mRNA (2). Similarly, mutation of the surrounding transla- in place of the upstream activator sequences. Plants are The publication costs of this article were defrayed in part by page charge This paper was submitted directly (Track II) to the Proceedings office.
payment. This article must therefore be hereby marked ‘‘advertisement'' in Abbreviations: Fed-1, Ferredoxin-1; DCMU, 3-(3,4-dichlorophenyl)- 1,1-dimethylurea; P accordance with 18 U.S.C. §1734 solely to indicate this fact.
35S, caulif lower mosaic virus 35S promoter; ActD, actinomycin D.
1998 by The National Academy of Sciences 0027-8424y98y959009-5$2.00y0 †To whom reprint requests should be addressed. e-mail: PNAS is available online at http:yywww.pnas.org.
Plant Biology: Petracek et al. Proc. Natl. Acad. Sci. USA 95 (1998) transformed with a construct encoding the tet repressor fused liquid on top of the nylon membrane was removed and to the VP16 transcriptional activator protein and with a replaced with 3 ml of 10 mgyliter tetracycline in Hoagland's construct containing the P-Top10 fused to the GUS reporter solution with or without 1 mM DCMU. Time zero was gene. In the absence of tetracycline, the tet repressor binds the designated as the time after 1 hr of tetracycline solution P-Top10 and VP16 activates GUS transcription. Upon addition uptake; at this time, covers were replaced and boxes were of tetracycline, the tetyVP16 fusion protein no longer binds either wrapped in foil for dark time points, or left in the light P-Top10, preventing transcription of GUS (16). Thus, this for light or DCMUylight time points. At appropriate time system can be used for a direct measurement of the half-life of points, leaves from all 15–20 plants grown in a single box were the GUS mRNA or any other mRNA driven by P-Top10. The harvested into liquid nitrogen.
Top10 system has been used successfully to measure half-life Octuplet Production. Plantlets derived from transgenic calli
of SAUR containing mRNAs in tobacco suspension culture were successively subdivided to produce eight clonal plantlets (octuplets) expressing Fed-1. Octuplets were rooted on rooting We have observed that a normal Fed-1 mRNA light re- medium, then transferred to Plant Cons (Sigma) containing sponse requires healthy, intact green plants. Previously, in approximately 20 ml of Hoagland's solution and placed in a studies of the half-life of GUS mRNA using the P-Top10 growth chamber illuminated at an intensity of 240 mmol system, tetracycline was introduced into detached leaves by m22zs21 supplied by a mixture of 6 incandescent and 12 vacuum infiltration (16). On the whole-plant level, the half-life fluorescent lights on a 12-hr lighty12-hr dark cycle. Plants were of Gus protein, not mRNA, was examined over a period of supplied with fresh Hoagland's solution every other day for a days, not hours, using low levels of tetracycline. To study the week to allow plants to grow and to adapt to the new light rapid Fed-1 mRNA light response, it was essential that we conditions. Half-life measurements were carried out as above develop conditions permitting rapid delivery of tetracycline to except that the entire 20 ml of Hoagland's solution in the Plant intact, healthy plants. Here we report measuring Fed-1 mRNA Cons was completely replaced with 20 ml of fresh Hoagland's half-life from intact plants by using P-Top10. We have deter- solution supplemented with 10 mgyliter tet andyor 1 mM mined that the half-life of the Fed-1 mRNA is significantly DCMU. A single member of the octuplet set was harvested for shortened in the dark and in the presence of photosynthetic each time point.
electron transport inhibitor DCMU. These results show that Polyribosome Profiles. Plants were grown on nylon mem-
light regulates Fed-1 mRNA abundance by affecting its sta- branes as above, treated with the solutions and light regime indicated, and harvested into liquid nitrogen. Polyribosomes then were isolated on a 15–65% sucrose gradient as previously described, and the absorbance of the fractions at UV 254 nm was monitored and recorded during fractionation.
Gene Construct. The P-Top10::Fed-1 construct was derived
from the ‘‘message'' construct as described in ref. 1. An XbaI and EcoRI (blunt-ended with Klenow) fragment, which in- cluded the Fed-1 transcribed region (749 bp) and 70 bp of the To make the P-Top10 useful for the examination of Fed-1 39 flanking region, was substituted for the GUS-INTyoctopine half-life, we needed to develop a system for rapid and efficient synthase terminator fragment [in pTOP10 (16)], which was excised by using XbaIyHindIII (blunt-ended with Klenow).
uptake of tetracycline in healthy plants. Thus, we developed The resulting plasmid was transferred to Agrobacterium tume- the P-Top10 repressible system for use in two different types faciens strain LBA4404 by triparental mating (18).
of plant growth systems as follows: Tobacco Petite havana was transformed sequentially with DNA encoding the VP16 Plant Transformations. Nicotiana tabacum (SR-1, Petite
Havana) were transformed by using the leaf disc method (19).
activator and then with the P-Top10::Fed-1::Fed-1 ter con- Initially, plants were transformed with the transactivator plas- struct (Fig. 1). Transgenic plant lines were screened for mid pTetVP16 (16) and selected on kanamycin (300 mgyml).
expression of the Fed-1 mRNA, which indicates appropriate The transformant expressing the highest amount of TetVP16 expression of both transgenes because the VP16yTet gene mRNA was chosen for a second round of transformation with product is required for activation of the P-Top10. Most lines the P-Top10::Fed-1 construct described above, and transfor- had significant levels of expression. One line (5010) was chosen mants were selected on hygromycin (50 mgyml).
for further study, and T2 seeds were collected for the exper- Growth of Transgenic Tobacco on Nylon Membrane. Twen-
iments with seedlings grown hydroponically on nylon mem- ty-five milliliters of sterile MS medium was added to thor- branes. Fifteen other transgenic plant lines produced from a oughly rinsed and autoclaved magenta boxes with membrane raft units (Sigma V8380; Sigma M1917). T2 transgenic tobacco seeds (line 5010) were sterilized in 30% commercial bleach followed by five washes with sterile deionized water. Approx- imately 15–20 transgenic seeds were spread on each mem- brane, the magenta box lids were put back on the box, and the junction between the lid and box was wrapped with micropore tape (3M Co., 1530–0). Plants were grown sterilely for 3–4 weeks to at least a four-leaf stage in a 20°C growth chamber.
White light (120 mmol m22zs21) was supplied by a mixture of six incandescent and six fluorescent lights operating on a 12-hr lighty12-hr dark cycle.
Half-Life Measurements in Seedlings. Two hours after the
FIG. 1. Constructs used in the repressible promoter system. The beginning of the light cycle, transgenic tobacco plants were chimeric transactivator Tet-VP16 is expressed under control of the treated either with 3 ml of 1 mM DCMU in Hoagland's cauliflower mosaic virus 35S promoter (P35S) (Upper). Progeny of solutiony1% EtOH or with Hoagland's solutiony1% EtOH plants transformed with the transactivator construct have been re- transformed with Fed-1 under control of the synthetic Top10 promoter (control plants). These solutions were added to roots growing (P-Top10) (Lower). The Tet-VP16 protein binds P-Top10, allowing across the nylon membrane, and the Magenta Box covers transcription to Fed-1. Endogenously supplied tetracycline binds Tet- propped open on the top to permit air exchange and encourage VP16, making Tet-VP16 unable to bind P-Top10, thus inhibiting uptake of the solution by transpiration. One hour later all transcription of Fed-1.


Plant Biology: Petracek et al. Proc. Natl. Acad. Sci. USA 95 (1998) single VP16yTet line were subdivided in tissue culture during long-term exposure (15). However, an external concentration the plantlet stage to form clonal octuplets (see Methods) of 1 mgyliter was ineffective in quickly repressing transcription expressing Fed-1. Five independent lines then were used for of the P-Top10::Fed-1 transgene. Although we eventually each of the treatments indicated.
observed decay of the Fed-1 mRNA, the response was slow and To ensure the seedlings grown on nylon membrane exhibited inconsistent (ref. 20 and M.E.P., data not shown). Therefore, a proper light response, we asked whether Fed-1 mRNA to rapidly deliver sufficient amounts of tetracycline to the abundance decreased after transfer to darkness. The abun- plants, we increased the external concentration of tetracycline dance of Fed-1 mRNA decreased approximately 3-fold after a to 10 mgyliter. This concentration was used successfully for 24-hr dark treatment, indicating that these conditions were half-life measurements in N. tabacum suspension cultures (17) appropriate for measuring Fed-1 half-life (Fig. 2A). In addi- and for rapid (0.5 hr) regulation of GUS by vacuum infiltration tion, we asked what level of tetracycline was required to shut in tobacco leaves (21).
off the P-Top10 promoter. Previously, 1 mgyliter of tetracy- Because 10 mgyliter tetracycline potentially could affect cytoplasmic translation and thus the light response of the Fed-1 cline has been shown to effectively repress P-Top10 during a mRNA, we monitored the polyribosome profiles of total plant mRNA in the presence and absence of tetracycline. In un- treated plants we observed a significant shift from polyribo- some fractions after only 1 hr of dark treatment, similar to what has been seen in agar grown plants (Eric R. Hansen, personal communication). Addition of tetracycline did not change the total polyribosome profile from that seen in untreated plants with the same light treatment (Fig. 2B), suggesting that the concentration of tetracycline delivered to the plants does not alter significantly overall translation. To further ensure that tetracycline does not alter the normal Fed-1 mRNA disap- pearance, we applied 10 mgyliter tetracycline to transgenic plants containing the P35S::Fed-1 mRNA. As shown in Fig. 2A, tetracycline had no effect on the normal dark decline of the Fed-1 mRNA.
In addition to rapid delivery of tetracycline, we needed to ensure rapid delivery of DCMU so we could observe the effects of short-term inhibition of photosynthesis on Fed-1 mRNA half-life. We showed previously that Fed-1 mRNA is dissociated from polyribosomes after a 40-hr incubation with 1 mM DCMU (8). Therefore, we monitored the Fed-1 mRNA polyribosome profile 1 hr after delivery of 1 mgyml DCMU to membrane-grown plants. Fig. 2C shows that within 1 hr after DCMU treatment, the Fed-1 mRNA is dissociated from the polyribosome fractions. Similar results were obtained for Fed-1 mRNA in plants exposed to 1 hr of dark treatment (Eric R. Hansen, personal communication). These data suggest that inhibition of photosynthesis by DCMU rapidly alters the polyribosome loading pattern of Fed-1 mRNA.
After establishing conditions that allowed rapid introduc- tion of tetracycline and DCMU to intact, healthy plants, we asked whether light regulation of Fed-1 mRNA abundance occurs posttranscriptionally by a change in mRNA half-life. To determine the half-life of Fed-1 mRNA we used FIG. 2. Establishment of a hydroponic system to study Fed-1 P-Top10::Fed-1 transgenic tobacco seedlings (T2 generation) mRNA half-life. (A) Decline of Fed-1 mRNA abundance in the dark.
grown on nylon membranes and isolated RNA samples at the Fifteen to 20 transgenic nylon membrane grown seedlings from a transgenic line containing P-Top10::Fed-1 treated with (tet) or without times indicated in Fig. 3. To assay plants grown under different tetracycline (no tet) were either left in the light for 24 hr (L) or conditions, we simultaneously performed the same experi- wrapped in foil for dark treatment for 24 hr from the time the light ments on clonal plantlets. In both plant systems (see Methods), sample was harvested (D). Total RNA extracted and 5 mg was identical half-lives were observed.
separated in each lane of an agarose gel, blotted, and hybridized with The half-life data from both types of growth systems are 32P-labeled Fed-1. (B) Lack of effect on total polyribosome loading by combined and summarized in Fig. 3. For all three conditions, tetracycline. Transgenic P35S::Fed-1 tobacco seedlings were treated approximately the first 80% of the decay can be fit with a single with either 10 mgyliter tetracycline in Hoagland's solution (Tet) or first-order component, using the equation ln(CyC0) 5 2kdt with Hoagland's solution alone (No Tet). After 1 hr of uptake the and ln2ykd 5 t1/2, where C0 is the initial mRNA concentration, plants were subjected to continued illumination (Light) or 1 hr of dark treatment (Dark). Shown are absorbance tracings at 254-nm UV of the C is the mRNA concentration at time t, kd is the decay sucrose gradients containing extracts from these plants. The direction constant, and t1/2 is the half-life of the mRNA (22). The of the sedimentation is from left to right. (C) Effective DCMU uptake.
half-lives estimated for this major component are approxi- Fifteen to 20 transgenic nylon membrane-grown seedlings from a mately 2.4 hr for the light-treated plants and 1.2 hr for transgenic line containing P-Top10::Fed-1 (nylon membrane grown) dark-treated plants. Correlation coefficients are 0.989 and either were not treated (Light) or treated with 1 mM DCMU (DCMU 0.999, respectively. The remaining 20% of the mRNA appears Light) for 1 hr and then harvested into liquid nitrogen. RNAs from to decay more slowly, although variation in the data prevents each fraction of a sucrose gradient were resolved by gel electrophore- accurate analysis of this minor component. The apparently sis, blotted, and probed with antisense 32P-labeled RNA to either slower decay might indicate a subset of the Fed-1 mRNA Fed-1. Light to heavy sucrose fractions were loaded from left to right.
Amounts of hybridizing RNA cannot be quantitatively compared decays more slowly than the bulk mRNA. However, measure- between gradients because of variations in the amount of tissue used ments in this range are based on weak hybridization signals and and the efficiency of grinding.
may be influenced by background variations or a small amount Plant Biology: Petracek et al. Proc. Natl. Acad. Sci. USA 95 (1998) and thus we could not answer whether the decline in mRNA abundance was because of stress from a long-term dark treatment or because of a more immediate, specific signal. The immediate decrease in Fed-1 mRNA half-life after transfer of the plants to darkness or treatment with DCMU suggests that the response to the photosynthetic state of the plant is a result of a rapid signal rather than a relatively slow change, such as increases in abscisic acid (23).
Using the P-Top10 system, we can now more easily study elements of the Fed-1 mRNA that affect its stability because half-life measurements circumvent ‘‘position effect'' variation in absolute expression levels among different transgenic lines.
It is difficult to firmly determine whether the stability of mutant mRNAs is altered by comparing mRNA abundance because transgene expression in plants can vary greatly. For example, although we know the Fed-1 mRNA light response is abolished when the AUG is mutated, we do not know whether this results from increasing mRNA stability in the dark or decreasing it in the light. Using the P-Top10 system, we will now be able to make those determinations.
Previously, we showed that changes in Fed-1 mRNA abun- dance are correlated with the ability of the mRNA to be FIG. 3. The effect of photosynthesis on Fed-1 mRNA half-life.
translated (2). The data presented here support the suggestion Fifteen to 20 control or DCMU-treated (1 mM) transgenic seedlings that Fed-1 is preferentially degraded when it is not associated per sample from a transgenic line containing P-Top10::Fed-1 (nylon with polyribosomes. We propose that a signal generated by membrane grown) or clonal octuplets were treated with 10 mgyliter photosynthetic electron transport in the chloroplast travels to tetracycline for 1 hr and then exposed to light (h), dark (r), or DCMU the cytoplasm and signals a change in the cytoplasmic trans- and light (E) for the time indicated on the x axis. Total RNA (5 mg) lation rate of the Fed-1 mRNA. Furthermore, we suggest that was separated in each lane of an agarose gel, blotted, and hybridized a reduction in Fed-1 mRNA translation rate in the dark results with 32P-labeled Fed-1. Blots were rehybridized with 32P-labeled in a faster rate of degradation of the Fed-1 mRNA. Cis analysis antisense HIS probe to detect the endogenous His HI transcript of the Fed-1 59 UTR identified a region containing four (divided square). The resulting hybridization signals were quantitated by using a PhosphorImager (Molecular Dynamics). The mean percent tandem copies of a CATT motif that is apparently sensitive to reduction in RNA was calculated for each time point and plotted on nuclease degradation (7). When this region is mutated, Fed-1 a semi-log plot. Each time point is derived from at least eight separate mRNA accumulates to a similar level in both the light and the experiments, with the error bars representing SEM. A line was drawn dark. However, the mutated Fed-1 mRNA that is not degraded at 50% mRNA remaining.
in the dark still accumulates in the nonpolyribosomal fractions.
We suggest that darkness inhibits translation at the level of of residual transcription from the P-Top10. It is therefore initiation and that wild-type but not mutant Fed-1 mRNA is premature to draw any conclusions based on this minor degraded in the absence of polyribosome association. The component of the decay curve.
dark-induced decrease in Fed-1 mRNA half-life thus appears We also asked whether short-term inhibition of photosyn- to be the result of a two-step process. First, Fed-1 mRNA thesis by DCMU treatment would show the same effect on dissociates from polyribosomes and second, the Fed-1 mRNA half-life as a brief dark treatment. As shown in Fig. 3, the decay is degraded in the absence of polyribosome protection. It curve for Fed-1 mRNA in the presence of DCMU is similar to would be interesting to know whether there is in fact a ‘‘labile'' that for mRNA of dark-treated leaves. Fitting a first-order factor required for Fed-1 degradation in the dark as inferred component to the first 80% of the decay curve yields a half-life from the stabilization of Fed-1 mRNA in the presence of ActD.
of 1.2 hr (correlation coefficient 5 0.978), a value indistin- If so, what is the nature of this factor? For example, a labile guishable from the half-life of Fed-1 mRNA in darkness.
nuclease may interact with the CATT repeat; in this case we Finally, as a control, we asked whether tetracycline addition would predict that degradation would be inhibited when affected endogenous Histone HI mRNA levels. As expected, expression of the nuclease is inhibited. Alternatively, it is Histone HI RNA levels remained constant in the presence of possible that a labile factor is required to inhibit translational tetracycline, indicating that tetracycline does not inhibit global initiation in darkness. In either case, it will be interesting to transcription or turnover of other mRNAs. Taken together, sort out the specificity of Fed-1 degradation as well as the our data show that photosynthetic signals control the stability photosynthesis-responsive signaling pathways involved in the of Fed-1 mRNA.
regulation of Fed-1 mRNA half-life.
We acknowledge excellent technical assistance from Tyrone Hughes and Tamyra Ravenel. We are also grateful to Dr. Mark Longtine for Here we present direct evidence that Fed-1 mRNA is post- critical review of the manuscript. Controlled environment plant transcriptionally light-regulated at the level of mRNA stability.
growth space was provided by the Southeastern Plant Environment Previously, we had only indirect evidence that did not distin- Laboratory (Raleigh, NC). This project was supported by National guish between altered stability and altered transcriptional Institutes of Health Postdoctoral Fellowship Grant 1F32GM15510–01 elongation. Furthermore, in the absence of Fed-1 transcrip- to M.P., and National Science Foundation Grant MCB-9507396 and tion, a difference in Fed-1 mRNA abundance is detectable National Institutes of Health Grant GM43108 to W.F.T. and L.F.D.
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Topic Introduction Immersion Freezing of Biological Specimens: Rationale, Principles,and Instrumentation Guenter P. Resch,1,5 Marlene Brandstetter,1 Angela M. Pickl-Herk,2 Lisa Königsmaier,3Veronika I. Wonesch,1,4 and Edit Urban3 1IMP-IMBA-GMI Electron Microscopy Facility, Institute of Molecular Biotechnology, 1030 Vienna, Austria2Max F. Perutz Laboratories, Medical University of Vienna, 1030 Vienna, Austria3Institute of Molecular Biotechnology, 1030 Vienna, Austria4University of Applied Sciences Wiener Neustadt, 2700 Wiener Neustadt, Austria