The 5' end of the pea ferredoxin-1 mRNA mediates rapid and reversible light-directed changes in translation in tobacco

Abstract

Ferredoxin-1 (Fed-1) mRNA contains an internal light response element (iLRE) that destabilizes mRNA when light-grown plants are placed in darkness. mRNAs containing this element dissociate from polyribosomes in the leaves of transgenic tobacco (Nicotiana tabacum) plants transferred to the dark for 2 d. Here, we report in vivo labeling experiments with a chloramphenicol acetyl transferase mRNA fused to the Fed-1 iLRE. Our data indicate that the Fed-1 iLRE mediates a rapid decline in translational efficiency and that iLRE-containing mRNAs dissociate from polyribosomes within 20 min after plants are transferred to darkness. Both events occur before the decline in mRNA abundance, and polyribosome association is rapidly reversible if plants are re-illuminated. These observations support a model in which Fed-1 mRNA in illuminated leaves is stabilized by its association with polyribosomes, and/or by translation. In darkness a large portion of the mRNA dissociates from polyribosomes and is subsequently degraded. We also show that a significant portion of total tobacco leaf mRNA is shifted from polyribosomal to non-polyribosomal fractions after 20 min in the dark, indicating that translation of other mRNAs is also rapidly down-regulated in response to darkness. This class includes some, but not all, cytoplasmic mRNAs encoding proteins involved in photosynthesis.

Figure 1

Diagrams of the transgenes used. Shown are the gene constructs used to transform tobacco plants. Except for 35S::FedA, plants expressing these constructs were previously characterized, as indicated. A, 35S::CAT (Petracek et al., 1997); B, 35S::Fed-1 5′-UTR::CAT (Dickey et al., 1998); C, 35S::Fed-1 iLRE::CAT (Dickey et al., 1998); D, 35S::Fed-1 (Dickey et al., 1992); E, 35S::PetE::Nos (Helliwell et al., 1997); F, 35S::FedA was constructed to achieve transcription at the transcriptional start site reported for the endogenous gene (Somers et al., 1990; Vorst et al., 1990).

Figure 2

Autoradiograph of immunoprecipitated proteins from tobacco plants labeled for 2.5 h. Six-week-old plants grown on a 12-h light/dark cycle were labeled in the light (L) or dark (D) for 2.5 h. Sample volumes were adjusted to represent an equal number of counts taken up in the tissue. Fed-1 iLRE:CAT fusion protein has lower mobility than CAT, as predicted by its higher M_r. A, Radioactivity in immunoprecipitated CAT or CAT fusion proteins was measured with a Phosphorimager as described in “Materials and Methods.” The boxes around the Fed-1 iLRE:CAT and CAT protein bands represent the approximate area counted. Background was estimated by moving the boxes to a position immediately above the protein bands. Resulting total counts are as follows: Fed-1 iLRE::CAT L (462,000), D (65,000), CAT L (61,000), D (72,000), and Fed-1 5′-UTR::CAT L (47,000), and D (57,000). B, RNA was extracted from the light- and dark-treated samples shown in A and 5 μg of total RNA was analyzed by northern-blot analysis using ³²P-labeled antisense CAT.

Figure 3

Polyribosome association of Fed-1 iLRE::CAT mRNA in plants transferred from light to dark. Transgenic 35S::Fed-1 iLRE::CAT tobacco were transferred to the dark for the time indicated, beginning during the 4th h of the light phase (time 0) of the 12-h-light/12-h-dark cycle. A, Autoradiograms of northern blots of Fed-1 iLRE::CAT mRNA fractionated on a Suc density gradient and resolved by gel electrophoresis. Fractions are labeled 1 through 12 from the top to the bottom of the gradient. The resulting gels were blotted to nylon membrane and hybridized with ³²P-labeled antisense probe for CAT sequences. Fractions 1 to 5 from the top of the gradient contain mRNA not associated with polyribosomes, whereas fractions 6 to 12 contain polyribosomal mRNA. The time that plants were in darkness is indicated in minutes. Sample sizes were not the same on each gradient so signal strengths on the northern blots can be directly compared only within a given gradient. B, Percent polyribosome association of Fed-1 iLRE::CAT mRNA after 0, 20, 40, and 60 min in the dark. The percentage of mRNA in the polyribosomal fractions is calculated from Phosphorimager analysis of three separate experiments including the northern blots shown in 4A. C, Change in Fed-1 iLRE::CAT mRNA abundance after 0, 20, 40, and 60 min in the dark. Data was derived by Phosphorimager analysis of northern blots of 5 μg of total mRNA hybridized with ³²P-labeled CAT specific antisense RNA. Each time point represents the average of at least three different experiments. Total abundance is shown relative to the abundance at zero time. D, UV A₂₅₄ profiles of polyribosome fractionations were recorded after ultracentrifugation by pumping the gradient through a cuvette. Sample extracts included chloroplast ribosomes as well as cytoplasmic ribosomes, yielding multiple ribosomal subunit peaks (approximately fractions 2–4). The x axis is labeled with numbers indicating the approximate posi- tion of the fractions collected for the northern analysis shown in A through C. Polyribosomal and non-polyribosomal portions of the gradient are indicated. Fractions 4 and 5 represent the monosome peak. The sensitivity of the chart recorder was increased by a factor of two at the position indicated by the arrow. The y axis indicates the relative UV A₂₅₄.

Figure 4

Reversible dissociation/re-association of Fed-1 iLRE::CAT mRNA. Transgenic 35S:: Fed-1 iLRE::CAT tobacco were transferred to the dark for 1 h, beginning during the 4th h of the light phase as described for Figure 3. Plants were then transferred back to the light for the indicated times prior to harvest. A, Autoradiograms of northern blots of Fed-1 iLRE::CAT mRNA fractionated on a Suc density gradient. Fractions are labeled 1 through 12 from the top to the bottom of the gradient. RNA was purified from each fraction and analyzed by gel-blot hybridization using a³²P-labeled antisense probe for CAT sequences. Different gradients were not loaded equally so band intensities can be compared only within a given gradient. B, Percentage of polyribosome association of Fed-1 iLRE::CAT mRNA after 0, 30, 60, and 120 min of re-illumination. The percentage of mRNA in the polyribosomal fractions is calculated from Phosphorimager data for three separate experiments, including northern blots shown in A. C, Change in Fed-1 iLRE::CAT mRNA abundance following 1 h of dark and various periods of re-illumination. The relative amount of Fed-1 iLRE::CAT hybridizing RNA was calculated from at least three different experiments at each time point. Data were derived by Phosphorimager analysis of northern blots hybridized with a ³²P-labeled CAT -specific antisense RNA probe. Abundance is shown relative to that at zero time.

Figure 5

Polyribosome association of transgenic and endogenous mRNAs. Wild-type tobacco or plants containing various transgenes (35S::Fed-1, 35S::FedA, or 35S::PetE) or wild-type tobacco plants (for endogenous genes) were transferred to darkness for 20 min as described in Figure 3 or allowed to remain in the light before harvest. Following Suc gradient ultracentrifugation, RNA from the resulting gradient fractions 1 through 12, numbered from the top to bottom of the gradient, were resolved by gel electrophoresis. The resulting gels were blotted to nylon membrane and hybridized with ³²P-labeled antisense gene-specific probes as indicated. The polyribosome association profile for each mRNA is representative of two to three independent experiments. Different gradients were not loaded equally so signal strengths on the northern blots can be compared only within the same gradient.