Overexpression of poplar xylem sucrose synthase in tobacco leads to a thickened cell wall and increased height.

Sucrose synthase (SuSy) is considered the first key enzyme for secondary growth because it is a highly regulated cytosolic enzyme that catalyzes the reversible conversion of sucrose and UDP into UDP-glucose and fructose. Although SuSy enzymes preferentially functions in the direction of sucrose cleavage at most cellular condition, they also catalyze the synthetic reaction. We isolated a gene that encodes a SuSy from Populus simonii×Populus nigra and named it PsnSuSy2 because it shares high similarity to SuSy2 in Populus trichocarpa. RT-PCR revealed that PsnSuSy2 was highly expressed in xylem, but lowly expressed in young leaves. To characterize its functions in secondary growth, multiple tobacco overexpression transgenic lines of PnsSuSy2 were generated via Agrobacterium-mediated transformation. The PsnSuSy2 expression levels and altered wood properties in stem segments from the different transgenic lines were carefully characterized. The results demonstrated that the levels of PsnSuSy2 enzyme activity, chlorophyll content, total soluble sugars, fructose and glucose increased significantly, while the sucrose level decreased significantly. Consequently, the cellulose content and fiber length increased, whereas the lignin content decreased, suggesting that PsnSuSy2 plays a significant role in cleaving sucrose into UDP-glucose and fructose to facilitate cellulose biosynthesis and that promotion of cellulose biosynthesis suppresses lignin biosynthesis. Additionally, the noticeable increase in the lodging resistance in transgenic tobacco stem suggested that the cell wall characteristics were altered by PsnSuSy2 overexpression. Scanning electron microscopy was performed to study the cell wall morphology of stem, and surprisingly, we found that the secondary cell wall was significantly thicker in transgenic tobacco. However, the thickened secondary cell wall did not negatively affect the height of the plants because the PsnSuSy2- overexpressing lines grew taller than the wildtype plants. This systematic analysis demonstrated that PsnSuSy2 plays an important role in cleaving sucrose coupled with cellulose biosynthesis in wood tissue

Validation of <i>PsnSuSy2</i> integration into tobacco genome and expression in transgenic tobacco lines.

<p>a. Validation of <i>PsnSuSy2</i> tobacco transgenic lines using PCR amplification. Genomic DNA was extracted from the leaves of one-month-old transgenic tobacco. The PCR products were electrophoresed on a 1.2% agarose gel with DL15000 DNA marker (first lane). b. Relative expression of <i>PsnSuSy2</i> in two month-old stem segments of plastichron index (PI) 3–5 determined by real-time RT-PCR. C. SuSy enzyme activity of in two month-old stem segments of PI3–5. Expressed levels in both <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120669#pone.0120669.g003" target="_blank">Fig. 3b and 3c</a> were averaged from five different samples per line ± S.E. The <i>t</i>-test was used to examine the significance of difference between <i>PsnSusy2</i> transgenic and wildtype lines, and *denotes significance at <i>p</i> < 0.05. WT represents wildtype tobacco while all others labeled with line numbers are of different <i>PsnSusy2</i> transgenic lines.</p

Phenotypic changes in <i>PsnSusy2</i> transgenic lines.

<p>a. Phenotypic comparison of three-month-old tobacco transgenic line of L38 and wildtype tobacco. CK represents wildtype tobacco while T represents L38 transgenic line. b. Comparison of heights of three-month-old tobacco transgenic lines and wildtype. c. Stem diameters of three-month-old tobacco transgenic lines at 3 cm height above the root collar. d. Puncture strength of stem segments (plastichron index 5) in three-month-old transgenic lines. All measurements shown in a, b, and c were averaged from five different samples per line ± S.E. The <i>t</i>-test was used to examine the significance of difference between <i>PsnSusy2</i> transgenic and wildtype lines, and *denotes significance at <i>p</i> < 0.05. The WT represents wildtype tobacco while the others lines labeled with line numbers are of different <i>PsnSusy2</i> tobacco transgenic lines.</p

Changes in lignocellulosic components in <i>PsnSusy2</i> tobacco transgenic lines.

<p>a. Cellulose contents in the plastichron index (PI) 5~8 stem tissues of three-month-old <i>PsnSusy2</i> transgenic tobacco lines. b. Hemicellulose contents in the PI5~8 stem tissues of three-month-old <i>PsnSusy2</i> transgenic tobacco lines. c. Lignin contents in the stem the PI5~8 stem tissues of three-month-old <i>PsnSusy2</i> transgenic tobacco lines. d. Fiber length in the PI5~8 stem tissue of three-month-old <i>PsnSusy2</i> transgenic tobacco lines. All measurements are the means of five different samples ± S.E. The <i>t</i>-test was used to examine the significance of difference between <i>PsnSusy2</i> transgenic and wildtype lines, and *denotes significance at <i>p</i> < 0.05. The WT represents wildtype tobacco plants, whereas all the others are different <i>PsnSusy2</i> transgenic lines.</p

Tissue-specific expression of the <i>PsnSuSy2</i> in one-year-old hybrid <i>Populus simonii</i> × <i>Populus nigra</i> propagated from two years old hybrid trees.

<p>a. Tissue-specific expression pattern characterized by RT-PCR. b. Tissue-specific expression pattern characterized by real-time quantitative PCR. The expression levels for real-time quantitative PCR were averaged from three replicates.</p

Changes in chlorophyll contents and biomass of <i>PsnSusy2</i> tobacco transgenics.

<p>a. Chlorophyll contents of the third, fourth and fifth functional leaves of different <i>PsnSuSy2</i> tobacco transgenic lines of two-month-old plants. b. Fresh weights of three-month-old <i>PsnSuSy2</i> tobacco transgenic lines. c. Dry weights of three-month-old <i>PsnSuSy2</i> tobacco transgenic lines. d. Ratios of dry vs. fresh weight for of three-month-old <i>PsnSuSy2</i> tobacco transgenic lines. All measurements shown in 5a, 5b, and 5c were averaged from five different samples per line ± S.E. The <i>t</i>-test was used to examine the significance of difference between <i>PsnSusy2</i> transgenic and wildtype lines, and *denotes significance at <i>p</i> < 0.05. The WT represents wildtype tobacco while the others lines labeled with line numbers are of different <i>PsnSusy2</i> tobacco transgenic lines.</p

Scanning electron micrographs of the PI5–8 stem transverse sections of three-month-old tobacco transgenic lines.

<p>a. and c were from wildtype tobacco plants. b. and d are the L15 <i>PsnSusy2</i> tobacco transgenic line. Short red lines in Figure c and d depict the difference between the cell wall thickness in the tobacco transgenic lines and the wildtype plants. The magnification factor is 100X for Fig. 7a and 7b, and 3000X for 7c and 7d.</p

Changes in carbohydrates (sugars) in <i>PsnSusy2</i> tobacco transgenics.

<p>a. Total soluble sugar contents of stem tissues in different <i>PsnSuSy2</i> tobacco transgenic lines. b. Sucrose contents of stem tissues in different <i>PsnSuSy2</i> tobacco transgenic lines. c. Glucose contents of stem tissues in different <i>PsnSuSy2</i> tobacco transgenic lines. d. Fructose contents of stem tissue in different <i>PsnSuSy2</i> tobacco transgenic lines. All samples in 6a, 6b and 6c were harvested from tissues between PI3 and PI5. All measurements shown in 6a-d were averaged from five different samples per line ± S.E. The <i>t</i>-test was used to examine the significance of difference between <i>PsnSusy2</i> transgenic and wildtype lines, and *denotes significance at <i>p</i> < 0.05. The WT represents wildtype tobacco while the others lines labeled with line numbers are of different <i>PsnSusy2</i> tobacco transgenic lines. Note that the total soluble sugar contents were measured using two-month-old plants, whereas sucrose, glucose, and fructose were measured one week later.</p

Phylogenetic tree of selected full SuSy coding sequences derived using the neighbor-joining method.

<p>The sequences were obtained by translating the following 116 full-length cDNA sequences of SuSy isozymes downloaded from Genbank (<a href="http://www.ncbi.nlm.nih.gov/genbank" target="_blank">http://www.ncbi.nlm.nih.gov/genbank</a>): <i>Arabidopsis thaliana</i> (AtSuSy1–6) isozyme1–6 (At5g20830, At5g 49190, At4g02280, At3g43190, At5g37180, At1g73370), <i>Actinidia chinensis</i> (AcSuSy) isozyme (AFO84090.1,), <i>Actinidia deliciosa</i> (AdSuSy) isozyme (AFO84091.1), <i>Amorphophallus konjac</i> (AkSuSy1) isozyme (AEH27530.1), <i>Arachis hypogaea</i> (AhSuSy2) isozyme (AFS17278.1), <i>Arachis hypogaea</i> var.vulgaris (AhSuSy) isozyme (AEF56625.1), <i>Bambusa oldhamii</i> (BoSuSy1–4) isozyme1–4(AAV64256.2, AAL50571.1, AAL50570.1, AAL50572.2), <i>Betula luminifera</i> (BlSuSy1–3) isozyme 1–3(AAV64256.2,AGV22112.1, AGV22113.1), <i>Beta vulgaris</i> (BvSuSy1) isozyme1 (ABR87939.1), <i>Camellia sinensi</i>s (CasSuSy1–2) isozyme1–2 (AHL29281.1, AHL29282.1), <i>Chenopodium rubrum</i> (<i>Cr</i>SuSy) isozyme (CAA57881.1), <i>Cicer arietinum</i> (<i>Cia</i>SuSy) isozyme (AEE60913.1,), <i>Citrus unshiu</i> (CuSuSy) isozyme(BAA88904.1), <i>Citrullus lanatus</i> (ClSuSy) isozyme (BAA89232.1), <i>Coccomyxa sub ellipsoidea</i> C-169(CeSuSy) isozyme (XP_005643464.1), <i>Coffea arabica</i> (CaSuSy) isozyme (CAJ32597.1), <i>Coffea canephora</i> (CcSuSy) isozyme (ABI17891.1), <i>Craterostigma plantagineum</i> (CpSuSy1–2) isozyme1–2 (CAB38021.1, CAB38022.1), <i>Cucumis sativus</i> (CsSuSy1,3–5) isozyme1,3–5 (AGA95977.1 AEN83999.1 AGA95976.1,P_001267613.1), <i>Daucus carota</i> (DcSuSy1,2) isozyme1,2 (CAA53081.1, CAA76057.1), <i>Dianthus caryophyllus</i> (DicSuSy) isozyme (BAJ10424.1), <i>Dendrobium officinale</i> (DoSuSy) isozyme (ADY02961.1), <i>Eucalyptus grandis</i> (EgSuSy1,3) isozyme1,3(ABB53601.1, ABB53602.1), <i>Gossypium arboreum</i> (GaSuSy1–6) isozyme1–6(AEV40460.1, AEV40461.1, AEV40462.1, AEV40463.1, AEV40464.1, AEV40465.1), <i>Gossypium hirsutum</i> (GhSuSy) isozyme (AIE38018.1), <i>Gunnera manicata</i> (GmSuSy)SuSy isozyme (ADP88918.1), <i>Hevea brasiliensis</i> (HbSuSy1–6) isozyme1–6(AGM14946.1, AGM14947.1, AGM14948, AGM14949.1, AGM14950.1, AGM14951. 1), <i>Ipomoea batatas</i> (IbSuSy) isozyme (ACL00957.1), <i>Jatropha curcas</i> (JcSuSy) isozyme (AGH29112.1), <i>Lilium davidii</i> (LidSuSy) isozyme (AGW23638.1), <i>Lilium davidii</i> var. unicolor(LdSuSy1,2) isozyme1,2(AHM02468.1, AHN50409.1), <i>Litchi chinensis</i> (LcSuSy) isozyme (AFP23359.1), <i>Lolium perenne</i> (LpSuSy) isozyme (BAE79815.1), <i>Manihot esculenta</i> (MeSuSy) isozyme (ABD96570.1), <i>Manihot esculenta</i> (MeSuSy1,4) isozyme1,4 (AIJ28962.1, AIJ28961.1), <i>Manihot esculenta subsp</i>. <i>Flabellifolia</i> (MefSuSy1,4) isozyme1,4 (AIJ28960.1,AIJ28959.1), <i>Malus domestica</i> (MdSuSy) isozyme (AFU56881.1), <i>Medicago sativa</i> (MsSuSy) isozyme (AAC17867.1), <i>Medicago truncatula</i> (MtSuSy) isozyme (XP_003616166.1), <i>Musa acuminata</i> (MaSuSy) isozyme (AEO09338.2), <i>Nicotiana tabacum</i> (NtSuSy) isozyme (AHL84158.1), <i>Oncidium hybrid</i> (OhSuSy, OhSuSy1) isozyme,1(AAM95943.1, AEA76429.1), <i>Orobanche ramose</i> (OrSuSy1) isozyme1 (AEN79500.1), <i>Oryza sativa</i>(OsSuSy1,3–7) isozyme1,3–7(|AEX32874.1, AEX32876.1, AEX32876.1, AEX32877.1, AEX32878.1, AEX32880.1), <i>Oryza sativa</i> (OsSuSy2–1, OsSuSy2–2) isozyme2–1,2–2 (AEX32875.1, AFI71274.1), <i>Populus simonii</i>×<i>Populus nigra</i> (PsnSuSy2) isozyme2, <i>Populus trichocarpa</i>(PtSuSy1–7) isozyme1–7(ADV71183.1, ADV71184.1, ADV71185.1, ADV71186.1, ADV71187.1, ADV71188.1, ADV71189.1), <i>Populus deltoids</i> (PdSuSy) isozyme (AHA41509.1), <i>Populus tremuloides</i> (PtSuSy) isozyme (AAR03498.1), <i>Potamogeton distinctus</i> (PodSuSy1–2) isozyme1–2(BAE06058.1, BAE06059.1), <i>Phaseolus vulgaris</i> (PvSuSy) isozyme (AAN76498.1), <i>Pinus taeda</i> (PitSuSy) isozyme (ABR15470.1), <i>Prunus serrulata</i> (PsSuSy) isozyme (AIL23782.1), <i>Prunus persica</i> SuSy (PpSuSy2,4–6) isozyme 2,4–6(AHZ90138.1, AHZ90140.1, AHZ90141.1, AHZ90142.1), <i>Prunus persica</i> (PpSuSy) isozyme(AHZ90138.1), <i>Solanum tuberosum</i> (SotSuSy) isozyme (AAA33841.1), <i>Solanum tuberosum</i> (StSuSy, StSuSy2) isozyme,2 (AAO67719.1, AAO34668.1), <i>Solanum lycopersicum</i> (SlSuSy) isozyme (AAA34196.1), <i>Saccharum officinarum</i> (SoSuSy) isozyme (AGI56230.1), <i>Theobroma cacao</i> (TcSuSy-1, TcSuSy-2) isozyme-1,-2 (XP_007032183.1, XP_007032184.1), <i>Theobroma cacao</i> (TcSuSy2–2) isozyme 2–2 (XP_007035652.1), <i>Theobroma cacao</i>(TcSuSy3–2) isozyme 3–2 (XP_007050985.1), <i>Theobroma cacao</i> (TcSuSy5–4) isozyme 5–4 (XP_007032186.1), <i>Theobroma cacao</i> (TcSuSy6–1) isozyme 6–1 (XP_007037101.1), <i>Triticum polonicum</i> (TpSuSy2–2) isozyme2–2 (AIL88517.1), <i>Triticum polonicum</i> (TpSuSy3) isozyme (AIL88515.1), <i>Vigna radiata</i> (VrSuSy) isozyme (BAA01108.1), <i>Vigna angularis</i> (VaSuSy) isozyme (BAH56282.1). The tree was generated using MEGA, version 4, with bootstrap of 1,000.</p

Overexpression of Poplar Xylem Sucrose Synthase in Tobacco Leads to a Thickened Cell Wall and Increased Height

Sucrose synthase (SuSy) is considered the first key enzyme for secondary growth because it is a highly regulated cytosolic enzyme that catalyzes the reversible conversion of sucrose and UDP into UDP-glucose and fructose. Although SuSy enzymes preferentially functions in the direction of sucrose cleavage at most cellular condition, they also catalyze the synthetic reaction. We isolated a gene that encodes a SuSy from Populus simonii×Populus nigra and named it PsnSuSy2 because it shares high similarity to SuSy2 in Populus trichocarpa. RT-PCR revealed that PsnSuSy2 was highly expressed in xylem, but lowly expressed in young leaves. To characterize its functions in secondary growth, multiple tobacco overexpression transgenic lines of PnsSuSy2 were generated via Agrobacterium-mediated transformation. The PsnSuSy2 expression levels and altered wood properties in stem segments from the different transgenic lines were carefully characterized. The results demonstrated that the levels of PsnSuSy2 enzyme activity, chlorophyll content, total soluble sugars, fructose and glucose increased significantly, while the sucrose level decreased significantly. Consequently, the cellulose content and fiber length increased, whereas the lignin content decreased, suggesting that PsnSuSy2 plays a significant role in cleaving sucrose into UDP-glucose and fructose to facilitate cellulose biosynthesis and that promotion of cellulose biosynthesis suppresses lignin biosynthesis. Additionally, the noticeable increase in the lodging resistance in transgenic tobacco stem suggested that the cell wall characteristics were altered by PsnSuSy2 overexpression. Scanning electron microscopy was performed to study the cell wall morphology of stem, and surprisingly, we found that the secondary cell wall was significantly thicker in transgenic tobacco. However, the thickened secondary cell wall did not negatively affect the height of the plants because the PsnSuSy2- overexpressing lines grew taller than the wildtype plants. This systematic analysis demonstrated that PsnSuSy2 plays an important role in cleaving sucrose coupled with cellulose biosynthesis in wood tissue