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NTZIP antisense plants show reduced chlorophyll levels
Plant Physiology and Biochemistry 42 (2004) 321–327 www.elsevier.com/locate/plaphy
Original article
NTZIP antisense plants show reduced chlorophyll levels Ning Liu a,b, Yu-Tao Yang a, Han-Hua Liu a, Guo-Dong Yang a, Nai-Hua Zhang a, Cheng Chao Zheng a,* a
College of Life Sciences, Shandong Agricultural University, Taian, Shandong 271018, PR China b Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, PR China Received 17 September 2003; accepted 17 February 2004
Abstract We have isolated and characterized a new photosynthetic tissue-specific gene NTZIP (Nicotiana tabacum leucine zipper) from tobacco (N. tabacum). Its deduced amino acid sequence has two highly conserved regions, leucine zipper and [EXnDEXRH]2 motifs, which are related to the gene’s biochemical functions. NTZIP was expressed in leaves and stems, but was not detected in roots or flowers, suggesting that its physiological functions might be associated with photosynthesis. Northern blot analysis showed that NTZIP mRNA accumulation was induced by light signals, increased greatly under low temperatures and was repressed by strong light illumination. Furthermore, a number of homologs of NTZIP were isolated from cucumber (Cucumis sativus), rape (Brassica napus), clover (Trifolium repens), willow (Salix babylonica), rosebush (Rusa dovurica), wheat (Triticum aestivum) and spinach (Spinacia oleracea), proving the ubiquitous existence of the NTZIP-like genes in higher plants. Transgenic tobaccos constitutively expressing antisense RNA to NTZIP displayed chlorosis and a lack of ability to turn green even under normal growth conditions. The chlorophyll deficiency was further confirmed by chlorophyll content determination and gas exchange analysis. Based on these observations, we propose that NTZIP may be involved in chlorophyll biosynthesis, and might define a novel family of evolutionarily conserved proteins with its homologs in other plant species. © 2004 Elsevier SAS. All rights reserved. Keywords: Chlorophyll synthesis; Gene expression; Leucine zipper protein; NTZIP; Nicotiana tabacum leucine zipper; Transgenic tobacco
1. Introduction Photosynthesis is the most important source of energy on earth. In chloroplast, about 250–300 chlorophyll molecules bind to their neighbouring pigments. This comprises the reaction centre, in which special chlorophyll pairs in photosystem I and II are the primary electron donors that drive the conversion of light into chemical energy to be conserved in NADPH2 and ATP [29]. Since chlorophyll is the principal pigment that traps light energy, the biosynthesis of chlorophyll has presented a number of challenging topics in the field of plant molecular biology [30]. Significant research has been conducted to investigate the mechanisms by which
Abbreviations: AcsF, Rubrivivax gelatinosus aerobic cyclase system Fe-containing subunit gene; AT103, Arabidopsis thaliana 103 gene; Chl, chlorophyll; Crd1, Chlamydomonas reinhhardtii copper response defect gene; PFD, photon flux density; PNZIP, Pharbitis nil leucine zipper gene; PSI, photosystem I; PSII, photosystem II; ZIP, leucine zipper. * Corresponding author. E-mail address: [email protected] (C.C. Zheng). © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.plaphy.2004.02.007
photosynthesis is conducted in higher plants and other photosynthetic organisms. Many genes in photosynthetic organisms, such as algae, bacteria and higher plants involved in photosynthesis have been cloned and characterized in the last decades, including cab, rbcS, chlI, bchM, petJ, PPDK and PNZIP [6,18,31,33]. Our previous studies have demonstrated that expression of PNZIP (Pharbitis nil leucine zipper) is regulated by light and is restricted to photosynthetically active mesophyll tissues in P. nil [33]. The promoter analysis of PNZIP using GUS as reporter gene also confirmed that it is expressed specifically in photosynthetic tissue in transgenic tobaccos [32]. Recently, two homologs in a photosynthetic bacterium and a green alga, AcsF (aerobic cyclase system Fe-containing subunit gene) and Crd1 (copper response defect gene), also have been characterized by mutant analysis [19,21]. AcsF, in purple bacteria Rubrivivax gelatinosus, has been proved to be involved in the aerobic oxidative cyclization of Mgprotoporphyrin IX mono-methylester, one of the intermediates in the synthesis of bacterio-chlorophyll [21]. In Chlamydomonas reinhhardtii, Crd1 was identified as a putative
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diiron enzyme required for photosystem I accumulation in copper deficiency [19]. These results implied that this class of genes might be involved in biosynthesis of photosystem components in bacteria and algae, but their precise function in higher plants remained to be elucidated. To identify the biological role of those genes by antisense RNA strategy, we cloned NTZIP (Nicotiana tabacum leucine zipper) in tobacco by using a PCR method with degenerate primers on the basis of conserved sequences of PNZIP, AcsF and Crd1. Transgenic tobacco plants constitutively expressing antisense RNA displayed a chlorophyll deficient phenotype, indicating that NTZIP might play a vital role during chlorophyll biosynthesis in tobacco. To address whether NTZIP’s homologs exist ubiquitously in the plant kingdom, we also isolated a number of genes with high similarity to NTZIP from various plant species, suggesting that their products consist of a novel protein family.
2. Results 2.1. Isolation of NTZIP and its homologs from plants Using RT-PCR with degenerate primers and the RACE method, a 1323 bp cDNA clone including a full-length coding region was isolated from tobacco leaf tissue and named NTZIP (GenBank accession AY221168). The deduced amino acid sequence of NTZIP contains 371 amino acid residues with overall identities of 91.0%, 91.4%, and 40.2% to PNZIP, AT103 and AcsF, respectively. Analysis of NTZIP’s putative secondary structure using Anthepro software (Ver. 5.0) shows that it is comprised almost exclusively of a-helices with the exception of a b-sheet between them (Fig. 1). To determine whether NTZIP represents a single locus in the N. tabacum genome or whether it is a multicopy gene, a Southern blot analysis was performed using the NTZIP cDNA as a probe. The result showed that NTZIP hybridized to only one genomic restriction fragment, indicating that it represents a single-copy gene (Fig. 2). The similar results were obtained in Arabidopsis thaliana and P. nil [32,33], suggesting that NTZIP-like gene probably represents a single locus also in the genome of other plants. In order to test whether NTZIP-homologous genes are ubiquitous in higher plants, RT-PCR was performed using the same degenerate primers for NTZIP to amplify cDNA fragments in other plant species. Specific cDNA fragments of E RDEARHA
E QDENRHG
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Fig. 1. Prediction of NTZIP secondary structure using the Anthepro5.0 software. a-Helix is in blue, b-sheet is in yellow. The location of [EXnEXRH]2 consensus and the leucine zipper are marked with vertical lines and an arrow, respectively.
Fig. 2. Genomic Southern analyses of NTZIP genes. DNA was isolated from young leaves of tobacco plants and digested with PstI, SpeI and EcoRI. NTZIP has no restriction sites for PstI, SpeI, whereas one site was identified for EcoRI. DNA marker sizes are indicated on the left of the figure.
about 1 kb with high similarity to NTZIP were isolated and sequenced from cucumber, rape, clover, willow, rosebush, wheat and spinach (Fig. 3). Moreover, eight NTZIP homologous sequences in other photosynthetic organisms were obtained from the GenBank database (Fig. 3). All deduced amino acid sequences contained a leucine zipper domain for protein dimerization and a [(D/E)EX2H]2 motif which is present in soluble fatty acid desaturases from mammals, fungi, insects, higher plants and cyanobacteria, or hydroxylase and monooxygenase from bacteria [23,25,26]. They share high similarity in amino acid sequences but can be distinguished from other previously known proteins, indicating that the proteins encoded by these genes consist of a novel protein family (Fig. 3). 2.2. The expression of NTZIP is regulated by environmental conditions To determine the pattern of NTZIP expression in different tobacco organs, a northern blot hybridization analysis was performed. The results showed that NTZIP transcripts are easily detected in the leaves, less detected in the stems, and cannot be detected at all in flowers and roots (Fig. 4A), indicating that it is expressed especially in photosynthetic tissues, but not in non-photosynthetic tissues. Therefore, we propose that the NTZIP gene may have a role in the biosynthesis or function of the photosynthetic apparatus. In order to determine whether the NTZIP gene is regulated by light signals, tobacco seedlings were pre-treated with continuous darkness for 3 days, then transferred to continuous light illumination. The levels of NTZIP mRNA increased significantly after 10 h light exposure, and kept increasing gradually during the subsequent period of light treatment (Fig. 4B). However, strong light inhibited the accumulation of NTZIP mRNA in tobacco leaves, implying that photoinhibition might affect NTZIP expression (Fig. 4C). Interest-
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Consensus PNZIP AT103 NTZIP RDZIP CSZIP Higher OSZIP plants BNZIP EEZIP SBZIP SOZIP TAZIP TRZIP ZMZIP CRZIP Bacteria AcsF or algae Crd1 RPZIP Consensus PNZIP AT103 NTZIP RDZIP CSZIP OSZIP BNZIP EEZIP SBZIP SOZIP TAZIP TRZIP ZMZIP CRZIP AcsF Crd1 RPZIP Consensus PNZIP AT103 NTZIP RDZIP CSZIP OSZIP BNZIP EEZIP SBZIP SOZIP TAZIP TRZIP ZMZIP CRZIP AcsF Crd1 RPZIP Consensus PNZIP AT103 NTZIP RDZIP CSZIP OSZIP BNZIP EEZIP SBZIP SOZIP TAZIP TRZIP ZMZIP CRZIP AcsF Crd1 RPZIP
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Fig. 3. Comparison of the partial deduced amino acid sequences of NTZIP and its homologs. The motifs are marked in the consensus sequence. The Leu residues in the Leu zipper domain are in bold and boxed; the e position amino acid is underlined, the a position of the second heptad is shaded; The [EXnDEXRH]2 motif is in bold and underlined (n = ~30). AT103, A. thaliana (U38232); BNZIP, Brassica napus (AY322556); CPPZIP, Porphyra purpurea chloroplast (U38804); Crd, Chlamydomonas reinhardtii (AY057871); CSZIP, Cucumis sativus (AY221169); EEZIP, Euphorbia elsua (AF417577); NTZIP, N. tabacum (AY221168); OSZIP, Oryza sativa (AP000815); PNZIP, P. nil (U37437); RDZIP, Rusa dovurica (AY322555); AscF, Rubrivivax gelatinosus (AY057871); RPZIP, R. palustris (AF195122); SLZIP, Salix babylonica (AY322554); SOZIP, Spinacia oleracea (AY322553); TAZIP, Triticum aestivum (AY322552); TRZIP, Trifolium repens (AY322557); ZMZIP, Zea mays (AY108897).
ingly, we observed that the NTZIP mRNA accumulations were stimulated by a low temperature stress as well (Fig. 4C). 2.3. Both NTZIP mRNA and chlorophyll levels were reduced in transgenic tobaccos
To verify that the NTZIP gene is involved in chlorophyll synthesis, as is its homologous gene in photosynthetic bacteria, we constructed a transcriptional fusion between the CaMV35S promoter and the NTZIP coding region in antisense orientation. Thirty-two transformants were obtained. The transgenic tobaccos were further confirmed by PCR with 35S primers and Southern blot (data not shown). In most
transgenic tobaccos, northern blots indicated that NTZIP mRNA accumulation decreased compared with wild-type plants, which means antisense repression was effective and markedly reduced the NTZIP transcript levels. A positional effect also was found in our transgenic tobaccos, and NTZIP mRNA accumulation was either difficult to detect or was still expressed at high levels, which was also related to their phenotype. The more yellowish the transformants were, the less NTZIP mRNA they accumulated (Fig. 5). As in the case of chlorophyll, the level of chlorophyll in the leaves of the transgenic plants was 35% less than that of wild-type controls, the ratio of Chl a/b, however, did not change much (Table 1). This result demonstrated that a chlorophyll deficiency occurred in the transgenic lines. Since chlorophyll is
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Fig. 4. Northern blot analysis of NTZIP in tobacco plants. Each lane contains 30 µg of tobacco leaf total RNA. A photograph of 28S rRNA served as a control for equal loading of RNA in each lane (lower panel). (A) Northern blot analysis of NTZIP mRNA accumulation in different organs; (B) northern blot hybridization analysis of NTZIP mRNA accumulation during dark and light treatment; (C) northern blot analysis of NTZIP mRNA accumulation at low temperature (LT) and after strong light illumination (SLI).
In order to test our hypothesis, gas exchange was analyzed in leaves of transgenic tobaccos and wild-type controls. The result showed that the net photosynthetic rate (PN) per leaf area unit in transgenic tobaccos decreased to only 54.0% of the controls. However, the net PN values on a chlorophyll basis are similar in both wild-type and transgenic tobaccos, and a partial stomata closure occurred during reduction of the photosynthetic rate in transgenic tobacco. The leaf conductance decreased more slowly than the net photosynthesis rate, resulting in that the intercellular CO2 concentrations (Ci) were similar in both transgenic and wild-type tobacco leaves (Table 2). These results indicated that the capacity of CO2 fixation was not inhibited by the partial stomata closure. Therefore, the decrease of photosynthetic activity might mainly be caused by the loss in decrease of CO2 assimilation due to the decline of the content of chlorophyll in transgenic tobacco leaves.
3. Discussion 3.1. NTZIP and its homologs define a novel protein family
Fig. 5. Northern blot hybridization analysis of tobacco NTZIP mRNA accumulation in transgenic lines. Each lane contains 30 µg of tobacco leaf total RNA. A photograph of 28S rRNA served as a control for equal loading of RNA in each lane (lower panel). WT: Wild-type tobacco; T1-1, T1-2 and T1-3: independent transgenic tobacco lines. Table 1 Changes in leaf chlorophyll content and Chl a/b ratio in wild-type and transgenic tobaccos. Values indicated with different letters were significantly different at P = 0.05. Each value is an average of five measurements. Numbers in parenthesis are percentages Wild-type Transgenic tobaccos
Chl (mg g–1 FW) 2.02 ± 0.13 (100) 0.71 ± 0.03 (35)
Chl a/b 3.15 ± 0.27 2.88 ± 0.17
the component of PSI and PSII, a chlorophyll deficiency might lead to a decrease of the photosynthetic rate in transgenic tobaccos.
Comparison of NTZIP cDNA with the GenBank database of known sequences revealed no obvious similarity with any other gene of known function in higher plants. A multiple alignment of the NTZIP deduced amino acid sequence and the candidate homologs demonstrated that the NTZIP is about 90% identical to its homologous genes or cDNA fragments in higher plants, and 39.7–65.4% identical with its homologs in algae and photosynthetic bacteria (Fig. 2). Our data demonstrated that these amino acid sequences are highly conserved, especially among higher plants, and might define a novel family of evolutionarily conserved proteins. The leucine zipper is one of the most popular motifs in transcriptional factors and can help protein to form homodimers or heterodimers in many creatures, especially in eukaryotic organisms [3,9,10]. Many proteins with leucine zippers play important roles in plant growth and development [14,22,24]. Two polypeptides may homo- or heterodimerize to adjust the function of new dimers that may have different catalysis characteristics [20,27]. NTZIP contains a leucine zipper with four heptads and the leucine residue is replaced by tyrosine in the fourth heptad (Fig. 3), which is often
Table 2 Net photosynthetic rate (PN), stomatal conductance (Gs), intercellular CO2 concentration (Ci) of wild-type and transgenic tobaccos. Each point represents the mean ± S.E. of five replications. Stars indicate t-test comparison between wild-type and transgenic tobaccos under high light irradiance
Wild-type Transgenic tobaccos *
PN (µmol CO2 m–2 s–1) 7.0+0.6 * 3.8+0.3 *
P < 0.001; NS, not significant at P < 0.05.
PN (µmol CO2 mg–1 Chl s–1) 945+37.2 NS 933+28.2 NS
Gs (mmol m–2 s–1) 106.5+9.3 * 48.5+5.2 *
Ci (µmol mol–1) 267+24.6 NS 259+26.7 NS
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happened in other zipper proteins [1,12]. Thus, it is possible that the new NTZIP family might heterodimerize or homodimerize with other ZIP proteins. The leucine zipper dimerization domain forms a parallel coil that consists of four to five heptads, in which each heptad is composed of two a-helical turns or seven amino acids, labeled a, b, c, d, e, f and g. Amino acids in the a, d, e and g positions regulate leucine zipper oligomerization, dimerization stability, and dimerization specificity [1,13,28]. Recently, genetic studies showed that homodimerizing leucine zippers have two distinct properties. First, each ZIP family has a distinct pattern of attractive g ↔ e′ pairs (′ refers to the second a-helix in the dimer). Second, all have asparagines in a position of the second heptad [8,28]. We observed that amino residues at the e position were all acidic or neutral amino acids with a negative charge in the putative proteins encoded by NTZIP and its homologs, which may improve attractive g ↔ e′ pairs, drive the formation of homodimerization, and contribute to its stability. There were also asparagine residues in the second heptad position in most proteins except in R. gelatinosus and Rhodopseudomonas palustris, in which isoleucine and leucine replace asparagines [21]. By protein crystallographic methods, it was observed that the recombinant castor D9 desaturase monomer can form a homodimer [16], while each subunit of monooxygenase or hydroxylase in Methylococcus capsulatus also consists of two protomers [23]. On the basis of these facts, we propose that the new protein family might function in homodimerization. 3.2. The possible functions of NTZIP Most transgenic tobaccos expressing antisense NTZIP mRNA showed a chlorophyll deficiency, ranging from patchy yellow to total yellow, and retarded growth (Fig. 5). We also found that transformants that completely lacked chlorophyll failed to survive on MS medium under normal conditions. Earlier studies concluded that AcsF and Crd1, homologs of NTZIP might serve as enzymes involved in the biosynthesis of their photosynthetic components [19,21]. It seemed that NTZIP might play an important role in chlorophyll biosynthesis in higher plants. Analysis of gas exchange revealed that stomatal closure occurred when the rate of photosynthesis in transformants began to reduce, but the intercellular CO2 concentration did not decrease compared to the wild-type controls, suggesting that stomatal closure did not restrict enough CO2 entry into the leaf (Table 2). Since the decline of the photosynthetic rate was mainly caused by non-stomatal limitation, the CO2 assimilatory capacity of mesophyll cells might decrease in the leaf of transgenic tobaccos. These results are coincident with the earlier studies in salt-affected rice and bell pepper [2,4]. The loss in CO2 assimilatory capacity in transgenic tobacco on a leaf area basis is closely correlated to a slow-down of the dark reactions in the Calvin cycle, which might result from the enzyme being involved in photosynthesis [7,11,17]. Likewise, no significant difference in net photosynthetic rate
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(PN-values) between wild-type and transgenic tobaccos was detected on a total chlorophyll basis (µmol CO2 mg–1 Chl s–1). This probably was due to the changes in photosynthetic enzymes, including enzymes involved in chlorophyll biosynthesis, which might contribute to the reduced photosynthetic rate. A study of purple bacteria demonstrated that AcsF might encode an enzyme during bacterochlorophyll synthesis and may be involved in aerobic oxidative cyclization of Mgprotoporphyrin IX monomethylester, a precursor of bacterochlorophyll [21]. Because AcsF encodes the putative enzyme bchM in the synthesis of bacterochlorophyll, NTZIP might encode an enzyme involving in chlorophyll synthesis in tobacco. Taken together, our observations lead to the conclusion that the reduction of photosynthesis results from a chlorophyll deficiency in transgenic tobaccos. We propose that NTZIP might form a homodimeric protein involved in the chlorophyll biosynthesis in tobacco. However, its cellular substrates and localization remain to be investigated.
4. Methods 4.1. Plant materials Tobacco (N. tabacum L. cv. NC89) seedlings grown in a growth chamber for 1 month at 25 °C with a 16/8 h light/dark cycle (450 µmol photons m–2 s–1) were used in this experiment. For the dark and light treatments, the 1-month-old tobacco plants were transferred to darkness for 3 days and then returned to the growth chamber (450 µmol photons m–2 s–1) for 10 min, 1, 4, 8 and 12 h, respectively. For the low temperature and strong light illumination treatments, the tobacco plants were transferred to 4 °C refrigerator for 4 h or to the growth chamber with strong light (1200 µmol photons m–2 s–1) for 4 h. 4.2. Isolation and sequence analysis of NTZIP Total RNA isolated from tobacco leaves using Trizol reagent (Gibico Corporation, Madison, WI, USA) was used for reverse transcription polymerase chain reaction (RTPCR). Briefly, 10 µg total RNA were treated with 10 U RNase-free DNase I (Promega, USA) at 37 °C for 15 min to remove genomic DNA, then extracted with phenol/chloroform, and finally precipitated in absolute ethanol. A 2 µg sample of RNA was denatured at 70 °C for 5 min, and quickly ice-quenched; then 5 µl reaction buffer, 2 µl of 10 mM dNTP, 10 U RNase inhibitor, 1 µl of 10 mM oligo-dT primer (5′-GACTCGAGTCGACATCGATTTTTTTTTTTTTTT-3′), and 200 U AMV reverse transcriptase (Promega, USA) were added. After brief mixing, the transcription reaction was incubated at 42 °Cfor 1 h, and terminated at 85 °C for 10 min. To isolate the PNZIP homologs in N. tabacum, a PCR reaction was performed by using two degenerate primers (5′-GGC/TTCA/GAAC/TTCA/G/C/ TGCA/GAAA/GTC-3′ and 5′-GAT/CTAT/CAAT/CCAA/
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GACT/C/A/GCAT/CTTT/CGT-3′) designed from the consensus sequence of PNZIP, AT103 and AcsF in other organisms. After sequencing the specific PCR fragment, 5′-RACE and 3′-RACE were performed to achieve full-length cDNA using the GIBCO-BRL kit (GIBCO, USA). The nucleotide sequence was determined from overlapping clones and confirmed from a full-length cDNA clone obtained by PCR. The secondary structure of NTZIP deduced amino acid sequences was processed using Antheprot (Version 5.0, France). Several homologous sequences of NTZIP obtained from GenBank were analysed with the aid of DNAman (Ver. 4.0, USA). 4.3. Southern blot analysis DNA was extracted from leaf tissue using the procedure described by Yang et al. [32]. A 10 µg of genomic DNA was digested with HindIII, SpeI and ECORI (Takara, Japan), then separated on a 1% agarose gel, and blotted onto a Nytran membrane (Amersham). The NTZIP cDNA fragment used as a probe was labelled by the random priming method. Hybridization was performed at 65 °C in a solution of 0.5 M sodium phosphate (pH 7.2), 1 mM EDTA, 7% (V/V) SDS and 1% (V/V) BSA. After the blot was washed three times with 0.1× SSC, 0.1% SDS at 5565 °C, autoradiography was performed at –8065 °C using a Kodak X-ray film with one intensifying screen for 2 days. 4.4. Northern blot analysis RNA extraction and RNA gel–blot hybridization were carried out as previously described [33]. Total RNA (30 µg/lane) was separated on 0.8% formaldehyde agarose gel and blotted onto a Nytran membrane. Blots were hybridized as described above and washed three times for 20 min at 55, 60 and 65 ° with 0.2× SSC and 0.1% SDS solution, then exposed to X-ray film for 3 days. 4.5. Antisense repression in transgenic tobacco To repress the expression of NTZIP gene in tobacco, the full-length cDNA was amplified by RT-PCR using specific primers with BamHI and SacI sites (5′-GGATCCGGTTGGAGATGTCGAGCGCCACC-3′ and 5′-GAGCTCGATTACAATCAGACGCATTTTGT-3′). The PCR product was digested with BamHI and SacI, and subcloned into the pBI121 vector in antisense orientation under the control of CaMV35S promoter and the nopaline synthase 3′ termination sequences. The resulting antisense vector was introduced in the Agrobacterium tumefaciens strain LBA4404, which was used for transformation of tobacco by the leaf-disc method. Tobaccos were selected on a medium containing 50 mg l–1 kanamycin. After rooting, the seedlings were transferred to soil and grown in a greenhouse. 4.6. Determination of chlorophyll content The chlorophyll a and chlorophyll b of tobacco leaves at the same developmental stage were extracted with 80%
(V/V) acetone and measured by the method of Lichtenthaler [15]. Absorbance was recorded at 664 and 647 nm using a Shimadzu UV-1601 spectrophotometer (Shimadzu, Tokyo, Japan). 4.7. Gas exchange Gas exchange analysis was measured using an open system (Ciras-1; PP system, Norfolk, UK). Leaf net photosynthetic rate (PN), intercellular CO2 concentration (Ci) and stomatal conductance (Gs) were determined under outdoor conditions at about 21–24 °C and a PFD of 500 µmol photons m–2 s–1 on the leaf surface, with air humidity of 40–60% [5].
Acknowledgements This research work was supported by grants from the “863” project in China (Grant 2002AA224101) and the National Natural Science Foundation of China (Grant 30270145) to Zheng C.C.
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Original article
NTZIP antisense plants show reduced chlorophyll levels Ning Liu a,b, Yu-Tao Yang a, Han-Hua Liu a, Guo-Dong Yang a, Nai-Hua Zhang a, Cheng Chao Zheng a,* a
College of Life Sciences, Shandong Agricultural University, Taian, Shandong 271018, PR China b Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, PR China Received 17 September 2003; accepted 17 February 2004
Abstract We have isolated and characterized a new photosynthetic tissue-specific gene NTZIP (Nicotiana tabacum leucine zipper) from tobacco (N. tabacum). Its deduced amino acid sequence has two highly conserved regions, leucine zipper and [EXnDEXRH]2 motifs, which are related to the gene’s biochemical functions. NTZIP was expressed in leaves and stems, but was not detected in roots or flowers, suggesting that its physiological functions might be associated with photosynthesis. Northern blot analysis showed that NTZIP mRNA accumulation was induced by light signals, increased greatly under low temperatures and was repressed by strong light illumination. Furthermore, a number of homologs of NTZIP were isolated from cucumber (Cucumis sativus), rape (Brassica napus), clover (Trifolium repens), willow (Salix babylonica), rosebush (Rusa dovurica), wheat (Triticum aestivum) and spinach (Spinacia oleracea), proving the ubiquitous existence of the NTZIP-like genes in higher plants. Transgenic tobaccos constitutively expressing antisense RNA to NTZIP displayed chlorosis and a lack of ability to turn green even under normal growth conditions. The chlorophyll deficiency was further confirmed by chlorophyll content determination and gas exchange analysis. Based on these observations, we propose that NTZIP may be involved in chlorophyll biosynthesis, and might define a novel family of evolutionarily conserved proteins with its homologs in other plant species. © 2004 Elsevier SAS. All rights reserved. Keywords: Chlorophyll synthesis; Gene expression; Leucine zipper protein; NTZIP; Nicotiana tabacum leucine zipper; Transgenic tobacco
1. Introduction Photosynthesis is the most important source of energy on earth. In chloroplast, about 250–300 chlorophyll molecules bind to their neighbouring pigments. This comprises the reaction centre, in which special chlorophyll pairs in photosystem I and II are the primary electron donors that drive the conversion of light into chemical energy to be conserved in NADPH2 and ATP [29]. Since chlorophyll is the principal pigment that traps light energy, the biosynthesis of chlorophyll has presented a number of challenging topics in the field of plant molecular biology [30]. Significant research has been conducted to investigate the mechanisms by which
Abbreviations: AcsF, Rubrivivax gelatinosus aerobic cyclase system Fe-containing subunit gene; AT103, Arabidopsis thaliana 103 gene; Chl, chlorophyll; Crd1, Chlamydomonas reinhhardtii copper response defect gene; PFD, photon flux density; PNZIP, Pharbitis nil leucine zipper gene; PSI, photosystem I; PSII, photosystem II; ZIP, leucine zipper. * Corresponding author. E-mail address: [email protected] (C.C. Zheng). © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.plaphy.2004.02.007
photosynthesis is conducted in higher plants and other photosynthetic organisms. Many genes in photosynthetic organisms, such as algae, bacteria and higher plants involved in photosynthesis have been cloned and characterized in the last decades, including cab, rbcS, chlI, bchM, petJ, PPDK and PNZIP [6,18,31,33]. Our previous studies have demonstrated that expression of PNZIP (Pharbitis nil leucine zipper) is regulated by light and is restricted to photosynthetically active mesophyll tissues in P. nil [33]. The promoter analysis of PNZIP using GUS as reporter gene also confirmed that it is expressed specifically in photosynthetic tissue in transgenic tobaccos [32]. Recently, two homologs in a photosynthetic bacterium and a green alga, AcsF (aerobic cyclase system Fe-containing subunit gene) and Crd1 (copper response defect gene), also have been characterized by mutant analysis [19,21]. AcsF, in purple bacteria Rubrivivax gelatinosus, has been proved to be involved in the aerobic oxidative cyclization of Mgprotoporphyrin IX mono-methylester, one of the intermediates in the synthesis of bacterio-chlorophyll [21]. In Chlamydomonas reinhhardtii, Crd1 was identified as a putative
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diiron enzyme required for photosystem I accumulation in copper deficiency [19]. These results implied that this class of genes might be involved in biosynthesis of photosystem components in bacteria and algae, but their precise function in higher plants remained to be elucidated. To identify the biological role of those genes by antisense RNA strategy, we cloned NTZIP (Nicotiana tabacum leucine zipper) in tobacco by using a PCR method with degenerate primers on the basis of conserved sequences of PNZIP, AcsF and Crd1. Transgenic tobacco plants constitutively expressing antisense RNA displayed a chlorophyll deficient phenotype, indicating that NTZIP might play a vital role during chlorophyll biosynthesis in tobacco. To address whether NTZIP’s homologs exist ubiquitously in the plant kingdom, we also isolated a number of genes with high similarity to NTZIP from various plant species, suggesting that their products consist of a novel protein family.
2. Results 2.1. Isolation of NTZIP and its homologs from plants Using RT-PCR with degenerate primers and the RACE method, a 1323 bp cDNA clone including a full-length coding region was isolated from tobacco leaf tissue and named NTZIP (GenBank accession AY221168). The deduced amino acid sequence of NTZIP contains 371 amino acid residues with overall identities of 91.0%, 91.4%, and 40.2% to PNZIP, AT103 and AcsF, respectively. Analysis of NTZIP’s putative secondary structure using Anthepro software (Ver. 5.0) shows that it is comprised almost exclusively of a-helices with the exception of a b-sheet between them (Fig. 1). To determine whether NTZIP represents a single locus in the N. tabacum genome or whether it is a multicopy gene, a Southern blot analysis was performed using the NTZIP cDNA as a probe. The result showed that NTZIP hybridized to only one genomic restriction fragment, indicating that it represents a single-copy gene (Fig. 2). The similar results were obtained in Arabidopsis thaliana and P. nil [32,33], suggesting that NTZIP-like gene probably represents a single locus also in the genome of other plants. In order to test whether NTZIP-homologous genes are ubiquitous in higher plants, RT-PCR was performed using the same degenerate primers for NTZIP to amplify cDNA fragments in other plant species. Specific cDNA fragments of E RDEARHA
E QDENRHG
400 200 0 -200 -400 100
200
300
Leucine zipper
Fig. 1. Prediction of NTZIP secondary structure using the Anthepro5.0 software. a-Helix is in blue, b-sheet is in yellow. The location of [EXnEXRH]2 consensus and the leucine zipper are marked with vertical lines and an arrow, respectively.
Fig. 2. Genomic Southern analyses of NTZIP genes. DNA was isolated from young leaves of tobacco plants and digested with PstI, SpeI and EcoRI. NTZIP has no restriction sites for PstI, SpeI, whereas one site was identified for EcoRI. DNA marker sizes are indicated on the left of the figure.
about 1 kb with high similarity to NTZIP were isolated and sequenced from cucumber, rape, clover, willow, rosebush, wheat and spinach (Fig. 3). Moreover, eight NTZIP homologous sequences in other photosynthetic organisms were obtained from the GenBank database (Fig. 3). All deduced amino acid sequences contained a leucine zipper domain for protein dimerization and a [(D/E)EX2H]2 motif which is present in soluble fatty acid desaturases from mammals, fungi, insects, higher plants and cyanobacteria, or hydroxylase and monooxygenase from bacteria [23,25,26]. They share high similarity in amino acid sequences but can be distinguished from other previously known proteins, indicating that the proteins encoded by these genes consist of a novel protein family (Fig. 3). 2.2. The expression of NTZIP is regulated by environmental conditions To determine the pattern of NTZIP expression in different tobacco organs, a northern blot hybridization analysis was performed. The results showed that NTZIP transcripts are easily detected in the leaves, less detected in the stems, and cannot be detected at all in flowers and roots (Fig. 4A), indicating that it is expressed especially in photosynthetic tissues, but not in non-photosynthetic tissues. Therefore, we propose that the NTZIP gene may have a role in the biosynthesis or function of the photosynthetic apparatus. In order to determine whether the NTZIP gene is regulated by light signals, tobacco seedlings were pre-treated with continuous darkness for 3 days, then transferred to continuous light illumination. The levels of NTZIP mRNA increased significantly after 10 h light exposure, and kept increasing gradually during the subsequent period of light treatment (Fig. 4B). However, strong light inhibited the accumulation of NTZIP mRNA in tobacco leaves, implying that photoinhibition might affect NTZIP expression (Fig. 4C). Interest-
N. Liu et al. / Plant Physiology and Biochemistry 42 (2004) 321–327
Consensus PNZIP AT103 NTZIP RDZIP CSZIP Higher OSZIP plants BNZIP EEZIP SBZIP SOZIP TAZIP TRZIP ZMZIP CRZIP Bacteria AcsF or algae Crd1 RPZIP Consensus PNZIP AT103 NTZIP RDZIP CSZIP OSZIP BNZIP EEZIP SBZIP SOZIP TAZIP TRZIP ZMZIP CRZIP AcsF Crd1 RPZIP Consensus PNZIP AT103 NTZIP RDZIP CSZIP OSZIP BNZIP EEZIP SBZIP SOZIP TAZIP TRZIP ZMZIP CRZIP AcsF Crd1 RPZIP Consensus PNZIP AT103 NTZIP RDZIP CSZIP OSZIP BNZIP EEZIP SBZIP SOZIP TAZIP TRZIP ZMZIP CRZIP AcsF Crd1 RPZIP
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DYNQTHFVRNKEFKEAA----DKLQGPLRQIFVEFLERSCTAEFSGFLLYKELGRRLKKTNPVVAEIFSLMSRDEARHAG DYNQTHFVRNKEFKEAA----DRLQGPLRQIFVEFLERSCTAEFSGFLLYKELGRRPKKTNPVVAEIFSLMSRDEARHAG DYNQTHFVRNKEFKEAA----DKLQGPLRQIFVEFLERSCTAEFSGFLLYKELGRRLKKTNPVVAEIFSLMSRDEARHAG DYNQTHFVRNKEFKEAA----DKMQGALREIFVEFLERSCTAEFSGFLLYKELGRRLKKTNPVVAEIFSLMSRDEARHAG DYNQTHFVRNKEFKEAA----DKLQGPLRQIFVEFLERSCTAEFSGFLLYKELGRRLKKTNPVVAEIFSLMSRDEARHAG DYNQTHFVRNKEFKEAA----DKMQGALREIFVEFLERSCTAEFSGFLLYKELGRRLEN-QSSVAEIFSLMSRDEARHAG DYNQTHFVRNPEFKAAA----DKMEGPLRQIFVEFLERSCTAEFSGFLLYKELGRRLKKTNPVVAEIFSLMSRDEARHAG DYNQTHFVRNKEFKEAA----DKLQGPLRQIFVEFLERSCTAEFSGFLLYKELGRRLKKTNPVVAEIFSLMSRDEARHAG DYNQTHFVRNKEFKEAA----DKMQGPLRQIFVEFLERSCTAEFSGFLLYKELGRRLKKTNPVVAEIFSLMSRDEARHAG DYNQTHFVRNKEFKEAA----DRLQGPLRQIFVEFLERSCTAEFSGFLLYKELGRRPKKTNPVVAEIFSLMSRDEARHAG DYNQTHFVRNKEFKEAA----DKLQGPLRQIFVEFLERSCTAEFSGFLLYKELGKRLKKTNPVVAEIFSLMSRDEARHAG DYNQTHFVRNKEFKEAA----DKLQGPLRQIFVEFLERSCTAEFSGFLLYEELGRRLKKTNPVVAEIFSLMSRDEARHAG DYNQTHFVRNKEFKEAA----DKLDGPLRQIFVEFLERSCTAEFSGFLLYKELGRRLKKTNPVVAEIFSLMSRDEARHAG DYNQTHFVRNPEFKAAA----DRMEGPLRQIFVEFLERSCTAEFSGFLLYKELGRRLKKTNPVVAEIFSLMSRDEARHAG DYNKVHFVRNETFKAAA----DKVTGETRRIFIEFLERSCTAEFSGFLLYKELARRMKASSPEVAEMFLLMSRDEARHAG DNNHDHFQRTPEFAQEVAERFSQVSPELRQEFLDFLVSSVTSEFSGCVLYNEIQKNVE--NPDVKALMRYMARDESRHAG DYNSQHFIRGEEFNQSW----SNLETKTKSLFIEFLERSCTAEFSGFLLYKELSRKLKDSNPIIAECFLLMSRDEARHAG DYNKSHFKKTDEF----VNSDLDKLPPELRAEFKDFLVSSLTAEFSGCVLYAEIKKRIK-NPEIRELFGLLSRDEARHAG FLNKGLSDFNLALDLGFLTKARKYTFFKPKFIFYATYLSEKIGYWRYITIYRHLKQNPEFQCYPIFKYFENWCQDENRHG FLNKGLSDFNLALDLGFLTKARKYTFFKPKFIFYATYLSEKIGYWRYITIYRHLKQNPEFQCYPIFKYFENWCQDENRHG FLNKGLSDFNLALDLGFLTKARKYTFFKPKFIFYATYLSEKIGYWRYITIYRHLKENPEFQCYPIFKYFENWCQDENRHG FLNKGLSDFNLALDLGFLTKARKYTFFKPKFIFYATYLSEKIGYWRYITIYRHLKANPEFQCYPIFKYFENWCDDENRHG FLNKGLSDFNLALDLGFLTKARKYTFFKPKFIFYATYLSEKIGYWRYITIYRHLKQNPEFQCYPIFKYFENWCQDENRHG FLNKGYRISTWPLDLGFLTKARKYTFFKPKFIFYATYLSEKIGYWRYITIYRHLKANPEFQCYPIFKYFENWCQDENRHG FLNKGLSDFNLALDLGFLTKARKYTFFKPKFIFYATYLSEKIGYWRYITIFRHLKANPEYQVYPIFKYFENWCQDENRHG FLNKGLSDFNLALDLGFLTKARKYTFFKPKFIFYATYLSEKIGYWRYITIYRHLKQNPEFQCYPIFKYFENWCQDENRHG FLNKGLSDFNYALDLGFLTKARKYTFFKPKFIFYATYLSEKIGYWRYITIYRHLKENPEYQCYPIFKYFENWCQDENRHG FLNKGLSDFNLALDLGFLTKARKYTFFKPKFIFYATYLSEKIGYWRYITIYRHLKQNPEFQCYPIFKYFENWCQDENRHG FLNKGLSDFNLALDLGFLTKARKYTFFKPKFIFYATYLSEKIGYWRYITIYRHLKQNPEFQCYPIFKYFENWCQDENRHG FLNKGLSDFNLALDLGFLTKARKYTFFKPKFIFYATYLSEKIGYWRYITIYRHLKENPEFQCYPIFKYFENWCQDENRHG FLNKGLSDFNLALDLGFLTKARKYTFFKPKFIFYATYLSEKIGYWRYITIYRHLKANPEYQCYPIFKYFENWCQDENRHG FLNKGLSDFNLALDLGFLTKARKYTFFKPKFIFYATYLSEKIGYWRYITIFRHLKANPEYQVYPIFKYFENWCQDENRHG FLNKALSDFNLALDLGFLTKNRTYTYFKPKFIIYATFLSEKIGYWRYITIYRHLQRNPDNQFYPLFEYFENWCQDENRHG FINQALRDFGLGIDLGGLKRTKAYTYFKPKYIFYATYLSEKIGYARYITIYRQLERHPDKRFHPIFRWFERWCNDEFRHG FLNKAIGDFNLSLDLGFLTKTRKYTFFSPKFIFYATYLSEKIGYWRYITIYRHMEQYPEHRIYPIFKFFENWCQDENRHG FINEILKDHGIGVDLSFLTKVKKYTYFRPKFIFYATYLSEKIGYARYITIYRQMERHPERRFHPIFKWFERWCNDEFRHG DFFSALMKAQPQFLNDWQAKLWSRFFCLSVYVTMYLNDCQRTDFYEGIGLNTKEFDMHVIIETNRTTARIFPAVLDVENP DFFSALMKAQPQFLNDWQAKLWSRFFCLSVYVTMYLNDCQRTDFYEGIGLNTKEFDMHVIIETNRTTARIFPAVLDVENP DFFSALMKAQPQFLNDWQAKLWSRFFCLSVYVTMYLNDCQRTNFYEGIGLNTKEFDMHVIIETNRTTARIFPAVLDVENP DFFSALLKAQPQFLNDWKAKLWSRFFCLSVYVTMYLNDWQRTAFYEGIGLNTKEFDMHVIIETNRTTARIFPAVLDVENP DFFSALMKAQPQFLNDWQAKLWSRFFCLSVYVTMYLNDCQRTDFYEGIGLNTKEFDMHVIIETNRTTARIFPAVLDVENP DFFSALLKAQPQFLNDWKAKLWSRFFCLSVYVTMYLNDCQRTAFYEGIGLNTKEFDMHVIIETNRTTARIFPAVPDVENP DFFSALLKAQPQFLNDWKAKLWSRFFCLSVYVTMYLNDCQRTTFYEGIGLDTKEFDMHVIIETNRTTARIFPAVLDVENP DFFSALMKAQPQFLNDWQAKLWSRFFCLSVYVTMYLNDCQRTDFYEGIGLNTKEFDMHVIIETNRTTARIFPAVLDVENP DFFSALMKAQPQFLNDWKAKLWARFFCLSVYVTMYLNDCQRTAFYEGIGLDTKEFDMHVIIETNRTTARIFPAVLDVENP DFFSALMKAQPQFLNDWQAKLWSRFFCLSVYVTMYLNDCQRTDFYEGIGLNTKEFDMHVIIETNRTTARIFPAVLDVENP DFFSALMKAQPQFLNDWQAKLWSRFFCLSVYVTMYLNDCQRTDFYEGIGLNTKEFDMHVIIETNRTTARIFPAVLDVENP DFFSALMKAQPQFLNDWQAKLWSRFFCLSVYVTMYLNDCQRTSFYEGIGLNTKEFDMHVIIETNRTTARIFPAVLDVENP DFFSALMKAQPQFLNDWKAKLWSRFFCLSVYVTMYLNDWQRTDFYEGIGLNTKEFDMHVIIETNRTTARIFPAVLDVENP DFFSALLKAQPQFLNDWKAKLWSRFFCLSVYVTMYLNDCQRTAFYEGIGLDTKEFDMHVIIETNRTTARIFPAVLDVENP DFLAACLKAKPELLNTFEAKLWSKFFCLSVYITMYLNDHQRTKFYESLGLNTRQFNQHVIIETNRATERLFPVVPDVEDP ESFALILRAHPHLISGAN-LLWVRFFLLAVYATMYVRDHMRPQLHEAMGLESTDYDYRVFQITNEISKQVFPISLDIDHQ DFFAALLKSQPHFLNDWKAKMWCRFFLLSVFATMYLNDFQRIDFYNAIGLDSRQYDMQVIRKTNESAARVFPVALDVDNP EAFALLMRADPSLLSG-VNKLWIRFFLLAVFSTMYVRDHMRPAFYEALGVDATDYGMQVFRITTEISKQVFPVTINLDDP EFKRKLDRMVVINEKLMAVGETDDPSLVKNLKRIPLIAALVSEILAAYLMPPIESGSVDFAEFEP EFKRKLDRMVVIYEKLMAVGKTDDPSFVKNLKKVPLIAGLVSEILAAYLMPPVESGSVDFAEFE EFKRKLDRMVVSYEKLLAIGETDDASFIKTLKRIPLVTSLASEILAAYLMPPVESGSVDFAEFEP EFKRKLDRMVEINQKILAVGETDDIPLVKNFKRIPLIAALASELLAAYLMKPIESGSVDFAEFEP EFKRKLDRMVVIYEKLMAVGKTDDPSFVKNLKKVPLIAGLVSEILAAYLMPPVESGSVDFAEFE EFKRKLDRMVEINEKLIAVGETNDLPLVKSLKRIPLAAALVSEILAAYLMPPIESGSVDFAEFEP EFKRKLDRMVEINKKIIAIGESDDIPLVKNLKRIPHVAALVSEIIAAYLMPPIESGSVDFAEFEP EFKRKLDRMVVINEKLMAVGQTDDPSFVKNLKRIPLIAGLVSEILAAYLMPPVESGSTDFEEMEP EFKRKLDRMVVINQKLQAVGETEDNSVVKNLKRVPLIAALVSEILAAYLMPPIESGSVDFAEFEP EFKRKLDRMVVIYEKLMAVGKTDDPSFVKNLKKVPLIAGLVSEILAAYLMPPVESGSVDFAEFE EFKRKLDRMVVIYEKLMAVGKTDDPSFVKNLKKVPLIAGLVSEILAAYLMPPAESGSVDFAEFEP EFKRKLDRMVVSYEKLLAIGETDDASFIKTLKRIPLVTSLAPEILAAYLMPPVESGSVDFAEFEP EFKRKLDRMVEINQKILAIGESDDNSVVKNLKGIPLVAALVSELLAAYLMPPIESGSVDFAEFEP EFKRKLDRMVEINQKIIAVGESDEIPLVKNLKRIPLVASLVSEIIAAYLMPPIESGSVDFAEFEP RFFEILNKMVDVNAKLVELSASSSP--LAGLQKLPLLERMASYCLQLLFFKEKDVGSVDIAGSG AFRAGMERLVRVKTKVDAA--KARGGLVGRLQQAAWAAAGAATFARMYLIPVRRHALPAQVRMAP KFFKYLDSCACNNRALIDIDKKGSQPLVKTFMKAPLYMSLILNLIKIYLIKPIDSQAVW RFLQNLERLRIAAEKIDRS--HSQG-LLGKLKRPFYAASAALAFGRLFLLPAKRNELPRVIGLRP
Fig. 3. Comparison of the partial deduced amino acid sequences of NTZIP and its homologs. The motifs are marked in the consensus sequence. The Leu residues in the Leu zipper domain are in bold and boxed; the e position amino acid is underlined, the a position of the second heptad is shaded; The [EXnDEXRH]2 motif is in bold and underlined (n = ~30). AT103, A. thaliana (U38232); BNZIP, Brassica napus (AY322556); CPPZIP, Porphyra purpurea chloroplast (U38804); Crd, Chlamydomonas reinhardtii (AY057871); CSZIP, Cucumis sativus (AY221169); EEZIP, Euphorbia elsua (AF417577); NTZIP, N. tabacum (AY221168); OSZIP, Oryza sativa (AP000815); PNZIP, P. nil (U37437); RDZIP, Rusa dovurica (AY322555); AscF, Rubrivivax gelatinosus (AY057871); RPZIP, R. palustris (AF195122); SLZIP, Salix babylonica (AY322554); SOZIP, Spinacia oleracea (AY322553); TAZIP, Triticum aestivum (AY322552); TRZIP, Trifolium repens (AY322557); ZMZIP, Zea mays (AY108897).
ingly, we observed that the NTZIP mRNA accumulations were stimulated by a low temperature stress as well (Fig. 4C). 2.3. Both NTZIP mRNA and chlorophyll levels were reduced in transgenic tobaccos
To verify that the NTZIP gene is involved in chlorophyll synthesis, as is its homologous gene in photosynthetic bacteria, we constructed a transcriptional fusion between the CaMV35S promoter and the NTZIP coding region in antisense orientation. Thirty-two transformants were obtained. The transgenic tobaccos were further confirmed by PCR with 35S primers and Southern blot (data not shown). In most
transgenic tobaccos, northern blots indicated that NTZIP mRNA accumulation decreased compared with wild-type plants, which means antisense repression was effective and markedly reduced the NTZIP transcript levels. A positional effect also was found in our transgenic tobaccos, and NTZIP mRNA accumulation was either difficult to detect or was still expressed at high levels, which was also related to their phenotype. The more yellowish the transformants were, the less NTZIP mRNA they accumulated (Fig. 5). As in the case of chlorophyll, the level of chlorophyll in the leaves of the transgenic plants was 35% less than that of wild-type controls, the ratio of Chl a/b, however, did not change much (Table 1). This result demonstrated that a chlorophyll deficiency occurred in the transgenic lines. Since chlorophyll is
B
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2.4. The transgenic tobaccos display lower photosynthetic activity
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Fig. 4. Northern blot analysis of NTZIP in tobacco plants. Each lane contains 30 µg of tobacco leaf total RNA. A photograph of 28S rRNA served as a control for equal loading of RNA in each lane (lower panel). (A) Northern blot analysis of NTZIP mRNA accumulation in different organs; (B) northern blot hybridization analysis of NTZIP mRNA accumulation during dark and light treatment; (C) northern blot analysis of NTZIP mRNA accumulation at low temperature (LT) and after strong light illumination (SLI).
In order to test our hypothesis, gas exchange was analyzed in leaves of transgenic tobaccos and wild-type controls. The result showed that the net photosynthetic rate (PN) per leaf area unit in transgenic tobaccos decreased to only 54.0% of the controls. However, the net PN values on a chlorophyll basis are similar in both wild-type and transgenic tobaccos, and a partial stomata closure occurred during reduction of the photosynthetic rate in transgenic tobacco. The leaf conductance decreased more slowly than the net photosynthesis rate, resulting in that the intercellular CO2 concentrations (Ci) were similar in both transgenic and wild-type tobacco leaves (Table 2). These results indicated that the capacity of CO2 fixation was not inhibited by the partial stomata closure. Therefore, the decrease of photosynthetic activity might mainly be caused by the loss in decrease of CO2 assimilation due to the decline of the content of chlorophyll in transgenic tobacco leaves.
3. Discussion 3.1. NTZIP and its homologs define a novel protein family
Fig. 5. Northern blot hybridization analysis of tobacco NTZIP mRNA accumulation in transgenic lines. Each lane contains 30 µg of tobacco leaf total RNA. A photograph of 28S rRNA served as a control for equal loading of RNA in each lane (lower panel). WT: Wild-type tobacco; T1-1, T1-2 and T1-3: independent transgenic tobacco lines. Table 1 Changes in leaf chlorophyll content and Chl a/b ratio in wild-type and transgenic tobaccos. Values indicated with different letters were significantly different at P = 0.05. Each value is an average of five measurements. Numbers in parenthesis are percentages Wild-type Transgenic tobaccos
Chl (mg g–1 FW) 2.02 ± 0.13 (100) 0.71 ± 0.03 (35)
Chl a/b 3.15 ± 0.27 2.88 ± 0.17
the component of PSI and PSII, a chlorophyll deficiency might lead to a decrease of the photosynthetic rate in transgenic tobaccos.
Comparison of NTZIP cDNA with the GenBank database of known sequences revealed no obvious similarity with any other gene of known function in higher plants. A multiple alignment of the NTZIP deduced amino acid sequence and the candidate homologs demonstrated that the NTZIP is about 90% identical to its homologous genes or cDNA fragments in higher plants, and 39.7–65.4% identical with its homologs in algae and photosynthetic bacteria (Fig. 2). Our data demonstrated that these amino acid sequences are highly conserved, especially among higher plants, and might define a novel family of evolutionarily conserved proteins. The leucine zipper is one of the most popular motifs in transcriptional factors and can help protein to form homodimers or heterodimers in many creatures, especially in eukaryotic organisms [3,9,10]. Many proteins with leucine zippers play important roles in plant growth and development [14,22,24]. Two polypeptides may homo- or heterodimerize to adjust the function of new dimers that may have different catalysis characteristics [20,27]. NTZIP contains a leucine zipper with four heptads and the leucine residue is replaced by tyrosine in the fourth heptad (Fig. 3), which is often
Table 2 Net photosynthetic rate (PN), stomatal conductance (Gs), intercellular CO2 concentration (Ci) of wild-type and transgenic tobaccos. Each point represents the mean ± S.E. of five replications. Stars indicate t-test comparison between wild-type and transgenic tobaccos under high light irradiance
Wild-type Transgenic tobaccos *
PN (µmol CO2 m–2 s–1) 7.0+0.6 * 3.8+0.3 *
P < 0.001; NS, not significant at P < 0.05.
PN (µmol CO2 mg–1 Chl s–1) 945+37.2 NS 933+28.2 NS
Gs (mmol m–2 s–1) 106.5+9.3 * 48.5+5.2 *
Ci (µmol mol–1) 267+24.6 NS 259+26.7 NS
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happened in other zipper proteins [1,12]. Thus, it is possible that the new NTZIP family might heterodimerize or homodimerize with other ZIP proteins. The leucine zipper dimerization domain forms a parallel coil that consists of four to five heptads, in which each heptad is composed of two a-helical turns or seven amino acids, labeled a, b, c, d, e, f and g. Amino acids in the a, d, e and g positions regulate leucine zipper oligomerization, dimerization stability, and dimerization specificity [1,13,28]. Recently, genetic studies showed that homodimerizing leucine zippers have two distinct properties. First, each ZIP family has a distinct pattern of attractive g ↔ e′ pairs (′ refers to the second a-helix in the dimer). Second, all have asparagines in a position of the second heptad [8,28]. We observed that amino residues at the e position were all acidic or neutral amino acids with a negative charge in the putative proteins encoded by NTZIP and its homologs, which may improve attractive g ↔ e′ pairs, drive the formation of homodimerization, and contribute to its stability. There were also asparagine residues in the second heptad position in most proteins except in R. gelatinosus and Rhodopseudomonas palustris, in which isoleucine and leucine replace asparagines [21]. By protein crystallographic methods, it was observed that the recombinant castor D9 desaturase monomer can form a homodimer [16], while each subunit of monooxygenase or hydroxylase in Methylococcus capsulatus also consists of two protomers [23]. On the basis of these facts, we propose that the new protein family might function in homodimerization. 3.2. The possible functions of NTZIP Most transgenic tobaccos expressing antisense NTZIP mRNA showed a chlorophyll deficiency, ranging from patchy yellow to total yellow, and retarded growth (Fig. 5). We also found that transformants that completely lacked chlorophyll failed to survive on MS medium under normal conditions. Earlier studies concluded that AcsF and Crd1, homologs of NTZIP might serve as enzymes involved in the biosynthesis of their photosynthetic components [19,21]. It seemed that NTZIP might play an important role in chlorophyll biosynthesis in higher plants. Analysis of gas exchange revealed that stomatal closure occurred when the rate of photosynthesis in transformants began to reduce, but the intercellular CO2 concentration did not decrease compared to the wild-type controls, suggesting that stomatal closure did not restrict enough CO2 entry into the leaf (Table 2). Since the decline of the photosynthetic rate was mainly caused by non-stomatal limitation, the CO2 assimilatory capacity of mesophyll cells might decrease in the leaf of transgenic tobaccos. These results are coincident with the earlier studies in salt-affected rice and bell pepper [2,4]. The loss in CO2 assimilatory capacity in transgenic tobacco on a leaf area basis is closely correlated to a slow-down of the dark reactions in the Calvin cycle, which might result from the enzyme being involved in photosynthesis [7,11,17]. Likewise, no significant difference in net photosynthetic rate
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(PN-values) between wild-type and transgenic tobaccos was detected on a total chlorophyll basis (µmol CO2 mg–1 Chl s–1). This probably was due to the changes in photosynthetic enzymes, including enzymes involved in chlorophyll biosynthesis, which might contribute to the reduced photosynthetic rate. A study of purple bacteria demonstrated that AcsF might encode an enzyme during bacterochlorophyll synthesis and may be involved in aerobic oxidative cyclization of Mgprotoporphyrin IX monomethylester, a precursor of bacterochlorophyll [21]. Because AcsF encodes the putative enzyme bchM in the synthesis of bacterochlorophyll, NTZIP might encode an enzyme involving in chlorophyll synthesis in tobacco. Taken together, our observations lead to the conclusion that the reduction of photosynthesis results from a chlorophyll deficiency in transgenic tobaccos. We propose that NTZIP might form a homodimeric protein involved in the chlorophyll biosynthesis in tobacco. However, its cellular substrates and localization remain to be investigated.
4. Methods 4.1. Plant materials Tobacco (N. tabacum L. cv. NC89) seedlings grown in a growth chamber for 1 month at 25 °C with a 16/8 h light/dark cycle (450 µmol photons m–2 s–1) were used in this experiment. For the dark and light treatments, the 1-month-old tobacco plants were transferred to darkness for 3 days and then returned to the growth chamber (450 µmol photons m–2 s–1) for 10 min, 1, 4, 8 and 12 h, respectively. For the low temperature and strong light illumination treatments, the tobacco plants were transferred to 4 °C refrigerator for 4 h or to the growth chamber with strong light (1200 µmol photons m–2 s–1) for 4 h. 4.2. Isolation and sequence analysis of NTZIP Total RNA isolated from tobacco leaves using Trizol reagent (Gibico Corporation, Madison, WI, USA) was used for reverse transcription polymerase chain reaction (RTPCR). Briefly, 10 µg total RNA were treated with 10 U RNase-free DNase I (Promega, USA) at 37 °C for 15 min to remove genomic DNA, then extracted with phenol/chloroform, and finally precipitated in absolute ethanol. A 2 µg sample of RNA was denatured at 70 °C for 5 min, and quickly ice-quenched; then 5 µl reaction buffer, 2 µl of 10 mM dNTP, 10 U RNase inhibitor, 1 µl of 10 mM oligo-dT primer (5′-GACTCGAGTCGACATCGATTTTTTTTTTTTTTT-3′), and 200 U AMV reverse transcriptase (Promega, USA) were added. After brief mixing, the transcription reaction was incubated at 42 °Cfor 1 h, and terminated at 85 °C for 10 min. To isolate the PNZIP homologs in N. tabacum, a PCR reaction was performed by using two degenerate primers (5′-GGC/TTCA/GAAC/TTCA/G/C/ TGCA/GAAA/GTC-3′ and 5′-GAT/CTAT/CAAT/CCAA/
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GACT/C/A/GCAT/CTTT/CGT-3′) designed from the consensus sequence of PNZIP, AT103 and AcsF in other organisms. After sequencing the specific PCR fragment, 5′-RACE and 3′-RACE were performed to achieve full-length cDNA using the GIBCO-BRL kit (GIBCO, USA). The nucleotide sequence was determined from overlapping clones and confirmed from a full-length cDNA clone obtained by PCR. The secondary structure of NTZIP deduced amino acid sequences was processed using Antheprot (Version 5.0, France). Several homologous sequences of NTZIP obtained from GenBank were analysed with the aid of DNAman (Ver. 4.0, USA). 4.3. Southern blot analysis DNA was extracted from leaf tissue using the procedure described by Yang et al. [32]. A 10 µg of genomic DNA was digested with HindIII, SpeI and ECORI (Takara, Japan), then separated on a 1% agarose gel, and blotted onto a Nytran membrane (Amersham). The NTZIP cDNA fragment used as a probe was labelled by the random priming method. Hybridization was performed at 65 °C in a solution of 0.5 M sodium phosphate (pH 7.2), 1 mM EDTA, 7% (V/V) SDS and 1% (V/V) BSA. After the blot was washed three times with 0.1× SSC, 0.1% SDS at 5565 °C, autoradiography was performed at –8065 °C using a Kodak X-ray film with one intensifying screen for 2 days. 4.4. Northern blot analysis RNA extraction and RNA gel–blot hybridization were carried out as previously described [33]. Total RNA (30 µg/lane) was separated on 0.8% formaldehyde agarose gel and blotted onto a Nytran membrane. Blots were hybridized as described above and washed three times for 20 min at 55, 60 and 65 ° with 0.2× SSC and 0.1% SDS solution, then exposed to X-ray film for 3 days. 4.5. Antisense repression in transgenic tobacco To repress the expression of NTZIP gene in tobacco, the full-length cDNA was amplified by RT-PCR using specific primers with BamHI and SacI sites (5′-GGATCCGGTTGGAGATGTCGAGCGCCACC-3′ and 5′-GAGCTCGATTACAATCAGACGCATTTTGT-3′). The PCR product was digested with BamHI and SacI, and subcloned into the pBI121 vector in antisense orientation under the control of CaMV35S promoter and the nopaline synthase 3′ termination sequences. The resulting antisense vector was introduced in the Agrobacterium tumefaciens strain LBA4404, which was used for transformation of tobacco by the leaf-disc method. Tobaccos were selected on a medium containing 50 mg l–1 kanamycin. After rooting, the seedlings were transferred to soil and grown in a greenhouse. 4.6. Determination of chlorophyll content The chlorophyll a and chlorophyll b of tobacco leaves at the same developmental stage were extracted with 80%
(V/V) acetone and measured by the method of Lichtenthaler [15]. Absorbance was recorded at 664 and 647 nm using a Shimadzu UV-1601 spectrophotometer (Shimadzu, Tokyo, Japan). 4.7. Gas exchange Gas exchange analysis was measured using an open system (Ciras-1; PP system, Norfolk, UK). Leaf net photosynthetic rate (PN), intercellular CO2 concentration (Ci) and stomatal conductance (Gs) were determined under outdoor conditions at about 21–24 °C and a PFD of 500 µmol photons m–2 s–1 on the leaf surface, with air humidity of 40–60% [5].
Acknowledgements This research work was supported by grants from the “863” project in China (Grant 2002AA224101) and the National Natural Science Foundation of China (Grant 30270145) to Zheng C.C.
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