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Suping Zhou, Roger J. Sauve, and Abdulah Abdulah

Complementary Deoxyribonucleic Acid (cDNA) differential display and reverse Northern dot blot were used to identify genes in Pachysandra terminalis Sieb. & Zucc., a cold-tolerant plant, that are regulated by low temperatures. Rooted cuttings were obtained from stock plants that had been maintained in a greenhouse at 24 °C. These cuttings were subjected to the following cold treatments: 2 weeks at 12 °C, 48 hours at 4 °C, 48 hours at 0 °C, and 4 hours at –1 °C. Following leaf tissue analysis of treated and control plants, some stress-related genes and many novel genes were identified. Northern blot hybridization demonstrated that all novel genes were regulated by the cold treatments.

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Suping Zhou, Roger Sauve, and Fur-Chi Chen

A cysteine proteinase gene (DQ403257) with an open reading frame of 1125 base pairs was isolated from Pachysdandra terminalis. The primary translated peptide has a predicted length of 374 amino acids, pI (isoelectric point) of 5.70, and molecular mass of 40.9 kDa. The Peptidase_C1 domain is between residue 141 and 367. The proteinase has a conserved motif Gly-Xaa-Thy-Xaa-Phe-Xaa-Asn in the pro region. Sequence comparison shows that the deduced peptide shares 82% identity with the cysteine proteinase RD19a precursor (RD19) (accession P43296) from Arabidopsis thaliana (L.) Heynh. Real-time quantitative reverse-transcriptase–polymerase chain reaction revealed that the gene is induced by treatments of 1 to 7 days of darkness, 2 hours and 3 to 7 days at 5 °C, and 3 days at 38 °C.

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Roger J. Sauve, Suping Zhou, Yingchun Yu, and Wolfram George Schmid

A randomly amplified polymorphic DNA (RAPD) technique was used to identify and determine the phylogenetic relationships of 37 hosta accessions representing the major subgenera, sections and groups in the genus Hosta. Results of this study show that RAPD markers were able to differentiate not only the main groups, whose plants shared many genetic traits, but also cultivars within a species. Some accessions were identified by a single primer while others had high intercross linkage and required many markers for their separation. The phylogenetic clustering showed that H. plantaginea, the only night-blooming species, and H. ventricosa, the only known natural tetraploid, are unique and should be classified separately. The four species in the subgenus Bryocles, section Lamellatae H. venusta, H. minor, H. capitata, and H. nakaiana have very low genetic similarity since they do not share many amplified fragments. The other accessions were classified into four main clusters; cluster 1: H. venusta, H. tardiva, H. pycnophylla, H. tsushimensis `Ogon', H. montana, H. tibae, H. montana f. macrophylla, H. kikutii `Kikutii', H. longissima `Longifolia', H. rectifolia `Rectifolia', H. takahashii and H.`Undulata'; cluster 2: H. laevigata, H. sieboldiana, H. pycnophylla × H. longipes f. latifolia, H. longipes `Urajiro' and H. ibukiensis; cluster 3: H. capitata, H. kikutii `Polyneuron', H. nigrescens, H. kikutii `Yakusimensis', H. pachyscapa, H. kikutii `Caput-Avis', H. longipes f. latifolia, H. hypoleuca, H. okamotoi, H. densa and H. takiensis; and cluster 4: H. aequinoctiiantha, H. rupifraga, H. `Amanuma', H. minor and H. kikutii `Densa'.

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Suping Zhou, Roger J. Sauvé, Margaret T. Mmbaga, and Chaim Frenkel

Leucanthemum maximum `Silver Princess' plants, that were gradually acclimated for 7 days at 10 °C followed by 28 days at 7 °C, were subjected to the following cold treatments: 30 days at 4 °C; 4 or 5 days at 0 °C and for 3 hours at –1 °C to identify cold inducible proteins that may be responsible for cold tolerance in this cold tolerant species. Change in antioxidant enzymes activity in fully expanded leaves was assessed after each treatment. Catalase activity began to increase after 30 days at 4 °C and reached its peak after a 5-day exposure to 0 °C. The activity of cellular glutathione peroxidase and glutathione reductase significantly increased after a 4-day exposure to 0 °C. Changes in activity of four active superoxide dismutase isoforms, one basic guaiacol peroxidase and two o-dianisine peroxidase isoforms were also detected following the full series of cold treatments (30 days at 4 °C; 4 or 5 days at 0 °C and for 3 hours at –1 °C).

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Tingting Chen, Suping Zhou, Stephen Smith, and Roger Sauve

The goal of this study was isolate genes that are regulated by Al treatments of tomato roots growing in vitro. For Al treatment, germinating tomato seeds were plated on a MS agar medium supplemented with 0, 350, and 1200 μM AlCl3 for 30 days. Total RNA was extracted from root tissues and separated on denature formamide gel to check their quantity and quality. Equal amount of total RNA from treatment and control was treated with DNAse I (Genhunter, TN) to remove genomic DNA contamination. cDNA was obtained by reverse transcription using all the regents in RNA Image Kit (Genhunter, TN). The cDNA was amplified using the fluorescently labeled anchor primers (Oligo dT-A, C, G) and 16 random primers. Amplification products were separated by electrophoresis in 6% nondenaturing polyacrylamide gels and DNA bands were observed by scanning the gel on a FMBIOIII scanner. After comparing the band profile on the gel image, fragments of gene that showed changes in intensity compared to control (0 μM AlCl3) were isolated from the gel manually. These bands were re-amplified with the same pair of primers as the original amplification and cloned onto PCR-Trap cloning vector (Genhunter, TN). After DNA sequence analysis and homology comparison with NCBI database, we have identified clone # C01HBa0256E08 on L. esculentum chromosome 01, clone # C10HBa0111D09 on chromosome 10 and clone # LE_HBa-31H5 on chromosome 4.

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Suping Zhou, Fur-Chi Chen, Samuel Nahashon, and Tingting Chen

Photorespiration provides a protection mechanism in plants by diverting excessive energy accumulated from photochemical reaction, metabolizing toxic products and producing some protective molecules. The authors report cloning and characterization of a glycolate oxidase gene (GOX; NCBI accession DQ442286) and a NADH-dependent hydroxypyruvate reductase gene (HPR; NCBI DQ442287) from Pachysandra terminallis. The DQ442286 had the predicted GOX-like–Riboflavin-5′-phosphate (FMN) conserved domain and the DQ442287 had the predicted adenosine 5′-(alpha-thio)diphospho-5′-ribofuranosylnicotinamide nicotinamide adenine dinucleotide (NAD) binding domain (2-Hacid_DH_C). C-terminal peroxisome targeting signal was predicted to be -ARL for DQ442286 and –SKL for DQ442287. Both genes encoded enzyme proteins that are located in peroxisome and are involved in the photorespiration process. Real-time quantitative reverse-transcriptase polymerase chain reaction was performed to compare transcript level of the cloned genes after cold treatment. The 18s Ribosomal RNA (rRNA) was included to calibrate the data. The relative cycle threshold values (gene/18s rRNA) were 1.4, 1.5, and 1.5 for GOX and 1.2, 1.3, and 1.3 for HPR in the treatments of 4 °C 4 h, 4 °C 12 h, and control. The data revealed that gene expression was enhanced by only short-term (4-h) cold treatment. A ribulose-1, 5-biphosphate carboxylase/oxygenase (Rubisco) activase gene (DQ 486905) was also cloned and analyzed following the same procedure.

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Suping Zhou, Roger Sauve, Tara Fish, and Theodore W. Thannhauser

Tomato (Solanum lycopersicum cv. Money Maker) seedlings at the two-leaf stage were grown in one-half strength Hoagland solution supplemented with 50 mm NaCl for 4 days, with 100 mm NaCl for 4 days, with 150 mm NaCl for 4 days, and with a final concentration 200 mm NaCl for 2 days. Solutions were refreshed every 2 days for treated and untreated seedlings. Non-treated plants were grown in nonamended one-half strength Hoagland solution. Three biological replicates (BR) were included for treated and control experiments. At the end of treatments, the uppermost three newly expanded leaves from all 12 plants in each BR were collected and bulked to extract total protein. Proteomic analysis resulted in the identification of several salt-induced and salt-suppressed proteins. Salt-induced proteins were: vacuolar H+-ATPase A1 subunit isoform (1.6-fold), germin-like protein (1.5-fold), ferredoxin-NADP (+) reductase (1.2-fold), quinone oxidoreductase-like protein (4.4-fold), heat-shock protein (4.9-fold), and pyrophosphorylase (1.7-fold). Salt-suppressed proteins were: ATPase alpha subunit (−1.5-fold) and rubisco activase (−1.4-fold). Proteins identified in this study affect cellular activities for antioxidant, stress protection, carbon fixation, and carbohydrate partitioning in young tomato leaves under salt stress.

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Suping Zhou, Roger Sauve, Tingting Chen, Sara Bhatti, and Debrah Long

Identification of low temperature–regulated gene expression in Pachysandra terminalis: Pachysandra terminalis is a cold-hardy, evergreen plant species. In order to identify molecular mechanism of cold tolerance of this plant species, seedlings with four fully expanded leaves were subjected to 4, 0, and –1 °C low temperature treatments. Low temperature–induced genes were identified from treated plants using cDNA differential display. The cDNA fragments were cloned onto PCR-trap vectors. Low temperature regulation of these genes was confirmed by reverse-northern blot. Sequence analysis has identified that these genes can be classified into three groups, stress-related, photosystem-related. Most of the genes cannot find matching sequences in the database. To further study the regulation of these genes by temperature fluctuation, the plants were treated at 4, 0, and 40 °C. Northern blot analysis showed that several clones showed increased expression after cold and heat shock. Previous cold treatment at 4 °C can negate the effect of heat shock on expression of these genes. Complete sequence of these genes is cloned from the cDNA library and their temporal regulation by environmental stresses is analyzed using real-time PCR.

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Suping Zhou, Roger J. Sauvé, and Margaret T. Mmbaga

A cold acclimatization mechanism regulated by the accumulation of mRNAs and proteins has been tentatively identified in japanese spurge (Pachysandra terminalis Sieb. & Zucc.). Two polypeptides and several cDNA fragments were observed in leaf tissue after acclimation. When these proteins were probed with type III fish antifreeze antibodies, an immune-cross reaction occurred. Nonacclimatized young leaves and stems of japanese spurge survived 20-minute exposures at -5 °C. Although newly emerged leaves and stems were damaged, plants resumed growth at higher temperatures. After acclimation by gradual cold treatments (4 to -5 °C), new proteins began to accumulate in young leaves and plants were more tolerant to extended treatments at -5 °C. Changes in accumulation of proteins and mRNA in leaf tissue of japanese spurge appear to be an adaptation mechanism to subfreezing conditions. This is the first report of the immune-cross reaction between antibodies of type III fish antifreeze proteins and plant proteins

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Suping Zhou, Marsha Palmer, Jing Zhou, Sarabjit Bhatti, Kevin J. Howe, Tara Fish, and Theodore W. Thannhauser

A comparative proteomics study using isobaric tags for relative and absolute quantitation (iTRAQ) was performed on a mesophytic tomato (Solanum lycopersicum) cultivar and a dehydration-resistant wild species (Solanum chilense) to identify proteins that play key roles in tolerance to water deficit stress. In tomato ‘Walter’ LA3465, 130 proteins were identified, of which 104 (80%) were repressed and 26 (20%) were induced. In S. chilense LA1958, a total of 170 proteins were identified with 106 (62%) repressed and 64 (38%) induced. According to their putative molecular functions, the differentially expressed proteins belong to the following subgroups: stress proteins, gene expression, nascent protein processing, protein folding, protein degradation, carbohydrate metabolism, amino acid and nucleotide metabolism, lipid metabolism, signal transduction, and cell cycle regulation. Based on changes in protein abundance induced by the dehydration treatment, cellular metabolic activities and protein biosynthesis were suppressed by the stress. In S. chilense, dehydration treatment led to elevated accumulation of proteins involved in post-transcriptional gene regulation and fidelity in protein translation including prefoldin, which promotes protein folding without the use of adenosine-5′-triphosphate (ATP), several hydrophilic proteins, and calmodulin in the calcium signal transduction pathway. Those protein changes were not found in the susceptible tomato, ‘Walter’. Within each functional protein group, proteins showing opposite changes (dehydration induced vs. repressed) in the two species were identified and roles of those proteins in conferring tolerance to water deficit stress are discussed. Information provided in this report will be useful for selection of proteins or genes in analyzing or improving dehydration tolerance in tomato cultivars.