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Cloning and expression analysis of Zmglp1, a new germin-like protein gene in maize
BBRC Biochemical and Biophysical Research Communications 331 (2005) 1257–1263 www.elsevier.com/locate/ybbrc
Cloning and expression analysis of Zmglp1, a new germin-like protein gene in maize q Zhanmin Fan a, Hongya Gu a,b, Xiaowei Chen a, Hui Song a, Qian Wang a, Meihua Liu a, Li-Jia Qu a,b,*, Zhangliang Chen a,b a
Peking-Yale Joint Research Center for Plant Molecular Genetics and Agro-Biotechnology, National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, PR China b The National Plant Gene Research Center (Beijing), Beijing 100101, PR China Received 4 April 2005 Available online 19 April 2005
Abstract The cDNA and genomic DNA of a green tissue-specific gene were cloned from maize (Zea mays L.) using cDNA-amplified fragment length polymorphism (cDNA-AFLP) and library screening. The deduced protein was highly similar to Hordeum vulgare germin-like protein 1 (HvGLP1), and the maize gene was therefore designated Zmglp1. Northern blot specifically detected the mRNA of Zmglp1 in young whorl leaves at the early-whorl stage. However, at the late-whorl, tassel, and silk stages, Zmglp1 transcripts were highly abundant in young whorl leaves; less abundant in mature leaves, young tassels, and cobs; and not detectable in roots, immature kernels, and stalks. RNA in situ hybridization revealed that Zmglp1 expressed only in mesophyllous, phloem, and guard cells in the young whorl leaves. Deletion analysis of the promoter in transgenic Arabidopsis resulted in the identification of several regions containing important regulatory cis-elements controlling the expression levels and circadian rhythm-oscillated patterns of Zmglp1. Ó 2005 Elsevier Inc. All rights reserved. Keywords: cDNA-AFLP; Germin-like protein; Tissue-specific promoter; AGPPase
Leaves are the major sites of photosynthesis in higher plants. Excess glucose produced by photosynthesis is converted into polymeric forms for storage (mainly starch) and transport (sucrose). The biochemistry and molecular genetics of starch biosynthesis are well characterized in photosynthetic and sink tissues [1,2]. ADP-glucose pyrophosphorylase (AGPase) is thought to catalyze the important regulatory step in the biosynthesis of starch, producing the activated glucosyl donor
q Abbreviations: AGPase, ADP-glucose pyrophosphorylase; AGPPase, nucleotide-sugar pyrophosphatase/phosphodiesterase; cDNAAFLP, cDNA-amplified fragment length polymorphism; GUS, b-glucuronidase; MW, molecular weight; TDF, transcript derived fragment. * Corresponding author. Fax: +86 10 6275 1841. E-mail address: [email protected] (L.-J. Qu).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.04.045
ADP-glucose (ADPG) which serves as the direct precursor for starch synthesis [3,4]. The presence of various isozymes of AGPase and their regulation, together with the involvement of starch synthase (SS), starch branching enzyme (BE), and starch debranching enzyme (DBE), is considered to define both the quantity and quality of starch produced [5–7]. However, little is known about the enzymes catalyzing the break down of ADPG or the role of ADGP in starch biosynthesis. Recently a widely distributed nucleotide-sugar pyrophosphatase (AGPPase) that catalyzed the break down of ADPG was identified from leaves of barley, Hordeum vulgare L. [8]. This enzyme had a relatively low Km value (0.5 mM) for ADPG and could also hydrolyze UDP-glucose and CDP-glucose (Km 2–3 mM), although with lower affinity. Attempts to identify different isoforms of AGPPase have revealed
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that the activity of AGPPase declines concomitantly with the accumulation of starch during the development of sink organs, and with the occurrence of cell wallbound and starch granule-bound and soluble enzymes, the soluble enzymes being located in both intra- and extra-plastidial solutions [8–10]. The soluble AGPPase was highly abundant in young leaves, less abundant in older leaves, and absent in roots. However, the levels of the mRNA encoding AGPPase oscillated with a circadian rhythm, with the minimum and maximum mRNA abundance occurring at the ends of the dark and light periods [10,11]. The plastidial AGPPase isoform is fully active at pH 7.0 in the non-illuminated chloroplast and almost totally inactive at pH 8.0 or higher in the illuminated chloroplast during active starch biosynthesis [9]. In this study, we isolated cDNA and promoter regions of a green tissue-specific germin-like protein gene from maize (Zea mays L.). The transcript of this gene, designated Zmglp1 owing to its sequence similarity to barley AGPPase gene, was highly abundant in young leaves; less abundant in mature leaves, young tassels, and cobs; and not detectable in roots, immature kernels, and stalks. RNA in situ hybridization experiments revealed that Zmglp1 was only expressed in mesophyllous, phloem, and guard cells in the young whorl leaves. The RNA abundance of Zmglp1 was also found to oscillate with a circadian rhythm. To better understand the role of Zmglp1, the promoter was analyzed by deletion studies in transgenic Arabidopsis to identify the regions responsible for activity and tissue-specificity as well as the circadian rhythm.
Materials and methods DNA manipulation. DNA manipulations were conducted using standard procedures [12], unless otherwise specified. Escherichia coli DH5a was used as the host for plasmid amplification, and Agrobacterium tumefaciens GV3101 was used for Arabidopsis transformation. Plant materials. A maize cultivar from northern China (Z. mays L. var. Zhongdan 306) was used in this study. Arabidopsis thaliana (ecotype Columbia) was grown in a growth chamber at 22 °C with a 16/8 h light/dark cycle at a light intensity of ±45 lmol m2/s. cDNA-AFLP. Total RNA was isolated by the guanidium thiocyanate method [13]. After treatment with DNase I at 37 °C for 30 min to remove genomic DNA, the RNA concentration was determined by spectrophotometry. cDNA was synthesized from 50 lg of total RNA using a cDNA Synthesis kit (Boehringer–Mannheim, Mannheim, Germany). Starting with 250 ng of cDNA, subsequent restriction enzyme digestion, adapter ligation, and PCR amplification were performed according to the protocol provided with the Core AFLP kit (Gibco-BRL). After selective amplification, an equal volume of formamide dye was added, and 3 ll of each product was loaded on a 6% polyacrylamide gel on a GenomyxLR DNA Sequencer (Beckman). After electrophoresis, the gel was dried for 30 min and exposed to X-ray film (Kodak BIOMAX) at 70 °C for 48 h. The transcript-derived fragments (TDFs) identified as predominantly expressed in young whorls were excised from the gel, soaked in 50 ll TE buffer, and boiled in a water bath at 100 °C for 10 min. Aliquots of 1 ll were re-amplified using the same primer pairs and
conditions as used in the selective amplification. The PCR product was cloned into pUC. Northern and Southern blots. The total RNA samples (15 lg per lane) were loaded on 1.2%(w/v) agarose gels, and Northern blot and hybridization were carried out as described previously [14]. 10–15 lg of genomic DNA isolated from maize leaves was digested with a restriction enzyme, and Southern blot and hybridization were carried out as described [14]. Library screening and sequence analysis. A lambda FIX II genomic library constructed from the Missouri 17 inbred line was screened according to the manufacturerÕs instructions (Stratagene) using 32 P-labeled TDF 11-4 as the probe. The lambda phage DNA of the positive clone was extracted using a High Pure Lambda Isolation kit (Roche, Basel, Switzerland) and 100 ng of lambda DNA was used for Southern blot as described [14]. A 3.8-kb XbaI fragment with strong positive signal was sub-cloned into pBS and sequenced. Sequencespecific primers (primer A: 5 0 -63GTT GCC ATG GCC AAA ATG GTG T-3 0 and primer B: 5 0 -1067CAG ATT AAC AGC ATG CGG CAC T-3 0 ) were designed to amplify the full-length cDNA from young whorl leaves. Comparisons with sequences in the GenBank database were conducted using BLAST [15]. In situ hybridization. Probe preparation, leaf fixation, paraffin embedding, sectioning, pre-hybridization, and hybridization were carried out essentially as previously described [16], except for a few modifications described below. Young whorl leaves of four-week-old plants were selected for fixation. Eight-micrometer thick, horizontal sections were attached to slides coated with 1 mg/ml poly-lysine solution (MW > 150,000, Sigma). The RNase treatment step after hybridization and the high stringency wash were omitted. Deletion and GUS analysis in transgenic Arabidopsis. Promoter deletions of Zmglp1 were generated by PCR using Zmglp1 promoter as the template. The deletion promoter regions D13, D131, D637, D1027, D1215, and D1316 were amplified using primers 1363, 1245, 739, 349, 161, and 60 with 98, respectively. The primer sequences were as follows: 1363: 5 0 -1363ACC TCA CAT GTC ACG CAT GTA A-3 0 ; 1245: 5 0 -1245GGT GTG ACA GTG ATG GCG AGT A-3 0 ; 739: 5 0 -739TAG TGG AGA CCG TCG GAC ATA A-3 0 ; 349: 5 0 -349TAT CGC GTG TCC TAT CCA TAG C-3 0 ; 161: 5 0 -161TAG CCG CAT GCC TCT CAT CCTA-3 0 ; 60: 5 0 -60CAC TGA ATC ACT GAT ACA CTA C-3 0 ; and 98: 5 0 -98GAG CAC GCA GAG CAA CAC CAT T-3 0 . The amplified promoter fragments were fused with a promoterless b-glucuronidase (GUS) reporter gene on pCAMBIA1381Xa (CAMBIA) and the fusion was confirmed by sequencing. These constructs were transformed into Arabidopsis by the flower dip method [17]. The transgenic plants were screened with 25 mg/L hygromycin and confirmed by both PCR and Southern blot. The PCRs were carried out using primers 1363, 1245, 739, 349, 161, and 60 with GUS 906c (5 0 -GCC ACG TAA GTC CGC ATC TTC A-3 0 ), which annealed to the antisense strand of the GUS gene. The 0.75-kb GUS fragment, amplified from pCAMBIA1301 with GUS 906c and GUS 151 (5 0 -AAC GAT CAG TTC GCC GAT GCA G-3 0 ), was 32P-labeled and used as a probe in the Southern blot. The tissues (i.e., seedlings, rosette leaves, cauline leaves, stem, pedicels, flowers, and immature siliques) of T3 homozygotic, singlecopy, transgenic plants were GUS-stained in the staining buffer at 37 °C for 10 h as described [18]. Wild-type Arabidopsis plants were used as negative controls, while plants transformed with plasmid pCAMBIA1301 (CAMBIA), containing a CaMV 35S promoter, served as positive controls. Analysis of diurnal rhythms. The diurnal rhythms of GUS expression were assessed under different conditions with two lines of homozygotic, single-copy transgenic Arabidopsis plants containing each deletion promoter at principal growth stage 5.10 (about 4 weeks old) [19]. Five plants of each line were placed under normal conditions (16/8 h light/dark cycle), under conditions of constant dark at the end of dark or under conditions of constant light at the end of light. Three
Z. Fan et al. / Biochemical and Biophysical Research Communications 331 (2005) 1257–1263 days later, approximately 20 mg of rosette leaves was collected from each plant every 4 h over a period of 48 h. The leaves were ground in 1 ml extraction buffer on ice and centrifuged before 20 ll of the supernatant was assayed with a BioRad VersaFluor Fluorometer as described [20]. GUS activity was expressed as nmoles 4-MU (4methylumbelliferone) per min per mg total protein. The mean value was used for diurnal rhythm analysis. To examine the changes in activity during different developmental stages in Arabidopsis owing to the four deletion promoters (D131, D637, D1027, and D1215), five T3 transgenic plants containing each deletion promoter were used for GUS activity analysis. Eight hours after lights-on, 5–20 mg of rosette leaves was taken from each plant at principal growth stage 1.08 (20 days old), 5.1 (26 days old), 6.0 (30 days old), 6.3 (40 days old), and 6.9 (50 days old) [19], ground in 1 ml extraction buffer, and examined for GUS activity as described above.
Results Cloning and sequence analysis of Zmglp1 Roots, stalks, and young whorl leaves of four-weekold maize plants were used for total RNA isolation and cDNA-AFLP. Using four primer combinations,
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we detected eight TDFs present predominantly in the young whorl leaves (Fig. 1A). The eight TDFs were 32 P-labeled by PCR methods for Northern blot analyses. One of these (TDF 11-4) was detected only in young whorl leaves of maize plants (Fig. 1B). TDF 11-4 was cloned and sequenced. The fact that many ESTs from two-week-old maize shoots were found to be highly similar to TDF 11-4 suggests that this fragment represents an expressed gene rather than genomic DNA contamination. A maize lambda FIX II genomic library was screened with 32P-labeled TDF 11-4 and a bona fide positive plaque was found (data not shown). The 3.8-kb fragment from this positive clone was sub-cloned and sequenced (GenBank Accession No. AY394010). The sequence analysis revealed a complete open reading frame (ORF) (Fig. 1C). Cloning and sequencing of the cDNA showed that this gene has no intron (data not shown). The putative protein sequence deduced from the ORF was found to have high identity (P82%) with the sequences of H. vulgare germin-like protein 1 (HvGLP1, EMBL Accession No. AJ291451.1) and Oryza sativa germin-like protein 1 (OsGLP1, GenBank Accession No. AB015593). The gene was therefore
Fig. 1. Cloning and sequence of the maize leaf-specific gene Zmglp1. (A) cDNA-AFLP fingerprints showing the pattern of TDF from maize at the early-whorl stage. The numbers 1, 2, and 3 indicate the primer combinations used in this assay: 1, EcoRI-ACC and MseI-CTT; 2, EcoRI-ACC and MseI-CAC; and 3, EcoRI-AGG and MseI-CTG. R, roots; S, stalk; and L, young whorl leaves. Arrow indicates TDF 11-4. (B) Northern blot of TDF 11-4: total RNA extracted from root (R), stalk (S), and young whorl leaves (L) hybridized with 32P-labeled TDF 11-4 and 18 S rRNA. (C) DNA sequence of Zmglp1 and its deduced amino acid sequence. The putative TATA box and a basic promoter element are underlined and shaded in grey, respectively. The putative transcription initiation site is designated as +1 and indicated by an arrow.
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designated Z. mays germin-like protein gene 1 (Zmglp1). Southern blot showed that Zmglp1 exists as a singlecopy gene per haploid genome in maize (data not shown). Zmglp1 expresses predominantly in young whorl leaves In order to confirm the expression patterns of Zmglp1, Northern blots were performed. Zmglp1 mRNA was highly abundant in young whorl leaves; less abundant in mature leaves, young tassels, and cobs; and
not detected in roots, stalks, and immature kernels (Fig. 2A). To determine the cell types in which Zmglp1 was expressed, RNA in situ hybridization experiments were performed on young whorl leaves. Zmglp1 was expressed mainly in mesophyll, phloem, and guard cells in these leaves (Fig. 2B). Promoter of Zmglp1 functioned similarly in transgenic Arabidopsis The b-glucuronidase gene (GUS::intron, pCAMBIA1381Xa) was fused to the 1.4-kb promoter (D13) of Zmglp1 and transferred into Arabidopsis (Fig. 3A). The result showed that GUS activity was detected in filaments and most of the green tissues, i.e., rosette leaves, cauline leaves, stems, pedicels, sepals, stigmas, green carpel, and dissepiment (Fig. 3B). GUS expression was not detected in the roots of seedlings and adult plants (data not shown). These data suggest that the monocotyledon promoter of Zmglp1 was able to drive gene expression in a similar pattern (green tissue-specific pattern) in dicot plants. The histochemical analysis showed that the expression intensity varied in transgenic plants with different deletion promoters. The strongest expression was found in tissues with the deletion promoter D637, whereas the faintest expression was observed with D1316 (Fig. 3A). Interestingly, the tissue specificity changed with different
Fig. 2. Expression analysis of Zmglp1. (A) Northern blot analysis of Zmglp1 in different organs. R, roots; S, stalks; YL, young whorl leaves; ML, mature leaves; T, tassels; C, cobs; and IK, immature kernels. (B) Localization of Zmglp1 mRNA in transverse sections of maize young whorl leaf by in situ hybridization. The sections were viewed under light-field illumination, and the signal is blue. (Upper) Negative control with the TDF 11-4 sense strand as the probe; (middle and bottom) with the TDF 11-4 antisense strand as probes. UEC, upper epidermis cell; LEC, lower epidermis cell; VE, vessel element; GC, guard cell; SC, substomatic chamber; MC, mesophyll cell; PC, phloem cell; and BSC, bundle sheath cell. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
Fig. 3. Deletion analysis of the Zmglp1 promoter in transgenic Arabidopsis plants. (A) Schematic representation of constructs used in the development of transgenic plants. GUS activity values represent the mean values of eight different plants, examined in a period of 48 h at intervals of 4 h. The GUS activities with the CaMV 35S promoter were examined 8 h after lights-on. (B) Tissue-specificity analysis of GUS expression in transgenic plants. With promoter D13: panel 1, seedlings; panel 2, rosette leaves; panel 3, stems; panel 4, immature siliques; and panel 5, flowers. Panel 6, seedlings with promoter D1215.
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promoter deletions (i.e., deleted promoters D131, D637, D1027, D1215, and D1316). Similar to the results observed for D13, GUS activities with deletion promoters D131, D637, D1027, D1215, and D1316 were detected in stems and leaves of seedlings, rosette leaves, cauline leaves, stems, pedicels, sepals, filaments, stigmas, green carpel, and dissepiment (Fig. 3A). However, with deletion promoter D1215, GUS activity was detected not only in these organs but also in roots (Fig. 3B), suggesting that there might be a potential suppressor binding site in the region between 1245 and 161 for the root-specificity of expression. Expression of Zmglp1 is regulated by diurnal rhythms Northern blot analysis showed that Zmglp1 RNA abundance changed dramatically during a day, with the minimum abundance of RNA observed in the light and the maximum in the dark (Fig. 4A). In order to further analyze the Zmglp1 promoter, we examined GUS activities over a period of 48 h at 4h intervals. Like other germin-like proteins [21], the expression of Zmglp1 oscillated with a circadian rhythm (Fig. 4B). The analysis of these deletion promoter constructs identified several regions containing important regulatory cis-elements. For instance, a rhythm-responsive element and a negative-regulatory element were found located in the region between 1245 and 739, the loss of which resulted in a complete change of the diurnal rhythm pattern (i.e., phase) and greatly increased GUS activity (i.e., amplitude) under a 16/8 h light/dark cycle (Fig. 4B). A positive regulatory cis-element and another negative regulatory element might be located within the regions 739 to 349 and 349 to 161, respectively (Fig. 4B). However, when the region 161 to 60 was deleted, neither the diurnal rhythm-responsiveness nor high-level expression was maintained, suggesting that this region is important for efficient and proper transcription of Zmglp1 (Fig. 4B). Interestingly, GUS activity driven by the deletion promoter D637 was nearly threefold that driven by the CaMV 35S promoter (Fig. 3A). GUS activity analyses at different growth stages revealed that the activity of deletion promoter D637 remained at a steady and high level until principal growth stage 6.0. GUS activity then declined sharply, mainly after principal growth stage 6.3 (40 days old; Fig. 4C). Under conditions of constant dark or constant light, GUS levels directed by the six promoter constructs were as high as those under a 16/8 h light/dark cycle, except that the phases and amplitudes with D13, D131, D637, D1027, and D1215 were different (Fig. 4B). Under constant dark conditions, all GUS expression patterns oscillated synchronously with those of D13 and D131, with one peak and one trough in
Fig. 4. Analysis of Zmglp1 promoter activity in maize and transgenic Arabidopsis. (A) Abundance of Zmglp1 transcripts in young whorl leaves of maize during a day. The open and black boxes represent light and dark periods during the experiment. Numbers under the boxes indicate Zeitgeber time (ZT, hours after the last lights-on) of day. (B) The diurnal rhythm of GUS activity directed by the six deletion promoters in transgenic Arabidopsis plants. GUS activities were measured during the 16/8 h light/dark cycle, at 72 h after transfer to constant light or at 72 h after transfer to constant dark. Black box, darkness; open box, light; gray box, predicted dark intervals during constant light; olive-drab box, predicted light intervals during constant dark. Values represent means of GUS activity of eight Arabidopsis plants of two transgenic lines. Time is marked in Zeitgeber time (ZT, hours after the last lights-on). (C) GUS activity analysis at different growth stages. Values represent mean GUS activity of five Arabidopsis plants of one transgenic line. The principal growth stage was determined as described [19].
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24 h. Under constant light, GUS expression directed by the five constructs changed with the same diurnal rhythm observed with light/dark cycles. The phase and period difference under constant light conditions imply that the two rhythms are controlled by different oscillators.
Discussion We identified a gene, Zmglp1, that was predominantly expressed in the young leaves of maize. Zmglp1 is highly similar in sequence to those of germin-like proteins, which belong to the cupin superfamily [22]. Many seed storage proteins, and microbial and animal proteins belong to the cupin superfamily [22,23]. Germin-like proteins possess diversified functions. Some of the germinlike protein genes are inducible by biotic or abiotic stresses [22] and some act as oxalate oxidases in wheat [24]. The deduced protein sequence of Zmglp1 is highly similar to that of barley (H. vulgare) germin-like protein 1 (HvGLP1), which was found to encode a nucleotide-sugar pyrophosphatase/phosphodiesterase (AGPPase) [10]. The soluble isoform of HvGLP1 could play a role in controlling the levels of nucleotide-sugars, important intermediates in glycolysis and gluconeogenesis [25], whereas the cell wall-bound isoform may be involved in the scavenging of extracellular nucleotide sugars [10]. The mRNA of HvGLP1 was found to be highly abundant in young leaves, less abundant in older leaves, and absent in roots [11]. Similarly, we found in this study that transcripts of Zmglp1 were highly abundant in young whorl leaves; less abundant in mature leaves, young tassels, and cobs; and not detectable in roots, immature kernels, and stalks. Therefore, it is reasonable to presume that Zmglp1 represents a maize AGPPase gene. It was proposed that the activities of both AGPPase and AGPase might be coupled in the cytosol (endosperm) and plastids (endosperm and sycamore cells), forming a futile cycle [6]. Our results showed that Zmglp1 expression did not occur in bundle sheath cells and immature kernels (Fig. 2A), but occurred mainly in mesophyll, phloem, and guard cells (Fig. 2B) in a C4 plant, maize. Given that photosynthesis in the leaves of C4 plants involves two cell types (the mesophyll cells for CO2 fixation by the C4 pathway and bundle sheath cells for starch and sucrose biosynthesis by the C3 pathway; [25]), Zmglp1 expression in mesophyll cells may suggest a role in preventing the transitory biosynthesis of starch, saving the energy for hexose transportation. Zmglp1 is not expressed in bundle sheath cells, nor in the organs specialized in accumulating reserve starch (i.e., kernels), which allows the biosynthesis of starch and avoids energy waste in the futile cycle with AGPase, as assumed by Kleczkowski [6]. This could be a new process contributing to the high photosynthetic rates in C4 plants.
The GUS activity analysis demonstrated the following. First, the 1.4-kb promoter of Zmglp1 can drive GUS expression in a similar pattern in the dicotyledon plant Arabidopsis. Second, several putative positive and negative regulatory cis-elements were identified in the promoter, of which 60 bp were found to be necessary for minimum expression and 739 bp produced expression at maximum levels. Third, the activities of deleted promoters D131, D637, D1027, and D1215 declined with plant growth. Activities declined mainly after principal growth stage 6.3 (40 days old), with the exception of deletion promoters D637 and D1215, with which GUS activities were maintained at high levels even in growth stage 6.9 (Fig. 4C). Zmglp1 expression was also found to oscillate with a circadian rhythm. This circadian rhythm persists even under conditions of constant dark or light, and the different phases and periods observed under constant conditions may indicate that different oscillators control the two rhythms [21]. Interestingly, deletions in the promoter region between 1245 and 739 could shift the circadian rhythm. In the Zmglp1 promoter, we found two TAACTG elements in the regions of 739 to 349 and 349 to 161, which might be involved in gene activation. Since C1 and MYB proteins were reported to bind the TAACTG sequence [26,27], the putative binding sites found in the promoter of Zmglp1 imply that the expression of Zmglp1 is likely to be regulated by MYB genes. As the strength and activity of promoters largely depends on the additive and synergistic action of a series of regulation elements [28], more deletions and additional biochemical approaches are needed for more precise analysis of this promoter region and its expression.
Acknowledgments This research was supported by the National HighTech Program of China (GN SZ-02-01 and 2002AA2Z1001-07). The authors are grateful to Prof. Guoying Wang (China Agriculture University, Beijing) for providing maize materials.
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Cloning and expression analysis of Zmglp1, a new germin-like protein gene in maize q Zhanmin Fan a, Hongya Gu a,b, Xiaowei Chen a, Hui Song a, Qian Wang a, Meihua Liu a, Li-Jia Qu a,b,*, Zhangliang Chen a,b a
Peking-Yale Joint Research Center for Plant Molecular Genetics and Agro-Biotechnology, National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, PR China b The National Plant Gene Research Center (Beijing), Beijing 100101, PR China Received 4 April 2005 Available online 19 April 2005
Abstract The cDNA and genomic DNA of a green tissue-specific gene were cloned from maize (Zea mays L.) using cDNA-amplified fragment length polymorphism (cDNA-AFLP) and library screening. The deduced protein was highly similar to Hordeum vulgare germin-like protein 1 (HvGLP1), and the maize gene was therefore designated Zmglp1. Northern blot specifically detected the mRNA of Zmglp1 in young whorl leaves at the early-whorl stage. However, at the late-whorl, tassel, and silk stages, Zmglp1 transcripts were highly abundant in young whorl leaves; less abundant in mature leaves, young tassels, and cobs; and not detectable in roots, immature kernels, and stalks. RNA in situ hybridization revealed that Zmglp1 expressed only in mesophyllous, phloem, and guard cells in the young whorl leaves. Deletion analysis of the promoter in transgenic Arabidopsis resulted in the identification of several regions containing important regulatory cis-elements controlling the expression levels and circadian rhythm-oscillated patterns of Zmglp1. Ó 2005 Elsevier Inc. All rights reserved. Keywords: cDNA-AFLP; Germin-like protein; Tissue-specific promoter; AGPPase
Leaves are the major sites of photosynthesis in higher plants. Excess glucose produced by photosynthesis is converted into polymeric forms for storage (mainly starch) and transport (sucrose). The biochemistry and molecular genetics of starch biosynthesis are well characterized in photosynthetic and sink tissues [1,2]. ADP-glucose pyrophosphorylase (AGPase) is thought to catalyze the important regulatory step in the biosynthesis of starch, producing the activated glucosyl donor
q Abbreviations: AGPase, ADP-glucose pyrophosphorylase; AGPPase, nucleotide-sugar pyrophosphatase/phosphodiesterase; cDNAAFLP, cDNA-amplified fragment length polymorphism; GUS, b-glucuronidase; MW, molecular weight; TDF, transcript derived fragment. * Corresponding author. Fax: +86 10 6275 1841. E-mail address: [email protected] (L.-J. Qu).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.04.045
ADP-glucose (ADPG) which serves as the direct precursor for starch synthesis [3,4]. The presence of various isozymes of AGPase and their regulation, together with the involvement of starch synthase (SS), starch branching enzyme (BE), and starch debranching enzyme (DBE), is considered to define both the quantity and quality of starch produced [5–7]. However, little is known about the enzymes catalyzing the break down of ADPG or the role of ADGP in starch biosynthesis. Recently a widely distributed nucleotide-sugar pyrophosphatase (AGPPase) that catalyzed the break down of ADPG was identified from leaves of barley, Hordeum vulgare L. [8]. This enzyme had a relatively low Km value (0.5 mM) for ADPG and could also hydrolyze UDP-glucose and CDP-glucose (Km 2–3 mM), although with lower affinity. Attempts to identify different isoforms of AGPPase have revealed
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that the activity of AGPPase declines concomitantly with the accumulation of starch during the development of sink organs, and with the occurrence of cell wallbound and starch granule-bound and soluble enzymes, the soluble enzymes being located in both intra- and extra-plastidial solutions [8–10]. The soluble AGPPase was highly abundant in young leaves, less abundant in older leaves, and absent in roots. However, the levels of the mRNA encoding AGPPase oscillated with a circadian rhythm, with the minimum and maximum mRNA abundance occurring at the ends of the dark and light periods [10,11]. The plastidial AGPPase isoform is fully active at pH 7.0 in the non-illuminated chloroplast and almost totally inactive at pH 8.0 or higher in the illuminated chloroplast during active starch biosynthesis [9]. In this study, we isolated cDNA and promoter regions of a green tissue-specific germin-like protein gene from maize (Zea mays L.). The transcript of this gene, designated Zmglp1 owing to its sequence similarity to barley AGPPase gene, was highly abundant in young leaves; less abundant in mature leaves, young tassels, and cobs; and not detectable in roots, immature kernels, and stalks. RNA in situ hybridization experiments revealed that Zmglp1 was only expressed in mesophyllous, phloem, and guard cells in the young whorl leaves. The RNA abundance of Zmglp1 was also found to oscillate with a circadian rhythm. To better understand the role of Zmglp1, the promoter was analyzed by deletion studies in transgenic Arabidopsis to identify the regions responsible for activity and tissue-specificity as well as the circadian rhythm.
Materials and methods DNA manipulation. DNA manipulations were conducted using standard procedures [12], unless otherwise specified. Escherichia coli DH5a was used as the host for plasmid amplification, and Agrobacterium tumefaciens GV3101 was used for Arabidopsis transformation. Plant materials. A maize cultivar from northern China (Z. mays L. var. Zhongdan 306) was used in this study. Arabidopsis thaliana (ecotype Columbia) was grown in a growth chamber at 22 °C with a 16/8 h light/dark cycle at a light intensity of ±45 lmol m2/s. cDNA-AFLP. Total RNA was isolated by the guanidium thiocyanate method [13]. After treatment with DNase I at 37 °C for 30 min to remove genomic DNA, the RNA concentration was determined by spectrophotometry. cDNA was synthesized from 50 lg of total RNA using a cDNA Synthesis kit (Boehringer–Mannheim, Mannheim, Germany). Starting with 250 ng of cDNA, subsequent restriction enzyme digestion, adapter ligation, and PCR amplification were performed according to the protocol provided with the Core AFLP kit (Gibco-BRL). After selective amplification, an equal volume of formamide dye was added, and 3 ll of each product was loaded on a 6% polyacrylamide gel on a GenomyxLR DNA Sequencer (Beckman). After electrophoresis, the gel was dried for 30 min and exposed to X-ray film (Kodak BIOMAX) at 70 °C for 48 h. The transcript-derived fragments (TDFs) identified as predominantly expressed in young whorls were excised from the gel, soaked in 50 ll TE buffer, and boiled in a water bath at 100 °C for 10 min. Aliquots of 1 ll were re-amplified using the same primer pairs and
conditions as used in the selective amplification. The PCR product was cloned into pUC. Northern and Southern blots. The total RNA samples (15 lg per lane) were loaded on 1.2%(w/v) agarose gels, and Northern blot and hybridization were carried out as described previously [14]. 10–15 lg of genomic DNA isolated from maize leaves was digested with a restriction enzyme, and Southern blot and hybridization were carried out as described [14]. Library screening and sequence analysis. A lambda FIX II genomic library constructed from the Missouri 17 inbred line was screened according to the manufacturerÕs instructions (Stratagene) using 32 P-labeled TDF 11-4 as the probe. The lambda phage DNA of the positive clone was extracted using a High Pure Lambda Isolation kit (Roche, Basel, Switzerland) and 100 ng of lambda DNA was used for Southern blot as described [14]. A 3.8-kb XbaI fragment with strong positive signal was sub-cloned into pBS and sequenced. Sequencespecific primers (primer A: 5 0 -63GTT GCC ATG GCC AAA ATG GTG T-3 0 and primer B: 5 0 -1067CAG ATT AAC AGC ATG CGG CAC T-3 0 ) were designed to amplify the full-length cDNA from young whorl leaves. Comparisons with sequences in the GenBank database were conducted using BLAST [15]. In situ hybridization. Probe preparation, leaf fixation, paraffin embedding, sectioning, pre-hybridization, and hybridization were carried out essentially as previously described [16], except for a few modifications described below. Young whorl leaves of four-week-old plants were selected for fixation. Eight-micrometer thick, horizontal sections were attached to slides coated with 1 mg/ml poly-lysine solution (MW > 150,000, Sigma). The RNase treatment step after hybridization and the high stringency wash were omitted. Deletion and GUS analysis in transgenic Arabidopsis. Promoter deletions of Zmglp1 were generated by PCR using Zmglp1 promoter as the template. The deletion promoter regions D13, D131, D637, D1027, D1215, and D1316 were amplified using primers 1363, 1245, 739, 349, 161, and 60 with 98, respectively. The primer sequences were as follows: 1363: 5 0 -1363ACC TCA CAT GTC ACG CAT GTA A-3 0 ; 1245: 5 0 -1245GGT GTG ACA GTG ATG GCG AGT A-3 0 ; 739: 5 0 -739TAG TGG AGA CCG TCG GAC ATA A-3 0 ; 349: 5 0 -349TAT CGC GTG TCC TAT CCA TAG C-3 0 ; 161: 5 0 -161TAG CCG CAT GCC TCT CAT CCTA-3 0 ; 60: 5 0 -60CAC TGA ATC ACT GAT ACA CTA C-3 0 ; and 98: 5 0 -98GAG CAC GCA GAG CAA CAC CAT T-3 0 . The amplified promoter fragments were fused with a promoterless b-glucuronidase (GUS) reporter gene on pCAMBIA1381Xa (CAMBIA) and the fusion was confirmed by sequencing. These constructs were transformed into Arabidopsis by the flower dip method [17]. The transgenic plants were screened with 25 mg/L hygromycin and confirmed by both PCR and Southern blot. The PCRs were carried out using primers 1363, 1245, 739, 349, 161, and 60 with GUS 906c (5 0 -GCC ACG TAA GTC CGC ATC TTC A-3 0 ), which annealed to the antisense strand of the GUS gene. The 0.75-kb GUS fragment, amplified from pCAMBIA1301 with GUS 906c and GUS 151 (5 0 -AAC GAT CAG TTC GCC GAT GCA G-3 0 ), was 32P-labeled and used as a probe in the Southern blot. The tissues (i.e., seedlings, rosette leaves, cauline leaves, stem, pedicels, flowers, and immature siliques) of T3 homozygotic, singlecopy, transgenic plants were GUS-stained in the staining buffer at 37 °C for 10 h as described [18]. Wild-type Arabidopsis plants were used as negative controls, while plants transformed with plasmid pCAMBIA1301 (CAMBIA), containing a CaMV 35S promoter, served as positive controls. Analysis of diurnal rhythms. The diurnal rhythms of GUS expression were assessed under different conditions with two lines of homozygotic, single-copy transgenic Arabidopsis plants containing each deletion promoter at principal growth stage 5.10 (about 4 weeks old) [19]. Five plants of each line were placed under normal conditions (16/8 h light/dark cycle), under conditions of constant dark at the end of dark or under conditions of constant light at the end of light. Three
Z. Fan et al. / Biochemical and Biophysical Research Communications 331 (2005) 1257–1263 days later, approximately 20 mg of rosette leaves was collected from each plant every 4 h over a period of 48 h. The leaves were ground in 1 ml extraction buffer on ice and centrifuged before 20 ll of the supernatant was assayed with a BioRad VersaFluor Fluorometer as described [20]. GUS activity was expressed as nmoles 4-MU (4methylumbelliferone) per min per mg total protein. The mean value was used for diurnal rhythm analysis. To examine the changes in activity during different developmental stages in Arabidopsis owing to the four deletion promoters (D131, D637, D1027, and D1215), five T3 transgenic plants containing each deletion promoter were used for GUS activity analysis. Eight hours after lights-on, 5–20 mg of rosette leaves was taken from each plant at principal growth stage 1.08 (20 days old), 5.1 (26 days old), 6.0 (30 days old), 6.3 (40 days old), and 6.9 (50 days old) [19], ground in 1 ml extraction buffer, and examined for GUS activity as described above.
Results Cloning and sequence analysis of Zmglp1 Roots, stalks, and young whorl leaves of four-weekold maize plants were used for total RNA isolation and cDNA-AFLP. Using four primer combinations,
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we detected eight TDFs present predominantly in the young whorl leaves (Fig. 1A). The eight TDFs were 32 P-labeled by PCR methods for Northern blot analyses. One of these (TDF 11-4) was detected only in young whorl leaves of maize plants (Fig. 1B). TDF 11-4 was cloned and sequenced. The fact that many ESTs from two-week-old maize shoots were found to be highly similar to TDF 11-4 suggests that this fragment represents an expressed gene rather than genomic DNA contamination. A maize lambda FIX II genomic library was screened with 32P-labeled TDF 11-4 and a bona fide positive plaque was found (data not shown). The 3.8-kb fragment from this positive clone was sub-cloned and sequenced (GenBank Accession No. AY394010). The sequence analysis revealed a complete open reading frame (ORF) (Fig. 1C). Cloning and sequencing of the cDNA showed that this gene has no intron (data not shown). The putative protein sequence deduced from the ORF was found to have high identity (P82%) with the sequences of H. vulgare germin-like protein 1 (HvGLP1, EMBL Accession No. AJ291451.1) and Oryza sativa germin-like protein 1 (OsGLP1, GenBank Accession No. AB015593). The gene was therefore
Fig. 1. Cloning and sequence of the maize leaf-specific gene Zmglp1. (A) cDNA-AFLP fingerprints showing the pattern of TDF from maize at the early-whorl stage. The numbers 1, 2, and 3 indicate the primer combinations used in this assay: 1, EcoRI-ACC and MseI-CTT; 2, EcoRI-ACC and MseI-CAC; and 3, EcoRI-AGG and MseI-CTG. R, roots; S, stalk; and L, young whorl leaves. Arrow indicates TDF 11-4. (B) Northern blot of TDF 11-4: total RNA extracted from root (R), stalk (S), and young whorl leaves (L) hybridized with 32P-labeled TDF 11-4 and 18 S rRNA. (C) DNA sequence of Zmglp1 and its deduced amino acid sequence. The putative TATA box and a basic promoter element are underlined and shaded in grey, respectively. The putative transcription initiation site is designated as +1 and indicated by an arrow.
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designated Z. mays germin-like protein gene 1 (Zmglp1). Southern blot showed that Zmglp1 exists as a singlecopy gene per haploid genome in maize (data not shown). Zmglp1 expresses predominantly in young whorl leaves In order to confirm the expression patterns of Zmglp1, Northern blots were performed. Zmglp1 mRNA was highly abundant in young whorl leaves; less abundant in mature leaves, young tassels, and cobs; and
not detected in roots, stalks, and immature kernels (Fig. 2A). To determine the cell types in which Zmglp1 was expressed, RNA in situ hybridization experiments were performed on young whorl leaves. Zmglp1 was expressed mainly in mesophyll, phloem, and guard cells in these leaves (Fig. 2B). Promoter of Zmglp1 functioned similarly in transgenic Arabidopsis The b-glucuronidase gene (GUS::intron, pCAMBIA1381Xa) was fused to the 1.4-kb promoter (D13) of Zmglp1 and transferred into Arabidopsis (Fig. 3A). The result showed that GUS activity was detected in filaments and most of the green tissues, i.e., rosette leaves, cauline leaves, stems, pedicels, sepals, stigmas, green carpel, and dissepiment (Fig. 3B). GUS expression was not detected in the roots of seedlings and adult plants (data not shown). These data suggest that the monocotyledon promoter of Zmglp1 was able to drive gene expression in a similar pattern (green tissue-specific pattern) in dicot plants. The histochemical analysis showed that the expression intensity varied in transgenic plants with different deletion promoters. The strongest expression was found in tissues with the deletion promoter D637, whereas the faintest expression was observed with D1316 (Fig. 3A). Interestingly, the tissue specificity changed with different
Fig. 2. Expression analysis of Zmglp1. (A) Northern blot analysis of Zmglp1 in different organs. R, roots; S, stalks; YL, young whorl leaves; ML, mature leaves; T, tassels; C, cobs; and IK, immature kernels. (B) Localization of Zmglp1 mRNA in transverse sections of maize young whorl leaf by in situ hybridization. The sections were viewed under light-field illumination, and the signal is blue. (Upper) Negative control with the TDF 11-4 sense strand as the probe; (middle and bottom) with the TDF 11-4 antisense strand as probes. UEC, upper epidermis cell; LEC, lower epidermis cell; VE, vessel element; GC, guard cell; SC, substomatic chamber; MC, mesophyll cell; PC, phloem cell; and BSC, bundle sheath cell. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
Fig. 3. Deletion analysis of the Zmglp1 promoter in transgenic Arabidopsis plants. (A) Schematic representation of constructs used in the development of transgenic plants. GUS activity values represent the mean values of eight different plants, examined in a period of 48 h at intervals of 4 h. The GUS activities with the CaMV 35S promoter were examined 8 h after lights-on. (B) Tissue-specificity analysis of GUS expression in transgenic plants. With promoter D13: panel 1, seedlings; panel 2, rosette leaves; panel 3, stems; panel 4, immature siliques; and panel 5, flowers. Panel 6, seedlings with promoter D1215.
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promoter deletions (i.e., deleted promoters D131, D637, D1027, D1215, and D1316). Similar to the results observed for D13, GUS activities with deletion promoters D131, D637, D1027, D1215, and D1316 were detected in stems and leaves of seedlings, rosette leaves, cauline leaves, stems, pedicels, sepals, filaments, stigmas, green carpel, and dissepiment (Fig. 3A). However, with deletion promoter D1215, GUS activity was detected not only in these organs but also in roots (Fig. 3B), suggesting that there might be a potential suppressor binding site in the region between 1245 and 161 for the root-specificity of expression. Expression of Zmglp1 is regulated by diurnal rhythms Northern blot analysis showed that Zmglp1 RNA abundance changed dramatically during a day, with the minimum abundance of RNA observed in the light and the maximum in the dark (Fig. 4A). In order to further analyze the Zmglp1 promoter, we examined GUS activities over a period of 48 h at 4h intervals. Like other germin-like proteins [21], the expression of Zmglp1 oscillated with a circadian rhythm (Fig. 4B). The analysis of these deletion promoter constructs identified several regions containing important regulatory cis-elements. For instance, a rhythm-responsive element and a negative-regulatory element were found located in the region between 1245 and 739, the loss of which resulted in a complete change of the diurnal rhythm pattern (i.e., phase) and greatly increased GUS activity (i.e., amplitude) under a 16/8 h light/dark cycle (Fig. 4B). A positive regulatory cis-element and another negative regulatory element might be located within the regions 739 to 349 and 349 to 161, respectively (Fig. 4B). However, when the region 161 to 60 was deleted, neither the diurnal rhythm-responsiveness nor high-level expression was maintained, suggesting that this region is important for efficient and proper transcription of Zmglp1 (Fig. 4B). Interestingly, GUS activity driven by the deletion promoter D637 was nearly threefold that driven by the CaMV 35S promoter (Fig. 3A). GUS activity analyses at different growth stages revealed that the activity of deletion promoter D637 remained at a steady and high level until principal growth stage 6.0. GUS activity then declined sharply, mainly after principal growth stage 6.3 (40 days old; Fig. 4C). Under conditions of constant dark or constant light, GUS levels directed by the six promoter constructs were as high as those under a 16/8 h light/dark cycle, except that the phases and amplitudes with D13, D131, D637, D1027, and D1215 were different (Fig. 4B). Under constant dark conditions, all GUS expression patterns oscillated synchronously with those of D13 and D131, with one peak and one trough in
Fig. 4. Analysis of Zmglp1 promoter activity in maize and transgenic Arabidopsis. (A) Abundance of Zmglp1 transcripts in young whorl leaves of maize during a day. The open and black boxes represent light and dark periods during the experiment. Numbers under the boxes indicate Zeitgeber time (ZT, hours after the last lights-on) of day. (B) The diurnal rhythm of GUS activity directed by the six deletion promoters in transgenic Arabidopsis plants. GUS activities were measured during the 16/8 h light/dark cycle, at 72 h after transfer to constant light or at 72 h after transfer to constant dark. Black box, darkness; open box, light; gray box, predicted dark intervals during constant light; olive-drab box, predicted light intervals during constant dark. Values represent means of GUS activity of eight Arabidopsis plants of two transgenic lines. Time is marked in Zeitgeber time (ZT, hours after the last lights-on). (C) GUS activity analysis at different growth stages. Values represent mean GUS activity of five Arabidopsis plants of one transgenic line. The principal growth stage was determined as described [19].
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24 h. Under constant light, GUS expression directed by the five constructs changed with the same diurnal rhythm observed with light/dark cycles. The phase and period difference under constant light conditions imply that the two rhythms are controlled by different oscillators.
Discussion We identified a gene, Zmglp1, that was predominantly expressed in the young leaves of maize. Zmglp1 is highly similar in sequence to those of germin-like proteins, which belong to the cupin superfamily [22]. Many seed storage proteins, and microbial and animal proteins belong to the cupin superfamily [22,23]. Germin-like proteins possess diversified functions. Some of the germinlike protein genes are inducible by biotic or abiotic stresses [22] and some act as oxalate oxidases in wheat [24]. The deduced protein sequence of Zmglp1 is highly similar to that of barley (H. vulgare) germin-like protein 1 (HvGLP1), which was found to encode a nucleotide-sugar pyrophosphatase/phosphodiesterase (AGPPase) [10]. The soluble isoform of HvGLP1 could play a role in controlling the levels of nucleotide-sugars, important intermediates in glycolysis and gluconeogenesis [25], whereas the cell wall-bound isoform may be involved in the scavenging of extracellular nucleotide sugars [10]. The mRNA of HvGLP1 was found to be highly abundant in young leaves, less abundant in older leaves, and absent in roots [11]. Similarly, we found in this study that transcripts of Zmglp1 were highly abundant in young whorl leaves; less abundant in mature leaves, young tassels, and cobs; and not detectable in roots, immature kernels, and stalks. Therefore, it is reasonable to presume that Zmglp1 represents a maize AGPPase gene. It was proposed that the activities of both AGPPase and AGPase might be coupled in the cytosol (endosperm) and plastids (endosperm and sycamore cells), forming a futile cycle [6]. Our results showed that Zmglp1 expression did not occur in bundle sheath cells and immature kernels (Fig. 2A), but occurred mainly in mesophyll, phloem, and guard cells (Fig. 2B) in a C4 plant, maize. Given that photosynthesis in the leaves of C4 plants involves two cell types (the mesophyll cells for CO2 fixation by the C4 pathway and bundle sheath cells for starch and sucrose biosynthesis by the C3 pathway; [25]), Zmglp1 expression in mesophyll cells may suggest a role in preventing the transitory biosynthesis of starch, saving the energy for hexose transportation. Zmglp1 is not expressed in bundle sheath cells, nor in the organs specialized in accumulating reserve starch (i.e., kernels), which allows the biosynthesis of starch and avoids energy waste in the futile cycle with AGPase, as assumed by Kleczkowski [6]. This could be a new process contributing to the high photosynthetic rates in C4 plants.
The GUS activity analysis demonstrated the following. First, the 1.4-kb promoter of Zmglp1 can drive GUS expression in a similar pattern in the dicotyledon plant Arabidopsis. Second, several putative positive and negative regulatory cis-elements were identified in the promoter, of which 60 bp were found to be necessary for minimum expression and 739 bp produced expression at maximum levels. Third, the activities of deleted promoters D131, D637, D1027, and D1215 declined with plant growth. Activities declined mainly after principal growth stage 6.3 (40 days old), with the exception of deletion promoters D637 and D1215, with which GUS activities were maintained at high levels even in growth stage 6.9 (Fig. 4C). Zmglp1 expression was also found to oscillate with a circadian rhythm. This circadian rhythm persists even under conditions of constant dark or light, and the different phases and periods observed under constant conditions may indicate that different oscillators control the two rhythms [21]. Interestingly, deletions in the promoter region between 1245 and 739 could shift the circadian rhythm. In the Zmglp1 promoter, we found two TAACTG elements in the regions of 739 to 349 and 349 to 161, which might be involved in gene activation. Since C1 and MYB proteins were reported to bind the TAACTG sequence [26,27], the putative binding sites found in the promoter of Zmglp1 imply that the expression of Zmglp1 is likely to be regulated by MYB genes. As the strength and activity of promoters largely depends on the additive and synergistic action of a series of regulation elements [28], more deletions and additional biochemical approaches are needed for more precise analysis of this promoter region and its expression.
Acknowledgments This research was supported by the National HighTech Program of China (GN SZ-02-01 and 2002AA2Z1001-07). The authors are grateful to Prof. Guoying Wang (China Agriculture University, Beijing) for providing maize materials.
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