Introduction, Materials and Methods

From a published paper, I have taken and presented here the Title, Introduction, Materials and Methods, Figures (see separate files) and Tables. I have also included the reference list from the original paper.

For this assessment, I want you to write in your own words;

  1. an abstract which summarises the aims and findings of the study
  2. the text that you think would be included in a combined the results and discussion section,

This assessment is worth 10% and is due 5pm, 10th November.

The aim of this assessment is for you to look at the data in the figures, how the experiments were done and write down what you would conclude form the data.

You will notice that the end of the Introduction, suggest to you what the aims were of this study, then you should look at the data and summarise the findings. This is an important skill as a scientist and can be seen in all the scientific papers that you read in your career.

I have given you this task to do as you are more advanced than the undergraduate students that you studied with.

Title: Molecular evidence of sorbitol dehydrogenase in tomato, a non-Rosaceae plant

Introduction.

Sorbitol is important in translocating photosynthate in fruit trees of the Rosaceae family (Kanayama et al., 1992, Kanayama, 1998, Sakanishi et al., 1998). NAD-dependent sorbitol dehydrogenase (SDH) catalyzes the oxidation of sorbitol to fructose. SDH has been purified from Japanese pear fruit (Oura et al., 2000). The expression analysis of SDH cloned from apple cDNA demonstrated the importance of SDH in the metabolism of sorbitol that is translocated to fruit (Yamada et al., 1998, Yamada et al., 1999, Park et al., 2002).

SDH has also been found in plants that are not in the Rosaceae and that synthesize sucrose for translocation of photosynthate. SDH activity was detected in a crude extract from germinating soybean seeds (Kuo et al., 1990), and SDH was partially purified from developing maize endosperm (Doehlert, 1987) and from the shoot axes of Viscum album, a parasitic plant (Wanek and Richter, 1993).

Recently, plant genome mapping projects have revealed that SDH-like sequences are widespread in the plant kingdom, and are present in the expressed sequence tag (EST) databases of several plant species (Fig. 1). Nevertheless, little information exists about the physiological roles of the proteins encoded by these genes. The ultimate goal of this research was to understand the significance of the widespread SDH-like genes. As a first step, we now provide molecular evidence of SDH genes in non-Rosaceae plants. This study is a molecular and biochemical characterization of an SDH homolog in tomato, a species that utilizes sucrose to translocate photosynthate.

The results

Hint: (The order the results were presented in the paper were Figures 1,2,3,4,5, Tables 1,2,3 and then Figure 6. I have also given you a figure that the authors used in their abstract, you can chose to use it in the abstract you will write.)

Table 1. Substrate and coenzyme specificity of SDLa

Substrate Relative activity (%)
d-Sorbitol 100
l-Iditol 79
Ribitol 60
Xylitol 29
Erythritol 13
l-Arabitol 13
d-Mannitol 6
myo-Inositol n.d.c
Glycerol n.d.
Ethanol n.d.
NADPb n.d.

a

Enzyme activity was assayed in 400 mM of each substrate with 1 mM NAD+.

Table 2. Kinetic characteristics and pH optima of SDL, Japanese pear SDH, and maize ketose reductase (SDH)

Plant Substrate Km (mM) Vmax (μmol min−1 mg−1 protein) pH optimum Reference
Tomato Sorbitol 2.39 0.378 10.5 This study
Fructose 99.5 2.13 7.5






Japanese pear Sorbitol 96.4 64.8 9.0 Oura et al. (2000)
Fructose 4239 162.6 7.0






Maize Sorbitol 8.45 5.87 9.0 Doehlert (1987)
Fructose 136 21.2 6.0

Table 3. SDH activity in SDL antisense plants

Line Number of plants assayed SDH activitya (μmol min−1 mg−1 protein) % of control
Control 4 0.185(0.014)
Antisense 9 0.091 (0.013) 49.2

a

Each value represents the mean (the associated standard error).

Fig. 1. Multiple alignment of the deduced tomato SDL amino acid sequence, the apple SDH sequence, and the SDH-like sequences in EST from species of different plant families. Black shading indicates identical amino acids. Asterisks and plus signs indicate conserved amino acid residues in the catalytic zinc binding site and in the structural zinc binding site, respectively. The zinc-containing alcohol dehydrogenase signature is underlined. SDL (accession number AB183015); Malus domestica, apple SDH (AB016256); Arabidopsis thaliana (AF370161); Zea mays (BT016754); Citrus paradisi × Poncirus trifoliata (CX668813); Medicago sativa (CB894631); Gossypium raimondii (CO082515); Pinus taeda (CO361351).

Fig. 2. Genomic Southern blot analysis of tomato SDL. Genomic DNA was digested with the indicated restriction enzymes. The blot was probed with the SDL EST fragment.

Fig. 3. Northern blot analysis of tomato SDL mRNA. (A) Each lane was loaded with 10 μg of total RNA isolated from immature leaves (IL), mature leaves (ML), stems (ST), roots (RT), flowers (FL), fruit at 15, 30, and 45 days after flowering (15, 30, and 45), and ripe fruit (RF). (B) Each lane was loaded with 10 μg of total RNA isolated from individual floral organs: whole flowers (FL), sepals (SE), petals (PE), stamens (ST), and carpels (CA). The blots were probed with the SDL EST fragment. rRNA stained with ethidium bromide was used as a control for loading.

Fig. 4. Preparation of recombinant SDL monitored by SDS–polyacrylamide gel electrophoresis. Molecular weight markers (M); crude extract from E. coli before (1) and after (2) the induction of GST–SDL fusion protein expression; total fraction (3), soluble fraction (4), and insoluble fraction (5) after the lysis of E. coli, GST–SDL fusion protein solubilized from the insoluble fraction by urea (6); GST and SDL cleaved by protease (7); purified SDL after removal of GST (8). The protein equivalent of approximately 1% of each fraction was electrophoresed and stained with Coomassie Brilliant Blue.

Fig. 5. Gas chromatographic analysis of the products of the SDL reactions. Chromatograms before (A,C) and after (B,D) the reactions are shown. Sorbitol (A,B) or fructose (C,D) was used as a substrate. Xylitol was added before the trimethylsilylation of the products, as an internal standard for gas chromatography.

Fig. 6. PCR analysis for the transgene in SDL antisense plants. One example is shown. PCR bands in SDL antisense lines are shorter than those in control lines since the antisense insert, which is approximately 500 bp EST clone of SDL, is shorter than the insert of β-glucuronidase gene, which is approximately 1900 bp, in the control vector, pBI121. Each vector for the transformation was used as a positive control (P) for the PCR analysis.

The Methods.

3. Experimental

3.1. Plant materials

Lycopersicon esculentum Mill. cv. Momotaro was used for the molecular cloning of SDL, Southern and Northern blot analyses, and the production of recombinant protein. Lycopersicon esculentum Mill. cv. Alisa Craig was used for the antisense transformation.

3.2. Cloning of cDNA encoding SDL

The total RNA was extracted from mature tomato leaves by the SDS–phenol method (Kanayama et al., 1997). The SDH-like EST sequence (accession number BI203186) was amplified using an RNA PCR kit (AMV) Ver. 2.1 (Takara) and was cloned into pT7Blue vector (Novagen). Next, 5′-RACE was carried out with poly (A)+ RNA prepared using Oligotex-dT30 (Takara), and the cDNA that contained the open reading frame encoding SDL was cloned into pT7Blue vector. A high-fidelity enzyme was used for the 5′-RACE. The BigDye Terminator v3.0 Cycle Sequencing Ready Reaction kit and an ABI PRISM 310 Genetic Analyzer (Applied Biosystems) were used for sequencing.

3.3. Southern and Northern blot analyses

The genomic DNA extraction from tomato leaves and the Southern blot analysis were based on the method of Kanayama et al. (1997). The EST clone for SDL was labeled using the PCR DIG probe synthesis kit (Roche) for use as a probe. After hybridization, the membrane was washed in 0.2× SSC at 65 °C. The SDS–phenol method (Kanayama et al., 1997) was used to extract total RNA from various tomato plant organs for Northern blot analyses, which employed the method of Odanaka et al. (2002). The EST clone for SDL was labeled using the PCR DIG probe synthesis kit (Roche) for use as a probe.

3.4. Production of recombinant SDL protein

The open reading frame of SDL was cloned into the pGEX-6P-3 expression vector (Amersham Bioscience) to make pGEX-SDL. E. coli BL21 transformed with pGEX-SDL was cultured to an OD600 of 0.5 in Luria–Bertani medium supplemented with ampicillin at 37 °C, 0.2 mM isopropyl β-d-thiogalactopyranoside was added, and the culture was continued for 3 h more at 37 °C. The E. coli was collected by centrifugation and lyzed using BugBuster HT (Novagen). The GST–SDL fusion protein was found in the pellet after centrifugation; the fusion protein was extracted in 100 mM Tris–HCl (pH 8.0) containing 6 M urea, 1 mM EDTA, and 1 mM DTT for 1 h at 4 °C. After centrifugation, the supernatant was dialyzed against 100 mM Tris–HCl (pH 8.0) containing 2 M urea, 1 mM EDTA, 1 mM DTT, and 1 mM ZnSO4 for 1 h at 4 °C and then against 100 mM Tris–HCl (pH 8.0) containing 50 mM NaCl, 1 mM EDTA, 1 mM DTT, and 1 mM ZnSO4 for 16 h at 4 °C. After dialysis and centrifugation, the protein in the supernatant was bound to glutathione–Sepharose 4B (Amersham Bioscience) and the GST was removed from the recombinant SDL protein using PreScissin protease (Amersham Bioscience). The expression and purity of the recombinant protein were checked by SDS–polyacrylamide gel electrophoresis (Laemmli, 1970).

3.5. Enzyme assay using recombinant SDL protein

The products of the SDL reaction were examined by gas chromatography. The reaction mixture consisted of 50 mM Tris–HCl (pH 9.0), 10 mM sorbitol, and 20 mM NAD+ or of 50 mM Tris–HCl (pH 7.5), 10 mM fructose, and 40 mM NADH. After 4 h of incubation at 37 °C, the reaction was halted by boiling. Xylitol was added as an internal standard, and after trimethylsilylation, the reaction mixture was analyzed on a gas chromatograph G-300 (Hitachi, Tokyo, Japan).

The substrate specificity of SDL was examined in 50 mM Tris–HCl (pH 9.5) containing 1 mM NAD+ and 400 mM substrate, as described in Table 1. The coenzyme specificity of SDL was examined in 50 mM Tris–HCl (pH 9.5) containing 1 mM NADP+ and 400 mM sorbitol (1b). For the determination of the Km and Vmax values, the reaction mixture for sorbitol oxidation consisted of 50 mM glycine–NaOH (pH 10.5), 1 mM NAD+, and various concentrations of sorbitol. For fructose reduction, the reaction mixture consisted of 50 mM Tris–HCl (pH 7.5), 0.1 mM NADH, and various concentrations of fructose. These reactions were carried out at 37 °C.

The effect of pH on the SDL reaction was examined in 50 mM Tris–HCl (pH 8.0–9.5) and 50 mM glycine–NaOH (pH 9.0–11.0) for sorbitol oxidation, or in 50 mM Tris–acetate (pH 5.5–7.5) and 50 mM Tris–HCl (pH 7.0–9.0) for fructose reduction. Each reaction was carried out in a buffer that contained 400 mM substrate and 1 mM NAD+ or 0.1 mM NADH at 37 °C.

3.6. Transformation of tomato with antisense LeSDH

The β-glucuronidase gene in the binary vector pBI121 was replaced with the insert of the SDL EST clone in an antisense direction. This antisense SDL vector with the cauliflower mosaic virus 35S promoter was introduced into Agrobacterium tumefaciens LBA4404. The transformation was carried out using tomato cotyledons as described by Odanaka et al. (2002). After shoot and root formation, the independent transformants were acclimated and checked by PCR for the transgene (Fig. 6). Primers used for the PCR were 5′-CAAACCAAGGCAAGTAATAG-3′ in 35S promoter and 5′-CTATATTTTGTTTTCTATCGCG-3′ in nos terminator. When the first flower opened, the third and fourth leaves below the inflorescence were sampled for enzyme assays. The method described by Suzuki et al. (2001) was used to extract the crude enzyme and to determine SDH activity.

3.7. Protein determination

The protein determination was carried out by the method of Bradford (1976) using bovine serum albumin as a standard.

Introduction.

1. Introduction

Sorbitol is important in translocating photosynthate in fruit trees of the Rosaceae family (Kanayama et al., 1992, Kanayama, 1998, Sakanishi et al., 1998). NAD-dependent sorbitol dehydrogenase (SDH) catalyzes the oxidation of sorbitol to fructose. SDH has been purified from Japanese pear fruit (Oura et al., 2000). The expression analysis of SDH cloned from apple cDNA demonstrated the importance of SDH in the metabolism of sorbitol that is translocated to fruit (Yamada et al., 1998, Yamada et al., 1999, Park et al., 2002).

SDH has also been found in plants that are not in the Rosaceae and that synthesize sucrose for translocation of photosynthate. SDH activity was detected in a crude extract from germinating soybean seeds (Kuo et al., 1990), and SDH was partially purified from developing maize endosperm (Doehlert, 1987) and from the shoot axes of Viscum album, a parasitic plant (Wanek and Richter, 1993).

Recently, plant genome mapping projects have revealed that SDH-like sequences are widespread in the plant kingdom, and are present in the expressed sequence tag (EST) databases of several plant species (Fig. 1). Nevertheless, little information exists about the physiological roles of the proteins encoded by these genes. The ultimate goal of this research was to understand the significance of the widespread SDH-like genes. As a first step, we now provide molecular evidence of SDH genes in non-Rosaceae plants. This study is a molecular and biochemical characterization of an SDH homolog in tomato, a species that utilizes sucrose to translocate photosynthate.

References

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Y. Oura, K. Yamada, K. Shiratake, S. Yamaki Purification and characterization of a NAD+-dependent sorbitol dehydrogenase from Japanese pear fruit Phytochemistry, 54 (2000), pp. 567-572

S.W. Park, K.J. Song, M.Y. Kim, J.H. Hwang, Y.U. Shin, W.C. Kim, W. Chung Molecular cloning and characterization of four cDNAs encoding the isoforms of NAD-dependent sorbitol dehydrogenase from the Fuji apple Plant Sci., 162 (2002), pp. 513-519

U. Roessner-Tunali, H. Bjorn, A. Lytovchenko, F. Carrari, C. Bruediagam, D. Granot, A.R. Fernie Metabolic profiling of transgenic tomato plants overexpression hexokinase reveals that the influence of hexose phosphorylation diminishes during fruit development Plant Physiol., 133 (2003), pp. 84-99

K. Sakanishi, Y. Kanayama, H. Mori, K. Yamada, S. Yamaki Expression of the gene for NADP-dependent sorbtiol-6-phosphate dehydrogenase in peach leaves of various developmental stages Plant Cell Physiol., 39 (1998), pp. 1372-1374

H. Schluepmann, T. Pellny, A. van Dijken, S. Smeekens, M. Paul Trehalose 6-phosphate is indispensable for carbohydrate utilizaion and growth in Arabidopssi thaliana Proc. Natl. Acad. Sci. USA, 100 (2003), pp. 6849-6854

Y. Suzuki, S. Odanaka, Y. Kanayama Fructose content and fructose-related enzyme activity during the fruit development of apple and Japanese pear J. Jpn. Soc. Hort. Sci., 70 (2001), pp. 16-20

W. Wanek, A. Richter -iditol:NAD-oxidoreductase in Viscum album: utilization of host-derived sorbitol Plant Physiol. Biochem., 31 (1993), pp. 205-211

K. Yamada, Y. Oura, H. Mori, S. Yamaki Cloning of NAD-dependent sorbitol dehydrogenase from apple fruit and gene expression Plant Cell Physiol., 39 (1998), pp. 1375-137

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K. Yamada, N. Niwa, K. Shiratake, S. Yamakic DNA cloning of NAD-dependent sorbitol dehydrogenase from peach fruit and its expression during fruit development J. Hort. Sci. Biotech., 76 (2001), pp. 581-587

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