Untranslated regions of a mobile transcript mediate RNA metabolism

Abstract

BEL1-like transcription factors are ubiquitous in plants and interact with KNOTTED1 types to regulate numerous developmental processes. In potato (Solanum tuberosum subsp. andigena), the BEL1-like transcription factor StBEL5 and its Knox protein partner regulate tuber formation by targeting genes that control growth. RNA detection methods and heterografting experiments demonstrated that StBEL5 transcripts are present in phloem cells and move across a graft union to localize in stolon tips, the site of tuber induction. This movement of RNA originates in leaf veins and petioles and is induced by a short-day photoperiod, regulated by the untranslated regions, and correlated with enhanced tuber production. Assays for RNA mobility suggest that both 5' and 3' untranslated regions contribute to the preferential accumulation of the StBEL5 RNA but that the 3' untranslated region may contribute more to transport from the leaf to the stem and into the stolons. Addition of the StBEL5 untranslated regions to another BEL1-like mRNA resulted in its preferential transport to stolon tips and enhanced tuber production. Transcript stability assays showed that the untranslated regions and a long-day photoperiod enhanced StBEL5 RNA stability in shoot tips. Upon fusion of the untranslated regions of StBEL5 to a beta-glucuronidase marker, translation in tobacco (Nicotiana tabacum) protoplasts was repressed by those constructs containing the 3' untranslated sequence. These results demonstrate that the untranslated regions of the mRNA of StBEL5 are involved in mediating its long-distance transport, in maintaining transcript stability, and in controlling translation.

Figure 1.

Constructs for the functional analysis of the 5′ and 3′ UTRs of StBEL5 (A and B) and tuber yields (C) for transgenic lines expressing these constructs. Constructs were driven by either the CaMV 35S (A) or the leaf-abundant GAS (B) promoter in the binary vector pBI101.2 for movement assays. Transformation, regeneration, and screening of transgenic potato lines were performed as described by Banerjee et al. (2006b). The StBEL5 5′ UTR and 3′ UTRs are 146 and 505 nucleotides in length, respectively. Transgenic lines of potato that overexpress the chimeric constructs of StBEL5 listed in A (driven by the CaMV 35S promoter) were evaluated for their effect on tuberization (C). The rate of tuberization (number of long days [LD] to tuberize) was determined by the first appearance of tubers from among 20 replicates (C). The yield (g fresh weight plant⁻¹) of tubers was scored for four plants after 14 d of short-day (SD) conditions (8 h of light/16 h of dark). In vitro conditions were the same as described previously (Chen et al., 2003). For the soil-grown experiment, plants from one independent transgenic line per construct were grown in 10-cm pots under long days (16 h of light/8 h of dark) until they reached the 16-leaf stage and then transferred to short-day conditions. After 28 d under short days, four plants from a high-expressing independent line for each construct were evaluated for tuber yield (g fresh weight plant⁻¹). Wild-type plants were used as controls. se values are shown, and asterisks indicate significant differences from the control as determined by t test at a confidence interval of 95%. ND, Not detected; NOST, nopaline synthase terminator.

Figure 2.

GUS activity driven by the leaf-abundant promoter (Ayre et al., 2003) for GAS in transgenic potato plants detected by histochemical analysis. With transgenic potato plants containing a GAS::GUS construct, GUS activity was detected in leaves but not in roots or stolons. Activity is generally not detected in tubers, but in a few rare cases, GAS::GUS activity was detected in trace amounts on knobby structures of irregularly formed tubers. A, Transgenic plant grown in vitro under long-day conditions. B, Enlargement of leaf from the plant in A. C, Stolon tips from a short-day plant. D, Newly formed tubers. E, Primary root tip. F, Lateral root tips. C and D are from a soil-grown plant. E and F are from an in vitro-grown long-day plant. These samples are representative of numerous ones that were examined. Bars = 2.0 mm.

Figure 3.

Effects of UTRs on movement of the StBEL5 mRNA. Representative Mfold models (Zuker, 2003) of two RNA constructs assayed for movement and the full-length StBEL5 RNA (A; from left to right): 5′ UTR + cds, cds + 3′ UTR, and full-length RNA of StBEL5. In nearly all Mfold models, the full-length mRNA has both UTRs in close proximity (arrows, full-length model), forming a multifingered structure. The BEL5 UTRs for each model are highlighted by solid circles. Quantification of movement was performed on transgenic lines with the constructs shown in Figure 1, A and B, and on StBEL5 constructs with or without UTRs (D, FL and cds, respectively), all grown under short-day conditions (8 h of light/16 h of dark). Relative levels of RNA were quantified from total RNA extracted from both new leaves (white bars) and 0.5-cm samples from the tip of the stolon (black bars) from three separate plants for each construct. Homogenous RT-PCR products were quantified using ImageJ software (Abramoff et al., 2004) and normalized using rRNA values. se values of three clones from one independent transgenic line per construct are shown. A movement factor is provided for mobility assays above each set of constructs (B–D). The movement factor is equal to the relative stolon tip RNA quantity divided by the relative leaf RNA quantity. One set of constructs was driven by the GAS promoter of C. melo (B), whereas the other two sets were driven by the CaMV 35S promoter (C and D). The GAS promoter is most active in the minor veins of leaves (Ayre et al., 2003; Fig. 2). [See online article for color version of this figure.]

Figure 4.

A, Three RNA constructs driven by the leaf-abundant GAS promoter were tested for their capacity to move from leaf veins to stolon tips: FL-BEL14 (contains BEL14 cds plus both native BEL14 UTRs), BEL14 + both StBEL5 UTRs replacing the StBEL14 UTRs, and full-length StBEL5. StBEL14 was chosen as a test RNA because it is not abundant in stems or stolons (Chen et al., 2003). B, Relative RNA accumulation in new leaves and 0.5-cm samples from the tip of the stolon was quantified in transgenic lines expressing these chimeric transcripts. Transgenic plants were grown under short days (8 h of light/16 h of dark) for 12 d, and the RNA was extracted from new leaves (white bars) and from 0.5-cm stolon tips (black bars) from three separate plants for each construct. C, At the same time, tuber yields were scored for BEL14 lines with and without UTR fusions. Homogenous RT-PCR products were quantified using ImageJ software (Abramoff et al., 2004) and normalized using rRNA values. se values of three clones from one independent transgenic line per construct are shown. A movement factor is provided for mobility assays above each set of constructs (B). The movement factor is equal to the relative stolon tip RNA quantity divided by the relative leaf RNA quantity. D and E, Representative Mfold models of the two BEL14 RNA constructs assayed for movement. Models for the RNA structure of BEL14 + the BEL5 UTRs exhibited the same multifingered structure (D, arrow) formed by the UTRs (D, solid circles) of the full-length StBEL5 transcript (Fig. 3A, full-length model, solid circles). All predicted Mfold models for StBEL14 exhibited a stable, conserved motif arising from its cds (D and E, dotted circles). NOST, Nopaline synthase terminator. [See online article for color version of this figure.]

Figure 5.

Effects of the UTRs (A and B) and photoperiod (C) on the stability of StBEL5 RNA. Transcript stability time lines for three different RNA molecules, transgenic cds of StBEL5 (A), transgenic full-length StBEL5 (B), and native StBEL5 (C), were determined in potato plants. One-centimeter shoot tips from in vitro-grown plants were harvested, RNA was extracted, and quantitative RT-PCR with gene-specific primers was performed as described previously (Banerjee et al., 2006a). The percentage of RNA remaining for each sample is relative to the amount of RNA present when the experiment was started (0 h). Samples were harvested from plants grown under short-day (SD) conditions (A and B) or both long-day (LD) and short-day conditions (C). The arrows indicate the approximate half-life point for each experimental time line. The UTRs of StBEL5 (accession no. AF406697) include 146 nucleotides from the 5′ end and 505 nucleotides from the 3′ end. The CaMV 35S promoter was used for the transgenic lines in A and B.

Figure 6.

The effects of the UTRs of StBEL5 on the translation efficiency of GUS in tobacco protoplasts. A, Four constructs driven by the 35S promoter in pBI221 were analyzed: GUS + the 3′ UTR of StBEL5, the 5′ UTR of StBEL5 + GUS, GUS plus both UTRs attached, and the GUS cds alone. B, The LUC gene in pBI221 under the control of the CaMV 35S promoter was included as an internal control. A relative value for GUS expression was obtained by dividing the GUS activity by the specific LUC activity. Each transfection was performed three times. Data are means ± se. NOST, Nopaline synthase terminator. The levels of GUS translation have been adjusted to reflect differences in RNA accumulation for each of the constructs.

Figure 7.

Enlarged computer-generated version (Zuker, 2003) of the 3′ UTR of StBEL5 from nucleotide (nt) 2,215 to 2,674 (arrows). Three types of conserved motifs are highlighted: poly-U (solid line), a unique UAGUA motif (double-striped line), and the polypyrimidine tract, CUUCU (dotted line). The enlarged figure (at right) is derived from the Mfold model of full-length StBEL5 RNA (at left and Fig. 3A). Full-length StBEL5 RNA is 2,716 nucleotides in length. The 3′ UTR begins at nucleotide 2,215 (designated by the lower arrow). It should be noted, however, that the existence of these conserved motifs does not validate their function. [See online article for color version of this figure.]

Figure 8.

The formation of aerial tubers (black arrows) from stem axillary buds of a transgenic line of potato that constitutively overexpresses the cds (no UTRs) of StBEL5. Expression of the transgene was driven by the CaMV 35S promoter. This BEL5 transgenic line, designated no. 20, exhibited an extreme phenotype characterized by the formation of aerial stem tubers, stunted growth, and a reduction in the yield of underground tubers (Chen et al., 2003). A leaf has been excised (white arrow) to enhance visibility.