INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Despite our current understanding of the biochemistry of fatty acid and lipid synthesis in plants, the signals and factors that direct the expression of genes in these pathways remain largely unknown. Unlike other organisms, the enzymes responsible for de novo fatty acid synthesis in plants are not localized in the cytosol, rather they are in the plastid. Although a portion of the newly synthesized acyl chains is utilized for lipid synthesis within the plastid, a majority is exported into the cytosol for glycerolipid assembly at the endoplasmic reticulum (Somerville et al., 2000). In addition, some extraplastidial glycerolipids return to the plastid, and extensive lipid interchange exists between these two organelles (Ohlrogge and Jaworski, 1997). To adjust to changes in the demand of lipids during tissue development and environmental influences, plant cells must regulate and coordinate the expression of the many genes involved in fatty acid and lipid synthesis pathways. An attractive possibility is the existence of a global transcriptional control for these genes, perhaps comparable with the yeast (Saccharomyces cerevisiae) system (Schuller et al., 1992). For example, it has been shown that the expression of the different subunits of acetyl-coenzyme A carboxylase (ACCase) is orchestrated during tissue development (Ke et al., 2000). This enzyme catalyzes the first committed step in fatty acid synthesis and available evidence points to its key role as a major regulatory enzyme in fatty acid production (Ohlrogge and Jaworski, 1997). In addition, global post-transcriptional mechanisms have also been suggested for regulation of fatty acid synthesis genes in plants (Eccleston and Ohlrogge, 1998).

Analyses of gene expression under conditions where the demand for fatty acids is elevated may unravel part of the signals and mechanisms that participate in the regulation of genes involved in plant lipid biosynthesis. A time when the cells are fully engaged in fatty acid production is during leaf expansion in illuminated plants (Browse et al., 1981). In addition to cell division and growth, light induces the differentiation of the proplastid to the specialized, membrane-rich chloroplast (Kasemir, 1979). This transition involves not only a radical change in the structure and function of the plastid, but also its increase in size and number per cell. Therefore, de novo synthesis of glycerolipids is critical in providing chloroplasts with new lamellae to accommodate the photosynthetic apparatus. During the process of leaf expansion, light activates transcription and translation of a large number of genes that participate in chloroplast differentiation (Chory and Susek, 1994). Moreover, the interdependent relationship between chloroplast development and the activation of nuclear genes has been confirmed by mutant analyses in Arabidopsis (Susek et al., 1993). As a consequence of the plastidic location of the fatty acid synthesis machinery and its commitment to supplying lipids for new membrane synthesis, it seems possible that at least some of the genes participating in this pathway belong to the group of genes activated by light during leaf expansion. However, previous studies argue against this hypothesis and suggest a minor role of light on the regulation of the genes for fatty acid synthesis (Scherer and Knauf, 1987; Battey and Ohlrogge, 1990; Baerson and Lamppa, 1993). At present, the only gene known to participate in glycerolipid production and to be transcriptionally induced by light in Arabidopsis is the chloroplast -3 fatty acid desaturase (Nishiuchi et al., 1995).

A second condition where the fatty acid synthesis machinery is very active is during the exponential growth of plant cells in culture. When plant cells are grown in liquid culture in the presence of a carbon source they show rapid incorporation of labeled precursors into phospholipids and neutral lipids (Weber et al., 1992). The presence of an energy source such as carbohydrates in the media increases the cell growth rate and hence the need for new membranes. Sugars not only function as substrates for growth, but they also affect sugar-sensing systems that initiate changes in gene expression. In plant metabolism, carbohydrate depletion generally enhances the expression of genes involved in photosynthesis, reserve breakdown, and export, whereas abundant carbon resources favor genes for storage and utilization (Koch, 1996). For example, Graham et al. (1994) showed that in cucumber (Cucumis sativus), the mRNA levels for two enzymes of the glyoxylate cycle (malate-synthase and isocitrate-lyase) are coordinately induced not only during postgerminative seedling development, but also by a metabolic signal derived from sugars. The glyoxylate cycle plays an important role in the degradation of fatty acids during seedling germination. In animal pancreatic cells, a Glc-mediated signal activates the transcription of the ACCase gene (Brun et al., 1993). Thus, due to the metabolic regulation via sugars of several genes involved in primary biochemical pathways in plants, it is likely that at least some of the genes involved in fatty acid synthesis are also under the control of similar mechanisms.

The regulation of acyl carrier proteins (ACPs) has been the focus of several studies based on their central role in lipid biosynthesis in plants (Hannapel and Ohlrogge, 1988; Hlousek-Radojcic et al., 1992; Baerson and Lamppa, 1993). ACPs are small acidic proteins that carry the nascent acyl chains during the synthesis of 16- and 18-carbon acyl groups. In this report, we demonstrate that the transcripts for several isoforms of Arabidopsis ACPs are subject to differential regulation by light and also by metabolic and/or growth control. In addition, we provide evidence for the role of the ACP1 and ACP2 5'-untranslated regions (UTRs) in gene expression in different tissues of Arabidopsis plants.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

ACP4 mRNA Levels Are Increased by Light

The participation of light in the activation of fatty acid synthesis in young leaves is well established (e.g. Browse et al., 1981; Bao et al., 2000). Light regulation can be attributed in part to activation of ACCase by mechanisms that involve reduced thioredoxin, phosphorylation, substrate activation, cofactors, and pH changes (Sasaki et al., 1997; Hunter and Ohlrogge, 1998; Savage and Ohlrogge, 1999). Although light-mediated mechanisms of gene activation such as transcription are prominent during chloroplast biogenesis in leaves, there is no evidence available that connects these mechanisms to the activation of genes responsible for de novo fatty acid synthesis. Therefore, we first examined the influence of light on the mRNA steady-state levels of distinct isoforms of Arabidopsis ACPs. Based on protein analyses, ACP1, ACP2, and ACP3 have been identified in all tissues examined (Hlousek-Radojcic et al., 1992), whereas a fourth isoform (ACP4) is expressed predominantly in leaves (Shintani, 1996). As shown in Figure 1, the mRNA levels of ACP4 increased approximately 4-fold in leaf tissue when 2-week-old Arabidopsis seedlings were reilluminated for 4 h after a 24-h dark period. An even higher increase (5-fold) was observed at 8 h of illumination, with a decline to 4-fold again after 12 h (Fig. 1). It is interesting that the ACP4 mRNA presented similar kinetics of light-mediated induction to the Arabidopsis ferredoxin-A (FEDA) mRNA, a 4-fold increase after 4 h in white light with a subsequent decline after 12 h (Fig. 1). Previous studies demonstrated that the gene for FEDA is transcriptionally activated by light, and part of this induction is initiated by phytochromes (Caspar and Quail, 1993).

View larger version (19K): [in this window] [in a new window]   Figure 1.   Light-mediated induction of ACP4 mRNA levels in Arabidopsis leaf tissue. A, Northern-blot analyses of 2-week-old Arabidopsis plants dark treated for 24 h and reilluminated with fluorescent white light for the times indicated. Each lane of the blot contains 5 µg of total RNA from leaf tissue. The eIF4A probe was used as a loading control. B, The signal intensities were quantified by densitometer scanning and values represent the amount of mRNA for each gene relative to that present at time zero.

In contrast to ACP4 transcript levels, the signal presented by a probe corresponding to the ACP2 coding sequence did not show intensity variations in the conditions tested (Fig. 1). This result agrees with a previous study that showed that the Arabidopsis Acl1.2 gene promoter (ACP2) does not confer light responsiveness to a reporter gene in transgenic tobacco (Nicotiana tabacum) seedlings (Baerson and Lamppa, 1993). Because the ACP2 and ACP3 isoforms are 82% identical at the nucleotide level, we assumed that the signal conferred by the ACP2 fragment represented the abundance of ACP2 and ACP3 transcripts (ACP2 and 3 in Fig. 1). This assumption is also based on the observation that both proteins are expressed in similar amounts in Arabidopsis leaf tissue (Hlousek-Radojcic et al., 1992) and that the corresponding promoters confer similar levels of expression to a reporter gene in Arabidopsis leaves (Baerson et al., 1998).

Polyribosomal Association of ACP mRNAs Is Increased by Light

Previous studies suggested that the expression of ACPs could be regulated at the level of translation. First, transgenic oilseed rape (Brassica napus) plants overexpressing a 12:0-ACP thioesterase show an approximately 2-fold increase in the ACP protein level with no significant changes in the corresponding messenger abundance (Eccleston and Ohlrogge, 1998). Second, Hannapel and Ohlrogge (1988) reported that during seed development in soybean, the relative abundance of ACP and lectin mRNAs is, at most, 28-fold different, whereas the corresponding protein levels differ at least 200-fold, suggesting a differential translational efficiency between these two mRNAs. These observations led us to compare the polyribosomal distribution of ACP mRNAs with other transcripts highly expressed in leaf tissue of Arabidopsis plants. In addition, the fact that light has a major effect on the polyribosomal association of several mRNA-encoding plastidic proteins (Berry et al., 1990; Dickey et al., 1998) prompted us to investigate the influence of light on the association of ACP mRNAs with ribosomes. Therefore, we analyzed Arabidopsis leaf tissue from 24-h dark-treated plants or from 12-h reilluminated (with white fluorescent light) plants. The results demonstrated that the messengers for ACP2, 3, and 4 were associated with polyribosomes (fractions 10-15 of the gradient) after 12 h of illumination (Fig. 2). In contrast, after 24 h in the dark, the transcripts for the ACP isoforms appeared in the upper fractions (3-6) of the gradient, corresponding to low M_r polyribosomes or ribosome-free mRNAs (Fig. 2). The polyribosomal distribution of the FEDA mRNA was similar to that of ACP mRNAs in both conditions (Fig. 2). The eukaryotic initiation factor 4A (eIF4A) transcript also showed association with polyribosomes in the light, but in contrast to ACP and FEDA mRNAs, a significant proportion of the eIF4A transcript was still bound to polyribosomes after 24 h in the dark (Fig. 2). In summary, these observations indicate that the transcripts encoding for ACP isoforms are associated with polyribosomes in Arabidopsis leaf tissue in a light-dependent manner similar to FEDA, but they differ significantly with respect to eIF4A mRNAs in the dark.

View larger version (36K): [in this window] [in a new window]   Figure 2.   Light affects the polyribosome association of ACP mRNAs. Leaf tissue extracts from plants grown in the dark for 24 h (Dark) or reilluminated for 12 h after a 12-h dark period (Light) were spun in 15% to 60% Suc gradients. Sixty aliquots were collected at identical volume intervals and A260 was measured. Total RNA was extracted from 15 fractions of the gradients, loaded onto an agarose gel, and analyzed by northern blot. The blots were hybridized with probes corresponding to the genes indicated. Fractions 7 through 15 correspond to the polyribosomal fractions.

Conserved Motifs Occur in the 5'-Leader Region of ACP mRNAs

The examination of Arabidopsis genomic and cDNA ACP sequences has revealed two unusual motifs in the 5'-leader region of ACP mRNAs (Table I; Ohlrogge et al., 1991). One feature is the presence of short sequences rich in pyrimidines. The high cytosine and thymidine content is uncommon in that leader sequences of plant mRNAs tend to be rich in adenines and thymidines (Joshi, 1987). A second conserved motif is an element composed of seven nucleotides, CTCCGCC (Table I). In addition to Arabidopsis sequences, these features are also conserved in the 5'-leader region of ACP messengers from several diverse species (Table I). The importance of 5'-UTRs as essential regulators of gene expression in plants has been described (Bolle et al., 1996; Dickey et al., 1998).

To analyze the participation of the 5'-leader sequences of the ACP mRNAs in the regulation of ACP expression, we generated independent Arabidopsis transgenic lines where the 5'-UTR sequences of the Arabidopsis ACP1 (line atL-acp1) and ACP2 (line atL-acp2) mRNAs were fused to the luciferase reporter gene (LUC) under the control of the cauliflower mosaic virus (CaMV) 35S promoter (Fig. 3). To preserve the endogenous translation initiation site, a short portion of the corresponding ACP coding sequence was also included (see Fig. 3 for details). The 5'-leader regions of ACP1 and ACP2 mRNAs present sequence variations, but both have conserved CU-rich regions and include the heptanucleotide motif CTCCGCC (Table I). However, ACP1 and ACP2 transcripts contain distinct translation initiation sites (Fig. 3). The AUG context in the Acl1.1 gene (ACP1) matches with one of the most common translation initiation sequences found in plants, AAACAAUGGC whereas the translation initiation site in the Acl1.2 gene (ACP2), CUUCUAUGGC, is found in a smaller number of plant genes (Joshi et al., 1997). This suggests that the efficiency of AUG recognition by the translation machinery might also influence ACP expression. A third line of transgenic plants carrying the LUC gene fused to a deleted version of the ACP1 leader region (line atL-del1) was also generated. In this case, 58 bp of the 5'-UTR was removed, conserving the first 10 nucleotides toward the 5' end together with the ACP1 translation initiation site (see Fig. 3 for sequence details). As an internal control for position effect and copy number, all the T-DNA constructs also carried a cassette expressing the GUS gene under the CaMV35S promoter (Fig. 3).

View larger version (16K): [in this window] [in a new window]   Figure 3.   Constructs used for Arabidopsis transformation. A, Scheme of the T-DNA constructs carrying the LUC and -glucuronidase (GUS) genes. , T-DNA borders (L) left and (R) right ends; , nopaline synthase 3'-polyadenylation signal; NPTII, neomycin phosphotransferase coding sequence; , CaMV35S promoter; GUS, -glucuronidase coding sequence; B, BamHI; P, PstI, Bl, BglII; +1 denotes the transcription initiation site. B, Sequences of the 5'-UTRs derived from Arabidopsis ACP1 (pCaACP1), ACP2 (pCaACP2), and the deleted version of ACP1 (pCadel1) used in the three independent constructs. The ACP translation initiation codon is underlined. The displayed sequences start at the predicted transcription initiation site and, therefore, the leaders generated by these constructs have eight additional bases from the vector sequence (indicated in bold).

The 5'-Leader Sequences of ACP1 and ACP2 Increase Reporter Gene Expression

We analyzed the LUC- and GUS-specific activities in expanding leaves of 2-week-old transgenic plants. After 24 h in the dark, the atL-acp1 transgenic plants showed a 2-fold lower level of LUC/GUS activity ratio than atL-acp2 plants, and 10- and 20-fold higher LUC/GUS activity ratio than atL-del1 plants, respectively (Fig. 4A). Based on the polyribosomal distribution of the ACP transcripts (Fig. 2), we also asked whether the ACP 5'-UTRs could have a light regulatory role similar to other UTRs from plastid proteins (Bolle et al., 1994; Dickey et al., 1998). When the plants were reilluminated for 6 h, the LUC activity in atL-acp1 and atL-acp2 lines differentially increased over the GUS activity in the same lines compared with the absence of light (Fig. 4A). These results suggest that the leader could facilitate the expression of LUC in the light by differential translation or transcript stability. Alternatively, a decrease of GUS mRNA stability in the presence of light should be considered. Arabidopsis developing seeds are green and posses photosynthetic capacity. However, they are considered heterotrophic, as imported Suc is their major carbon source. As shown in Figure 4B, the LUC/GUS activity ratios of specific activities in mid-stage developing seeds differed from the ratios observed in leaves. The activity ratio in atL-acp1 transgenic lines was approximately 2.5-fold higher than the ratio in atL-acp2 plants (Fig. 4B). Thus, in contrast to leaf tissue, the 5'-UTR of ACP1 mRNA appeared to confer a preferential expression of the LUC gene in developing seeds compared with the 5'-UTR of ACP2 mRNA. The expression of the reporter genes was also evaluated in root tissue from 2-week-old transgenic plants. In contrast to leaf and seed tissue, the influence of the ACP1 and ACP2 5'-UTRs was less pronounced, and for atL-acp1 plants the ratio of reporter gene activity was similar to the ratio in atL-del1 plants (Fig. 4C). For atL-acp2 plants, the LUC/GUS activity ratio was 4-fold higher than the ratio in atL-acp1 plants (Fig. 4C). In conclusion, the results presented in Figure 4 provide evidence that ACP 5'-UTRs do not function as negative elements of gene expression, as these leaders increase LUC expression compared with a truncated leader (see "Discussion"). In addition, the observation of a differential effect on LUC expression in the tissues analyzed also indicates a role of ACP mRNA leaders in control of tissue-specific gene expression (Fig. 4). The participation of 5'-UTR sequences in differential tissue expression has been previously observed for the enoyl-ACP reductase gene. Deletion of a CT-rich tract from the 5' leader of the respective mRNA reduces gene expression in young leaves, but not in seeds and roots (Jan de Boer et al., 1999).

View larger version (17K): [in this window] [in a new window]   Figure 4.   Reporter gene expression in different tissues of Arabidopsis transgenic plants. atL-del1, atL-acp1, and atL-acp2 indicate the transgenic lines transformed with pCadel1, pCaACP1, and pCaACP2, respectively (see Fig. 3). A, Transgenic plants were dark treated for 24 h (Dark) or reilluminated for 6 h (Light) with white fluorescent light. LUC- and GUS-specific activities were measured in leaves of 10 independent transgenic plants for atL-del1, atL-acp1, and atL-acp2. LUC/GUS activity ratios are the average of the 10 individual ratios from each transgenic line. The atL-acp1 and atL-acp2 LUC/GUS activity ratios are expressed with respect to atL-del1 LUC/GUS activity ratios (set arbitrary to one). The bars denote the SD of the average. B, LUC- and GUS-specific activities were measured in roots of 10 independent transgenic plants for atL-del1, atL-acp1, and atL-acp2. C, Reporter gene activity was measured in developing seeds from 10 independent transgenic plants for atL-del1, atL-acp1, and atL-acp2.

ACP mRNA Levels Are Affected by a Suc-Derived Signal and/or Growth Control in Cell Suspension Cultures

In bacteria and yeast, the expression of several genes involved in fatty acid and lipid synthesis is tightly coupled to growth (Jiang and Cronan, 1994; Carman and Henry, 1999). Cells growing in the presence of a carbon source such as Suc show high rates of transcription of these genes (Carman and Henry, 1999). In addition to their role as energy sources, sugars have been demonstrated to control the expression of plant genes involved in diverse processes such as starch metabolism (Nakamura et al., 1991), storage protein accumulation (Hattori et al., 1990), and lipid degradation (Graham et al., 1994). These observations led us to ask whether the expression of some of the fatty acid synthesis genes in plants might also be under metabolic and/or growth control by sugars. For this purpose, we analyzed the mRNA levels of ACP2, ACP3, ACP4, and the endoplasmic reticulum-associated -12-desaturase (FAD2) in Arabidopsis mesophyll-derived cell suspension cultures. To distinguish between the mRNAs corresponding to ACP2 and ACP3 in this experiment, we synthesized a messenger-specific probe based on the 3'-UTR sequences of these genes (see "Materials and Methods"). The transcript levels of ACP2 and ACP3 isoforms declined approximately 2-fold after 48 h of starvation (Fig. 5). In contrast, the relative mRNA abundance of ACP4 and FAD2 transcripts did not change significantly during the starvation period (Fig. 5). Thus, the absence of Suc in the media and/or the reduced growth rate preferentially affected the expression of the ACP2 and ACP3 mRNAs. In the same experiment, the transcript levels of the Rubisco1A small subunit (RBCS1A) increased approximately 2-fold after 12 h of starvation, and slightly declined afterward (Fig. 5). This result was in agreement with the fact that the gene for the small subunit of Rubisco is activated by light and is repressed by sugars (Terzaghi and Cashmore, 1995). To evaluate the effect of addition of Suc to starved cells in the absence of light, the cells were grown in the dark and in the presence of Suc. The transcript levels of ACP2 and ACP3 increased approximately 2.5-fold after 24 h (Fig. 6). In contrast, if the cells were kept in an osmotic control media in the dark, the level of the same mRNA remained steady (Fig. 6). It is interesting that we observed that the incubation of starved cells in the dark and Suc had a negative effect on the ACP4 mRNA levels, decreasing its abundance more than 2.5-fold after 12 h. Conversely, no variations in the levels of the same mRNA were observed with the osmotic control media in the dark (Fig. 6). Thus, the absence of light and the presence of Suc were responsible for the down-regulation of the ACP4 transcript levels. The same mRNA profile was observed for RBCS1A, and similar mechanisms of regulation might be operating for both genes (Fig. 6). In the case of FAD2 mRNAs, no significant differences in the relative levels of the corresponding messenger were found in the conditions tested (Figs. 5 and 6). Thus, in contrast to ACP, starvation and light did not alter FAD2 mRNA abundance in liquid cell culture.

View larger version (29K): [in this window] [in a new window]   Figure 5.   Differential regulation of ACP mRNA levels in starved Arabidopsis cells. A, Cells were starved for 48 h in the presence of light and total RNA was extracted at the times indicated. Each lane of the northern blots contains 2.5 µg of total RNA. The blots were hybridized with probes corresponding to the genes indicated and eIF4A was used as a loading control. B, The signals in A were quantified by scanning densitometry from a range of film exposures and the values are represented as in Figure 1B.

View larger version (47K): [in this window] [in a new window]   Figure 6.   Differential regulation of ACP mRNA levels in Arabidopsis cells grown in the presence of Suc. A, Cells were starved for 48 h in the light and were subsequently transferred to Suc-containing media (dark/Suc) or to an osmotic control media (dark/mannitol) and were then incubated in the dark. Total RNA was extracted at the times indicated. Each lane of the northern blots contains 2.5 µg of total RNA. The blots were hybridized with probes corresponding to the genes indicated and eIF4A was used as a loading control. B, The signals corresponding to the dark/Suc treatment were quantified by scanning densitometry from a range of film exposures and the values are represented as in Figure 1B.

To investigate whether sugars could have a direct role in the regulation of ACP transcript levels, we examined if their expression could be altered by uncoupling growth from the presence of sugars in the media. For this purpose, cells were starved in an osmotic control media (58 mM mannitol) for 48 h and were subsequently transferred into media containing 58 mM 3-O-methyl-glucopyranose (3-OMG) or 0.2 mM 2-deoxy-Glc. The effect of these two Glc analogs on gene expression has been previously studied in Arabidopsis cell suspension cultures (Fujiki et al., 2000). The first Glc analog is taken up by cells but is not phosphorylated, whereas the second is phosphorylated but is not further metabolized (Dixon and Webb, 1979). The presence of 58 mM 3-OMG in the media did not show a positive effect on ACP2 and 3 transcript levels and it did not decrease the ACP4 mRNA level after 24 h (data not shown). The presence of 0.2 mM 2-deoxy-Glc presented cytotoxic effects on Arabidopsis mesophyll cells, as previously observed in other systems (Graham et al., 1994). The Arabidopsis cells turned yellowish and showed a decrease in total RNA content even after short periods of time (data not shown). The results obtained with 3-OMG suggest that cell growth and/or a metabolic signal generated by Suc or downstream of Glc are necessary to control ACP mRNA levels in Arabidopsis cell liquid culture.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

One objective addressed by the current work is the nature of the signals that give rise to changes in the expression of genes involved in fatty acid synthesis. Although a limited number of previous studies suggest a minor role for light on the expression of these genes (Scherer and Knauf, 1987; Battey and Ohlrogge, 1990; Baerson and Lamppa, 1993), the mutually dependent relation between chloroplast biogenesis and de novo production of glycerolipids implies a closer connection between light and at least some of the genes for this pathway. In this report, we demonstrated that light affects the expression of mRNAs encoding ACP isoforms from Arabidopsis.

First, we demonstrated that the levels of the messenger for ACP4 increased 4- to 5-fold after light treatment of dark-grown plants. The similarity in the kinetics of mRNA induction between ACP4 and FEDA in Arabidopsis suggests similar mechanisms of activation for both genes. Nevertheless, it remains to be examined whether the increase of the ACP4 transcript levels is direct due to light (e.g. via phytochromes) or indirect via cell growth. The analyses of 1 kb of genomic sequence upstream of the Acl1.4 gene disclosed the presence of several GATA-like motifs. The GATA (or I) boxes are regulatory elements that are functionally important in many light-regulated promoters. The core element is defined as GATAA, and related GATA motifs with variable flanking sequences are found in several promoters (Terzaghi and Cashmore, 1995). For instance, the 1-kb upstream region of the FEDA gene contains seven copies of related GATAA elements. Although the actual transcription initiation site for the ACP4 gene has not been mapped yet, we localized the GATA-like motifs respective to the AUG initiation codon of ACP4. Thus, the upstream region of this gene presents two AAGATAA elements at 715 and 812, one AGATAA at 505, and three GATAA at 70, 526, and 908 bp respective to the translation initiation codon. The presence of these elements in the upstream region of the gene for ACP4 suggests the direct role of light on the transcription of this gene. In contrast, only one and two copies (expected by chance) of these elements are present in the upstream regions (1 kb) of Acl1.2 and Acl1.3 genes, respectively. This observation is consistent with results that demonstrate that light has no impact on mRNA levels of ACP2 and most likely ACP3 (Fig. 1; Baerson and Lamppa, 1993).

A second instance of the influence of light on ACP gene expression was observed at the level of ribosome association (Fig. 2). A general effect of light on polysome formation has been previously described in several studies (Giles et al., 1977; Mosinger and Schopfer, 1983). These works show that the proportion of ribosomes present as polyribosomes increases substantially in response to light. Moreover, continuous far-red light mediates a strong increase in the relative level of polysomes, demonstrating the participation of phytochromes in the response. Despite this general effect on translation, there is evidence that particular mRNAs are less affected by the decrease in the translation rate in the absence of light. For example, Berry et al. (1990) showed that in Amaranth (Amaranthus hypochondriaws) cotyledons, the expression of the small and large subunits of Rubisco are not found associated with polyribosomes in cotyledons of dark-grown plants, but are rapidly recruited onto them upon illumination. In contrast, mRNAs encoding non-light-regulated proteins are associated with polysomes regardless of the light/dark treatment (Berry et al., 1990; Petracek et al., 1997). Our results (Fig. 2) demonstrated that light-regulated mRNAs such as ACP4 and FEDA and non-light-regulated plastidic proteins such as ACP2 and ACP3 were differentially dissociated from polyribosomes in the dark.

In chloroplasts of Chlamydomonas reinhardtii and plants, translation and mRNA stability are enhanced by nuclear-encoded factors that bind to the 5'-UTR of several chloroplast-encoded RNAs (Danon and Mayfield, 1991; Staub and Maliga, 1994). Likewise, several studies confirmed the major role of the 5'-UTR in translation and mRNA stability of cytoplasmic mRNAs (Dickey et al., 1998; Lukaszewicz et al., 1998). A previous report demonstrated that these regions can also be important for transcription activation (Bolle et al., 1994, 1996). Analyses of the 5'-leader sequences of several genes encoding for thylakoid proteins in spinach disclosed the presence of CT-rich regions, designated CT-leader boxes (Bolle et al., 1994). The deletion of these elements from the PsaF (subunit III of photosystem I) and PetH (plastocyanin) upstream gene sequences severely reduces the transcription of a reporter gene in transgenic tobacco (Bolle et al., 1994). It is noteworthy that the CT-leader box is also present in the 5'-UTR of the spinach ACP II (Bolle et al., 1996). In mammalian cells, the presence of polypyrimidine tracts in the 5'-UTR of a family of mRNAs has been reported, and these elements function as negative regulators of translation (Morris et al., 1993). Our analysis of expression of ACP leader:luciferase constructs in different tissues of transgenic Arabidopsis suggests that the CT-rich and CTCCGCC boxes have an active role in control of tissue-specific gene expression. The 5'-UTR of Arabidopsis ACP1 conferred preferential reporter gene expression in seeds, whereas the ACP2 leader favored expression in roots (Fig. 4).

In leaf tissue, the 10- to 20-fold differences in LUC expression between transgenic plants containing the ACP or truncated leaders were most likely brought about by differences in translation efficiency (Fig. 4). In this regard, Gallie and Walbot (1992) demonstrated that 5'-leader length influences the expression of a reporter gene in carrot (Daucus carota) protoplasts. In particular, a 74-base leader construct is expressed 6-fold more highly than a 29-base leader construct. However, Bolle et al. (1994) found that in transgenic tobacco, the expression of a reporter gene under CaMV35S promoter is not altered when the 5' leader of PsaF (188 bases) is added to the 5'-UTR (24 bases) of the native reporter gene mRNA. Thus, although a more passive effect due to leader length can explain the reduced reporter gene expression observed with the 58-bp deletion in leaves, the effect of the leader length in roots was minor. Thus, the different expression patterns observed in leaf and seed argue in favor of an active role of ACP leaders in gene regulation via translation. However, at this point we cannot completely rule out transcriptional or mRNA stability mechanisms to explain the differences in the expression of the reporter genes.

In summary, results of this study demonstrate that expression of genes involved in plant fatty acid synthesis is under multiple levels of control. Light clearly has a major impact on ACP4 mRNA levels, but this impact does not extend to the other ACP isoforms examined here. The different responses to light on ACP are probably transcriptional, and may be based on the presence of GATA boxes in the ACP4 but not other ACP upstream regions. Light control is also exerted post-transcriptionally because all ACP isoforms are more highly associated with polysomes in the light than the dark. Experiments with suspension cultures indicate that differences in expression patterns of ACP isoforms are also observed in their response to sugar supplements. A common element in the leaf and tissue culture experiments is that ACP4, the most leaf-specific isoform, behaves in a manner similar to mRNA for genes involved in photosynthesis (FEDA or RBCS1A). In contrast, expression of the other ACP isoforms may be most responsive to demands for fatty acid synthesis brought about by enhanced growth. This level of complexity demonstrated by multiple ACP genes under different controls may reflect the need of plant cells to tightly regulate the amount and the cellular destination of fatty acids produced in plastids (Ohlrogge and Browse, 1995) and to match the supply of fatty acids to different tissue and environmental demands.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Nomenclature

There are five genes for plastidial ACP in the Arabidopsis genome (Mekhedov et al., 2000). In this study, we report results for Acl1.1, Acl1.2, Acl1.3, and Acl1.4 genes corresponding to the gene products ACP1, 2, 3, and 4, respectively. Acl1.1 (GenBank accession no. X13708) was previously described in Post-Beittenmiller et al. (1989), and Acl12.2 and Acl1.3 (GenBank accession nos. X57698 and X57699, respectively) were described in Lamppa and Collen (1991). We propose that the ACP major leaf isoform (Shintani, 1996) gene be named Acl1.4 (isoform ACP4) and that Acl1.5 refer to GenBank accession no. A_TM021B04.

Plant Growth and Lighting Conditions

Wild-type Arabidopsis plants (ecotype Columbia) were grown on soil at 20°C in a 12-h light/12-h dark photoperiod (80-100 µmol m² s¹). For the analyses of light-mediated induction of mRNA levels, 2-week-old plants were left in the dark for 24 h and were reilluminated with white fluorescent light (80-100 µmol m² s¹) for the times indicated in Figure 1. Leaf tissue was harvested and immediately frozen in liquid N prior to RNA extraction. For polyribosomal association analyses, 2-week-old plants were dark incubated for 12 h and were then incubated in the dark for an additional 12 h or illuminated for 12 h. Leaf tissue was harvested at the end of each period and was processed as indicated above.

Constructs and Plant Transformation

Sequences corresponding to the 5'-UTRs of ACP1 (GenBank accession no. X13708) and ACP2 (GenBank accession no. X57698) mRNAs were obtained by PCR amplification of Arabidopsis (ecotype Columbia) genomic DNA using the primers: 5'-CATGCTCGAGCTCTTTGTACACTCCGCC- CT-3' and 5'-CATGCCATGGCGCTGAATTGAGTCGCC- AT-3' for ACP1 (gene Acl1.1); and 5'-CATGCTCGAGACTGT-TTCTCTATCTCTTTG-3' and 5'-CGCGCCATGGCAATGG-AAGCCATAGAAGAAT-3' for ACP2 (gene Acl1.2). The PCR amplification products were digested with XhoI and NcoI and were cloned in frame to the firefly LUC in pUC-LUC-BT2 (Weisshaar et al., 1991) to give pTACP1 and pTAC2, respectively. A truncated version of the ACP1 5'-leader sequence was generated by in vitro annealing of two synthetic oligonucleotides: 5'-TCGAGCTCTTTGTAC- AAACAATGGCGA-CTCAATTCAGCGC-3' and 5'-CATGGCGCTGAATTGAGT-CGCCATTGTTTGTACAAAGAGC-3'. The double-stranded fragment was cloned in frame to the LUC-coding region in pUC-LUC-BT2 to give pTdel1. All constructs were confirmed by sequencing. The vector pTACP1 was digested with BamHI and the vectors pTACP2 and pTdel1 with BglII. The resulting fragments were cloned independently in the BamHI site of the binary vector pCAMBIA-2201 (Hajdukiewicz et al., 1994) to generate pCaACP1, pCaACP2, and pCadel1, respectively (Fig. 3). The orientation of the inserts was confirmed by restriction mapping using PstI. Agrobacterium tumefaciens strain C58C1 (pM90; Koncz and Schell, 1986) was transformed with the pCaACP1, pCaACP2, and pCadel1 binary vectors by the freeze/thaw transformation protocol (An, 1987). The leaf-vacuum infiltration method was used for Arabidopsis transformation (Bechtold et al., 1993). The transgenic lines transformed with pCaACP1, pCaACP2, and pCadel1 were named atL-acp1, atL-acp2, and atL-del1, respectively.

Selection of Transgenic Plants and Tissue Collection

Seeds (T₁) from transformed Arabidopsis plants were surface-sterilized and sown on seed germination media in the presence of 50 µg mL¹ kanamycin. After 1 week, resistant seedlings (T₁ plants) were transplanted individually to soil and were grown at 20°C in a 18-h light/6-h dark period until they set seeds. For reporter gene analyses in leaf tissue, seeds from T₂ plants were selected in germination media under 50 µg mL¹ kanamycin and were transferred to soil. Plants from 10 independent transgenic lines for each construct (see Fig. 4) were grown in a 12-h light/12-h dark cycle for 2 weeks. On the last day, the plants were kept in the dark for 24 h and the leaf tissue from one-half of the plants per line was collected and immediately frozen in liquid N for subsequent enzyme analyses. Leaf tissue from the remaining plants was harvested as above after 6 h of reillumination with white fluorescent light (80-100 µmol m² s¹). For reporter gene analyses in developing seeds, 10 independent transgenic T₂ seedlings for each construct (see Fig. 4) were selected as above and transferred to soil. Plants were grown in a 16-h light/8-h dark cycle. Flowers from the primary stem were tagged in the morning every day for 1 week. Flower anthesis was considered as day zero in our experiment, and mid-stage siliques (8-10 d after anthesis) were dissected and the seeds immediately frozen in liquid N for subsequent enzyme analyses.

Cell Suspension and Root Liquid Cultures

Arabidopsis (ecotype Columbia) suspension cultures (Axelos et al., 1992) were a gift from Dr. N. Raikhel's laboratory (Department of Energy Plant Research Laboratory, Michigan State University). Suspension cultures were maintained in a 12-h light/12-h dark photoperiod at 22°C on a rotary shaker (120 rpm) in cell suspension media-Suc media (0.32% [w/v] Gamborg's with minimal organics [G5893; Sigma, St. Louis], 58 mM Suc, 0.05% [w/v] MES [2-(N-morpholino)ethanesulfonic acid], and 1.1 µg mL¹ 2,4-dichloro-phenoxyacetic acid, pH 5.7). For the analyses of mRNA levels in different growth conditions, 100 mL of cell cultures were grown for 3 d in CSM-Suc in the presence of white light (100 µmol m² s¹). After the 3rd d, the cells were pelleted at 700 rpm and washed twice with CSM-ma (0.32% [w/v] Gamborg's with minimal organics [G5893; Sigma], 58 mM mannitol, 0.05% [w/v] MES, and 1.1 µg mL¹ 2,4-dichlorophenoxyacetic acid; pH 5.7). Cells were finally resuspended in 150 mL of CSM-ma and were incubated in the presence of light for 2 d (starved cells). An aliquot of 10 mL was taken at the times indicated in Figure 5, and the cell pellet was stored at 80°C until RNA extraction. The remaining 100 mL of the culture was pelleted as before, resuspended in 50 mL of CSM-Suc or 50 mL of CSM-ma, and incubated in the dark for 24 h. An aliquot of 10 mL was taken at the times indicated in Figure 6, and the cell pellet was stored at 80°C until RNA extraction. In the experiment with Glc analogs, cells were starved with CSM-man for 2 d and were subsequently transferred to CSM supplemented with 58 mM 3-O-methyl-D-glucopyranose (Sigma) or 0.2 mM 2-deoxy-D-Glc (Sigma). Cells were incubated in the dark, an aliquot of 10 mL was taken at 0, 3, 6, 12, and 24 h, and the cell pellet stored at 80°C until RNA extraction.

For root liquid culture, approximately 50 seeds from 10 independent transgenic T₂ plants for each construct (see Fig. 4) were surface sterilized and incubated in root liquid media (0.43% [w/v] Murashige and Skoog salts, 0.05% [w/v] MES, 0.1% [v/v] 1,000× B5 vitamin stock, and 50 µg mL¹ kanamycin, pH 5.7) at 4°C for 2 d. The seeds were then incubated at 22°C in continuous fluorescent white light on a rotary shaker (120 rpm) for 2 weeks. Root tissue from approximately 20 seedlings per transgenic line was collected under the dissecting microscope and frozen in liquid N for subsequent enzyme analyses.

RNA Extraction and Northern Analyses

Total RNA was isolated from plant tissue and cell pellets by standard phenol/chloroform extraction and lithium chloride precipitation (Sambrook et al., 1989). The RNA was fractionated in formaldehyde-agarose gels and was blotted onto Hybond-N filters (Amersham Biosciences AB, Uppsala). In all cases hybridization reactions were done at 42°C in 50% (v/v) formamide with [³²P]dCTP-radio-labeled fragments corresponding to ACP2 (X57698), ACP3 (X57699), ACP4 (RXW18), FEDA (M35868), eIF4A (X65052), FAD2 (L26296), and RBCS1A (X13611). A range of different autoradiographic exposures of each filter was made to allow hybridizing bands of different intensity to produce signals in the linear range of the x-ray film. Relative transcript levels were quantified by densitometer scanning (Molecular Dynamics, Sunnyvale, CA) using a dilution series of RNA from light-grown plants or Suc-grown cells as a calibration standard. Filters were stripped to allow reprobing by incubating in 1% (w/v) SDS at 100°C for 30 min. Specific probes for ACP2 and ACP3 transcripts were generated by PCR amplification of Arabidopsis (ecotype Columbia) genomic DNA corresponding to the 3'-UTR of these transcripts. The primers used were: 5'-TGAAAA-GGCCAAGTAGAAT-3' and 5'-GTCAGATACAAGCCTT-GTA-3' for ACP2; and 5'-GGAAAAGGCCAAGTAG-AAA-3' and 5'-CAAGCCTTGTAATAATTATC-3' for ACP3.To evaluate the specificity of the probes for their respective transcripts, 5 µg of double-stranded DNA from each PCR reaction was spotted onto Hybond-N filters (Amersham Biosciences AB). Hybridizations were performed as indicated previously using [³²P]dCTP-radiolabeled fragments corresponding to the 3'-UTR sequences of ACP2 and ACP3. The signals were revealed by autoradiography with x-ray films (data not shown; Eastman-Kodak, Rochester, NY).

Polyribosome Analyses

The protocol for polyribosome isolation was adapted from Petracek et al. (1997) with the following modifications: approximately 1 g of Arabidopsis leaf tissue was homogenized in 4 mL of U buffer (200 mM Tris-HCl, pH 8.5, 50 mM potassium chloride, 25 mM magnesium chloride, 2 mM EGTA, 100 µg mL¹ heparin, 2% [w/v] polyoxyethylene, and 1% [w/v] deoxycholic acid), centrifuged at 13,000g for 15 min at 4°C, and the complete supernatant (4 mL) was loaded onto 30-mL linear Suc gradients (15%-60%). After gradient centrifugation, 15 2-mL fractions were collected by dripping directly into 2 mL of phenol-chloroform, 50 µL of 10% (w/v) SDS, 40 µL of 0.5 M EDTA, and 10 µL of 100 mM aurin-tricarboxylic acid (Sigma). The final RNA pellets were washed with 70% (w/v) ethanol, dried, and resuspended in 10 µL of water, 20 µL formamide, 6.5 µL formaldehyde, and 3.5 µL of MOPS [3-(N-morpholino)-propanesulfonic acid] buffer (0.2 M MOPS, pH 7, 50 mM sodium acetate, and 5 mM EDTA). Samples were heated for 15 min at 68°C and were loaded onto a formaldehyde-agarose gel. Northern analyses were performed as described above. For gradient UV profile analyses, two gradients were spun in parallel and one was used to measure UV absorbance. Sixty 100-µL samples were taken at identical volume intervals and their absorbance was measured at 260 nm.

Tissue Extraction and Enzyme Assays in Arabidopsis Transgenic Plants

Leaf tissue (approximately 0.25 g) was ground with a mortar and pestle and was resuspended in 300 µL of GUS buffer (200 mM Trizma-HCL, pH 7.0, 10 mM -mercaptoethanol, 10 mM EDTA, 0.01% [w/v] SDS, and 0.1% [v/v] Triton X-100) or 300 µL of LUC buffer (100 mM potassium phosphate, pH 7.8, 1 mM EDTA, 7 mM -mercaptoethanol, and 10% [v/v] glycerol) and further homogenized on ice. Root tissue (approximately 0.2 g) was homogenized and resuspended in 100 µL of GUS buffer or LUC buffer. Developing seeds (approximately 0.1 g) were homogenized in 200 µL of GUS buffer or LUC buffer. In all cases, cell debris was removed by centrifugation for 15 min at 13,000g at 4°C. The supernatant was transferred to a clean tube and was kept on ice. For GUS activity, 50 µL of the sample supernatant was diluted in 400 µL of GUS buffer and was then mixed with 50 µL of 10 mM 4-methylumbelliferyl-b-D-glucuronide (Sigma). The reaction was incubated at 37°C and a 100-µL aliquot was diluted in 0.2 M Na₂CO₃ at time 0 and every 15 min for 60 min. UV fluorescence was measured at 365 nm excitation and 455 nm emission with an F-2000 fluorometer (Hitachi, Tokyo). GUS-specific activity is expressed as nanomoles methylumbelliferone produced min¹ mg¹ protein. For LUC analyses, a 25-µL aliquot of the sample was mixed with 100 µL of LUC reagent (20 mM Tricine, pH 7.8, 5 mM MgCl₂, 0.1 mM EDTA, 3.3 mM dithiothreitol, 270 µM coenzyme A, 500 µM luciferin [Promega, Madison, WI]), and 500 µM ATP). The linear range of the reaction was determined by mixing increasing extract volumes with 100 µL of LUC reagent. Luminescence was measured (3-s delay and 10-s integration time) in a TD-20e luminometer (Turner, Sunnyvale, CA). LUC-specific activity is expressed as light units mg¹ protein. In all cases, the GUS- and LUC-specific activities and LUC/GUS activity ratio were measured in triplicate and independently for each transgenic line. Protein concentration was determined using a protein assay kit (Bio-Rad, Hercules, CA) with bovine serum albumin (Sigma) as the standard.