Results Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol158.Issue1-2.Sept2000:

2 . 2 . cDNA library construction and differential screening cDNAs were generated using oligo-dT primers and the cDNA synthesis kit purchased from Stratagene. The cDNAs were cloned into lambda Zap II vector DNA linearized by digestion with EcoRI and XhoI. The library of recombinant clones was screened by differential hybridization of duplicate nitrocellulose filters [23] using a- 32 P- dCTP labeled cDNA as probe [24]. Probes used for screening consisted of radioactively labeled cDNAs prepared from tobacco root and leaf RNAs, and root RNA from tomato as a het- erologous control probe. cDNAs showing strong hybridization with probes prepared from roots of topped plants were recovered and their nucleotide sequence determined. 2 . 3 . DNA sequencing and analysis Nucleotide sequencing was carried out manually using the Sequenase Version 2.0 protocols accord- ing to the manufacturer’s protocol United States Biochemical or with an ABI 310 Genetic Ana- lyzer PE Applied Biosystems using double- stranded plasmid DNA templates prepared utilizing the Qiaprep Spin Plasmid Kit Qiagen. The nucleotide and predicted amino acid se- quences of the various cDNAs were analyzed us- ing BLAST sequence analysis programs [25,26] and protein sequence alignments were carried out using the PILEUP program Genetics Computer Group Sequence Analysis Package, Version 9.0, Madison, WI and the various gene sequences available in the NCBI National Center for Bio- technology Information, Bethesda, MD nucle- otide and protein sequence database. Manual adjustment of the sequence alignments was carried out as necessary. 2 . 4 . RNA gel blot analysis Total RNA was extracted from tobacco roots, leaves, and floral parts using Tri-Reagent Molec- ular Research Center according to the manufac- turer’s protocol. For RNA gel blot analysis, aliquots 10 mg of total RNA extracted from the various tissues were fractionated by electrophore- sis through a 1.2 agarose – formaldehyde gel and blotted onto Nytran nylon membranes Schleicher and Schuell using 10 × SSC [24]. The transferred RNA was UV cross-linked to the membrane using a UV Stratalinker Stratagene and the mem- branes were prehybridized in 7 SDS, 0.25 M Na 2 HPO 4 , pH 7.2 for 2 – 4 h at 65°C. Hybridiza- tion was carried out in the same buffer in the presence of a- 32 P-dCTP labeled probes for 16 h at 65°C. The membranes were washed under high stringency conditions and subject to autoradiogra- phy at − 80°C for 48 h. Restriction fragments derived from cDNA clones of interest were separated by agarose gel electrophoresis, the DNA was purified, and quantified spectrophotometrically. The a- 32 P- dCTP labeled probes were prepared from 25 – 50 ng of insert DNA by random primed labeling Random Primed Labeling Kit, Boehringer Mannheim, IN. As a control probe used to quan- tify and normalize RNA levels in each lane, blots were hybridized with a 400-bp portion of the cDNA encoding the b-subunit of mitochondrial ATPase [27] 2 . 5 . Genomic DNA isolation and gel blot analysis Tobacco genomic DNA was isolated from to- bacco leaf tissue by the method of Junghans and Metzlaff [28]. Total genomic DNA 15 mg was digested to completion with EcoRI or HindIII, the digestion products were fractionated by elec- trophoresis through a 0.8 wv agarose gel, and transferred onto Nytran nylon membrane Schle- icher and Schuell in the presence of 0.4 N NaOH [24]. Following transfer, the membrane was rinsed in 2 × SSC, the DNA was UV cross-linked to the membrane, and the membrane was prehybridized and hybridized as described above. Following hy- bridization and washing, the membranes were sub- jected to autoradiography at − 80°C.

3. Results

3 . 1 . Analysis of differentially expressed gene products in the roots of topped tobacco plants Using a subtractive hybridization strategy, a group of 60 cDNAs were isolated that showed differential levels of expression in the roots of tobacco N. tabacum cv. Burley 21 plants before and 3 days after topping i.e. removal of the flower head and upper leaves and stem of the plant. The nucleotide sequence of the individual cDNAs was determined and both the nucleotide and predicted amino acid sequences of the various cDNAs were analyzed using BLAST sequence analysis programs [25,26]. In most cases, the complete nucleotide sequence of the individual cDNA was determined. Where multiple clones were available, only full length clones were fully sequenced. Following our analysis, the majority of the cDNAs isolated could be placed into one of several categories based upon their similarity to known genes or their encoded products already present in the NCBI databases. The results of our study are presented in Table 1. Among the largest subset of related cDNAs recovered were clones encoding enzymes involved in polyamine biosynthesis and alkaloid formation Group I. Among this group were both full length and partial cDNAs encoding ADC, ODC, and S-adenosylmethionine synthetase SAMS. The fur- ther characterization of these gene products is described in greater detail below. cDNAs were also identified that encode ho- mologs of various cell wall-associated proteins or enzymes involved in wall formation Group II. Among these are three distinct extensins and two different proline-rich proteins PRPs. The recovery of homologs of extensin and PRPs is not surprising. Members of both gene families have been reported to be expressed during root growth and regenera- tion, and the steady state levels of both classes of transcripts have been shown to increase in response to wounding [29]. The third recognizable category of cDNAs Group III contains homologs of proteins previously shown to be involved in tran- scription or translation, or to be components of signal transduction pathways. Notable among these clones is the ethylene responsive element binding EREB protein. The role of ethylene in the control of gene expression both independently and in con- junction with other phytohormones e.g. auxin is well documented [30]. cDNAs encoding enzymes known to be involved in general cellular processes i.e. cell structure, intra- and intercellular communication and transport, protein turnover, etc. constitute Group IV. In- cluded among these are transmembrane proteins with similarity to phosphate transporters and intrin- sic water channel proteins e.g. PR12 and PR16 whose activities have been previously suggested to be modulated in response to various stress responses or changes in phytohormone levels or ratios. Changes in some of these components, such as fructose-1,6-bisphosphate aldolase and ribose-5- phosphate isomerase, may simply reflect the gross alteration in whole plant physiology upon topping. A number of cDNAs encode proteins for which no function has yet to be defined in plants Group V, or for which no match of any significance could be found within the databases Group VI. There- fore, the function of these cDNAs and their impor- tance to the regulation of alkaloid formation and distribution remains unknown. 3 . 2 . Characterization of ADC, ODC, and SAMS proteins from N. tabacum L. c6. Burley 21 Within the differentially expressed gene products cloned in our investigation were both partial and full-length cDNAs encoding ADC, ODC, and SAMS. PR24 encodes the full-length N. tabacum ADC cDNA. The cDNA contains an open reading frame of 2163 bp coding for a protein 720 amino acids in length. The ADC transcript contains an unusually long 5-untranslated region UTR of 431 nucleotides and a short 3-UTR of 84 nucleotides with a near consensus polyadenylation signal AATAATA 30 nucleotides upstream of the polyA n tract. The N. tabacum ADC is 82 identical to the ADC of its evolutionary progenitor species N. syl6estris [Genbank Accession No. AB012873] and 86 identical to the ADC from tomato Lycopersi- con esculentum [31], another member of the Solanaceae family Fig. 1. As might be expected, the N. tabacum ADC shares considerably less sim- ilarity to ADCs isolated from species more distantly related evolutionarily, such as Arabidopsis, 67 identical [32,33]; soybean, 67 identical [34]; oat, 42 identical [35]; and Escherichia coli, 29 identi- cal [36]. The predicted protein coding region for the N. tabacum ADC is substantially longer than those reported for the ADC proteins of N. syl6estris and L. esculentum [31], but is similar in length to those reported in other higher plant species e.g. Ara- bidopsis, oat, soybean [32 – 35] and for the E. coli enzyme [36]. The difference in overall length ap- pears to arise from an apparent nucleotide dele-tion in the N. syl6estris and tomato cDNA sequences relative to the PR24 sequence and those in other plants. In the nucleotide sequences reported Table 1 Summary of differentially expressed cDNAs isolated from roots of wild-type Burley 21 tobacco after topping Insert size bp HomologyIdentity blast score Plasmid Accession no. designation amt. seq. Group I. Alkaloid biosynthesis PR-1 SAMS, partial cds. 6e-84 1400 219 1200 168 SAMS, partial cds. 8e-29 PR-2 PR-3 1300 701 SAMS, partial cds. 1e-138 AF127243 1636 1636 SAMS, full length coding PR-6 PR-7 SAMS, partial cds. 600 600 1600 198 SAMS, partial cds. 2e-58 PR-8 1300 135 PR-9 SAMS, partial cds. 4e-27 1400 174 SAMS, partial cds. 2e-25 PR-10 1600 299 PR-11 SAMS, partial cds. 1e-102 PR-17 U59812 959 959 ODC partial clone 1000 201 SAMS, partial cds. 2e-08 PR-21 PR-23 SAMS, partial cds. 1e-77 228 228 AF127239 2694 2694 ADC, full length coding PR-24 PR-37 1600 140 SAMS, partial cds AF127242 ODC, full length coding 1596 1596 PR-46 Group II. Cell wall related proteins AF156371 3500 559 Extensin 1, partial cds. 0.96 PR-29 659 659 PR-38 AF154651 Extensin 2, partial cds. 3e-89 AF154653 696 696 Extensin 3, partial cds. 11e-3 PR-41 AF154654 PR-42 Extensin precursor, partial cds. 1e-17 734 734 AF154655 661 661 Lignin-forming anionic peroxidase, partial cds. 3e-72 PR-45 PR-59 832 832 AF154667 Proline-rich cell wall-associated protein, partial cds. 2e-94 AF154669 1500 901 Proline cell wall-associated protein, partial cds. 7e-26 PR-64 Group III. Transcription, translation, signal transduction 673 673 PR-5 AF154636 40S ribosomal S4 protein, partial cds. 1e-56 AF154644 705 705 Glycine-rich RNA-binding protein, ABA-inducible, partial cds 3e-31 PR-20 AF156367 PR-22 Glycine-rich RNA binding protein, partial cds. 4e-19 128 128 500 172 26S ribosomal RNA PR-31 1078 1078 PR-47 AF154656 Putative ethylene responsive element binding EREB protein 3e-22 AF154657 1122 1122 Putative serinethreonine protein kinase, partial cds. 2e-08 PR-48 708 708 PR-50 AF154659 40S ribosomal S12 protein, partial cds. 8e-42 AF154660 PR-51 Putative elongation factor EF-1a; vitronectin-like adhesion protein 948 948 5e-65 AF154663 PR-55 60S ribosomal L15 protein, partial cds. 2e-99 888 888 PR-57 687 687 AF154665 Glycine-rich RNA-binding protein wound repressed, partial cds. 1e-35 AF156372 60S cytoplasmic ribosomal protein L2, partial cds. 2e-71 PR-60 600 440 Group IV. General metabolic function housekeeping and structural genes AF154637 Putative inorganic phosphate transporter, partial cds. 9e-08 PR-12 450 430 AF154640 1451 1451 Actin, partial cds 1e-180 PR-15 AF154641 Intrinsic plasmamembrane protein water channel, partial cds. 1e-106 PR-16 1100 1100 AF154647 637 637 Poly-ubiquitin 2e-76 PR-32 AF154648 PR-33 Fructose-1,6-bisphosphate aldolase, partial cds. 1e-160 1313 1313 AF154650 638 638 Ubiquitin conjugating enzyme E2, partial cds. 7e-69 PR-35 PR-36 X00945 737 737 a-1 Protease inhibitor antitrypsin, partial cds. 8e-49 AF154658 Ribose-5-phosphate isomerase, partial cds. 8e-50 1084 1084 PR-49 Group V. Unknown function in plants AF154635 685 685 Putative N7 protein homolog, partial cds. 2e-19 PR-4 PR-13 722 722 AF154638 dnaJ homolog, partial cds. 2e-37 AF154642 057 1057 CF2 protein homolog; partial cds. 4e-20 PR-18 1034 1034 PR-19 AF154643 Formamidopyrimidine-DNA glycosylase, partial cds. 2e-79 AF156369 a-2-HS-glycoprotein homolog, partial cds. 3e-58 PR-27 159 159 AF154652 Auxin regulated mRNA glycine max, partial cds. 2e-90 824 824 PR-39 Table 1 Continued Insert size bp HomologyIdentity blast score Plasmid Accession no. amt. seq. designation 1083 1083 AF154666 PR-58 Putative auxin-regulated mRNA, partial cds. 4e-15 Group VI. Unique no matches in database 1085 1085 PR-14 AF154639 Hypothetical topping-induced protein Hypothetical topping-induced protein AF156363 PR-25 900 171 1141 1141 PR-26 AF154645 Hypothetical topping-induced protein Hypothetical topping-induced protein AF156370 PR-28 1200 106 1217 1217 PR-M AF154646 Hypothetical topping-induced protein 750 536 AF154659 PR-34 Hypothetical topping-induced protein Hypothetical topping-induced protein AF154661 PR-52 871 871 429 429 AF154662 PR-53 Hypothetical topping-induced protein 429 429 same as PR53 PR-54 Hypothetical topping-induced protein 900 705 PR-56 AF154664 Hypothetical topping-induced protein PR-63 AF154668 Hypothetical topping-induced protein 1500 986 Fig. 1. Comparison of the predicted amino acid sequences of arginine decarboxylases ADCs from various species. Shown is a PILEUP alignment of the predicted amino acid sequences of the N. tabacum cv Burley 21 ADC encoded on plasmid PR24 AF127239 with the ADCs from N. syl6estris AB12873, Arabidopsis thaliana AF009647, A6ena sati6a oat X56802, Lycopersicon esculentum tomato L16582 and E. coli M31770. Amino acid residues conserved among the various ADC are shaded. for both the N. syl6estris and tomato cDNAs, a guanine residue position 2295 in the N. syl6estris sequence and 1531 in the tomato se- quence is missing [Genbank Accession No. AB012873]. This deletion changes the reading frame and introduces a premature termination to the predicted coding region. Using the sequence infor- mation available in the NCBI database, correcting for this error allowed us to extend the predicted C-terminus of the both ADC proteins, yielding the alignment to the N. tabacum ADC and those of other plant ADCs as indicated in Fig. 1. We have also included in the alignment shown in Fig. 1, the correction at the N-terminus of the predicted tomato ADC protein sequence noted by Pe´rez- Amado et al. [37], allowing better alignment of all of the higher plant sequences. Shown in Fig. 2 is the predicted amino acid sequence for the N. tabacum ODC encoded by the full-length cDNA PR46 in a PILEUP alignment with ODC proteins isolated from other plant and animal species. The predicted amino acid sequence for the N. tabacum ODC protein encoded in PR 46 is identical to the partial N. tabacum ODC cDNA sequence PR17 reported earlier [38], but differs at eight amino acids 98 identity from the protein sequence of an ODC isolated from the high alkaloid cultivar, N. tabacum cv. SC58 [Genbank Accession No. Y10472.1]. The two tobacco proteins are 88 – 89 identical to the ODC from tomato and jimson- weed Datura stramonium [39,40], but substantially less similar to other ODCs from non-photosynthetic eukaryotes and prokaryotes e.g. C. elegans, 32 similarity; Xenopus lae6is, 31. All eukaryotic ODCs share several structural features in common, including a conserved lysine involved in binding of pyridoxyl phosphate cofactor LYS-96 in the N. tabacum ODC sequence and a conserved cysteine CYS-378, which is the attach- ment site for DMFO, a potent inhibitor of enzyme function [41]. The ODCs characterized from eu- karyotic animal cells also contain a C-terminal extension relative to the enzymes present in prokaryotes that is thought to be involved in the rapid degradationturnover of the protein [41]. As evidenced from the alignment shown in Fig. 3, the tobacco ODC lacks this extension, as do the ODCs from other plant species [39]. It has been previously noted that plant ADCs and ODCs share domains in common and, therefore, it is likely that they share a common evolutionary origin [5,41]. Among the more highly conserved Fig. 2. Comparison of the predicted amino acid sequences of ornithine decarboxylases ODCs from various species. Shown is a PILEUP alignment of the predicted amino acid sequences of the N. tabacum cv. Burley 21 ODC encoded on plasmid PR46 AF127242 with the ODCs from N. tabacum cv. SC58 Y10472, Lycopersicon esculentum tomato AF029349, Datura stramonium jimsonweed X87847, and C. elegans U03059. Amino acid residues conserved among the various ODCs are shaded. Fig. 3. Comparison of the predicted amino acid sequences of S-adenosylmethionine synthetases SAMS from various spe- cies. Shown is a PILEUP alignment of the predicted amino acid sequences of the N. tabacum cv Burley 21 SAMS en- coded on plasmid PR6 AF127243, with the SAMS from Lycopersicon esculentum tomato Z24743, Catharanthus roseus Z71272, Arabidopsis thaliana M55077, Pisum sa- ti6um pea L36681, Oryza sati6a rice Z26867, and Homo sapiens humans X68836 . Amino acid residues conserved among the various SAMS are shaded. regions of the ADC and ODC proteins of N. tabacum is the proposed active site involved in decarboxylation of arginine and orinithine, respec- tively. This sequence, DTGGGL in ADC and DVGGGF in ODC is similar to the consensus motif DIVGGGLF observed in ADCs, ODCs, and the related protein diaminopimelic acid decarboxylases, in other species of plants, animals, and microbes [5,41,42]. The largest number of cDNAs recovered by our differential hybridization screening were partial and full-length clones encoding SAMS. Based upon a comparison of available nucleotide sequences from the coding and 3-UTRs of the various clones, it appears that there are at least five different ex- pressed gene products in N. tabacum roots. Shown in Fig. 3 is the predicted amino acid sequence for the full-length N. tabacum SAMS encoded in PR6 compared to the predicted protein sequences of SAMS from other species. The N. tabacum SAMS is most similar to the SAMS of tomato 90 identical[43] and has between 85 and 88 identity to the SAMS from plant species, including pea [44], Arabidopsis [45] and Catharanthus [46]. Significantly less sequence similarity is found between the SAMS of N. tabacum and those present in non-photosyn- thetic eukaryotes e.g. 60 identity to SAMS from yeast [47] and humans [48]. It has been reported previously that the members of the SAMS gene families present within various plant species can be divided into evolutionary groupings based upon the conserved na-ture of specific amino acid residues within the SAMS primary protein sequence [46]. Comparison of the predicted amino acid sequence for PR6 encoded SAMS from tobacco with SAMS from other plant species indicates that the PR6-encoded protein falls into the Type II cluster, as defined by Schru˚der et al. [46]. Unfortunately, the partial cDNAs encoding SAMS isolated in our studies do not provide sufficient sequence information and therefore we can not predict their phylogenetic placement with any accuracy. 3 . 3 . Genomic complexity of the ADC, ODC, and SAMS gene families Both ADC and ODC are encoded by small gene families in the N. tabacum genome. Gel blots of total genomic DNA hybridized with radioactively-la- beled ADC cDNA probes detected two major bands and several minor bands in DNA samples digested with either EcoRI or HindIII. Five to seven major bands and several minor bands of sufficient size to encode full-length genes were detected when the same blots were hybridized with radioactively-la- beled cDNA probes encoding ODC. It has been reported previously that ADC is encoded by a single gene or low-copy number nuclear gene in other plants species [31 – 33,42]. For example, tomato and soybean are reported to contain a single ADC gene [31,42], two copies of ADC are present in many Brassicaceae [49], including Ara- bidopsis [33]. ODC has also been reported to be encoded by a small gene family in other plant species, such as Datura, where three to five family members are reported to be present [39]. The presence of multiple genes encoding ADC and ODC in N. tabacum is consistent with its evolu- tionary origin from the hybridization of three different progenitor species i.e. N. syl6estris, N. tomentosiformis, and N. otophora, each of which could contribute a locus to the N. tabacum genome [50,51]. The recovery of multiple expressed SAMS cD- NAs is consistent with our genomic DNA gel blot analysis Fig. 4 showing eight to ten hybridizing bands in both EcoRI- or HindIII- digested total genomic DNA samples probed with a radioac- tively-labeled SAMS coding region probe. The gene family encoding SAMS activity in N. tabacum appears to be of greater complexity than that observed for either the ADC or ODC gene family in this species. The existence of multiple genes encoding SAMS in N. tabacum is consistent with previous reports of multiple expressed SAMS genes in other plant species, including pea [44], Arabidopsis [45], and tomato [43], although it should be noted that the SAMS gene family in tobacco appears to be substantially larger than those present in the other plant species. 3 . 4 . RNA gel blot analysis of ADC, ODC, and SAMS expression in tobacco The distribution and abundance of ADC, ODC, and SAMS transcripts in mature tobacco plants was analyzed by gel blots using total RNA pre- pared from the roots, stems, leaves, and various floral parts of mature Burley 21 tobacco plants. As shown in Fig. 5, transcripts encoding ODC were highly expressed in the roots and present to a lesser extent in floral portions of the plant. Little or no expression of these transcripts was detected in other tissues. Only very low levels of ADC transcripts were present in roots and among floral tissues significant levels were only observed in sepals. The tissue specific distribution of ODC and ADC transcripts was similar but not identical to that found for transcripts encoding putrescine N- methyl transferase PMT, an enzyme controlling the flow of precursors between polyamine and nicotine biosynthesis [6,50]. In contrast, transcripts encoding SAMS were easily detected in all tissues with slightly higher levels observed in photosyn- thetic versus non-photosynthetic tissues. It has been previously shown that removal of the flower head and several young leaves i.e. topping leads to activation of nicotine formation in the roots of decapitated plants [6,10]. It has also been reported that coincident with nicotine accu- mulation over the subsequent 24 h period, there is an increase in the levels of transcripts encoding PMT and spermidine synthase SPS in wild-type plants [6,50]. To determine the effects of topping on ADC, ODC, and SAMS expression in roots, Burley 21 plants were grown in the greenhouse to the bud stage at which point the upper third of the plant was removed and samples of roots tissues were collected before and at various times post- topping. As shown in Fig. 5B, low levels of the ODC and ADC transcripts were found in roots Fig. 4. Gel blot analysis of genomic DNA from N. tabacum cv Burley 21 probed with radioactively-labeled cDNA encod- ing ADC, ODC, and SAMS. Total genomic DNA 30 mg was digested with EcoRI or HindIII, fractionated by agarose gel electrophoresis, transferred to nylon membranes and hy- bridized with a- 32 P-dCTP labeled probes encoding tobacco ADC, ODC and SAMS as described in the Materials and methods. The mobility of molecular weights standards are given to the right of the figure in kilobases kb. Fig. 5. Gel blot analysis of the ADC, ODC, and SAMS transcript levels in various tissues of mature tobacco plants and in the roots before and after topping. Total RNA was isolated from various tissues of mature N. tabacum cv. Burley 21 and analyzed by gel blot analysis using a- 32 P-dCTP labeled coding region probes for ADC PR24, ODC PR46, and SAMS PR10. As a control, the blots were also probed with radioactively labeled probes encoding the alkaloid biosynthe- sis enzyme putrescine PMT [50], the b-subunit of coupling factor CF 1 -ATPase b-ATPase [27] and 26S rRNA PR31. Panel A. Transcript levels in various organs of wild-type tobacco. R, Root; St, Stem; ML, Mature Leaf; YL, Young Leaf; Se, Sepal; C, Carpel; MS, Mature Stamen; P, Petal; Et Br, ethidium bromide stained. Panel B. Transcript levels in roots of Burley 21 tobacco plants before and after topping. under stress will show higher levels of ODC prior to topping. Low but detectable amounts of mR- NAs encoding SAMS are present in the roots of untopped plants, and the level of SAMS tran- scripts changed little over the 24 h period after topping. No significant expression of the ODC and ADC transcripts was observed in leaf tissues before or after topping. In contrast, transcripts encoding SAMS were moderately abundant in young leaves and present at low levels in the mature leaves and other photosynthetic tissues. This observation is consistent with increased requirement for methyl groups during light-induced leaf development and the general requirement of SAM for a wide range of cellular processes.

4. Discussion