- Email: [email protected]
Isolating Soil Drought-Induced Genes from Maize Seediing Leaves Through Suppression Subtractive Hybridization
Available online at www.sclencedtrect.com
Agricultural Sciences in China 2007, 6(6): 647-651
4
* s*e
* r
ScienceDirect
June 2001
Isolating Soil Drought-Induced Genes from Maize Seedling Leaves Through Suppression Subtractive Hybridization LI Hui-yongl+2,HUANG Su-hua2, SHI Yun-su2, SONG Yan-chun2, ZHAO Jiu-ran3, WANG Feng-ge3, WANG Tian-yu* and LI Yu2 1
Institute of Food Crops, Henan Academy of Agricultural Sciences, Zhengzhou 450002, P.R.China
2
Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China
3
Maize Research Institute, Beijing Academy of Agricultural and Forestry Sciences, Beijing 100081, P.R.China
Abstract In this study, a forward cDNA library was constructed by suppression subtractive hybridization using seedling leaves of CN165, a drought-tolerant maize inbred line. In the suppression subtractive hybridization (SSH) library, 672 positive clones were picked up randomly. After polymerase chain reaction (PCR) of each clone, all the single clones were sequenced. Totally 598 available sequences were obtained. After cluster analysis of the EST sequences, 80 uniESTs were obtained, among which 57 uniESTs were contigs and 23 uniESTs were singlets. The results of BLASTN showed that all the uniESTs had homologous sequences in the nr database. The BLASTX results indicated that 68 uniESTs had significant protein homology, 8 uniESTs with homology of unknown proteins and putative proteins, and 4 uniESTs without protein homology. Those drought stress-induced genes were involved in many metabolism pathways to regulate plant growth and development under drought stress.
Key words: maize, drought stress, suppression subtractive hybridization (SSH), uniESTs
INTRODUCTION Maize is an important crop because not only maize feed both the world's people and livestock but also its products are necessary for many industries. For example, in the manufactureof diverse commoditiesincludingalcohol, glue, soap, paint, insecticides,toothpaste, shaving cream, rubber tires, rayon, molded plastics and others (Fussell 1999). However, maize is often subjected to several harsh environmental stresses that adversely affect growth, metabolism, and finally the yield. Among these environmental factors, drought is a major abiotic factor limiting agricultural production. Although the general
effects of drought on plant growth are fairly well known, the primary effects of water deficit at the biochemical and the molecular levels are not well understood (Zhu 2002; Chaitanya et al. 2003; Chaves et al. 2003). In many plants, drought stress often activates cell signaling pathways (Shinozaki et al. 2000; Knight H .and Knight M R 2001) and cellular responses, such as the production of stress proteins, up-regulation of antioxidants and accumulation of compatible solutes (Cushman and Bohnert 2000). These drought inducedgenes include three major categories: (1) genes that are involved in signaling cascades and in transcriptional control, such as some protein kinases and transcriptional factors; (2) genes that function directly in the
This paper is translated from its Chinese version in Sciantia Agricultura Sinica. LI Hui-yong, Ph D, E-mail [email protected];Correspondence LI Yu,Tel +86-10-62131196, E-mail: [email protected],WANG Tian-yu, Tel: +86-10-62186632, E-mail: wangty@mail,caas,net.cn
92007.CMS. All Mhtsreserved. PuMihedby E l h r Lid.
648
protection of membranes and proteins; and (3) genes that are involved in water and ion uptake and transport (Wang er al. 2003). Although many important genes are isolated, it is difficult to construct a clear metabolism network for explaining the plant drought resistance mechanism. Therefore, it is important to isolate drought induced genes and understand the function of genes and interaction of genes to develop maize varieties with drought resistance. In this study, a number of drought induced-genes were isolated from the leaves of maize seedlings throughout the suppression subtractive hybridization method, to Understand the mechanisms of drought tolerance in maize. Those specific stress-relatedgenes may be used in engineering more tolerant plants.
MATERIALS AND METHODS Plant materials and drought treatment Seeds of maize inbred CN165 with strong drought tolerance were allowed to imbibe overnight in distilled water before being transferred to trays of soil mix (soil: vermicu1ite:organic fertilizer = 3:2: l), under a rainout shelter. Before drought stress treatment, equal volume of water was applied to each tray, every other day to keep the plants in all trays under uniform water status. Water stress was applied at the five-leaf stage. The leaves from the control and treated plants were sampled at 2, 4, 6, 8, 10 and 12 d after stress application, respectively. At the same time, the leaf water relative water content (RWC), was measured as described by Barr and Weatherley (1962) (RWC = (Fresh wt - Dry wt)/(Turgid wt - Dry wt) x 100.
RNA extraction Total RNAs for drought treated plants were extracted from the samples harvested at six different time points (2, 4,6, 8, 10, 12 d), and then mixed equally as tester samples. Total RNAs for the control plants at the corresponding time points were also extracted and mixed equally as driver samples. The extraction method using Invitrogen Trizol and the isolation of poly (A) RNA using oilgotex were according to the manufacturer's protocol (Qiagen. Germany).
LI Hui-yong er al.
Construction of subtracted cDNA library Suppression subtractive hybridization was carried out using the PCR-selected cDNA subtractive hybridization kit according to the manufacturer's protocol (Clontech, USA). The water-stress plants were used as testers and the control plants were used as drivers. The tester and driver cDNAs of the leaves were prepared from 2 pg of mRNA extracted from the treated and the control samples. The tester cDNA was digested with Rsa I at 37°C for 1.5 h and then ligated to adaptors 1 and 2 in separate reactions at 16"C, overnight. Then driver cDNA was added to each of the tester samples, which were subsequently resuspended in the hybridization buffer. After heat-denaturation,the mixture was allowed to anneal at 68°C for 8 h. Then, the two samples from the first hybridization were mixed together and freshly denatured driver cDNA was added to the sample followed by incubation at 68°C overnight for the second hybridization. PCR amplification with two different nested primers was conducted to amplify the differentially expressed cDNAs. cDNA fragments for differentially expressed genes were inserted into the pMD18-T vector (Takara, Japan), and transformed into E. coli JMP109 cells. The recombinant white clones were randomly picked to construct the subtracted cDNA library.
EST clustering and sequence analysis Those white positive clones selected from the SSH library were PCR screened. The clones that contained the insert fragment were sequenced with the 3730 Automatic DNA sequencer (ABIPrism, USA). Sequences obtained were first extracted with PHRED and the lowquality (=lo0 bp), vector and adapter sequences were then removed. Cluster analysis of those sequences was done using the TGICL program. On the basis of all known information, the clones were further annotated and analyzed by the BLAST in NCBI database.
RESULTS Performance of CN165 under drought stress The maize inbred line CN165 has been identified as an inbred with strong drought tolerance. Within the first
Qm7, CAAS. All fights tmerved. Publishedby EIsmbr Ltd.
Isolating Soil Drought-Induced Genes from Maize Seedling Leaves Through Suppression Subtractive Hybridization
four days after water stress the plants were not easy to distinguish phenotypically from the well-watered plants. However visible symptoms of the stressed plants such as leaf rolling appeared as the stress continued. The leaves RWC of stress plants also showed a trend to decrease (Fig.).
2
4
6
8
1 0 1 2
Days without water
Fig. Changes of leaf relative water content (RWC) in maize at the seeding stage when plants were subjected to drought stress treatment for 12 days. Bars are mean & SD of five samples.
The subtracted cDNA library and sequence analysis of ESTs The PCR-selected cDNA subtraction library was constructed with two micrograms of mRNAs from the ear and silk mix. The forward subtractive PCR products were ligated into pMD18 T-easy vectors and then transferred into E. coli (JM109). A total of 672 white colonies were picked from the plates randomly. PCR amplification with nested primers from recombinant clones revealed an insert size of approximately 200-1 000 bp (the figure not shown). Thus, a putative drought stress specific forward subtracted cDNA library of maize seedlings was constructed. After the positive clones tested by PCR were sequenced, 598 available EST sequences were obtained, resulting in 80 uniESTs including 23 singlets and 57 contigs after assembling. Those uniESTs were analyzed using BLASTX to NCBI database. The results showed that 68 uniESTs had highly homologouesamino acid sequencesto known function proteins. Eight uniESTs were unknown function protein and putative protein, four uniESTs had no homologoues sequence in the NCBI database. It could be because those several uniESTs were new genes or located in 3’-UTR or 5’-UTR. In those known function
649
proteins, many drought-related genes were found. The partial clones in the SSH library are shown in Table.
DISCUSSION Suppression subtractive hybridization (SSH) is a polymerase chain reaction (PCR)-based method, aiming to enrich rare transcripts and low-abundance genes and isolate different express genes (Diatchenko et al. 1996). In the present study, an SSH library was constructed and many drought stress-induced genes from maize seedling leaves were obtained. In the SSH library, there are many genes that are involved in signaling and regulatory pathways. For example, DREB2A was induced under dehydration and high salinity. The transcript factors of DREB can activate a series of genes which include cis-acting elements, for example, rd29A, corl5a, rdl7 and kinl. These products of those genes have important functions in plant resistance to abiotic stress (Liu 1998). Zinc finger protein with zinc finger domain is one of the important transcription factors. Of these, C2H2 type zinc finger protein is the most clearly identified zinc finger transcription factor, which can be induced under salt stress and low temperature. In Arabidopsis thaliana, AZF2 was induced under ABA treatment, AF1, A F 3 and STZ, were independent-ABA pathway (Huang et al. 2004; Sakamoto et al. 2000). Carbonic anhydrase (CA) was found in the SSH library. CA from higher plants traces its origin with prokaryotes and exhibits compartmentalizationamong their organs, tissues, and cellular organelles commensurate with specific functions. In leaves, CA represents 1-20% of total soluble protein and abundance next only to ribulose- 1,5-bisphosphate carboxylase/oxygenase (RuBPCO) in chloroplasts, facilitating CO, supply to phosphoenolpyruvate carboxylase in C4 and CAM plants and RuBPCO in C3 plants (Tiwari et al. 2005; Lee 2001). The genes encode proteins conferring stress tolerance include HSP, ERD4, and so on. Heat shock proteins are generated when cells are exposed to elevated temperatures, or to other kinds of environmental stresses, such as drought, ethanol, trace metals, or oxygen deprivation. Increasing expression of these proteins can protect organisms from stress-induced damage. They can combine with denatured proteins
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650
LI Hui-yong et al.
and maintain the state of solubility, and then fold and assist in the establishment of proper protein configuration and prevention of unwanted protein aggregation with the existing of Mg" and ATP. In the SSH library established here, HSP, HSP82 and HSP70 were found. Of those, HSP70 in plants has important chaperoning roles during high-temperature stress and is involved in folding newly translated polypeptides. Moreover, it also has functions in growth and development, and high and low temperature tolerance (Zhang et al. 2005; Netzer
and Hart1 1998). This SSH library consisted of a number of genes encoding enzymes associated with pathways, leading to the synthesis of functional and structural metabolites. For example, serine palmitoyltransferase, a key enzyme in generating endogenous phytosphingosine, prevents proteolysis during heat stress. Phytosphingosine, a putative sphingolipid second messenger, mediates heat stress signaling and activates ubiquitin-depndent proteolysis via the endocytosis vacuolar degradation and
Table Part of the genes isolated from maize seeding leaves under drought stress Clone No. Accession No. Functional annotation Genes involved in signaling and regulatory pathways L 13-C I AAV90624.1 DREB 2A L8-HI AAS873'1 Zinc finger protein L13-BS AAA86945.1 Carbonic anhydrase L.8-F7 AAV90623 Rab7 L13-B5 BAD331ll.l Putative chlorophyll aib-binding protein L 13-C6 XP-468022.1 Putative KOW domain-containing transcription factor LK-FS AAC24574 Shaggy kinase homolog L8-C 1 I AAR20887 Circadian oscillator component L2-H9 XP-482865.1 Putative Hecl protein Lh- A1 2 ABA9986 1.1 Putative protease, chloroplast precursor L7-E12 BAD33787.1 BRII-KD interacting protein 120 L8-t.11 BAB882 15 Putative secretory acid phosphatase precursor 1.8-C? XP-482468. I Putative methylcrotonyl-CoA carboxylase beta chain, mitochondria1 precursor Genes encoded protein confemng stress tolerance L7-AS BAD73668.1 Putative heat shock protein 82 L7-CI2 CAA5SIX4.1 Heat shock protein 70 kDa Ll-A7 AAL83988.1 Putative heat shock protein L?-G5 AAV59379.1 Putative early-responsive to dehydration stress protein (ERD4) LX-A 1 I XP-480189. I Putative LHY protein L7-A1 AAF91388.1 SocE LO-c 1 NP-565965.1 Hydrolase L8-F4 NP-914832 Putative selenium binding protein L2-DY XP-549860.1 Putative prolyl endopeptidaye L6-A6 AAX96187.1 Similar to seven transmembrane protein Mlo.1 L8-H3 AAT751.15 Putative cullin protein L13-FI XP-469'39.1 Putative RNA-binding protein Enzymes present in pathways leading to the synthesis of functional and structural metabolites L8-FIO AAK98692 Putative serine palmitoyltransferase L13-Dh NP-922378.3 Putative trehalase L2-B4 BAD82666.1 Putative aldose reductase L16-CI CAA81349.1 Triose phosphate/phosphate translocator L7-CI AAC80271.2 Glyceraldehyde phosphate dehydrogenase L13-C2 AAW50989.1 Ribosomal protein L7 L13-A6 XP-470900.1 Putative RNA polymerase Ill LlZ-BIl XP-463028.1 Putative SMC3 protein L.13-F9 NP-178634.? RNA binding/aconitate hydratase/hydro-lyase/iron ion bindingllyase L7-3 NP-567950.1 MCCB (3-methylcrotonyl-coa carboxylase); biotin carboxylase L2-A1 NP-564'XI . I Ubiquitin-protein ligasdzinc ion binding L?-El0 CAD23 143.1 Ht-transporting ATP synthase LZ-Hi BAB02957.1 Zinc metalloprotease (insulinase family) L6-B9 ~ ~ ~ 9 6 3I 7 2 . Arabinoxylan arabinofuranohydrolase isoenzyme AXAH- 11 L6-E4 AAV440-14.I Putative diphosphate-fructose-6-phosphate1-phosphotransferase L;'-EI 1 NP-922.IO2.1 Kinesin-like protein AAG40328.1 Myo-inasitol 1-phosphate synihase L;'-H12 LXA12 AAL'44107.1 Putative ketol-acid reductoisomerase LR-A3 AAC63962.1 Pyruvate dehydrogenase kinase isoform 2 ; PDKZ L&BI AAA33498.1 Pyruvate,orthophosphate dikinase LS-B4 NP-9 19692 Putative transposase L8-C8 AAC62456.1 Alanine aminotransferase L8-C9 XP-476534 Immunopbilin/FKBP-type peptidyl-prolyl cis-trans isomerase-like protein L8-F8 XP-466819 Putative lysyl-tRNA synthetase L8-G4 XP-468293 GCNS-related N-acetyltransferase (GNAT) family protein-like
Species
E-value
~~
Pennisetum glaucum Oryza saiiva Zea mays Pennisetum glaucum Oryza sativa Oryza sativa Zea mays Oryia sativa Orvza sativa Oryza sativa Oryza sativa Oryza sativa Orvza sativa
3.008-27 1.00E-57 48-59 4.00E-94 IE-37 5.00E-73 8.00E-36 2.00E-46 4.00E-23 4.00E-95 5.00E-2 1 6.00E-82 1E-103
Oryza sativa Zea mays Oryza sativa Oryza saiiva Oryza sativa Myxococcus xanthus A rahidopsis thaliana Oryza sativa Oryza sativa Oryza sativa Oryza sativa Oryza sativa
1.00E-89 5.00E-24 4.00E-42 1.008-114 7E-69 4.00E-18 7.00E-27 4.00E-7 I 3.00E-18 7.00E-25 2.00E-92 3.00E-84
Oryza sativa Oryza sativa Oryza sativa Zea mays Vibrio fischeri Triticum aestivum Oryza sativa Oryza sativa Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Oryza sariva Arabidopsis thaliana Oryza sativa Oryza sativa Oryza sativa Zea mays Oryza sativa Zea mays Zea mays Oryza sativa Zea mays Oryza sativa Oryza sativa Orvra sativa
3.00E-26 5E-113 1.00E-27 3.00E- 133 0.001 2.00E-44 1.OOE- 19 4.00E- 108 4.00E-59 3.00E-46 4.00E-68 2.00E-24 1.00E-120 3.00E-157 1.00E-129 8.00E-2 1 9.00E-32 7E-48 5E-119 1E-129 1.OOE- 16 4E-86 6.00E-24 6.00E- 162 3.00E-69
I -
8 2007, CAAS. All fightsreserved. Publishedby Elsevier Ltd.
Isolating Soil Drought-Induced Genes from Maize Seedling Leaves Through Suppression Subtractive Hybridization
26 S proteasome pathways (Namjin 2000). Trehalose is a non-reducing disaccharide, serving as a protectant of enzymes and membranes, particularly under conditions of heat and desiccation stress. Trehalases have been found in various tissues, particularly in pollen and legume root nodules with high activities (Joachim et al. 1995). It was also found in the SSH library, which probably has new functions in maize seedling leaves. In addition, other gene products, such as, H+-transporting ATP synthase, hydrolase, prolyl endopeptidase and PDK2, are also involved in molecular regulated mechanism to abiotic stresses, but the functions have not been clearly understood in stress tolerance. The analyses of the uniESTs generated in the present study indicated that molecular responses of plants to drought stress possibly crosstalk to other abiotic stresses to some extent since they share some common metabolic pathways. This crosstalk was due to that most of the abiotic stresses at the subcellular level have ion stress, oxidative stress and osmotic stress. Those specific stresses might result in ionic and osmotic imbalance, destruction of functional proteins, structural proteins and cell membranes. However, it should be pointed out that there were many unknown functional proteins and nonhomologoues proteins in the SSH library, which need to be further studied. I
Acknowledgements This work was supported by the National Natural Science Foundation of China (30571133), also supported partly by the Beijing Agricultural Innovative PlatformBeijing Natural Science Fund Program “Yzpt02-06”, and the Project for International Scientific and Technological Cooperation-Discovery and Utilization of Drought-Resistant Genes and Allele Gene in Maize Germplasm Resources, Ministry of Science and Technology of China (0502).
References Ban H D, Weatherley P E. 1962. A re-examination of the relative turgidity technique for estimating water deficit in leaves. Australian Journal of Biological Science, 15,413-428. Chaitanya K V, Sundar D, Jutur P P, Ramachandra Reddy A. 2003. Water stress effects on photosynthesis in different mulberry cultivars. Plant Growth Regulation, 40, 75-80. Chaves M M, Maroco J P, Periera S. 2003. Understanding plant responses to drought from genes to the whole plant. Functional Plant Biology, 30,239-264.
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Cushman J C, Bohnert H J. 2000. Genomic approaches to plant stress tolerance. Current Opinion in Plant Biology, 3, 117124. Diatchenko L, ChrisLau Y F, Campbell A P, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov E, Siebert P D. 1996. Suppression subtractive hybridization: A method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proceedings of the National Academy of Sciences of the USA, 93,6025-6030. Fussell B. 1999. The Story of Corn. 2nd ed. North Point Press, New York, NY. Huang J, Wang J F, B a n g H S . 2004. The structure and function of plant C2H2 zinc finger protein. Hereditas (Beijing). 26, 414-418. (in Chinese) Joachim M, Thomas B, Andres W. 1995. Trehalose and trehalase in plants: recent developments. Plant Science, 112, 1-9. Knight H, Knight M R. 2001. Abiotic stress signalling pathways: specificity and cross-talk. Trends Plant Science, 6,262-267. Lee R C, Burton R A, Hrmova M, Fincher G B. 2001. Barley arabinoxylan arabinofuranohydrolases: purification, characterizationand determinationof primary structures from cDNA clones. Biochemistry Journal, 356, 181-189. Liu Q,Kasuga M, Sakuma Y. 1998.Two transcription factors, DREBl and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsivegene expression, respectively, in Arabidopsis. Plant Cell, 10,1391-1406. Namjin C. 2000. Sphingolipids signal heat stress-induced ubiquitin-dependent proteolysis. Journal of Biological Chemistry, 275, 17229-17232. Netzer W J, Hart1 F U. 1998. Protein folding in the cytosol: chaperonin- dependent and -independentmechanisms. Trends in Biochemical Sciences, 23,68-73. Sakamoto H, Araki T, Meshi T, Iwabuchi M. 2000. Expression of a subset of the Arabidopsis Cys2IHis2-type zinc-finger protein gene family under water stress. Gene, 248.23-32. Shinozaki K, Yamaguchi-Shinozaki K. 2000. Molecularresponses to dehydration and low temperature: differences and crosstalk between two stress signalling pathways. Current Opinion in Plant Biology, 13,217-223. Tiwari A, Kumar P, Singh S, Ansari S A. 2005. Carbonic anhydrase in relation to higher plants. Photosynthetica, 43, 1-11 . Wang W X, Vinocur B, Altman A. 2003. Plant responses to drought, salinity and extreme temperatures: towards genetic engineering for stress tolerance. Planta, 218, 1-14. Zhang J G, He C Y, Duan Ai G, Yin J Y. 2005. Advances in research on plant heat shock proteins. Journal of Fujian College of Forestry, 25, 187-192. (in Chinese) Zhu J K. 2002. Salt and drought stress signal transduction in plants. Annual Review of Plant Biology, 53,247-73, (Edited by ZHANG Yi-min)
02007,CAAS. All rights reserved.PuMishedby ElsevierLM.
Agricultural Sciences in China 2007, 6(6): 647-651
4
* s*e
* r
ScienceDirect
June 2001
Isolating Soil Drought-Induced Genes from Maize Seedling Leaves Through Suppression Subtractive Hybridization LI Hui-yongl+2,HUANG Su-hua2, SHI Yun-su2, SONG Yan-chun2, ZHAO Jiu-ran3, WANG Feng-ge3, WANG Tian-yu* and LI Yu2 1
Institute of Food Crops, Henan Academy of Agricultural Sciences, Zhengzhou 450002, P.R.China
2
Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China
3
Maize Research Institute, Beijing Academy of Agricultural and Forestry Sciences, Beijing 100081, P.R.China
Abstract In this study, a forward cDNA library was constructed by suppression subtractive hybridization using seedling leaves of CN165, a drought-tolerant maize inbred line. In the suppression subtractive hybridization (SSH) library, 672 positive clones were picked up randomly. After polymerase chain reaction (PCR) of each clone, all the single clones were sequenced. Totally 598 available sequences were obtained. After cluster analysis of the EST sequences, 80 uniESTs were obtained, among which 57 uniESTs were contigs and 23 uniESTs were singlets. The results of BLASTN showed that all the uniESTs had homologous sequences in the nr database. The BLASTX results indicated that 68 uniESTs had significant protein homology, 8 uniESTs with homology of unknown proteins and putative proteins, and 4 uniESTs without protein homology. Those drought stress-induced genes were involved in many metabolism pathways to regulate plant growth and development under drought stress.
Key words: maize, drought stress, suppression subtractive hybridization (SSH), uniESTs
INTRODUCTION Maize is an important crop because not only maize feed both the world's people and livestock but also its products are necessary for many industries. For example, in the manufactureof diverse commoditiesincludingalcohol, glue, soap, paint, insecticides,toothpaste, shaving cream, rubber tires, rayon, molded plastics and others (Fussell 1999). However, maize is often subjected to several harsh environmental stresses that adversely affect growth, metabolism, and finally the yield. Among these environmental factors, drought is a major abiotic factor limiting agricultural production. Although the general
effects of drought on plant growth are fairly well known, the primary effects of water deficit at the biochemical and the molecular levels are not well understood (Zhu 2002; Chaitanya et al. 2003; Chaves et al. 2003). In many plants, drought stress often activates cell signaling pathways (Shinozaki et al. 2000; Knight H .and Knight M R 2001) and cellular responses, such as the production of stress proteins, up-regulation of antioxidants and accumulation of compatible solutes (Cushman and Bohnert 2000). These drought inducedgenes include three major categories: (1) genes that are involved in signaling cascades and in transcriptional control, such as some protein kinases and transcriptional factors; (2) genes that function directly in the
This paper is translated from its Chinese version in Sciantia Agricultura Sinica. LI Hui-yong, Ph D, E-mail [email protected];Correspondence LI Yu,Tel +86-10-62131196, E-mail: [email protected],WANG Tian-yu, Tel: +86-10-62186632, E-mail: wangty@mail,caas,net.cn
92007.CMS. All Mhtsreserved. PuMihedby E l h r Lid.
648
protection of membranes and proteins; and (3) genes that are involved in water and ion uptake and transport (Wang er al. 2003). Although many important genes are isolated, it is difficult to construct a clear metabolism network for explaining the plant drought resistance mechanism. Therefore, it is important to isolate drought induced genes and understand the function of genes and interaction of genes to develop maize varieties with drought resistance. In this study, a number of drought induced-genes were isolated from the leaves of maize seedlings throughout the suppression subtractive hybridization method, to Understand the mechanisms of drought tolerance in maize. Those specific stress-relatedgenes may be used in engineering more tolerant plants.
MATERIALS AND METHODS Plant materials and drought treatment Seeds of maize inbred CN165 with strong drought tolerance were allowed to imbibe overnight in distilled water before being transferred to trays of soil mix (soil: vermicu1ite:organic fertilizer = 3:2: l), under a rainout shelter. Before drought stress treatment, equal volume of water was applied to each tray, every other day to keep the plants in all trays under uniform water status. Water stress was applied at the five-leaf stage. The leaves from the control and treated plants were sampled at 2, 4, 6, 8, 10 and 12 d after stress application, respectively. At the same time, the leaf water relative water content (RWC), was measured as described by Barr and Weatherley (1962) (RWC = (Fresh wt - Dry wt)/(Turgid wt - Dry wt) x 100.
RNA extraction Total RNAs for drought treated plants were extracted from the samples harvested at six different time points (2, 4,6, 8, 10, 12 d), and then mixed equally as tester samples. Total RNAs for the control plants at the corresponding time points were also extracted and mixed equally as driver samples. The extraction method using Invitrogen Trizol and the isolation of poly (A) RNA using oilgotex were according to the manufacturer's protocol (Qiagen. Germany).
LI Hui-yong er al.
Construction of subtracted cDNA library Suppression subtractive hybridization was carried out using the PCR-selected cDNA subtractive hybridization kit according to the manufacturer's protocol (Clontech, USA). The water-stress plants were used as testers and the control plants were used as drivers. The tester and driver cDNAs of the leaves were prepared from 2 pg of mRNA extracted from the treated and the control samples. The tester cDNA was digested with Rsa I at 37°C for 1.5 h and then ligated to adaptors 1 and 2 in separate reactions at 16"C, overnight. Then driver cDNA was added to each of the tester samples, which were subsequently resuspended in the hybridization buffer. After heat-denaturation,the mixture was allowed to anneal at 68°C for 8 h. Then, the two samples from the first hybridization were mixed together and freshly denatured driver cDNA was added to the sample followed by incubation at 68°C overnight for the second hybridization. PCR amplification with two different nested primers was conducted to amplify the differentially expressed cDNAs. cDNA fragments for differentially expressed genes were inserted into the pMD18-T vector (Takara, Japan), and transformed into E. coli JMP109 cells. The recombinant white clones were randomly picked to construct the subtracted cDNA library.
EST clustering and sequence analysis Those white positive clones selected from the SSH library were PCR screened. The clones that contained the insert fragment were sequenced with the 3730 Automatic DNA sequencer (ABIPrism, USA). Sequences obtained were first extracted with PHRED and the lowquality (=lo0 bp), vector and adapter sequences were then removed. Cluster analysis of those sequences was done using the TGICL program. On the basis of all known information, the clones were further annotated and analyzed by the BLAST in NCBI database.
RESULTS Performance of CN165 under drought stress The maize inbred line CN165 has been identified as an inbred with strong drought tolerance. Within the first
Qm7, CAAS. All fights tmerved. Publishedby EIsmbr Ltd.
Isolating Soil Drought-Induced Genes from Maize Seedling Leaves Through Suppression Subtractive Hybridization
four days after water stress the plants were not easy to distinguish phenotypically from the well-watered plants. However visible symptoms of the stressed plants such as leaf rolling appeared as the stress continued. The leaves RWC of stress plants also showed a trend to decrease (Fig.).
2
4
6
8
1 0 1 2
Days without water
Fig. Changes of leaf relative water content (RWC) in maize at the seeding stage when plants were subjected to drought stress treatment for 12 days. Bars are mean & SD of five samples.
The subtracted cDNA library and sequence analysis of ESTs The PCR-selected cDNA subtraction library was constructed with two micrograms of mRNAs from the ear and silk mix. The forward subtractive PCR products were ligated into pMD18 T-easy vectors and then transferred into E. coli (JM109). A total of 672 white colonies were picked from the plates randomly. PCR amplification with nested primers from recombinant clones revealed an insert size of approximately 200-1 000 bp (the figure not shown). Thus, a putative drought stress specific forward subtracted cDNA library of maize seedlings was constructed. After the positive clones tested by PCR were sequenced, 598 available EST sequences were obtained, resulting in 80 uniESTs including 23 singlets and 57 contigs after assembling. Those uniESTs were analyzed using BLASTX to NCBI database. The results showed that 68 uniESTs had highly homologouesamino acid sequencesto known function proteins. Eight uniESTs were unknown function protein and putative protein, four uniESTs had no homologoues sequence in the NCBI database. It could be because those several uniESTs were new genes or located in 3’-UTR or 5’-UTR. In those known function
649
proteins, many drought-related genes were found. The partial clones in the SSH library are shown in Table.
DISCUSSION Suppression subtractive hybridization (SSH) is a polymerase chain reaction (PCR)-based method, aiming to enrich rare transcripts and low-abundance genes and isolate different express genes (Diatchenko et al. 1996). In the present study, an SSH library was constructed and many drought stress-induced genes from maize seedling leaves were obtained. In the SSH library, there are many genes that are involved in signaling and regulatory pathways. For example, DREB2A was induced under dehydration and high salinity. The transcript factors of DREB can activate a series of genes which include cis-acting elements, for example, rd29A, corl5a, rdl7 and kinl. These products of those genes have important functions in plant resistance to abiotic stress (Liu 1998). Zinc finger protein with zinc finger domain is one of the important transcription factors. Of these, C2H2 type zinc finger protein is the most clearly identified zinc finger transcription factor, which can be induced under salt stress and low temperature. In Arabidopsis thaliana, AZF2 was induced under ABA treatment, AF1, A F 3 and STZ, were independent-ABA pathway (Huang et al. 2004; Sakamoto et al. 2000). Carbonic anhydrase (CA) was found in the SSH library. CA from higher plants traces its origin with prokaryotes and exhibits compartmentalizationamong their organs, tissues, and cellular organelles commensurate with specific functions. In leaves, CA represents 1-20% of total soluble protein and abundance next only to ribulose- 1,5-bisphosphate carboxylase/oxygenase (RuBPCO) in chloroplasts, facilitating CO, supply to phosphoenolpyruvate carboxylase in C4 and CAM plants and RuBPCO in C3 plants (Tiwari et al. 2005; Lee 2001). The genes encode proteins conferring stress tolerance include HSP, ERD4, and so on. Heat shock proteins are generated when cells are exposed to elevated temperatures, or to other kinds of environmental stresses, such as drought, ethanol, trace metals, or oxygen deprivation. Increasing expression of these proteins can protect organisms from stress-induced damage. They can combine with denatured proteins
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LI Hui-yong et al.
and maintain the state of solubility, and then fold and assist in the establishment of proper protein configuration and prevention of unwanted protein aggregation with the existing of Mg" and ATP. In the SSH library established here, HSP, HSP82 and HSP70 were found. Of those, HSP70 in plants has important chaperoning roles during high-temperature stress and is involved in folding newly translated polypeptides. Moreover, it also has functions in growth and development, and high and low temperature tolerance (Zhang et al. 2005; Netzer
and Hart1 1998). This SSH library consisted of a number of genes encoding enzymes associated with pathways, leading to the synthesis of functional and structural metabolites. For example, serine palmitoyltransferase, a key enzyme in generating endogenous phytosphingosine, prevents proteolysis during heat stress. Phytosphingosine, a putative sphingolipid second messenger, mediates heat stress signaling and activates ubiquitin-depndent proteolysis via the endocytosis vacuolar degradation and
Table Part of the genes isolated from maize seeding leaves under drought stress Clone No. Accession No. Functional annotation Genes involved in signaling and regulatory pathways L 13-C I AAV90624.1 DREB 2A L8-HI AAS873'1 Zinc finger protein L13-BS AAA86945.1 Carbonic anhydrase L.8-F7 AAV90623 Rab7 L13-B5 BAD331ll.l Putative chlorophyll aib-binding protein L 13-C6 XP-468022.1 Putative KOW domain-containing transcription factor LK-FS AAC24574 Shaggy kinase homolog L8-C 1 I AAR20887 Circadian oscillator component L2-H9 XP-482865.1 Putative Hecl protein Lh- A1 2 ABA9986 1.1 Putative protease, chloroplast precursor L7-E12 BAD33787.1 BRII-KD interacting protein 120 L8-t.11 BAB882 15 Putative secretory acid phosphatase precursor 1.8-C? XP-482468. I Putative methylcrotonyl-CoA carboxylase beta chain, mitochondria1 precursor Genes encoded protein confemng stress tolerance L7-AS BAD73668.1 Putative heat shock protein 82 L7-CI2 CAA5SIX4.1 Heat shock protein 70 kDa Ll-A7 AAL83988.1 Putative heat shock protein L?-G5 AAV59379.1 Putative early-responsive to dehydration stress protein (ERD4) LX-A 1 I XP-480189. I Putative LHY protein L7-A1 AAF91388.1 SocE LO-c 1 NP-565965.1 Hydrolase L8-F4 NP-914832 Putative selenium binding protein L2-DY XP-549860.1 Putative prolyl endopeptidaye L6-A6 AAX96187.1 Similar to seven transmembrane protein Mlo.1 L8-H3 AAT751.15 Putative cullin protein L13-FI XP-469'39.1 Putative RNA-binding protein Enzymes present in pathways leading to the synthesis of functional and structural metabolites L8-FIO AAK98692 Putative serine palmitoyltransferase L13-Dh NP-922378.3 Putative trehalase L2-B4 BAD82666.1 Putative aldose reductase L16-CI CAA81349.1 Triose phosphate/phosphate translocator L7-CI AAC80271.2 Glyceraldehyde phosphate dehydrogenase L13-C2 AAW50989.1 Ribosomal protein L7 L13-A6 XP-470900.1 Putative RNA polymerase Ill LlZ-BIl XP-463028.1 Putative SMC3 protein L.13-F9 NP-178634.? RNA binding/aconitate hydratase/hydro-lyase/iron ion bindingllyase L7-3 NP-567950.1 MCCB (3-methylcrotonyl-coa carboxylase); biotin carboxylase L2-A1 NP-564'XI . I Ubiquitin-protein ligasdzinc ion binding L?-El0 CAD23 143.1 Ht-transporting ATP synthase LZ-Hi BAB02957.1 Zinc metalloprotease (insulinase family) L6-B9 ~ ~ ~ 9 6 3I 7 2 . Arabinoxylan arabinofuranohydrolase isoenzyme AXAH- 11 L6-E4 AAV440-14.I Putative diphosphate-fructose-6-phosphate1-phosphotransferase L;'-EI 1 NP-922.IO2.1 Kinesin-like protein AAG40328.1 Myo-inasitol 1-phosphate synihase L;'-H12 LXA12 AAL'44107.1 Putative ketol-acid reductoisomerase LR-A3 AAC63962.1 Pyruvate dehydrogenase kinase isoform 2 ; PDKZ L&BI AAA33498.1 Pyruvate,orthophosphate dikinase LS-B4 NP-9 19692 Putative transposase L8-C8 AAC62456.1 Alanine aminotransferase L8-C9 XP-476534 Immunopbilin/FKBP-type peptidyl-prolyl cis-trans isomerase-like protein L8-F8 XP-466819 Putative lysyl-tRNA synthetase L8-G4 XP-468293 GCNS-related N-acetyltransferase (GNAT) family protein-like
Species
E-value
~~
Pennisetum glaucum Oryza saiiva Zea mays Pennisetum glaucum Oryza sativa Oryza sativa Zea mays Oryia sativa Orvza sativa Oryza sativa Oryza sativa Oryza sativa Orvza sativa
3.008-27 1.00E-57 48-59 4.00E-94 IE-37 5.00E-73 8.00E-36 2.00E-46 4.00E-23 4.00E-95 5.00E-2 1 6.00E-82 1E-103
Oryza sativa Zea mays Oryza sativa Oryza saiiva Oryza sativa Myxococcus xanthus A rahidopsis thaliana Oryza sativa Oryza sativa Oryza sativa Oryza sativa Oryza sativa
1.00E-89 5.00E-24 4.00E-42 1.008-114 7E-69 4.00E-18 7.00E-27 4.00E-7 I 3.00E-18 7.00E-25 2.00E-92 3.00E-84
Oryza sativa Oryza sativa Oryza sativa Zea mays Vibrio fischeri Triticum aestivum Oryza sativa Oryza sativa Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Oryza sariva Arabidopsis thaliana Oryza sativa Oryza sativa Oryza sativa Zea mays Oryza sativa Zea mays Zea mays Oryza sativa Zea mays Oryza sativa Oryza sativa Orvra sativa
3.00E-26 5E-113 1.00E-27 3.00E- 133 0.001 2.00E-44 1.OOE- 19 4.00E- 108 4.00E-59 3.00E-46 4.00E-68 2.00E-24 1.00E-120 3.00E-157 1.00E-129 8.00E-2 1 9.00E-32 7E-48 5E-119 1E-129 1.OOE- 16 4E-86 6.00E-24 6.00E- 162 3.00E-69
I -
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Isolating Soil Drought-Induced Genes from Maize Seedling Leaves Through Suppression Subtractive Hybridization
26 S proteasome pathways (Namjin 2000). Trehalose is a non-reducing disaccharide, serving as a protectant of enzymes and membranes, particularly under conditions of heat and desiccation stress. Trehalases have been found in various tissues, particularly in pollen and legume root nodules with high activities (Joachim et al. 1995). It was also found in the SSH library, which probably has new functions in maize seedling leaves. In addition, other gene products, such as, H+-transporting ATP synthase, hydrolase, prolyl endopeptidase and PDK2, are also involved in molecular regulated mechanism to abiotic stresses, but the functions have not been clearly understood in stress tolerance. The analyses of the uniESTs generated in the present study indicated that molecular responses of plants to drought stress possibly crosstalk to other abiotic stresses to some extent since they share some common metabolic pathways. This crosstalk was due to that most of the abiotic stresses at the subcellular level have ion stress, oxidative stress and osmotic stress. Those specific stresses might result in ionic and osmotic imbalance, destruction of functional proteins, structural proteins and cell membranes. However, it should be pointed out that there were many unknown functional proteins and nonhomologoues proteins in the SSH library, which need to be further studied. I
Acknowledgements This work was supported by the National Natural Science Foundation of China (30571133), also supported partly by the Beijing Agricultural Innovative PlatformBeijing Natural Science Fund Program “Yzpt02-06”, and the Project for International Scientific and Technological Cooperation-Discovery and Utilization of Drought-Resistant Genes and Allele Gene in Maize Germplasm Resources, Ministry of Science and Technology of China (0502).
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