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. 1999 Nov;121(3):731–741. doi: 10.1104/pp.121.3.731

Light Induction of Cell Type Differentiation and Cell-Type-Specific Gene Expression in Cotyledons of a C4 Plant, Flaveria trinervia1

Guoping Shu 1,2,*, Vincenza Pontieri 1, Nancy G Dengler 1, Laurens J Mets 1
PMCID: PMC59435  PMID: 10557221

Abstract

In Flaveria trinervia (Asteraceae) seedlings, light-induced signals are required for differentiation of cotyledon bundle sheath cells and mesophyll cells and for cell-type-specific expression of Rubisco small subunit genes (bundle sheath cell specific) and the genes that encode pyruvate orthophosphate dikinase and phosphoenolpyruvate carboxylase (mesophyll cell specific). Both cell type differentiation and cell-type-specific gene expression were complete by d 7 in light-grown seedlings, but were arrested beyond d 4 in dark-grown seedlings. Our results contrast with those found for another C4 dicot, Amaranthus hypochondriacus, in which light was not required for either process. The differences between the two C4 dicot species in cotyledon cell differentiation may arise from differences in embryonic and post-embryonic cotyledon development. Our results illustrate that a common C4 photosynthetic mechanism can be established through different developmental pathways in different species, and provide evidence for independent evolutionary origins of C4 photosynthetic mechanisms within dicotyledonous plants.

In contrast to C3 photosynthesis, in which the whole process of primary CO2 assimilation takes place autonomously within a single cell, C4 CO2 assimilation requires metabolic cooperation between bundle sheath cells (BSC) and mesophyll cells (MC) (Edwards and Walker, 1983; Anderson and Deardall, 1991; Suzuki et al., 1999). CO2 is first fixed by phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) in MC. The C4 acid formed diffuses to BSC, where it is decarboxylated and the CO2 released is refixed by Rubisco (EC 4.1.1.39). The substrate for PEPCase in the initial CO2 fixation, PEP, is regenerated by pyruvate phosphate dikinase (PPDK, EC 2.7.9.1) in MC (Hatch 1987, 1997; Furbank and Taylor, 1995). The foundation of this metabolic cooperation is the cell-type-specific compartmentation of photosynthetic enzymes and expression of the corresponding enzyme-coding genes.

Cell type specificity of the proteins and their mRNAs has been demonstrated in several monocot and dicot C4 species (Bauwe, 1984; Martineau and Taylor, 1985; Sheen and Bogorad, 1985, 1986; Langdale et al., 1988a, 1988b; Hudson et al., 1992; Wang et al., 1992; Dengler et al., 1995; Ramsperger et al., 1996; Shu, 1996; Drincovich et al., 1998). The genetic mechanisms that control the cell-type- and tissue-specific expression of genes encoding C4 enzymes have been studied with both molecular and transgenic approaches in C4 species (Berry et al., 1985, 1986; Martineau et al., 1989; Sheen, 1991; Matsuoka et al., 1993; Chitty et al., 1994; Bilang and Bogorad, 1996; Furbank et al., 1997; Marshall et al., 1997; Stockhaus et al., 1997; Taylor et al., 1997; Westhoff et al., 1997; Mann, 1999).

Light is one of the most important environmental signals regulating leaf development in plants, including leaf cellular differentiation and photosynthetic gene expression (Tobin and Silverthorne, 1985; Nelson and Langdale, 1992; Fankhauser and Chory, 1997). Light perception and the various light signal transduction pathways that regulate leaf development are currently under intensive study in C3 plants (Fankhauser and Chory, 1997). In foliage leaves of the C4 plant maize, light has been shown to play important roles in C4 cell type differentiation, direct activation of C4 photosynthetic genes at the transcriptional level, and establishment of cell type specificity of C4 gene expression (Nelson et al., 1984; Sheen and Bogorad, 1987a, 1987b; Langdale, et al., 1988a; Maroco et al., 1998). However, the role of light in cell type differentiation and cell-type-specific gene expression in C4 dicot foliage leaves is largely unknown. Previous studies have investigated the pattern of cell-type-specific gene expression in developing foliage leaves of several C4 dicots (Wang et al., 1992, 1993b; Dengler et al., 1995; Ramsperger et al., 1996; Berry et al., 1997), but the role of light in these processes was not directly addressed.

An important experimental approach in studying the light regulation of plant development is to compare changes in developmental and gene expression patterns between dark- and light-grown foliage organs or whole plants. In the C4 grass maize, several immature, primordial foliage leaves are present in the embryo and expand under dark-grown conditions. Thus, the role of light in regulating C4 gene expression and cell type differentiation can be assessed by comparing developing light- and dark-grown leaves (Nelson et al., 1984; Sheen and Bogorad, 1985, 1986, 1987a, 1987b; Langdale et al., 1988a, 1988b). However, in most dicot species, foliage leaves will not initiate or expand in dark-grown seedlings, so the comparative ontogenetic analysis used in C4 grasses cannot be applied (Dengler et al., 1997). For this reason, studies of the role of light in the development of photosynthetic tissues in C3 dicots have often exploited post-germination cotyledons, which can develop in both dark- and light-grown conditions and also have simpler developmental patterns than dicot foliage leaves (Tobin and Silverthorne, 1985; Tsukaya et al., 1994; Kretsch et al., 1995; Fankhauser and Chory, 1997).

In cotyledons of the dicot C4 plant Amaranthus hypochondriacs (Berry et al., 1985, 1986; Wang et al., 1993a), Wang et al. (1993a) found that light is not required for either C4 cell type differentiation or cell-type-specific gene expression. This finding contrasts with the results from studies of the foliage leaves of the C4 grass maize, in which light is required for differentiation between BSC and MC (Sheen and Bogorad, 1985; Langdale et al., 1988b). It is not clear whether the discrepancy found between maize foliage leaves and amaranth cotyledons reflects differences between foliage leaves and cotyledons, between monocot leaves and dicot leaves in general, or simply species-specific variation in developmental patterns. More comparative studies of cotyledon development among different C4 dicot species are needed to address these questions.

In this study, we assessed the role of light in the regulation of cell type differentiation and cell-type-specific expression of C4 photosynthetic genes in cotyledons of a dicot C4 plant, Flaveria trinervia. We examined cotyledon anatomical development and temporal and spatial mRNA accumulation of three C4 photosynthetic genes that encode the small subunit of Rubisco (RbcS), PPDK, and PEPCase in cotyledons of both dark- and light-grown seedlings of F. trinervia from d 0 to d 12 after sowing. We found that light is essential for full morphological differentiation of cotyledon BSC and MC and for cell-type-specific expression of C4 genes. Cell-type-specific gene expression was established between d 4 and d 7 after germination, coincident with maturation of Kranz anatomy in cotyledons. The light dependence of bundle sheath and mesophyll maturation in F. trinervia is more similar to that of maize than to that of amaranth. We suspect that the difference in light responsiveness and in patterns of C4 gene expression between the two C4 dicots F. trinervia and amaranth reflect species-specific differences in embryonic and post-embryonic cotyledon development.

MATERIALS AND METHODS

Plant Material

Seeds were harvested from a self-pollinated Flaveria trinervia plant. Seeds for dark- and light-grown seedlings were soaked in deionized, distilled water for 24 h at 24°C in either regular transparent (light-grown d 0) or light-proof glass beakers (dark-grown d 0). The imbibed seeds were sown in pots with commercial potting soil (PRO-MIX, Premier Horticulture, Red Hill, PA), and covered with 5 mm of vermiculite. The light-grown seedlings were grown in a growth room with 14 h of light/d (approximately 180 μE m−2 s−1) at 24°C. Twelve pots for dark-grown seedlings were prepared and sealed in an air-fluent dark box and kept in a dark room at 24°C. One pot was randomly sampled each day (every 24 h) after sowing; the pot was opened and the seedlings were fixed in complete darkness.

Tissue Preparation

Whole seedlings were fixed in 4% (w/v) paraformaldehyde for 24 h at 4°C and stored at 4°C in 70% (v/v) ethanol. The cotyledons for in situ hybridization were embedded in paraffin (Paraplast, Oxford Labware, St. Louis) and 6-μm sections were mounted on poly-d-Lys (Sigma, St. Louis)-coated slides. Sections from seedlings of different developmental stages were placed side by side on the same slides for hybridization with the same in situ hybridization probes. Cotyledons used for anatomical observations were dehydrated in an ethanol and acetone series, embedded in Spurr's resin, and sectioned at 2 μm with a diamond knife on an ultramicrotome (MT-7, RMC, Tucson, AZ). The sections were mounted on poly-d-Lys-coated slides and stained in 0.5% (w/v) toluidine blue O in 0.1% (w/v) sodium carbonate (O'Brien and McCully, 1981). Sections were photographed using a microscope (Polyvar, Reichert, Vienna, Austria) and Tmax film (Eastman-Kodak, Rochester, NJ).

mRNA in Situ Hybridization

The DNA templates used for generating sense and antisense RNA probes were as follows: RbcS was from a 400-bp cDNA fragment that covers exons II and III of the RbcS-R1 gene from Flaveria ramosissima; it was cloned in pBlueScript II SK(+) (Shu, 1996), Ppc was a 1.82-kb cDNA fragment from the 3′ region of the gene (3 kb) that encodes a C4 isoform of PEPCase from F. trinervia (Hermans and Westhoff, 1992) Pdk was from a 1.3-kb cDNA fragment of the 5′ region of the gene (3.1 kb from F. trinervia (Hermans and Westhoff, 1992; Rosche and Westhoff, 1995). Both Pdk and Ppc cDNA clones were kind gifts from Dr. Peter Westhoff.

Digoxigenin-labeled sense and antisense RNA probes were generated by in vitro transcription using T3 and T7 RNA polymerase (Boehringer Mannheim, Basel). Probe hydrolysis followed Langdale et al. (1988a). Slide pretreatment, prehybridizaton, and hybridization were modified from Langdale et al. (1988a) and Wang et al. (1992). Proteinase K treatment was 20 μg/mL for 20 min at 37°C, and RNase A treatment was 5 μg/mL for 20 min at 37°C. The prehybridization and hybridization solution (1,000 μL) contained 125 μL of 10× in situ salts (3.0 m NaCl, 0.1 m Tris, pH 6.8, 0.1 m sodium phosphate, pH 6.8, and 50 mm EDTA), 500 μL of deionized formamide, 250 μL of 50% (w/v) dextran sulfate, 25 μL of 20 μg/mL tRNA, 60 μL of 5 μg/mL poly(A+), and 40 μL of distilled, deionized water. Hybridization was at 50°C overnight. The highest washing stringency was 0.25× SSC at 42°C for 30 min. Immunodetection and colorimetric reaction followed protocols from Boehringer Mannheim. The color substrates nitroblue tetrazolium and X-phosphate were used. Slides were photographed using dark-field microscopy (Labophoto, Nikon, Tokyo) on Ektachrome 64T or 160T (Kodak).

RESULTS

Seedling Morphology, Cotyledon Development, and Cell Type Differentiation

In the light (see “Materials and Methods” for growth conditions used), the radicle of a F. trinervia seedling emerged from the seed coat by d 3 (48 h after sowing in soil). Two cotyledons emerged from the soil surface by d 4 (after 72 h). Newly emerged cotyledons were 2 mm long and yellowish opaque. Greening was first observed on the upper or adaxial side of the cotyledons and then on the lower or abaxial side by d 5 (after 96 h). The cotyledons expanded to 4 mm long by d 7 (after 144 h) and remained green through d 25 before undergoing senescence. The first foliage leaf was visible by d 10 and became fully expanded by d 15. When the seeds were germinated in the dark, radicle protrusion was delayed by 12 h. The 4-d-old seedling had an elongated hypocotyl and yellowish-white cotyledons about 1.5 mm long that were partly enclosed in the seed coat. Beyond d 4, the hypocotyl remained elongated and hooked, and cotyledon expansion was arrested. The whole seedling started to shrink by d 9 and died by d 18. Foliage leaves did not expand in dark-grown seedlings.

Figure 1, A to C, shows cotyledon anatomical development in light-grown seedlings. Kranz anatomy was present in a rudimentary form in the cotyledons of d 0 seedlings (after 24 h of soaking the seeds in distilled, deionized water) (Fig. 1A). Cotyledon vasculature was still forming at this stage, but veins already present had a well-defined bundle sheath layer. The mesophyll was five layers thick and strongly dorsiventral, with a well-defined adaxial palisade layer (layer 1). BSC and MC had numerous storage bodies and lacked differentiated plastids (Fig. 1A); proplastids were present but lacked well-developed thylakoids (V. Pontieri and N.G. Dengler, unpublished results). After 4 d in the light, BSC and MC became somewhat enlarged, storage bodies disappeared, and plastids were visible at the light microscope level. The intercellular space also expanded and some stomata appeared mature (Fig. 1B).

Figure 1.

Figure 1

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Transverse sections of post-germination cotyledons of light- and dark-grown seedlings of F. trinervia. A, d 0, Light-grown; B, d 4, light-grown; C, d 7, light-grown; D, d 0, dark-grown; E, d 5, dark-grown; F, d 10, dark-grown. Bar = 50 μm. am, Abaxial MC; bs, BSC; i, intercellular space; pm, palisade MC; s, stomata; sm, spongy MC; unlabeled arrows, chloroplasts.

BSC showed a clear differentiation from other MC, with larger, centripetally located chloroplasts. This pattern of plastid localization contrasts with the centrifugal location seen in NADP-malic enzyme-type C4 grass species (Hattersley and Browning, 1981; Dengler et al., 1996). By 7 d in the light, the Kranz anatomy became mature, with BSC and MC undergoing further enlargement and intercellular space becoming extensive (Fig. 1C). MC not directly adjacent to BSC did not develop plastids visible under the light microscope. Chloroplasts of both cell types had well-developed thylakoid membranes. Chloroplasts in MC formed grana, while these were inconspicuous or lacking in BSC (V. Pontieri and N.G. Dengler, unpublished results).

Figure 1, D and E, shows that in dark-grown seedlings, cotyledon anatomical development was similar to that of the light-grown seedlings between d 0 and d 4. In the dark, development is arrested at this stage, and from d 7 to 10, dark-grown BSC and MC remained small, with small and undifferentiated pro-plastids (Fig. 1F). The cellular expansion characteristic of the maturation phase of Kranz anatomy development in the light did not occur in the dark. There was also very little overall cotyledon expansion in dark-grown seedlings.

While MC expansion was evident in both light- and dark-grown cotyledons, it occured earlier and to a greater extent in the light (Fig. 1). The length to width ratio of palisade-like MC decreased from d 4 to d 7 and was more pronounced in light-grown cotyledons (Fig. 1). The isodiametric form of non-palisade MC was maintained during cell expansion through d 7 (Fig. 1). The dorsiventral mesophyll was moderately developed in the foliage leaves and cotyledons of F. trinervia, and was also evident in both cotyledons and foliage leaves of a C3 species in the same genus, Flaveria pringlei (Fig. 2F; V. Pontieri and N.G. Dengler, unpublished results). This dorsiventral mesophyll pattern was less conspicuous in the cotyledons and foliage leaves of the C4 dicot Amaranthus hypochondriacus (Wang et al., 1992, 1993a).

Figure 2.

Figure 2

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RbcS mRNA accumulation patterns detected by mRNA in situ hybridization to transverse sections of the post-germination cotyledons of light- and dark-grown seedlings of F. trinervia (C4) (A–E) and F. pringlei (C3) (F). A, d 4, Light-grown, RbcS RNA antisense; B, d 7, light-grown, RbcS RNA antisense; C, d 9, light-grown, RbcS RNA antisense; D, d 4, dark-grown, RbcS RNA antisense, the seed coat is visible; E, d 7, dark-grown, RbcS RNA antisense; F, d 9, light-grown, RbcS RNA antisense, a section from F. pringlei (C3). Cotyledon pairs were appressed while embedding in paraffin so that their adaxial sides were adjacent to each other. Transverse sections (6 mm) were taken from a region midway between the apex and base of cotyledons. Bar = 320 μm.

Light Induction of BSC-Specific RbcS mRNA Accumulation

We examined the temporal and spatial accumulation patterns of RbcS mRNA in the cotyledons of both dark- and light-grown seedlings d 0 to d 12 after seed sowing, and the results are shown in Figure 2. The cotyledons of light- and dark-grown seedlings from d 0 to d 3 lacked detectable RbcS mRNA by our in situ hybridization techniques (data not shown).

In light-grown seedlings, accumulation of RbcS mRNAs in BSC was observed at d 4 and the level continued to increase up to d 7 (Fig. 2, A and B). Abundant accumulation of RbcS mRNA was also detected in MC at d 4 (Fig. 2A), but the level continuously decreased and became undetectable in the cotyledons of d 7 and older seedlings (Fig. 2, B and C). At d 4, dark-grown seedlings showed a low level of RbcS mRNA in both BSC and MC (Fig. 2D); the level of RbcS mRNA accumulation remained unchanged by d 6 and was found to decline slightly in both BSC and MC by d 7 (Fig. 2E). RbcS mRNA was not detectable in the cotyledons of d 9 and older seedlings under dark-grown conditions (data not shown). The RbcS mRNA accumulation patterns observed in our in situ hybridization studies fell into a similar temporal scheme to that reported for light- and dark-grown foliage leaves of maize seedlings (Nelson et al., 1984). For comparison, we also examined the pattern of RbcS mRNA accumulation in the cotyledons of a C3 Flaveria species, F. pringlei. We detected abundant RbcS mRNA accumulation in all cotyledon photosynthetic cell types of light-grown seedlings, with the highest level in adaxial palisade MC (Fig. 2F). This resembles the pattern observed in the dark-grown cotyledons of F. trinervia (Fig. 2, D and E), although the accumulation level was much higher.

The above results suggest that in the cotyledons of F. trinervia, the expression of RbcS genes is developmentally controlled, so that RbcS mRNAs accumulate in both cell types independent of light conditions. However, in the presence of light, RbcS gene expression was either down-regulated or repressed in MC and up-regulated in BSC in d 5 and older cotyledons. The RbcS mRNA accumulation pattern in dark-grown cotyledons was C3 like in terms of cell specificity and remained unchanged after d 5.

Light Induction of MC-Specific Pdk and Ppc mRNA Accumulation

We also examined the accumulation of Pdk and Ppc mRNA by mRNA in situ hybridization and daily sampling from d 0 to d 12 (Fig. 3). In MC of the light-grown cotyledons, we detected a dorsiventral gradient of mRNA accumulation for both Pdk and Ppc at d 4: high levels of mRNA accumulated in palisade-like MC (layer 1) and very low levels in other MC (layers 2–4) (Figs. 1 and 3, A and D). By d 5, high levels were detected in the MC of layers 2 to 4 (Fig. 3B). The level of mRNA accumulation decreased after d 5 and reached the steady state at d 7. From d 7 to d 12, the mRNA transcripts were detected in all MC except in the abaxial mesophyll (layer 5), which is adjacent to the lower epidermal cells and lacks direct contact with the BSC (Figs. 1C and 3, C and E). The absence of Pdk and Ppc expression in this mesophyll layer has also been observed in the foliage leaves of this species (Shu, 1996). The absence of Ppc mRNA accumulation in MC distal from BSC has also been reported for husk leaves of maize and foliage leaves of Atriplex rosea (Langdale et al., 1988b; Dengler et al., 1995).

Figure 3.

Figure 3

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Pdk and Ppc mRNA accumulation patterns in F. trinervia cotyledons. A to C and F to H, Pdk mRNA accumulation patterns. D, E, I, and J, Ppc mRNA accumulation patterns. A, d 4, Light-grown, Pdk RNA antisense; B, d 5, light-grown, Pdk RNA antisense; C, d 7, light-grown, Pdk RNA antisense; D, d 4, light-grown, Ppc RNA antisense; E, d 7, light-grown, Ppc RNA antisense; F, d 4, dark-grown, Pdk RNA antisense; G, d 4, dark-grown, Pdk RNA sense probe; H, d 7, dark-grown, Pdk RNA antisense; I, d 7, dark-grown, Ppc RNA antisense; J, d 7, light-grown, Ppc RNA sense. Bar = 320 μm. For details, see Figure 2 legend. Under dark-field microscopy, the abundance of mRNA accumulation corresponds to the color saturation: yellowish red indicates low abundance and dark red indicates high abundance.

In BSC of light-grown cotyledons, low levels of accumulation were observed from d 5 to d 6 for Pdk mRNAs (Fig. 3B), but were undetectable by d 7 (Fig. 3C). A low level of RNA accumulation remained detectable in BSC of the d 7 and older seedlings using both the sense and antisense endogenous Ppc probes (Fig. 3E) at the same high hybridization stringency (Fig. 3, E and J).

We found that the temporal and spatial patterns of Ppc mRNA accumulation were very similar to those of Pdk mRNA, with two exceptions: (a) the steady-state level of Ppc mRNA in the MC of the expanded cotyledons (from d 7 to d 12) was much higher than that of Pdk mRNA (Fig. 3, C and E), which is consistent with the estimates from RNA blotting analysis of mRNA from the maize leaf blade (Sheen and Bogorad, 1987b; Langdale and Kidner, 1994); (b) a low level of accumulation of unknown RNA transcripts was detected in the BSC of light-grown cotyledons with both sense and antisense endogenous Ppc probes, whereas the mRNA accumulation detected with endogenous Pdk gene probes was clearly MC specific in the 7 d and older cotyledons of light-grown seedlings.

In the cotyledons of dark-grown seedlings, no mRNA accumulation for either gene was detected between d 0 and d 3. The level of Pdk and Ppc mRNA accumulation in the palisade mesophyll was higher than in the other mesophyll layers or in BSC between d 4 and d 7 (Fig. 3, F, H, and I).

The accumulation patterns of RbcS, Pdk, and Ppc mRNAs in the expanded light-grown cotyledons of 7 d and older seedlings were essentially the same as those detected in the expanded foliage leaves of this species (Shu, 1996). Studies of both anatomy and photosynthetic physiology have shown that the foliage leaves of F. trinervia are typical C4 organs (Ku et al., 1991). Although no data on photosynthetic physiology are available for the cotyledons, our results concerning anatomy and gene expression patterns indicate that the expanded cotyledons of light-grown F. trinervia could function as typical C4 photosynthetic organs and undergo C4 CO2 assimilation.

DISCUSSION

Light Is Required for Full Differentiation of Photosynthetic Cell Types in C4 F. trinervia Cotyledons

Post-germination cotyledon development in F. trinervia appears to include two phases: a light-independent phase, or post-germination I, and a light-dependent phase, or post-germination II. From d 0 to d 4, both light- and dark-grown seedlings shared similar developmental patterns, such as nutrient reserve breakdown, repositioning and differential growth of plastids so that BSC have enlarged, and centripetally placed plastids, while MC had small, peripherally positioned plastids and C3-like photosynthetic gene expression. Light is therefore apparently not required for these developmental processes. We refer to this phase as post-germination I (Fig. 4A).

Figure 4.

Figure 4

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Differences in cotyledon and seedling development between two C4 dicotyledonous species, F. trinervia (A) and A. hypochondriacus (B). Embryogenesis, Formation of the embryo proper and endosperm; the two species represent two types of embryogenesis patterns in angiosperms. Post-embryogeny, Absorption of endosperm and seed formation in both species; cotyledon expansion in A, perisperm formation in B; cotyledon cell differentiation and light-induced chloroplast formation take place. Maturation, Nutrient reserve deposition; lipid and protein deposition in cotyledons for both species, starch deposition in cotyledons for A and in perisperm for B. Desiccation, Dehydration and dormancy of seedlings. Germination, From seed imbibition to rupture of testa by radicle. Post-germination I, C3-like development such as mobilization of cotyledon nutrient reserves, resumption of cell type differentiation, and non-cell-type-specific C3 and C4 gene expression. Post-germination II, Full C4 development such as maturation of Kranz anatomy and establishment and maintenance of cell-type-specific C4 gene expression. The two species employ two different subtypes of decarboxylation pathways and show different developmental patterns in light- and dark-grown conditions (shaded). C4 competence, Hypothetical period or developmental window when essential light-induced signals for C4 development and cell-type-specific C4 gene expression are generated.

From d 5 to d 7, light- and dark-grown seedlings showed divergent development patterns. In light-grown seedlings, we observed arrest of hypocotyl elongation, continued BSC and MC expansion and redifferentiation, and high-level, cell-type-specific mRNA accumulation of the three C4 photosynthetic genes in the cotyledons. In contrast, we observed continuous hypocotyl elongation, arrest of cell expansion and redifferentiation, and low and nonspecific mRNA accumulation for all three genes in dark-grown seedlings. Therefore, light is essential for the full differentiation of cotyledon BSC and MC. We refer to this phase as post-germination II (Fig. 4A).

Our division of post-germination cotyledon development into two phases is based solely on the observation that some development can proceed in both light- and dark-grown seedlings. It is known that regulatory proteins and mRNAs synthesized in the post-embryogeny phase can be stored in various forms in mature and desiccated seeds and be mobilized in the post-germination phases to regulate gene activities (Goldberg et al., 1989; Kretsch et al., 1995). Thus, it is possible that light-independent developmental processes in dark-grown seedlings of post-germination I are actually light dependent and regulated by light-induced signals synthesized during the post-embryogeny phase of seed development.

We found that some veins with morphologically distinctive BSC and associated mesophyll layers were already present in d 0 imbibed embryos, but that fully mature Kranz anatomy, including substantial cell expansion and the formation of extensive intercellular space in the abaxial mesophyll did not develop until d 7. Thus, it is likely that C4 cell type development is initiated in either late embryogenesis or in the post-embryogeny phase of the parental plant, but that differentiation processes are arrested by nutrient storage and seed desiccation. Further development of Kranz anatomy is resumed during the post-germination I phase and is completed during the post-germination II phase only in light-grown cotyledons.

Based on our observations, the completion of the transition from post-germination I to post-germination II in the cotyledons of F. trinervia requires light-induced signals that contribute to the competence for further C4 development (Fig. 4). C4 competence is likely attained around d 5 under light-grown conditions. Previous studies indicate that C4 competence is also required for foliage leaf development in both monocot and dicot C4 species, since young leaves that have not obtained C4 competence always show C3-like gene expression patterns even in light-grown conditions (Sheen and Bogorad, 1985; Langdale et al., 1988b; Wang et al., 1992, 1993b; Dengler et al., 1995).

Establishment of Cell-Type-Specific C4 Gene Expression Is Light Dependent

In the present study, we found that light is essential for inducing both high-level and cell-type-specific accumulation of RbcS, Pdk, and Ppc mRNAs in post-germination cotyledons of light-grown seedlings. The two events, up-regulation and the cell-type-specificity of C4 gene expression, are likely regulated by different light-induced signals. While up-regulation of the three C4 genes was observed by d 4 (right after seedling emergence), initial mRNA accumulation patterns were not cell type specific. Complete cell-type-specific patterns for RbcS and Pdk genes were only finalized at d 7. The difference of 3 d between the two events indicates that the cell type specificity of C4 gene expression is likely regulated by light-induced developmental signals or conditions, rather than by direct light action on C4 gene transcription. A study of maize foliage leaves using biolistic gene transfer methods showed that red light, though important in inducing rapid up-regulation of C4 genes and chloroplast development, is not sufficient to suppress RbcS gene transcription in improper cell types such as maize epidermal cells (Bilang and Bogorad, 1996).

Recently, transgenic studies using GUS fusion constructs from different sequences at the 5′ and 3′ ends of Pdk genes, Ppc, and NADP-malic enzyme genes have shown that control of cell type specificity and the level of gene expression use different cis-acting elements (Taylor et al., 1997; Westhoff et al., 1997). The cis-acting elements involved in controlling cell-type-specific expression of the C4-enzyme-coding genes are not found in the genes that encode enzymes of C3 isoforms (Sheen, 1991; Chitty et al., 1994; Stockhaus et al., 1994, 1997; Furbank et al., 1997; Marshall et al., 1997). All of the above results suggest that the regulation of C4 gene expression is 2-fold: regulation of the steady-state level of gene activity, a common theme shared with C3 plants, and regulation of cell type specificity of gene expression, a unique feature of C4 genes.

We detected a low level of hybridization signal in cotyledon BSC with the antisense Ppc mRNA probe (Fig. 3E). We also detected a similar level of hybridization signal in both BSC and MC at the same high hybridization stringency with the sense riboprobe of Ppc (Fig. 3J). Therefore, it is likely that the signal detected in BSC with both Ppc sense and antisense probes are background noise, and that the accumulation of Ppc and Pdk mRNAs are MSC specific.

We found that an increase in Pdk and Ppc mRNA levels in MC of light-grown cotyledons coincided with cotyledon greening. High levels of mRNA accumulation for both genes begins in the palisade MC (layer 1) by d 4 and extends to the abaxial side of the cotyledons by d 5. Dorsiventral chlorophyll gradients that cause the dorsiventral cotyledon greening patterns have also been reported in the post-germination cotyledons of a C3 plant, Cucurbita pepo (Knapp et al., 1988), and have been suggested to be due to differential exposure to blue light along the dorsiventral axis of the cotyledons (Knapp et al., 1988). It remains to be investigated whether the Pdk and Ppc mRNA gradients seen in F. trinervia might have been caused by a blue light gradient.

Cell-Type-Specific C4 Gene Expression Occurs during Maturation of Morphological Cell Type Differentiation

An important question in understanding C4 development is whether light induction of C4 gene expression is coupled with leaf development and leaf cell type differentiation (Nelson and Dengler, 1992; Liu and Dengler, 1994; Berry et al., 1997). Our results show that in F. trinervia, accumulation of mRNA for genes involved in C4 carbon metabolism occurs as morphological cell type differentiation becomes mature. BSC and MC differentiation proceeded simultaneously in post-germination cotyledons and full differentiation of BSC and MC were observed by d 7, which is also the time when cell-type-specific mRNA accumulation for all three genes takes place. In contrast to our findings for F. trinervia cotyledons, in young foliage leaves of this species the establishment of a MC-specific pattern of Ppc and Pdk expression was conspicuously delayed in relation to the establishment of a BSC-specific pattern of RbcS gene expression. The same delay of Ppc and Pdk expression relative to that of RbcS is seen in the young foliage leaves of maize, amaranth, and Atriplex rosea (Langdale et al., 1988a; Schäffner and Sheen, 1992; Wang et al., 1992; Dengler et al., 1995; Shu, 1996). This distinction between cotyledons and foliage leaves may reflect a difference in the timing of cell proliferation relative to differentiation. In cotyledon development, cell proliferation only occurs during embryogenesis, and post-germination cotyledon cells do not divide (Tsukaya et al., 1994; Kretsch et al., 1995). In contrast, developing foliage leaves undergo considerable cell proliferation, and BSC surrounding the veins may be delimited before cell division within the MC ceases (Dengler et al., 1996).

Our data support the view that signals involved in cell-type-specific regulation of C4 gene expression are positional relative to bundle sheath or vascular tissues (Langdale and Nelson, 1991; Nelson and Langdale, 1992). It remains unclear whether the up-regulation of Pdk and Ppc and the suppression of RbcS genes in the same MC are regulated by the same positional signals. In maize husk leaves, the absence of Ppc mRNAs and the presence of rbcL and RbcS mRNAs are found to coincide in the MC that do not have direct contact with the BSC (Langdale, 1988b; Langdale and Nelson, 1991). We did not observe the same coincidence in the cotyledons of F. trinervia. No RbcS mRNA accumulation was detected in the fifth layer of MC, where the Pdk and Ppc mRNAs are absent. This observation suggests that up-regulation of Pdk and Ppc and suppression of RbcS genes in MC, though normally coincident, may be controlled by different signals. Results from promoter analyses, mutant studies, and transgenic studies using different C4 photosynthetic genes in Flaveria sp. also suggest that there is no universal regulation mechanism among different C4 photosynthetic genes (Langdale and Kidner, 1994; Furbank et al., 1997; Taylor et al., 1997; Westhoff et al., 1997).

F. trinervia and A. hypochondriacus Differ in C4 Gene Expression Pattern and Cotyledon Development

Our results demonstrate that in F. trinervia, cell-type-specific expression of genes involved in C4 carbon metabolism in the post-germination cotyledons only takes place in light-grown seedlings, not in dark-grown seedlings. This is true in spite of the existence of clear morphological differentiation between BSC and MC in the cotyledons. This finding emphasizes the fact that light is essential for the establishment and maturation of differential expression of these genes. The light requirement for differential expression of rbcS, PPDK, and PEPCase mRNA in F. trinervia is in marked contrast to the observed light independence of their differential expression in the cotyledons of another C4 dicot, A. hypochondriacus (Amaranthaceae) (Wang et al., 1993a). Wang et al. (1993a) found that the accumulation of PPDK mRNA and protein and PEPCase protein are MC specific in cotyledons of both dark- and light-grown 2-d-old seedlings. RbcS mRNAs and polypeptides were not cell type specific at d 2, but became BSC specific at d 5 in both dark- and light-grown cotyledons. Thus, light is not required for the establishment of cell-type-specific C4 gene expression in this species (Wang et al., 1993a). The underlying mechanisms that lead to this difference between the two dicot species is unclear.

It is possible that the apparent difference in light requirement for cell-type-specific C4 gene expression between the two species is associated with their difference in seedling development and cotyledon cell type differentiation. Previous studies have shown that F. trinervia and A. hypochondriacus have strikingly different patterns of embryogenesis, post-embryogenic development, and post-germination development, and these are summarized in Figure 4. Embryogenesis in angiosperms is classified into six types (Johri et al., 1992): F. trinervia is an Asterad type, whereas A. hypochondriacus is a Chenopodiad type (Misra, 1964; Coimbra and Salema, 1994). Dicot plants are also classified into at least three types based on seed nutrient storage location: cotyledon storage, such as A. hypochondriacus endosperm storage, or perisperm storage. The three types show distinctive temporal patterns of cotyledon development (Johri et al., 1992; Bewley and Black, 1994; Kaplan and Cooke, 1997).

Both Flaveria and Amaranthus spp. have nuclear endosperm that starts to form at the heart stage of embryogenesis. At the post-embryogeny phase, endospermis is gradually absorbed by the growing embryo. In amaranth, a portion of the nucellus converts into a perisperm storage tissue (Fig. 4B). Carbohydrates are stored as starch grains in perisperm plastids, but starch grains are absent from cotyledon plastids (Coimbra and Salema, 1994). In Flaveria, cotyledons and hypocotyl convert to nutrient storage organs during post-embryogenic development, and cotyledon cells are filled with protein bodies, lipids, and plastids with starch grains (Misra, 1964).

Previous studies have shown that the presence of carbohydrates suppresses C4 gene transcription in isolated maize leaf MC (Sheen, 1990). In developing foliage leaves of A. hypochondriacus, a tight coordination between cell-type-specific C4 gene expression and the state of carbon metabolism or sink-source transition have been reported (Wang et al., 1993b; Berry et al., 1997). It is plausible that amaranth cotyledons, which lack starch deposition, could have faster photosynthetic development than the Flaveria cotyledons in the post-embryogeny phase, to become more leaf-like and obtain light-induced C4 competence before seed desiccation (Fig. 4). Further detailed comparative study is needed to confirm this speculation.

The temporal differences in cotyledon development and C4 gene expression between A. hypochondriacus and F. trinervia are likely an evolutionary adaptation of each species to its germination environment. The great diversity among different plant species in seed structure, germination strategy, and corresponding temporal control of light-regulated genes is well documented (Stebbins, 1974; Johri et al., 1992; Bewley and Black, 1994).

The differences between these two C4 dicot species in C4 development of cotyledons provide evidence that independent evolutionary origins of C4 photosynthetic mechanisms need not arise by the evolution of common developmental control pathways. A third distinctive developmental pattern has been recognized in species of Haloxylon (Chenopodiaceae) that show C4 photosynthesis in the single large subepidermal BSC/MC ring surrounding assimilatory shoots (Pyankov et al., 1999). The reduced leaves of these plants are nonphotosynthetic, but the cotyledons are C3 and show no morphological differentiation among the photosynthetic cells at any stage of development (Pyankov et al., 1999).

Evidence of independent evolution of C4 photosynthetic pathways has been reported and reviewed for grass species (Hattersley and Browning, 1981; Hattersley, 1984; Dengler et al., 1985, 1986; Sinha and Kellogg, 1996). Extensive comparative studies of C4 gene expression and post-germination cotyledon development across different taxa of C4 dicots are promising to bring new insight into the evolution of developmental mechanisms in general and the evolution of this complex adaptation in particular.

ACKNOWLEDGMENTS

We are grateful to Peter Westhoff for providing the Pdk and Ppc cDNA clones and Jane Langdale and Jing-liang Wang for the in situ hybridization protocols. Our thanks also go to our colleagues Hewson Swift, Gayle Lamppa, Martin Kreitman, and Manfred Ruddat for helpful discussion. Aida Pascual, Mary Crane, and Maya Moody provided valuable technical assistance. We thank Sue Yamins and her greenhouse staff for taking care of seedstock plants. We thank J. Sheen and E.A. Kellog and anonymous reviewers for their thoughtful comments.

Footnotes

1

This work was supported by National Science Foundation and Department of Energy grants to L.J.M., a Natural Sciences and Engineering Research Council of Canada grant to N.G.D., and a Hutchins Plant Biology Predoctoral Fellowship to G.S. G.S. is a trainee of a National Institutes of Health Genetics and Regulation Training Grant.

LITERATURE CITED

  1. Anderson JW, Deardall J. Molecular Activities of Plant Cells: An Introduction to Plant Biochemistry. Oxford, UK: Blackwell Scientific Publications; 1991. [Google Scholar]
  2. Bauwe H. Photosynthetic enzyme activities and immunofluorescence studies on the localization of ribulose-1,5-bisphosphate carboxylase/oxygenase in leaves of C3, C4, and C3-C4 intermediate species of Flaveria (Asteraceae) Biochem Physiol Pflanz. 1984;179:253–268. [Google Scholar]
  3. Berry JO, McCormac DJ, Long JJ, Boinski J, Corey AC. Photosynthetic gene expression in amaranth, an NAD ME type C4 dicot. Aust J Plant Physiol. 1997;24:423–428. [Google Scholar]
  4. Berry JO, Niklou JP, Carr JP, Klessig DF. Transcriptional and post-transcriptional regulation of ribulose-1,5-bisphosphate carboxylase gene expression in light- and dark-grown amaranth cotyledons. Mol Cell Biol. 1985;5:2238–2246. doi: 10.1128/mcb.5.9.2238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berry JO, Niklou JP, Carr JP, Klessig DF. Translational regulation of light-induced ribulose-1,5-bisphosphate carboxylase gene expression in amaranth. Mol Cell Biol. 1986;6:2347–2353. doi: 10.1128/mcb.6.7.2347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bewley JD, Black M. Seeds: Physiology of Development and Germination. Ed 2. New York: Plenum Press; 1994. [Google Scholar]
  7. Bilang R, Bogorad L. Light-dependent developmental control of RbcS genes expression in epidermal cells of maize leaves. Plant Mol Biol. 1996;31:831–841. doi: 10.1007/BF00019470. [DOI] [PubMed] [Google Scholar]
  8. Chitty JA, Robert TF, Jerry SM, Chen Z, Taylor WC. Genetic transformation of the C4 plant Flaveria bidentis. Plant J. 1994;6:949–956. [Google Scholar]
  9. Coimbra S, Salema R. Amaranthus hypochondriacus: seed structure and localization of seed reserves. Ann Bot. 1994;74:373–379. [Google Scholar]
  10. Dengler NG, Dengler RE, Donnelly PM, Filosa MF. Expression of the C4 pattern of photosynthetic enzymes in Atriplex rosea (Chenopodiaceae) Am J Bot. 1995;82:318–327. [Google Scholar]
  11. Dengler NG, Dengler RE, Hattersley PW. Differing ontogenetic origins of PCR (“Kranz“) sheaths in leaf blades of C4 grasses (Poaceae) Am J Bot. 1985;72:284–302. [Google Scholar]
  12. Dengler NG, Dengler RE, Hattersley PW. Comparative bundle sheath and mesophyll differentiation in the leaves of the C4 grass Panicum effusum and P. bulbosum. Am J Bot. 1986;73:1431–1442. [Google Scholar]
  13. Dengler NG, Donnelly PM, Dengler RE. Differentiation of bundle sheath mesophyll, and distinctive cells in the C4 grass Arundinella hirta (Poaceae) Am J Bot. 1996;83:1391–1405. [Google Scholar]
  14. Dengler NG, Woodvine MA, Donnelly PM, Dengler RE. Formation of vascular pattern in developing leaves of the C4 grass Arundinella hirta. Int J Plant Sci. 1997;158:1–12. [Google Scholar]
  15. Drincovich MF, Casati P, Andreo CS, Chessin SJ, Franceschi CV, Edwards GE, Ku MSB. Evolution of C4 photosynthesis in Flaveria species: isoforms of NADP-malic enzyme. Plant Physiol. 1998;117:733–744. doi: 10.1104/pp.117.3.733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Edwards G, Walker D. C3, C4: Mechanism, and Cellular and Environmental Regulation of Photosynthesis. Oxford, UK: Blackwell Scientific Publications; 1983. [Google Scholar]
  17. Fankhauser C, Chory J. Light control of plant development. Annu Rev Cell Dev Biol. 1997;13:203–229. doi: 10.1146/annurev.cellbio.13.1.203. [DOI] [PubMed] [Google Scholar]
  18. Furbank RT, Chitty JA, Jenkins CLD, Taylor WC, Trevanion SJ, Von Caemmerer S, Ashton AR. Genetic manipulation of key photosynthetic enzymes in the C4 plant Flaveria bidentis. Aust J Plant Physiol. 1997;24:477–485. [Google Scholar]
  19. Furbank RT, Taylor WC. Regulation of photosynthesis in C3 and C4 plants: a molecular approach. Plant Cell. 1995;7:797–807. doi: 10.1105/tpc.7.7.797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Goldberg RB, Barker SJ, Perez-Grau L. Regulation of gene expression during plant embryogenesis. Cell. 1989;56:149–160. doi: 10.1016/0092-8674(89)90888-x. [DOI] [PubMed] [Google Scholar]
  21. Hatch MD. C4 photosynthesis: a unique blend of modified biochemistry, anatomy, and ultrastructure. Biochim Biophys Acta. 1987;895:81–106. [Google Scholar]
  22. Hatch MD. Resolving C4 photosynthesis: trials, tribulations and other unpublished stories. Aust J Plant Physiol. 1997;24:413–422. [Google Scholar]
  23. Hattersley PW. Characterization of C4 type leaf anatomy in grasses (Poaceae) Aust J Plant Physiol. 1984;13:399–408. [Google Scholar]
  24. Hattersley PW, Browning AJ. Occurrence of the suberized lamella in leaves of grasses of different photosynthetic types in parenchymatous bundle sheaths and PCR (“Kranz”) sheaths. Protoplasma. 1981;109:371–401. [Google Scholar]
  25. Hermans J, Westhoff P. Homologous genes for C4 isoform of phosphoenolpyruvate carboxylase in a C3 and a C4 Flaveria species. Mol Gen Genet. 1992;234:275–284. doi: 10.1007/BF00283848. [DOI] [PubMed] [Google Scholar]
  26. Hudson GS, Dengler RE, Huttersley PW, Dengler NG. Cell-specific expression of Rubisco small subunit and Rubisco activase genes in C3 and C4 species of Atriplex. Aust J Plant Physiol. 1992;19:89–96. [Google Scholar]
  27. Johri BM, Ambegaokar KB, Srivastava PS. Comparative Embryology of Angiosperms. Berlin: Springer-Verlag; 1992. [Google Scholar]
  28. Kaplan DR, Cooke TJ. Fundamental concept in the embryogenesis of dicotyledons: a morphological interpretation of embryo mutants. Plant Cell. 1997;9:1903–1910. doi: 10.1105/tpc.9.11.1903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Knapp AK, Vogelman TC, Mclean TM, Smith WK. Light and chlorophyll gradients within Cucurbita coyledons. Plant Cell Environ. 1988;11:257–263. [Google Scholar]
  30. Kretsch T, Emmler K, Schäfer E. Spatial and temporal pattern of light-regulated gene expression during tobacco seedling development: the photosystem II-related genes Lhcb (Cab) and PsbP (Oee2) Plant J. 1995;7:715–729. [Google Scholar]
  31. Ku MSB, Wu J, Dai Z, Scott RA, Chu C, Edwards GE. Photosynthetic and photorespiratory characteristics of Flaveria species. Plant Physiol. 1991;96:519–528. doi: 10.1104/pp.96.2.518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Langdale J, Nelson T. Spatial regulation of photosynthetic development in C4 plants. Trends Genet. 1991;7:191–196. doi: 10.1016/0168-9525(91)90435-s. [DOI] [PubMed] [Google Scholar]
  33. Langdale JA, Kidner A. Bundle sheath defective, a mutation that disrupts cellular differentiation in maize leaves. Development. 1994;120:673–681. [Google Scholar]
  34. Langdale JA, Rothermal BA, Nelson T. Cellular pattern of photosynthetic gene expression in the developing maize leaves. Gen Dev. 1988a;2:106–115. doi: 10.1101/gad.2.1.106. [DOI] [PubMed] [Google Scholar]
  35. Langdale JA, Zelitch I, Miller E, Nelson T. Cell position and light influence C4 versus C3 patterns of photosynthetic gene expression in maize. EMBO J. 1988b;7:3643–3651. doi: 10.1002/j.1460-2075.1988.tb03245.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Liu YQ, Dengler NG. Bundle sheath and mesophyll cell differentiation in the C4 dicotyledon Atriplex rosea: quantitative ultrastructure. Can J Bot. 1994;72:644–657. [Google Scholar]
  37. Mann CC. Genetic engineers aim to soup up crop photosynthesis. Science. 1999;283:314–316. doi: 10.1126/science.283.5400.314. [DOI] [PubMed] [Google Scholar]
  38. Marshall JS, Stubbs JD, Chitty JA, Surin B, Taylor WC. Expression of the C4 Me1 gene from Flaveria bidentis requires an interaction between 5′ and 3′ sequences. Plant Cell. 1997;9:1515–1525. doi: 10.1105/tpc.9.9.1515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Martineau B, Smith HJ, Dean C, Dunsmuir P, Bedbrook J, Mets LJ. Expression of a C3 plant Rubisco SSU gene in regenerated C4 Flaveria plants. Plant Mol Biol. 1989;13:419–426. doi: 10.1007/BF00015554. [DOI] [PubMed] [Google Scholar]
  40. Martineau B, Taylor WC. Photosynthetic gene expression and cellular differentiation in developing maize leaves. Plant Physiol. 1985;78:399–404. doi: 10.1104/pp.78.2.399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Maroco V, Ku MSB, Furbank RT, Lea PJ, Leegood RC, Edwards GE. CO2 and O2 dependence of PS II activity in C4 plants having genetically produced deficiencies in the C3 or C4 cycle. Photosynth Res. 1998;58:91–101. [Google Scholar]
  42. Matsuoka M, Tada Y, Fujimura T, Kano-Murakami Y. Tissue-specific light-regulated expression directed by the promoter of a C4 genes, maize pyruvate orthophosphate dikinase, in a C3 plant, rice. Proc Natl Acad Sci USA. 1993;90:9586–9590. doi: 10.1073/pnas.90.20.9586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Misra S. Floral morphology of the family Compositae II: development of the seed and fruit in Flaveria repanda. Bot Mag. 1964;77:290–296. [Google Scholar]
  44. Nelson T, Dengler NG. Photosynthetic tissue differentiation in C4 plants. Int J Plant Sci Suppl. 1992;153:S93–S105. [Google Scholar]
  45. Nelson T, Harpster MH, Mayfied SP, Taylor WC. Light-regulated gene expression during maize leaf development. J Cell Biol. 1984;98:558–564. doi: 10.1083/jcb.98.2.558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Nelson T, Langdale JA. Developmental genetics of C4 photosynthesis. Annu Rev Plant Physiol Plant Mol Biol. 1992;43:25–47. [Google Scholar]
  47. O'Brien TP, McCully ME. The Study of Plant Structure: Principles and Selected Methods. Melbourne, Australia: Termarcharphi Pty; 1981. [Google Scholar]
  48. Pyankov VI, Black CC, Jr, Artyusheva EG, Voznesenskaya EV, Ku MSB, Edwards GE. Features of photosynthesis in Haloxyon species of Chenopodiaceae that are dominant plants in central Asian deserts. Plant Cell Physiol. 1999;40:125–134. [Google Scholar]
  49. Ramsperger VC, Summers RG, Berry JO. Photosynthetic gene expression in meristems and during initial leaf development in a C4 dicotyledonous plant. Plant Physiol. 1996;111:999–1010. doi: 10.1104/pp.111.4.999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Rosche E, Westhoff P. Genomic structure and expression of the pyruvate, orthophosphate dikinase gene of the dicotyledonous C4 plant Flaveria trinervia (Asteraceace) Plant Mol Biol. 1995;29:663–678. doi: 10.1007/BF00041157. [DOI] [PubMed] [Google Scholar]
  51. Schäffner AR, Sheen J. Maize C4 photosynthesis involves differential regulation of phosphoenolpyruvate carboxylase genes. Plant J. 1992;2:221–232. doi: 10.1046/j.1365-313x.1992.t01-44-00999.x. [DOI] [PubMed] [Google Scholar]
  52. Sheen J. Metabolic repression of transcription in higher plants. Plant Cell. 1990;2:1027–1038. doi: 10.1105/tpc.2.10.1027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Sheen J. Molecular mechanisms underlying the differential expression of maize pyruvate, orthophosphate dikinase genes. Plant Cell. 1991;3:225–245. doi: 10.1105/tpc.3.3.225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Sheen JY, Bogorad L. Differential expression of the ribulose bisphosphate carboxylase large subunit genes in bundle sheath and MC of developing maize leaves is influenced by light. Plant Physiol. 1985;79:1072–1076. doi: 10.1104/pp.79.4.1072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Sheen JY, Bogorad L. Expression of the ribulose bisphosphate carboxylase large subunit gene and three small subunit genes in two cell types of maize leaves. EMBO J. 1986;5:3417–3422. doi: 10.1002/j.1460-2075.1986.tb04663.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Sheen JY, Bogorad L. Differential expression of C4 pathway genes in mesophyll and BSC of greening maize leaves. J Biol Chem. 1987a;262:11726–11730. [PubMed] [Google Scholar]
  57. Sheen JY, Bogorad L. Regulation of levels of nuclear transcripts for C4 photosynthesis in bundle sheath and MC of maize leaves. Plant Mol Biol. 1987b;8:227–238. doi: 10.1007/BF00015031. [DOI] [PubMed] [Google Scholar]
  58. Shu G. Cell type-specific expression of C4 photosynthetic genes in Flaveria species: developmental and genetic control. PhD dissertation. The University of Chicago; 1996. [Google Scholar]
  59. Sinha NR, Kellogg EA. Parallelism and diversity in multiple origins of C4 photosynthesis in the grass family. Am J Bot. 1996;83:1458–1470. [Google Scholar]
  60. Stebbins GL. Flowering Plants: Evolution above the Species Level. Cambridge, MA: Harvard University Press; 1974. [Google Scholar]
  61. Stockhaus J, Poetsch W, Steinmuller K, Westhoff P. Evolution of C4 phosphoenolpyruvate carboxylase promoter of the C4 dicot Flaveria trinervia: an expression analysis in the C3 plant tobacco. Mol Gen Genet. 1994;245:286–293. doi: 10.1007/BF00290108. [DOI] [PubMed] [Google Scholar]
  62. Stockhaus J, Schlue U, Koczor M, Chitty JA, Taylor WC, Westhoff P. The promoter of the gene encoding the C4 form of phosphoenolpyruvate carboxylase directs mesophyll specific expression in transgenic C4 Flaveria spp. Plant Cell. 1997;9:479–489. doi: 10.1105/tpc.9.4.479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Suzuki T, Takio S, Tanaka K, Yamamoto I, Satoh T. Differential light regulation of the rbcS gene expression in two cell lines of the liverwort Marchantia paleacea var. diptera. Plant Cell Physiol. 1999;40:100–103. doi: 10.1093/oxfordjournals.pcp.a029465. [DOI] [PubMed] [Google Scholar]
  64. Taylor WC, Rosche E, Marshall JS, Ali S, Chastain CJ, Chitty JA. Diverse mechanisms regulate the expression of genes coding for C4 enzymes. Aust J Plant Physiol. 1997;24:437–442. [Google Scholar]
  65. Tobin EM, Silverthorne J. Light regulation of gene expression in higher plants. Annu Rev Plant Physiol Plant Mol Biol. 1985;36:734–737. [Google Scholar]
  66. Tsukaya H, Tsuge T, Uchimiya H. The cotyledon: a superior system for studies of leaf development. Planta. 1994;195:309–312. [Google Scholar]
  67. Wang JL, Klessig DF, Berry JO. Regulation of C4 gene expression in developing amaranth leaves. Plant Cell. 1992;4:173–184. doi: 10.1105/tpc.4.2.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Wang JL, Long JJ, Hotchkiss T, Berry JO. C4 photosynthetic gene expression in light- and dark-grown amaranth cotyledons. Plant Physiol. 1993a;102:1085–1093. doi: 10.1104/pp.102.4.1085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Wang JL, Turgeon R, Carr JP, Berry JO. Carbon sink-to-source transition is coordinated with establishment of cell-specific gene expression in a C4 plant. Plant Cell. 1993b;5:289–296. doi: 10.1105/tpc.5.3.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Westhoff P, Svensson P, Ernst K, Blasing O, Burscheidt J, Stockhaus J. Molecular evolution of C4 phosphoenolpyruvate carboxylase in the genus Flaveria. Aust J Plant Physiol. 1997;24:429–436. [Google Scholar]

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