The Plant Cell, Vol. 5, 289-296, March 1993 O 1993 American Society of Plant Physiologists

Carbon Sink-to-Source Transition 1s Coordinated with Establishment of Cell-Specif ic Gene Expression in a C4 Plant

Jing-Liang Wang,’ Robert Turgeon,b John P. Caqc and James O. Ber$>’

a Department of Biological Sciences, State University of New York, Buffalo, New York 14260 Section of Plant Biology, Cornell University, Ithaca, New York 14853 Department of Plant Pathology, Cornell University, Ithaca, New York 14853

Plants that use the highly efficient C4 photosynthetic pathway possess two types of specialized leaf cells, the mesophyll and bundle sheath. In mature C4 leaves, the C02 fixation enzyme ribulose-l,5-bisphosphate carboxylase (RuBPCase) is specifically compartmentalized to the bundle sheath cells. However, in very young leaves of amaranth, a dicotyledon- ous C4 plant, genes encoding the large subunit and small subunit of RuBPCase are initially expressed in both photosynthetic cell types. We show here that the RuBPCase mRNAs and proteins become specifically localized to leaf bundle sheath cells during the developmental transition of the leaf from carbon sink to carbon source. Bundle sheath cell-specific ex- pression of RuBPCase genes and the sink-to-source transition began initially at the leaf apex and progressed rapidly and coordinately toward the leaf base. These findings demonstrated that two developmental transitions, the change in photoassimilate transport status and the establishment of bundle sheath cell-specific RuBPCase gene expression, are tightly coordinated during C4 leaf development. This correlation suggests that processes associated with the accumula- tion and transport of photosynthetic compounds may influence patterns of photosynthetic gene expression in C4 plants.

INTRODUCTION

Ribulose-1 5bisphosphate carboxylase (RuBPCase) is located in the chloroplasts of all higher plants and is the principal en- zyme in photosynthetic carbon fixation. This enzyme is composed of two subunits, the chloroplast-encoded large subunit (LSU) and the nuclear-encoded small subunit (SSU) (Ellis, 1981; Miziorko and Lorimer, 1983). In plant species that utilize the C3 photosynthetic pathway, RuBPCase is found in all of the photosynthetic cells of the leaf. In mature leaves of plants that utilize the more complex C4 photosynthetic path- way, RuBPCase is specifically localized to one specialized cell type, the bundle sheath cells (Hatch and Slack, 1970; Edwards and Huber, 1981). Bundle sheath cells in C4 leaves occur as one or two rings around each of the leaf veins. Mesophyll cells, the other photosynthetic cell type in C4 plants, surround each ring of bundle sheath cells in one or more layers.

The two types of photosynthetic cells compartmentalize the different reactions of the C4 pathway (Hatch and Slack, 1970; Gutierrez et al., 1974; Edwards and Huber, 1981). In C4 plants, the initial fixation of atmospheric COe is accomplished by the enzyme phosphoenolpyruvate carboxylase, which is found only in mesophyll cells. The carboxylation phase of the C4 path- way in mesophyll cells produces C4 acids, which are then transported to neighboring bundle sheath cells. In bundle sheath cells, decarboxylation of the C4 acids releases COn To whom correspondence should be addressed.

for refixation by RuBPCase. These reactions cause COn to be concentrated in bundle sheath cells in the vicinity of RuBPCase, thereby increasing photosynthetic efficiency and reducing metabolically wasteful photorespiration caused by the oxygen- ase activity of this enzyme.

The developmental signals and molecular mechanisms that control cell type-specific expression of RuBPCase and other C4 pathway enzymes are not well understood. In the C4 monocot maize, both cell position and light influence the bun- dle sheath cell-specific expression of RuBPCase genes (Sheen and Bogarad, 1985, 1986; Langdale et al., 1988a, 1988b; Nelson and Langdale, 1989). Recently, we reported that in the C4 dicot amaranth, the developmental stage of the leaf, rather than light, appears to control bundle sheath cell-spe- cific expression of the RuBPCase LSU and SSU genes (Wang et al., 1992). In very young (5-mm-long) amaranth leaves, the RuBPCase genes were expressed in a pattern similar to that observed in plants that use the C3 photosynthetic pathway, with LSU and SSU proteins and mRNAs present in both bun- dle sheath and mesophyll cells. The RuBPCase proteins and mRNAs became specifically localized to bundle sheath cells in the characteristic C4-type pattern as the leaves expanded to 10 mm in length over the time period of 24 to 36 hr.

It is probable that C3-like expression of RuBPCase genes in both photosynthetic cell types represents an initial “default”

290 The Plant Cell

%-K'*rv%'!

Figure. 1. Cellular Localization of RuBPCase LSU and SSU Polypeptides and mRNAs in Apical and Basal Regions of a 7-mm-long Amaranth Leaf.

Sink-to-Source Transition in a C4 Plant 291

state (Langdale et al., 1988b; Langdale and Nelson, 1991; Wang et al., 1992). Specific signals are then required to suppress the expression of RuBPCase genes in mesophyll cells while allowing their continued expression in bundle sheath cells. To identify developmental processes that might accompany and possibly signal the C3-to-C4 transition in RuBPCase gene ex- pression, we examined developing amaranth leaves during the short time period when this transition is in progress. We report here that changes in the pattern of RuBPCase gene expression are tightly coordinated with changes in carbon balance that occur during the early stages of dicot leaf development.

RESULTS

For these studies, the first and second leaves to emerge after the cotyledons were analyzed during the time they were un- dergoing the C3-to-C4 transition, at m7 mm in length. Using immunofluorescence microscopy and in situ hybridization (Wang et al., 1992), we observed that patterns of RuBPCase gene expression in the apical and basal regions of these leaves were different. In Figures 1A to lD, a series of cross-sections from a typical 7-mm-long leaf were reacted with polyclonal antisera against RuBPCase LSU and SSU, then with R-phyco- erythrin-conjugated second antibody. Near the leaf apex, the RuBPCase LSU (Figure 1A) and SSU (Figure 16) polypeptides were specifically localized to bundle sheath cells in the charac- teristic C4 pattern. Near the base of the leaf, these polypeptides were found in both bundle sheath and mesophyll cells in a C3-like pattern (Figures 1C and 1D). Similarly, in situ hybrid- ization analysis using labeled LSU and SSU antisense RNA probes indicated that both RuBPCase transcripts were bun- dle sheath cell-specific near the leaf apex (Figures 1E and

1F) and were present in both cell types near the leaf base (Figures 1G and 1H). An examination of seria1 cross-sections from severa1 apical and basal regions of these leaves indicated that the change from the C3-like pattern of localization to the C4-type pattern in RuBPCase gene expression (C3-to-C4 tran- sition) occurs in the basipetal (apex-to-base) direction. In each region of the leaf, patterns of mRNA and protein accumula- tion correlated very closely.

A specific region showing a change in the pattern of cell- specific RuBPCase gene expression can be clearly observed in a longitudinal section of the entire length of a 7-mm leaf hy- bridized to a labeled LSU antisense RNA probe, as shown in Figure 2. In this longitudinal section, the C3-to-C4 transition zone is approximately one-third of the way up the leaf from the base. ldentical patterns of cellular localization were ob- served when adjacent longitudinal sections were hybridized with an SSU antisense RNA probe, or when the LSU or SSU proteins were detected using specific antisera (data not shown). Taken together, these results demonstrate that the develop- mental processes that determine the cell-specific localization of RuBPCase LSU and SSU show polarity from the leaf tip to the leaf base. Furthermore, changes in mRNA and protein levels appear to occur simultaneously.

The developmental polarity of the C3-to-C4 transition in amaranth leaves suggested similarity to another transition event that occurs during early leaf development in dicotyle- donous plants. Dicot leaves undergo a sink-to-source transition, in which a leaf is converted from a net importer to a net ex- porter of photoassimilate (Turgeon, 1989). The basipetal direction of the C3-to-C4 transition in amaranth leaves and the basipetal direction of the sink-to-source transition observed in the leaves of other dicots suggested that these early de- velopmental processes might be related. Therefore, we followed the movement of 14C-labeled photoassimilate as the develop- ing leaves progressed through the C3-to-C4 transition. These

Figure 1. (continued).

Apical sections were taken from regions in the upper quarter of the leaf; basal sections were taken from the lower quarter of the leaf. Bundle sheath (b), mesophyll (m), upper epidermal (ue), and lower epidermal (le) cells are indicated. The bundle sheath cells can be observed as a single layer of cells forming a ring around each vein, with one or more layers of mesophyll cells in between. (A) to (D) lmmunolocalization of LSU and SSU polypeptides. Specific reaction of the antibodies is detected in these photographs as yellow-orange fluorescence. (A) Section from apical region reacted with RuBPCase LSU antiserum. (6) Apical region reacted with RuBPCase SSU antiserum. (C) Basal region reacted with RuBPCase LSU antiserum. (D) Basal region reacted with RuBPCase SSU antiserum. (E) to (H) LSU and SSU mRNA accumulation. Sections were prepared and hybridized with digoxigenin-labeled LSU or SSU transcripts generated using T7 or T3 polymerase. Hybridized transcripts were detected using anti-digoxigenin antisera conjugated to alkaline phosphatase in combina- tion with a color detection system. In these micrographs, specific hybridization is observed as a dark purple color. (E) Section from apical region hybridized to LSU antisense RNA. (F) Apical region hybridized to SSU antisense RNA. (G) Basal region hybridized to LSU antisense FINA. (H) Basal region hybridized to SSU antisense RNA. Scale is the same for all micrographs. Bar = 100 pm.

292 The Plant Cell

Figure 2. C3-to-C4 Transition for LSD mRNA Accumulation in 7-mm-long Amaranth Leaf.

The apex (A) and base (B) of the leaf are indicated. In this composite micrograph of a longitudinal leaf section, the C3-to-C4 transition zone(TZ) can be observed approximately one-third of the way up the leaf from the base. The USD antisense RNA probe was labeled and hybridized,as described in the legend to Figure 1. Bar = 500 urn.

experiments were performed by pulse labeling one of thecotyledons of the amaranth seedlings with 14CO2 for 5 min(Turgeon and Webb, 1973; Leisner et al., 1992). After allowing45 min for translocation of labeled photoassimilate from thecotyledon into the first and second leaves, one lateral half ofeach leaf was removed for analysis by immunolocalization andin situ hybridization. Carbon-14 distribution in the rest of theshoot was analyzed by autoradiography (Weisberg et al., 1988).

Figures 3A and 3B show the 14C-labeling patterns for firstand second emerging leaves at different developmental stages.In Figure 3A, the larger 8-mm-long first emerging leaf was moreheavily labeled in its basal half than its apical half, indicatingthat it was approximately midway through the sink-to-sourcetransition. The smaller 3-mm-long second leaf on the sameplant showed uniform labeling throughout, demonstrating thatat this early developmental stage, the entire leaf was a carbonsink that imported photoassimilate. For the older seedlingshown in Figure 3B, the larger 12-mm-long first emerging leafwas heavily labeled only at its base, indicating that it had nearlycompleted the sink-to-source transition. The smaller 6-mm-longleaf on the same plant was just beginning the sink-to-sourcetransition, as indicated by the small unlabeled region at theextreme tip (arrow). These experiments demonstrated that thefirst and second leaves of young amaranth seedlings beginthe developmental transition from carbon sink to carbon sourcewhen they are ~5 to 6 mm in length and complete the transi-tion at ~12 to 13 mm in length.

The sink-to-source transition period corresponded with theperiod when these leaves were undergoing the C3-to-C4 tran-sition. Immunolocalization and in situ hybridization analysisof sections taken from the detached lateral halves of theseleaves showed that bundle sheath cell-specific localizationof RuBPCase occurred only in regions of the leaves that wereunlabeled, whereas in the heavily labeled regions RuBPCasewas present in both bundle sheath and mesophyll cells. Theclose temporal and spatial correlation between patterns of pho-toassimilate transport and patterns of cell-specific RuBPCasegene expression is illustrated in Figures 3C to 3F. In Figure3C, an autoradiograph of one lateral half of a 7-mm-long leafshows that it is approximately midway through the sink-to-source transition. In the unlabeled apical (source) regions,

RuBPCase was specifically localized to bundle sheath cellsin the characteristic C4-type pattern (Figure 3D). Approximatelyone-third up from the base of the leaf, at the point betweenthe labeled and unlabeled regions, is the sink-to-source tran-sition zone. It was in this transition zone that RuBPCaseshowed an intermediate localization pattern, with levels in bun-dle sheath cells comparable to those in C4 regions and withgreatly reduced (but still detectable) levels in mesophyll cells(Figure 3E). Just below the transition zone, in the heavily la-beled basal (sink) regions, RuBPCase was present in bothbundle sheath and mesophyll cells in a C3-like localizationpattern (Figure 3F). These results indicate that two significantdevelopmental processes, the change in photoassimilate trans-port status and the development of C4 photosynthetic capacity,are tightly coordinated during C4 leaf development.

DISCUSSION

In leaves of amaranth, a dicotyledonous C4 plant, RuBPCaseLSD and SSU gene expression changes during leaf develop-ment from the initial default "C3-like" developmental pattern(RuBPCase is present in both bundle sheath and mesophyllcells; gene expression is not cell-type specific) to the morespecialized C4-type pattern of expression (RuBPCase is spe-cifically localized only to bundle sheath cells; gene expressionhas become cell-type specific). The findings presented heredemonstrate that this transition in the pattern of photosyntheticgene expression has developmental polarity, occurring fromleaf tip to leaf base, and is coupled to developmental changesin photosynthetic carbon metabolism.

In general, dicot leaves do not show strong developmentalpolarity in their overall patterns of cell growth and expansion(Sunderland, 1960; Scott and Possingham, 1982; Poethig, 1987;Steeves and Sussex, 1988). In dicot leaves, cell division gener-ates files of cells that are somewhat developmentally polarizedfrom the base to the leaf edge, but with further intercalary an-ticlinal divisions occurring in all directions. The processes ofcell division and expansion overlap in such a way that at anystage of development the dicot leaf consists of a mosaic of

Sink-to-Source Transition in a C4 Plant 293

Figure 3. Sink-to-Source Transition and C3-to-C4 Transition in Young Amaranth Leaves.

(A) to (C) Autoradiographs showing transition leaves that imported "C-labeled photoassimilate from cotyledons. Dark areas indicate the pres-ence of carbon-14 and define the sink regions; light regions indicate source tissue that did not import. For each seedling, the older and largerfirst emerging leaf had progressed further through the sink-to-source transition than the younger second emerging leaf, as indicated by differ-ences in labeling.(A) The larger 8-mm-long first leaf on this seedling was more heavily labeled in its basal than its apical half, indicating that it was approximatelymidway through the sink-to-source transition. The entire 3-mm-long second leaf on the same plant is still a sink and is uniformly labeled.(B) The larger 12-mm-long first leaf on this slightly older seedling has nearly completed the sink-to-source transition, while the transition is justbeginning in the smaller 6-mm-long second leaf on the same plant (indicated by arrow).(C) One lateral half of 7-mm-long leaf approximately midway through the sink-to-source transition.(0) Section from apical source region of the leaf shown in (C) reacted with RuBPCase LSU antiserum.(E) Section from sink-to-source transition zone of the leaf shown in (C) reacted with RuBPCase LSU antiserum.(F) Section from sink region of the leaf shown in (C), just below the transition zone, reacted with RuBPCase LSU antiserum.For analysis of photoassimilate transport, one cotyledon on each of the seedlings was labeled for 5 min with "CO2. After allowing either 25 or45 min for translocation (identical translocation patterns were observed for both labeling periods), the cotyledons and one lateral half of eachleaf were removed and processed for sectioning. The seedlings were then processed for autoradiography. Immunolocalization of the LSU wasperformed, as described in the legend to Figure 1. Bar = 100 urn.

cells of varying volumes and in different phases of develop-ment. Developmental polarity does occur in dicot leaveshowever, particularly during the differentiation of the vascularsystem. In dicotyledonous leaves, both acropetal (base-to-tip)and basipetal (tip-to-base) patterns of vascular development

have been shown to occur (Avery, 1933; Turgeon, 1989). Matu-ration of the midrib and the major veins occurs in the acropetaldirection, opposite to the direction of the C3-to-C4 transitionobserved in amaranth leaves, while maturation of the minorveins occurs in the basipetal direction. Developmental polarity

294 The Plant Cell

is also observed as dicot leaves undergo the transition from being carbon sinks, where they import and utilize carbon im- ported from other regions of the plant, to being carbon sources, where they produce an excess of photoassimilate and export it to other regions (Turgeon, 1989). The sink-to-source transi- tion occurs in the basipetal direction and is concurrent with the maturation of the minor veins.

The young amaranth leaves progressed through the sink- to-source transition during the same period when they were undergoing the C3-to-C4 transition, when they were at m5 to 10% of their final expansion. In contrast, the leaves of C3 dicots normally undergo the sink-to-source transition at a later de- velopmental stage, when they are at 30 to 60% of their final expansion (Turgeon, 1989). One possible explanation for the difference in timing of the sink-to-source transition in amaranth leaves versus the leaves of C3 dicots is that higher photosyn- thetic efficiency associated with the C4 pathway allows these leaves to begin producing and exporting photoassimilate much earlier than the leaves of C3 dicots.

The change in LSU and SSU gene expression occurred in tight coordination with the sink-to-source transition, so that as specific regions of the leaves went through the developmen- tal transition from carbon sink to carbon source, the cellular localization of RuBPCase gene expression in these regions shifted as well. What is the developmental signal that induces the basipetal development of differential C4-type gene expres- sion? If the sink-to-source transition and minor vein maturation are concurrent in amaranth leaves, as they are in the leaves of other dicots (Turgeon, 1989), then one possibility is that maturing veins in the transition zone might produce a signal that initiates the Wtype pattern of RuBPCase gene expression. In the leaves of maize, a C4 monocot, vascular differentiation has been correlated with the initial morphological differentia- tion of bundle sheath and mesophyll cells and with correct patterns of C4 gene expression (Langdale et al., l987,1988a, 1988b; Langdale and Nelson, 1991). In leaflike organs of maize that possess fewer and more widely separated veins, cells that are close to a vein show C4-type localization of RuBPCase to bundle sheath cells, whereas cells that are more distant show a C3-like pattern of RuBPCase localization. These observa- tions suggest that, in maize, the veins might produce or transmit factors required for the differentiation of the two cell types and for the cell-specific localization of the C4 enzymes.

In amaranth leaves, we have observed that C4-type expres- sion of RuBPCase genes does not correlate with the morphological differentiation of the bundle sheath cells and mesophyll cells, which occurs much earlier in leaf develop- ment. While the C3-to-C4 transition might be related to maturation of the smaller veins in the sink-to-source transition zone, it is clearly not correlated with the earlier acropetal matu- ration patterns of the larger high-order veins. Therefore, if C4 signaling factors in amaranth leaves are produced or trans- mitted from differentiating vascular centers, they would need to be specific to later basipetally maturing minor veins in the transition zone.

Another possibility is that the changes in patterns of RuBPCase gene expression might be directly related to

changes in carbon balance brought about by the sink-to-source transition. During the earlier "sink" phase of growth, a dicot leaf has a negative carbon balance and requires imported car- bohydrate, which is unloaded through the larger veins (Turgeon, 1989). As the leaf progresses through the sink-to-source tran- sition, the rate of photosynthesis increases and eventually reaches a leve1 that can meet and exceed the requirements of growth and respiration. This change in carbon balance is accompanied by changes in the accumulation of many pho- tosynthetic intermediates and end products, some of which could influence the expression of RuBPCase genes.

Severa1 studies have suggested that the expression of genes encoding photosynthetic enzymes, including RuBPCase, can be affected by levels of photosynthetic compounds. First, in maize mesophyll protoplasts, the presence of high concen- trations of sucrose and glucose in the medium greatly repressed transcription from severa1 C4 photosynthetic gene promoters, including the RuBPCase SSU (Sheen, 1990). For two of these C4 genes, pyruvate orthophosphate dikinase and chlorophyll alb binding protein, metabolic repression of tran- scription by photosynthetic end products was due to distinct upstream regulatory elements in their promoters. Second, transgenic tobacco plants that constitutively expressed yeast- derived invertase showed altered patterns of photoassimilate accumulation and photosynthetic activity (Stitt et al., 1990). In these transgenic plants, increased levels of carbohydrate led to decreased levels of RuBPCase as well as other pho- tosynthetic enzymes. These lines of evidence indicate that photosynthetic gene expression is sensitive to changes in levels of photosynthetic compounds, especially sucrose. Changes in carbon metabolism have been shown to affect the expres- sion of other plant genes as well. For example, in lines of transgenic potato where starch biosynthesis has been disrupted by expression of an antisense ADP-glucose pyrophosphory- lase gene, there are major changes in expression of sucrose synthesizing enzymes and tuber protein genes (Müller-Rober et al., 1992). We show here that the change to a positive car- bon balance during the sink-to-source transition in amaranth leaves is coordinated with the C3-to-C4 transition in the ex- pression of RuBPCase genes. Therefore, it is possible that changes in photoassimilate accumulation or transport are in- volved in signaling the initiation of C4-type RuBPCase gene expression during leaf development in amaranth.

In contrast to amaranth, light appears to be a major signal that influences C4-type gene expression in the C4 monocot maize (Sheen and Bogarad, 1985; Langdale et al., 1988b; Langdale and Nelson, 1991). In dark-grown (etiolated) maize seedlings, RuBPCase mRNAs and proteins are found in both bundle sheath and mesophyll cells in a C3-like pattern. When these seedlings are illuminated, RuBPCase decreases in mesophyll cells and increases in bundle sheath cells in a man- ner similar to that observed in the C3-to-C4 transition zone in amaranth. Monocot leaves do not undergo a basipetal sink- to-source transition as do dicot leaves, and it is possible that different signals influence C4 patterns of gene expression in these two plant groups. However, another interpretation might be that changes in photoassimilate accumulation or transport

Sink-to-Source Transition in a C4 Plant 295

brought about by the initiation of photosynthesis in the light could be used as a signal to initiate C4-type photosynthetic gene expression in monocots as well. Our experiments demon- strated that developmental changes in patterns of C4 photosynthetic gene expression are tightly coordinated with changes in photosynthetic transport status. Furthermore, they suggest that photosynthetic metabolism is linked to develop- mental processes that establish patterns of gene expression in higher plants.

METHODS

Plant Material and Gmwth Conditions

Seeds of Amaranthus hypochondriacus var 1023 were germinated and plants were grown in a Conviron growth chamber at 24% with 14-hr day illumination at an approximate intensity of 170 to 200 pE sec-l. Leaves were harvested from the plants at the appropriate time points.

lmmunolocallzation Analysls

Antibodies raised against the large subunit (LSU) and small subunit (SSU) of ribulose-l,5-bisphosphate carboxylase (RuBPCase) (Berry et ai., 1985) have been previously described. Leaf samples were fixed, embedded in paraffin, sectioned, and reacted with antisera as previ- ously described (Wang et al., 1992). Briefly, primary antiserum against the RuBPCase LSU or SSU was applied to the sections, followed by the application of R-phycoerythrin-conjugated secondary antibody (Sigma). Sections were visualized and photographed using a 20x ob- jective with an Axiovert 10 microscope (Zeiss, Oberkochen, Germany) using a 450-490, Fr510, LP520 filter systeni.

In Sltu Localizatlon of RuBPCase mRNAs

Plasmids used for generating sense and antisense RNA probes for the RuBPCase SSU and LSU have been previously described (Wang et al., 1992). Sense and antisense transcripts were synthesized and labeled in vitro with digoxigenin-11-UTP (Boehringer Mannheim) using T7 or T3 polymerase. Sections were prepared for in situ hybridization analysis according to the methods of Langdale and coworkers (1987, 1988a) and hybridized as previously described (Wang et ai., 1992). Hybridized transcripts were detected using anti-digoxigenin antisera conjugated to alkaline phosphatase in combination with a phosphatase detection system according to the manufacturer‘s recommendations (Boehringer Mannheim). Hybridizations were photographed with a BH-2 microscope using a 1Ox objective (Olympus, New Hyde Park, NY).

Photoassimilate Transport

The pattern of photoassimilate translocation was determined using a modification of previously described procedures (líurgeon and Webb, 1973; Leisner et ai., 1992). Briefly, one cotyledon was enclosed in a 0.5” microcentrifuge tube and sealed with modeling clay. The %Oz was generated inside the barrel of a 1-mL syringe by the addition of excess 80% lactic acid to 2 pL (106 mBq) NaZ14CO2 (ICN, Irvine, CA)

and injected into the sealed tube. The cotyledon was exposed to l4CO2 in the sealed tube for 5 min.

After allowing either 25 or 45 min for translocation (identical trans- location patterns were observed for both labeling periods), the cotyledons and one lateral half of each leaf were removed and processed for sectioning. The seedlings were then processed for au- toradiography as described previously (Weisberg et al., 1988). In brief, labeled seedlings were quickly placed between two stainless-steel screens to keep them flat and frozen by covering with powdered dry ice. Frozen seedlings were lyophilized (Virtis freeze dryer; Virtis Co., Gardiner, NY) for 3 days at -30% (condenser at -60%) and then flattened between polished steel plates in a large vise. The flattened seedlings were exposed for 3 days to x-ray film (Hyper film-Dmax, Amersham).

ACKNOWLEDGMENTS

We thank lan Baldwin, Mary Bisson, and Gerald Koudelka for critica1 review of the manuscript and Jim Stamos for preparing the illustra- tions. This work was supported by U.S. Department of Agriculture Grant NO. 91-37306-6323 to J.O.B.

Received December 7, 1992; accepted January 18, 1993.

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DOI 10.1105/tpc.5.3.289 1993;5;289-296Plant Cell

J. L. Wang, R. Turgeon, J. P. Carr and J. O. BerryExpression in a C4 Plant.

Carbon Sink-to-Source Transition Is Coordinated with Establishment of Cell-Specific Gene

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