Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M604162200 on June 27, 2006

J. Biol. Chem., Vol. 281, Issue 35, 25485-25491, September 1, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
281/35/25485    most recent
M604162200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Patel, M.
Right arrow Articles by Berry, J. O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Patel, M.
Right arrow Articles by Berry, J. O.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Untranslated Regions of FbRbcS1 mRNA Mediate Bundle Sheath Cell-specific Gene Expression in Leaves of a C4 Plant*Formula

Minesh Patel1, Alan J. Siegel, and James O. Berry2

From the Department of Biological Sciences, State University of New York, Buffalo, New York 14260

Received for publication, May 1, 2006 , and in revised form, June 14, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
C4 photosynthesis typically requires two specialized leaf cell types, bundle sheath (bs) and mesophyll (mp), which provide the foundation for this highly efficient carbon assimilation pathway. In leaves of Flaveria bidentis, a dicotyledonous C4 plant, ribulose 1,5-bisphosphate carboxylase (rubisco) accumulates only in bs cells surrounding the vascular centers and not in mp cells. This is in contrast to the more common C3 plants, which accumulate rubisco in all photosynthetic cells. Many previous studies have focused on transcriptional control of C4 cell type-specificity; however, post-transcriptional regulation has also been implicated in the bs-specific expression of genes encoding the rubisco subunits. In this current study, a biolistic leaf transformation assay has provided direct evidence that the 5'- and 3'-untranslated regions (UTRs) of F. bidentis FbRbcS1 mRNA (from a nuclear gene encoding the rubisco small subunit), in themselves, confer strong bs cell-specific expression to gfpA reporter gene transcripts when transcribed from a constitutive CaMV promoter. In transformed leaf regions, strong bs cell-specific GFP expression was accompanied by corresponding bs cell-specific accumulation of the constitutively transcribed FbRbcS1 5'-UTR-gfpA-3'-UTR mRNAs. Control constructs lacking any RbcS mRNA sequences were expressed in all leaf cell types. These findings demonstrate that characteristic cell type-specific FbRbcS1 expression patterns in C4 leaves can be established entirely by sequences contained within the transcribed UTRs of FbRbcS1 mRNAs. We conclude that selective transcript stabilization (in bs cells) or degradation (in mp cells) plays a key role in determining bs cell-specific localization of the rubisco enzyme.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Plants that utilize the highly efficient C4 pathway of carbon fixation typically possess a Kranz-type leaf anatomy that consists of two distinct photosynthetic cell types (1-4). Bundle sheath (bs)3 cells occur as a layer around each leaf vein, while mesophyll (mp) cells occur in one or more layers surrounding the vascular associated rings of bs cells. This specialized leaf anatomy compartmentalizes two sets of photosynthetic reactions that make up the C4 pathway. In the C4 dicot Flaveria bidentis, atmospheric CO2 entering via the leaf stomata is initially incorporated into four carbon acids by phosphoenolpyruvate carboxylase, an enzyme found only in the leaf mp cells. The C4 acids diffuse from mp cells to bs cells, where they are decarboxylated by a photosynthetic malic enzyme. Within bs chloroplasts, the released CO2 is incorporated into the Calvin cycle by ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco), the primary enzyme of photosynthetic carbon fixation.

The specialized anatomical framework and compartmentalized reactions of the C4 pathway function as a "CO2 pump" that concentrates CO2 in bs cells, where rubisco is specifically localized (1, 2, 5, 6). As a result, the carboxylase activity of this enzyme is increased, while its oxygenase activity, which can decrease photosynthetic productivity, is essentially eliminated. This two-cell compartmentalization of carboxylation/decarboxylation reactions does not occur in leaves of the more common C3 plants, which possess only one photosynthetic cell type and use rubisco as the initial enzyme of biological carboxylation. The result is that C4 plants are much more efficient at using biological energy (derived from photosynthetic electron transport) for CO2 fixation, particularly in warm and arid environments where carbon assimilation in C3 plants can be severely limited (1-3, 6, 7).

In mature C4 leaves, rubisco and other photosynthetic proteins (such as the mp cell-specific phosphoenolpyruvate carboxylase) are confined to only one of the two photosynthetic cell types, and transcripts encoding these enzymes are correspondingly bs or mp cell-specific (8, 9). For this reason, many previous studies have focused on regulation of transcription as the primary determinant of bs and mp cell specificity. In some cases transcriptional control has been demonstrated, and transcriptional regulatory elements involved in determining cell type specificity have been identified (8-15).

Genes encoding both small and large subunits of the rubisco enzyme (RbcS in the nucleus and rbcL in the plastid) often show patterns of expression that are distinct from other C4 enzymes. For example, in many C4 species RbcS and rbcL genes are initially expressed in all leaf cell types, similar to their expression in C3 plant species (8, 9, 16, 17). To achieve cell type-specific localization of rubisco, which is essential for the function of the C4 pathway, RbcS and rbcL genes become selectively down-regulated in leaf mp cells while continuing to be expressed abundantly in bs cells. This C3-C4 transition in rubisco gene expression occurs during leaf development and is influenced by light and/or photosynthetic metabolism (8, 9, 17-19). Some studies have indicated that unlike other C4 enzymes, the initiation of bs cell-specific rubisco gene expression during leaf development and its maintenance in mature leaves of C4 plants is due, at least in part, to post-transcriptional regulation; control of translation and mRNA stability have both been implicated (8, 9, 19, 20). Of particular significance, transcriptional run-on studies have shown that, while the rubisco mRNAs accumulate only in bs cells of mature C4 leaves, their corresponding genes are transcribed in the nuclei and plastids of both bs and mp cells, suggesting cell type-specific control of transcript stability (9, 21). A recent study showed that untranslated regions (UTRs) from a heterologous rubisco transcript (AhRbcS1 from amaranth, another C4 dicot), in themselves, conferred partial bs cell-specific expression to a gusA reporter gene at the level of mRNA accumulation when transcribed from a constitutive promoter in transgenic F. bidentis plants (22). This provided further evidence that post-transcriptional regulation contributes to bs cell-specific gene expression.

To better understand the role of post-transcriptional gene expression in determining the bs cell-specific localization of rubisco in C4 leaves, we made use of a rapid and efficient biolostic transient expression assay. For this assay, transcripts with 5'- and 3'-untranslated regions (UTRs) from an F. bidentis RbcS gene (FbRbcS1) were linked in frame with a gfpA reporter gene and placed under the control of a constitutive promoter. When introduced into intact F. bidentis leaves, these constructs produced striking patterns of bs cell-specific GFP fluorescence and transcript localization within the transformed leaf regions. In contrast, gfpA transcripts lacking the FbRbcS1 UTRs produced no cell-type specificity in fluorescence or mRNA localization. Because GFP transcript accumulation, as well as protein accumulation, was found to be highly specific to bs cells in the intact transformed leaves, we conclude that this strong cell type-specific expression occurred at the level of mRNA stability and was mediated by the FbRbcS1 UTRs.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Material and Growth Conditions—F. bidentis seedlings were germinated and grown under sterile conditions on Murashige and Skoog media (Sigma) in PlantCon (ICN) growth containers maintained in a growth chamber with 14 h/day illumination at 170-200 mmol photons m-2 s-1.

FbRbcS cDNAs—cDNAs of F. bidentis FbRbcS transcripts were prepared by 5'- and 3'-RACE PCR (FirstChoice RLM-RACE Kit, Ambion) according to manufacturers protocols, using primers provided with the kit together with gene-specific primers corresponding to sequences within the FbRbcS coding regions (see supplemental material). 5'-Amplifications (performed under conditions that required the presence of a cap to ensure authentic 5' ends) revealed the presence of two classes of cDNA, designated FbRbcS1 and FbRbcS2 (accession numbers AY267350 [GenBank] and AY267351 [GenBank] , respectively), based on the length and sequence of the 5'-UTRs. Full-length cDNAs were isolated using 5' end sense primers specific to each class of FbRbcS cDNA, together with a 3' primer provided with the kit.

Primer extension analysis was performed (Promega AMV Reverse Transcriptase-Primer Extension System and manufacturers protocols) using total RNA isolated from F. bidentis leaves and cotyledons, together with a 32P-end-labeled primer corresponding to a conserved region within the FbRbcS1 and FbRbcS2 coding regions. Primer extension products were analyzed using a 6% acrylamide-urea sequencing gel.

Post-transcriptional Expression Vectors—The experimental and control expression constructs were carried in the transient expression vector pBI221 (Clonetech), downstream of the CaMV promoter, and upstream of the nopaline synthase transcription terminator. The control transient expression vector CaMV-gfpA was based on the pBI221XS expression cassette described by Patel et al. (22) and was created by replacing the beta-glucuronidase (gusA) gene located within the SmaI and XhoI restriction sites of that vector, with the gfpA gene. To prepare the FbRbcS1 5'-UTR-gfpA-3'-UTR expression vector, two chimeric gene fragments were initially amplified. The 5'-fragment corresponded to the FbRbcS1 5'-UTR plus the 5' portion of the gfpA open reading frame and was amplified from a gfpA DNA template (using primers 5GFPFor and GFPRev; supplemental material). The forward primer incorporated a SmaI restriction site at start of the FbRbcS1 5'-UTR. The 3'-fragment was amplified from the FbRbcS1 cDNA template (primers GFP49_3FbFor and G3FbRev; supplemental material) and contained 49 bases from the 3' end of the gfpA coding sequence, plus the FbRbcS1 3'-UTR and an XhoI site at the 3' end. These 5'-UTR-gfpA and gfpA3'-UTR fragments overlapped by 49 bases within the gfpA open reading frame. The full-length FbRbcS1 5'-UTR-gfpA-3'-UTR expression cassette was prepared by denaturing and annealing these overlapping fragments and then extending the overlapping DNA strands with Klenow DNA polymerase (New England Biolabs). The transient expression vector FbRbcS1 5'-UTR-gfpA-3'-UTR was created by replacing the gusA fragment of pBI221XS (22) with this expression cassette, using the restriction sites SmaI and XhoI.

Biolistic Transformation—Leaves (2-3 cm in length) from 6-week-old F. bidentis plants were detached and placed into a Petri dish lined with H2O-moistened filter paper (Whatman 3MM). Four to five leaves were used per dish. For particle bombardment, gold particles (1.0 µm in size) were washed, prepared, and coated with DNA (either FbRbcS1 5'-UTR-gfpA-3'-UTR or CaMV-gfpA) according to manufacturer's protocols (Biolistic PDS-1000/He Particle Delivery System, Bio-Rad). Leaves were bombarded once at 1100 p.s.i. using a PDS-1000/He Particle Delivery System (Bio-Rad). Each bombardment was performed using 10 µl gold/plasmid DNA suspension, using macrocarriers and stopping plates (Bio-Rad) placed ~6 cm from the leaves. After bombardment, the leaves were incubated in the dishes with illumination at 25 °C for 16 h and analyzed by microscopy.

Microscopy and Tissue Preparation—Following incubation, the bombarded leaves were initially placed on microscope slides over a thin layer of H2O and visually scanned under 4x and 10x objectives of an Axiovert 10 microscope (Zeiss, Oberkochen, Germany), using a 450-490 FT510, LP520 filter system. Localized regions of leaf transformation (foci) showing GFP fluorescence were carefully excised on the microscope stage using a scalpel, and these were used immediately for capturing leaf surface images of the foci and subsequent tissue processing. All of the images shown were captured using a DM IRE2 inverted compound microscope (Leica Microsystems, Wetzlar, Germany) equipped with fluorescent and brightfield imaging systems and a Retiga Exi cooled CCD camera (QImaging, Burnaby, British Columbia, Canada). Immediately after leaf surface images were taken, the foci were placed either in optimal cutting temperature compound (O.C.T., TissueTek) for cryosectioning or processed for paraffin embedding/sectioning (13, 16, 22). 10-µm cryosections were prepared using a Reichert-Jung Cryocut 1800 (Leica Microsystems) at 18 °C. 15-µm-thick paraffin-embedded sections were prepared using a rotary microtome and fixed to glass slides treated with Sta-On (Surgipath, Richmond, IL).

In Situ Hybridization and Immunolocalization—In situ hybridization analysis of paraffin-embedded sections from GFP-expressing foci was performed as described previously (13, 16, 22). Sections were hybridized to a gfpA antisense RNA probe that was transcribed in vitro using biotin-16 UTP (Roche Applied Science). Hybridized transcripts were detected using a streptavidin-alkaline phosphatase conjugate (NeutrAvidin; Pierce Chemical) with an enzymatic color reaction (SigmaFast 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium tablets).

Immunolocalization analysis on serial paraffin-embedded sections of GFP-expressing foci was performed as described (13, 16, 22), using GFP antiserum (Assay Designs, Inc.). Fluorescent imaging of the antibody reactions was performed using biotinylated goat anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA) together with an R-phycoerythrin/streptavadin conjugate (Molecular Probes).


Figure 1
View larger version (123K):
[in this window]
[in a new window]
  		FIGURE 1.
Fluorescent images taken from the surface of C4 F. bidentis leaves biolistically transformed with experimental and control gfpA expression constructs. A-C, GFP-expressing foci on leaves bombarded with the FbRbcS1 5'-UTR-gfpA-3'-UTR construct. D-F foci on leaves bombarded with a CaMV-gfpA control construct, which lacked the FbRbcS1 UTRs. For A and D, images taken with a GFP imaging filter system were merged with corresponding bright-field images for clear visualization of transformed and untransformed leaf structures (leaf veins, bs, and mp cells). Images in other panels were captured using the GFP imaging filters alone. v = leaf veins, gbs = GFP-expressing bs cells. (Bar = 100 µm.)

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The goal of this investigation was to determine the role of 5'- and 3'-UTRs of a F. bidentis RbcS mRNA in mediating bs cell-specific gene expression in the leaves of a C4 plant. Therefore, to ensure that FbRbcS cDNA used for vector construction contained a full-length 5'-UTR, we used a RACE PCR protocol that required the presence of a 5'-cap. Approximately 100 clones were characterized, some of which contained small variations within their coding regions that identified them as being derived from individual members of an FbRbcS gene family. These comprised two distinct classes, based on the length and sequence of their 5'-UTRs, and by some differences in their 3'-UTRs. FbRbcS1 and FbRbcS2 (supplemental Fig. S1, A and C) are representative of these two classes. Primer extension analysis confirmed that two main classes of FbRbcS mRNA accumulate in F. bidentis in vivo, with 5' termini corresponding to the 5' ends of these two classes of cDNA (supplemental Fig. S1B).

The post-transcriptional expression construct derived from FbRbcS1 (supplemental Fig. S2) was prepared by linking the full-length 5'- and 3'-UTRs of this transcript (nucleotides 1-26 and 552-735, respectively) in frame with a gfpA reporter gene. To ensure that expression patterns were due entirely to post-transcriptional control, transcription of experimental, as well as control mRNAs (which lacked the FbRbcS1 UTRs, supplemental Fig. S2), was placed under the control of the constitutive CaMV promoter. These were inserted into the plasmid pBI221 for biolistic transformation.


Figure 2
View larger version (85K):
[in this window]
[in a new window]
  		FIGURE 2.
Fluorescent images of cryogenic cross-sections prepared from GFP-expressing foci excised from biolistically transformed F. bidentis leaves. A-C, sections from foci expressing the FbRbcS1 5'-UTR-gfpA-3'-UTR construct. D-F, sections from foci expressing the control CaMV-gfpA construct. bs = bundle sheath cells; gbs = GFP-expressing bs cells; mp = mesophyll cells; gmp = GFP-expressing mp cells; w = a wound/entry point for the DNA-coated mircoprojectiles. Blue autofluorescence at the epidermis is observable in some images. (Bar = 100 µm.)

 
Following bombardment, images taken from the leaf surface clearly show bs cell-specific GFP fluorescence within transformed regions (foci) expressing the FbRbcS1 5'-UTR-gfpA-3'-UTR construct (Fig. 1, A-C). These three images show GFP expression that is highly specific to bs cells that surround the leaf veins, with no GFP fluorescence observable outside of a single layer of cells surrounding the leaf vascular centers or within the veins themselves. In contrast, leaves bombarded with a CaMV-gfpA control construct, which lacked the FbRbcS1 UTRs, showed GFP fluorescence distributed throughout all of the cell types observable from the leaf surface (Fig. 1, D-F). Note that for A and D, images taken with a GFP imaging filter system were merged with the corresponding bright-field images, for clear visualization of transformed and untransformed leaf structures (leaf veins and bs cells). Images in other panels were captured using the GFP imaging filters alone and clearly show the differences in fluorescence between GFP-expressing and non-expressing cells.

To observe patterns of expression for the FbRbcS1-UTR and control constructs in more detail, cryogenic cross-sections were prepared from GFP-expressing foci that were excised from the intact leaves. Due to higher levels of GFP expression from FbRbcS1-UTR constructs relative to control constructs (similar findings were reported for AhRbcS1-UTR versus control constructs in ref. 22), exposures were adjusted for equal visualization of GFP fluorescence. In cross-sections, the FbRbcS1 5'-UTR-gfpA-3'-UTR construct clearly shows strong bs cell-specific expression (Fig. 2, A-C), whereas the control construct showed expression in all of the leaf cell types (D-F). With both the experimental and control constructs, transformed as well as non-transformed cells were observed within the foci. For example, both GFP-expressing (gbs) and non-expressing (bs) bundle sheath cells are observable in Fig. 2, A-C; expressing and non-expressing examples of all leaf cell types are observable in Fig. 2, D-F. Such cell to cell variations in transformation within a foci were most likely due to the nature of the biolistic protocol, which normally results in random clusters of cell transformations by the DNA-coated biolistic beads.

The images in Figs. 1 and 2 are representative of more than 60 independent biolistic transformation events. In all cases, the FbRbcS1 5'-UTR-gfpA-3'-UTR construct produced transformation foci showing strong GFP fluorescence only in bs cells, whereas transformation foci produced by the control construct showed GFP fluorescence in all cell types. Because both experimental and control constructs were transcribed from the same constitutive promoter, we conclude that bs cell-specific GFP expression mediated by the FbRbcS1-UTRs was due to post-transcriptional regulation.


Figure 3
View larger version (69K):
[in this window]
[in a new window]
  		FIGURE 3.
Bs cell-specific GFP mRNA in cross-sections prepared from paraffin-embed GFP foci excised from biolistically transformed F. bidentis leaves. For in situ hybridizations, leaf sections from GFP-expressing leaf regions were prepared and hybridized with biotin-labeled GFP antisense RNA probe (see "Experimental Procedures"). A-C, in situ hybridization of sections prepared from regions transformed with FbRbcS1-UTR/GFP constructs. E-G, in situ hybridization of sections prepared from regions transformed with the control (no RbcS UTR) constructs. Note that F shows both biolistically transformed (GFP-expressing) and non-transformed (no GFP expression) regions. D and H, adjacent serial sections corresponding to C and G, respectively, reacted with GFP antisera and visualized using an R-phycoerythrin fluorescent detection system. Bar = 100 µm.

 
In situ hybridizations were performed to determine whether the strong bs cell specificity mediated by the FbRbcS1 UTRs was due to regulation at the level of translation (gfpA mRNA would be present in all transformed cell types) or mRNA stability (gfpA mRNA would be present only in transformed bs cells). It was necessary to use paraffin-embedded sections for in situ localization of GFP mRNAs, since it was not possible to maintain the integrity of frozen sections from biolistically transformed F. bidentis leaf foci through the in situ hybridization procedure. These sections were hybridized to a biotin-labeled GFP antisense RNA probe, and hybridization was detected using streptavidin-alkaline phosphatase and an enzymatic color reaction.

Fig. 3, A-C, show representative cross-sections of foci from F. bidentis leaves bombarded with constructs containing the FbRbcS1 5'-UTR-gfpA-3'-UTR construct (all of which showed strong bs cell-specific GFP expression when viewed from the leaf surface). In these images (and in more than 15 additional sections), hybridization to the GFP antisense probe occurred primarily in leaf bs cells; only minimal signal was observed in mp cells of the biolistically transformed leaves. The very low levels of hybridization signal observed in some leaf mp cells was most likely due to low levels of mRNA accumulation from the FbRbcS1 5'-UTR-gfpA-3'-UTR construct, since the GFP antisense probe did not normally produce detectable background signal in non-expressing leaf cells (for example, see Fig. 3, right portion of section shown in F). In contrast, in sections from foci of leaves bombarded with the control construct (all of which showed GFP expression in all cell types when viewed from the leaf surface), hybridization to the GFP antisense probe occurred in all transformed leaf cell types, including bs and mp cells (Fig. 3, E-G). Note that Fig. 3F shows hybridization to a section from near the edge of an excised foci that contains both transformed cells (which showed hybridization to the gfpA probe) and non-transformed cells (which showed no hybridization). This observation is consistent with control hybridization reactions (data not shown) indicating that the gfpA antisense probe did not hybridize to sections from non-transformed F. bidentis leaves under conditions used here.

To co-localize GFP mRNA and protein accumulation within the same paraffin-embedded transformed leaf foci, serial sections were reacted with GFP antiserum; imaging of the antibody reactions was performed using an R-phycoerythrin/streptavadin detection system. Fig. 3D (serial section corresponding to 3C) shows the same highly specific pattern of GFP protein accumulation in bs cells that was observed for GFP transcripts. Similarly, GFP protein accumulation in leaf regions bombarded with the control construct (Fig. 3H, serial section corresponding to Fig. 3G) shows the same nonspecific accumulation patterns as the control GFP mRNAs.

It should be noted that differences between in situ hybridization versus immunolocalization protocols can lead to slight variations in tissue morphology, accounting for the apparent incomplete overlap between serial sections shown in Fig. 3, D and C and H and G).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This current study provides strong evidence for post-transcriptional control of bs cell-specific RbcS gene expression in the leaves of a C4 dicot. Taken together, the data shown in Figs. 1, 2, 3 show that constitutively transcribed reporter gene transcripts containing both 5'- and 3'-UTR regions from endogenous FbRbcS1 mRNAs display strong bs cell-specific patterns of expression in biolistically transformed F. bidentis leaves. Because gfpA transcript accumulation as well as expression of the GFP protein were both highly specific to bs cells, we conclude that this regulation occurred at the level of mRNA stability. This conclusion is supported by initial observations of regenerating F. bidentis T0 plants stably transformed with the FbRbcS1 5'-UTR-gfpA-3'-UTR construct, which also show GFP fluorescence that is highly specific to leaf bs cells in a pattern very similar to the foci shown here.4

Findings presented here differ from those of our previous study (22), in which 5'- and 3'-UTRs from heterologous amaranth AhRbcS1 mRNAs conferred only partial bs cell specificity to a gusA reporter gene, a pattern that was also likely mediated by post-transcriptional regulation of mRNA accumulation. The difference in expression patterns between these two systems is likely due to the heterologous origin of the AhRbcS1 5'- and 3'-UTRs sequences (these share 65 and 69% similarity, respectively, with the endogenous FbRbcS1 UTRs, see supplemental Fig. S1, A and C), which might not have been fully recognized by the F. bidentis post-transcriptional regulatory processes, resulting in "leaky" bs cell specificity. In consideration of our current findings, we conclude that 5'- and 3'-UTRs of endogenous F. bidentis RbcS transcripts are capable of mediating strong bs cell-specific protein as well as mRNA accumulation in C4 leaves and thus contain the information needed to generate the bs cell-specific expression patterns that characterize these plants. In initial studies, we have found that neither UTR by itself is capable of mediating bs-specific gfp expression, suggesting that interactions between the 5'- and 3'-UTRs may be required for this process.

Rubisco genes are known to be highly regulated; in many plants their expression is modulated by light (18, 23, 24), development (20, 25, 26), cell type (6, 9, 16), photosynthetic metabolism (17, 19, 27-29), and even pathogen infection (30). While regulation of transcription has been implicated in some of these processes, post-transcriptional control is often a primary regulatory determinant (21, 23, 26, 29, 31, 32). This current study indicates that post-transcriptional control was utilized as a primary mechanism when RbcS gene expression was adapted for the specialized cell-type specific expression that provides the foundation for the C4 pathway in F. bidentis leaves.

Post-transcriptional regulation of FbRbcS1 transcript accumulation would appear to be fully sufficient for mediating bs cell-specific gene expression. However, additional regulatory mechanisms might also contribute to this process in F. bidentis and other C4 systems. Separate studies from our laboratory and others have also implicated regulation of transcription and translation in determining bs cell-specificity (8, 9, 19, 20). For example, bs cell-specific localization of the rubisco proteins is separable from the specific localization of their corresponding transcripts in amaranth leaves during specific stages of leaf or photosynthetic development, implicating regulation at the level of translation (19, 20). Therefore, the establishment of full bs cell-specific RbcS gene expression in C4 leaves might in fact result from the integration of multiple levels of regulation. Each mechanism could act independently during different developmental stages (as in Refs. 19 and 20) or perhaps at some times in synergy, ultimately leading to full bs cell-specific localization of the rubisco proteins. Emerging evidence suggests that close interactions between different phases of transcriptional and post-transcriptional regulation may be a common occurrence in eukaryotic gene expression (33). Such redundancy and overlap for mechanisms determining bs cell specificity would ensure that full C4 photosynthetic capacity occurs in the leaves of these plants, at all stages of development and under diverse environmental conditions.

A less likely alternative explanation for bs cell-specific localization of FbRbcS1 mRNA is that sequences within the UTRs have an effect on transcriptional elongation. Inhibition or termination of transcription by downstream control elements is known to occur in eukaryotic viruses and more rarely in cellular genes (in both plants and animals; Refs. 34-37). Measurements of gfpA mRNA transcription and turnover in stable transgenic F. bidentis lines will provide mechanistic details about UTR-mediated control of bs cell-specific FbRbcS1 mRNA accumulation.

Direct evidence that bs cell specificity is controlled by sequences within the FbRbcS1 UTRs will focus future C4 studies (38) on an analysis of post-transcriptional mechanisms involved in this process. Control of gene expression through differential transcript stabilization occurs in all organisms (39-46); transcripts targeted for regulated turnover often contain specific sequences within their 5'- and 3'-UTRs that interact with cytoplasmic RNA-binding proteins to mediate their specific decay (40, 41, 44, 46-48). In many cases, these mRNAs contain AU-rich elements within their 3' UTRs that mediate turnover (41, 42, 44, 46-48). RbcS mRNAs from the C4 dicots F. bidentis and amaranth contain multiple AU-rich elements within their 3'-UTR (supplemental Fig. S1C); interestingly, one of these repeats, UUUAUU, is identical to a repeat in human mRNAs that become destabilized when this sequence interacts with a specific class of RNA binding protein (46). Identification of regulatory sequences within the RbcS 5'- and 3'-UTRs, as well as the RNA-binding proteins they interact with, will aid in the functional analysis of specific molecular recognition processes responsible for post-transcriptional, bs cell-specific transcript accumulation in C4 leaves.


   FOOTNOTES
 
* This work was supported by Grants 2001-01825 from United States Department of Agriculture/National Research Initiative and MCB 0110411 from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental primer material and supplemental Figs. S1 and S2. Back

1 Current address: USDA ARS Cereal Crops Research Unit, Barley and Malt Laboratory, 501 Walnut St., Madison, WI 53726. Back

2 To whom correspondence should be addressed: Dept. of Biological Sciences, State University of New York, Buffalo, NY 14260. Tel.: 716-645-2363 (ext. 145); Fax: 716-645-3369; E-mail: camjob{at}buffalo.edu.

3 The abbreviations used are: bs, bundle sheath; mp, mesophyll; rubisco, ribulose 1,5-bisphosphate carboxylase; UTR, untranslated region; GFP, green fluorescent protein; RACE, rapid amplification of cDNA ends. Back

4 M. Patel and J. O. Berry, unpublished data. Back


   ACKNOWLEDGMENTS
 
We are grateful to Jim Stamos for preparing the illustrations and Michael Farkas for preparing antisense transcripts used for in situ hybridizations. We thank Sejal Patel for excellent technical assistance and Paul Cullen, Gerry Koudelka, and Stephen Free for critical reading of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hatch, M. D. (1987) Biochim. Biophys. Acta 895, 81-106
  2. Kanai, R., and Edwards, G. E. (1999) in C4 Plant Biology (Sage, R. F., and Monson, R. K., eds) pp. 49-88, Academic Press, San Diego, CA
  3. Furbank, R. T., Hatch, M. D., and Jenkins, C. L. D. (2000) in Advances in Photosynthesis. Photosynthesis: Physiology and Metabolism (Leegood, R. C., Sharkey, T. D., von Caemmerer, S., and Kennedy, R., eds) pp. 459-469, Kluwer Academic Publishers, Dorecht, the Netherlands
  4. Edwards, G. E., Franceschi, V. R., Ku, M. S. B., Voznesenskaya, E. V., Pyankov, V. I., and Andreo, C. S. (2001) J. Exp. Bot. 52, 577-590[Abstract/Free Full Text]
  5. von Caemmerer, S., and Furbank, R. T. (2003) Photosynth. Res. 77, 191-207[CrossRef][Medline] [Order article via Infotrieve]
  6. Sage, R. F. (2004) New Phytol. 161, 341-370[CrossRef]
  7. Ehleringer, J. R., Cerling, T. E., and Helliker, B. R. (1997) Oecologia 112, 285-299[CrossRef]
  8. Furbank, R. T., and Taylor, W. C. (1995) Plant Cell 7, 797-807[CrossRef][Medline] [Order article via Infotrieve]
  9. Sheen, J. (1999) Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 187-217[CrossRef]
  10. Langdale, J. A., Taylor, W. C., and Nelson, T. (1991) Mol. Gen. Genet. 225, 49-55[Medline] [Order article via Infotrieve]
  11. Bansal, K. C., Virett, J.-F., Haley, J., Khan, B. M., Schantz, R., and Bogorad, L. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 3654-3658[Abstract/Free Full Text]
  12. Viret, J.-F., Mabrouk, Y., and Bogorad, L. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8577-8581[Abstract/Free Full Text]
  13. Long, J. J., and Berry, J. O. (1996) Plant Physiol. 112, 473-482[Abstract]
  14. Nomura, M., Sentoku, N., Nishimura, A., Lin, J.-H., Honda, C., Taniguchi, M., Ishida, Y., Ohta, S., Komari, T., Miyao-Tokutomi, M., Kano-Murakami, Y., Tajima, S., Ku, M. S., and Matsuoka, M. (2000) Plant J. 22, 211-221[CrossRef][Medline] [Order article via Infotrieve]
  15. Gowik, U., Burscheidt, J., Akyildiz, M., Schlue, U., Koczor, M., Streubel, M., and Westhoff, P. (2004) Plant Cell 16, 1077-1090[Abstract/Free Full Text]
  16. Wang, J.-L., Klessig, D. F., and Berry, J. O. (1992) Plant Cell 4, 173-184[Abstract/Free Full Text]
  17. Wang, J.-L., Turgeon, R., Carr, J. P., and Berry, J. O. (1993) Plant Cell 5, 289-296[Abstract]
  18. Wang, J.-L., Long, J. J., Hotchkiss, T., and Berry, J. O. (1993) Plant Physiol. 102, 1085-1093[Abstract]
  19. McCormac, D. J., Boinski, J. J., Ramsperger, V. C., and Berry, J. O. (1997) Plant Physiol. 114, 801-815[Abstract]
  20. Ramsperger, V. C., Summers, R. G., and Berry, J. O. (1996) Plant Physiol. 111, 999-1010[Abstract]
  21. Boinski, J. J., Wang, J.-L., Xu, P., Hotchkis, T., and Berry, J. O. (1993) Plant Mol. Biol. 22, 397-410[CrossRef][Medline] [Order article via Infotrieve]
  22. Patel, M., Corey, A.C., Yin, L.-P., Ali, S., Taylor, W. C., and Berry, J. O. (2004) Plant Physiol. 136, 3550-3561[Abstract/Free Full Text]
  23. Berry, J. O., Breiding, D. E., and Klessig, D. F. (1990) Plant Cell 2, 795-803[Abstract/Free Full Text]
  24. Zhou, J., Ma, L., Zhang, S., Zhu, Y., and Sun, D. (2001) Plant Cell Physiol. 42, 1049-1055[Abstract/Free Full Text]
  25. Wanner, L. A., and Gruissem, W. (1991) Plant Cell 3, 1289-1303[Abstract/Free Full Text]
  26. Hensel, L. L., Grbic, V., Baumgarten, D. A., and Bleecker, A. B. (1993) Plant Cell 5, 553-564[Abstract]
  27. Jiang, C. Z., Rodermel, S. R., and Shibles, R. M. (1993) Plant Physiol. 101, 105-112[Abstract]
  28. Cheng, S.-H., Moore, B. D., and Seemann, J. R. (1998) Plant Physiol. 116, 715-723[Abstract/Free Full Text]
  29. Sinha, A. K., Hofmann, M. G., Römer, U., Köckenberger, W., Elling, L., and Roitsch, T. (2002) Plant Physiol. 128, 1480-1489[Abstract/Free Full Text]
  30. Berger, S., Papadopoulos, M., Schreiber, U., Kaiser, W., and Roitsch, T. (2004) Physiol. Plant. 122, 419-428[CrossRef]
  31. Thompson, D. M., and Meagher, R. B. (1990) Nucleic Acids Res. 18, 3621-3629[Abstract/Free Full Text]
  32. Rodermel, S., Haley, J., Jiang, C.-Z., Tsai, C.-H., and Bogorad, L. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 3881-3885[Abstract/Free Full Text]
  33. Orphanides, G., and Reinberg, D. (2002) Cell 108, 439-451[CrossRef][Medline] [Order article via Infotrieve]
  34. Wright, S. (1993) Mol. Biol. Cell. 4, 661-668[Medline] [Order article via Infotrieve]
  35. Curie, C., and McCormick, S. (1997) Plant Cell 9, 2025-2036[Abstract]
  36. He, X., Fütterer, J., and Hohn, T. (2002) Nucleic Acids Res. 30, 497-506[Abstract/Free Full Text]
  37. Hu, W.-W., Gong, H., and Pua, E. C. (2005) Plant Physiol. 138, 276-286[Abstract/Free Full Text]
  38. Brown, N. J., Parsley, K., and Hibberd, J. M. (2005) Trends Plant Sci. 10, 215-221[CrossRef][Medline] [Order article via Infotrieve]
  39. Shirley, B. W., and Meagher, R. B. (1990) Nucleic Acids Res. 18, 3377-3385[Abstract/Free Full Text]
  40. Seeley, K. A., Byrne, D. H., and Colbert, J. T. (1992) Plant Cell 4, 29-38[Abstract/Free Full Text]
  41. Green, P. J. (1993) Plant Physiol. 102, 1065-1070[Medline] [Order article via Infotrieve]
  42. Zubiaga, A. M., Belasco, J. G., and Greenberg, M. E. (1995) Mol. Cell. Biol. 15, 2219-2230[Abstract]
  43. Staton, J. M., Thomson, A. M., and Leedman, P. J. (2000) J. Mol. Endocrinol. 25, 17-34[Abstract]
  44. Fedoroff, N. V. (2002) Curr. Opin. Plant Biol. 5, 452-459[CrossRef][Medline] [Order article via Infotrieve]
  45. Gutierrez, R. A., Ewing, R. M., Cherry, J. M., and Green, P. J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11513-11518[Abstract/Free Full Text]
  46. Blackshear, P. J., Lai, W. S., Kennington, E. A., Brewer, G., Wilson, G. M., Guan, X., and Zhou, P. (2003) J. Biol. Chem. 278, 19947-19955[Abstract/Free Full Text]
  47. Laroia, G., Cuesta, R., Brewer, G., and Schneider, R. J. (1999) Science 284, 499-502[Abstract/Free Full Text]
  48. Cheng, Y., and Chen, X. (2004) Curr. Opin. Plant Biol. 7, 20-25[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J Exp BotHome page
M. Patel and J. O. Berry
Rubisco gene expression in C4 plants
J. Exp. Bot., May 1, 2008; 59(7): 1625 - 1634.
[Abstract] [Full Text] [PDF]

Home page
 Plant Physiol.Home page
S. Engelmann, C. Wiludda, J. Burscheidt, U. Gowik, U. Schlue, M. Koczor, M. Streubel, R. Cossu, H. Bauwe, and P. Westhoff
The Gene for the P-Subunit of Glycine Decarboxylase from the C4 Species Flaveria trinervia: Analysis of Transcriptional Control in Transgenic Flaveria bidentis (C4) and Arabidopsis (C3)
Plant Physiology, April 1, 2008; 146(4): 1773 - 1785.
[Abstract] [Full Text] [PDF]

Home page
 Plant CellHome page
M. Akyildiz, U. Gowik, S. Engelmann, M. Koczor, M. Streubel, and P. Westhoff
Evolution and Function of a cis-Regulatory Module for Mesophyll-Specific Gene Expression in the C4 Dicot Flaveria trinervia
PLANT CELL, November 1, 2007; 19(11): 3391 - 3402.
[Abstract] [Full Text] [PDF]

Home page
 J Exp BotHome page
S. Thomas-Hall, P. R. Campbell, K. Carlens, E. Kawanishi, R. Swennen, L. Sagi, and P. M. Schenk
Phylogenetic and molecular analysis of the ribulose-1,5-bisphosphate carboxylase small subunit gene family in banana
J. Exp. Bot., July 11, 2007; (2007) erm129v3.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
281/35/25485    most recent
M604162200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Patel, M.
Right arrow Articles by Berry, J. O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Patel, M.
Right arrow Articles by Berry, J. O.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement