Identification of trans-resveratrol in the seedlings and seeds of transgenic and wild-type rice by HPLC analysis.

<p>The chromatograms of standard <i>trans</i>-resveratrol (<b>A</b>), the extracts from L3 seedlings (<b>B</b>), wild-type rice seedlings (<b>C</b>), L3 seeds (<b>D</b>), wild-type rice seeds (<b>E</b>) and L3 seedlings treated by UV-C (<b>F</b>, introduced in the later section), were detected at 306 nm under the same conditions. The marker “↙”indicates the peak of <i>trans</i>-resveratrol.</p

Evaluating the Effect of Expressing a Peanut Resveratrol Synthase Gene in Rice

<div><p>Resveratrol (Res) is a type of natural plant stilbenes and phytoalexins that only exists in a few plant species. Studies have shown that the Res could be biosynthesized and accumulated within plants, once the complete metabolic pathway and related enzymes, such as the key enzyme resveratrol synthase (RS), existed. In this study, a RS gene named <i>PNRS1</i> was cloned from the peanut, and the activity was confirmed in <i>E</i>. <i>coli</i>. Using transgenic approach, the <i>PNRS1</i> transgenic rice was obtained. In T₃ generation, the Res production and accumulation were further detected by HPLC. Our data revealed that compared to the wild type rice which <i>trans</i>-resveratrol was undetectable, in transgenic rice, the <i>trans</i>-resveratrol could be synthesized and achieved up to 0.697 μg/g FW in seedlings and 3.053 μg/g DW in seeds. Furthermore, the concentration of <i>trans</i>-resveratrol in transgenic rice seedlings could be induced up to eight or four-fold higher by ultraviolet (UV-C) or dark, respectively. Simultaneously, the endogenous increased of Res also showed the advantages in protecting the host plant from UV-C caused damage or dark-induced senescence. Our data indicated that Res was involved in host-defense responses against environmental stresses in transgenic rice. Here the results describes the processes of a peanut resveratrol synthase gene transformed into rice, and the detection of <i>trans</i>-resveratrol in transgenic rice, and the role of <i>trans</i>-resveratrol as a phytoalexin in transgenic rice when treated by UV-C and dark. These findings present new outcomes of transgenic approaches for functional genes and their corresponding physiological functions, and shed some light on broadening available resources of Res, nutritional improvement of crops, and new variety cultivation by genetic engineering.</p></div

Detection of trans-resveratrol contents in transgenic rice lines grown under UV-C or dark treatments.

<p>The Data of Res content of each line detected under normal grpwth condition, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136013#pone.0136013.t001" target="_blank">Table 1</a>, was used as relevant control. Bars represent the mean values ± SD from three independent experiments (30 seedlings each). Statistical analysis was performed by using one-way ANOVA. <b>*</b> Significant variances existed under different conditions in each transgenic line (p<0.01). WT wild-type rice “Shengdao 13”; EV empty-vector transformed rice; L1–L3 <i>PNRS1</i> transgenic rice lines.</p

Different phenotypes of wild-type and RS transgenic rice under UV-C treatment.

<p><b>A</b>, <b>B</b> two-week-old wild-type rice and L3 seedlings were treated by short UV-C radiation, separately. “→” indicates the brown patches appeared on the rice leaves. <b>C</b> six-week-old rice leaves were treated by UV-C radiation. <b>D</b> DAB coloration using fragments of six-week-old rice leaves under different UV-C treatment conditions. 12 h and 24 h were the time after UV-C treatment. <b>E</b> Chlorophyll determination of six-week-old rice leaves after treated by UV-C radiation. Each value represents the mean ± SD from three independent experiments (0.2 g leaves from 10 plants each). WT wild-type rice “Shengdao 13”; EV empty-vector transformed rice; L1–L3 <i>PNRS1</i> transgenic rice lines.</p

Different phenotypes of wild-type and RS transgenic rice under dark treatment.

<p><b>A</b>, <b>B</b> two-week-old wild-type rice and L3 seedlings were treated solely in the dark for 6 days. <b>C</b> six-week-old rice leaves incubated solely in the dark for 6 days. <b>D</b> The 6 days dark treated six-week-old rice leaves were stained with DAB. <b>E</b> Changes of chlorophyll in six-week-old rice leaves after being treated solely in the dark. Each value represents the mean ± SD from three independent experiments (0.2 g leaves from 10 plants each). WT wild-type rice “Shengdao 13”; EV empty-vector transformed rice; L1–L3 <i>PNRS1</i> transgenic rice lines.</p

Determination of RS enzyme activity.

<p><b>A</b> Co-expression of <i>PNRS1</i> and <i>At4CL2</i> in <i>E</i>. <i>Coli</i>. Two vectors, pET-28a and pMAL-c2x, were used for prokaryotic expression, and the cell lysates of BL21(DE3) transformed with empty or gene recombinanted vectors were analyzed by SDS-PAGE. Molecular weight standard of protein was shown on the left. The proteins of RS coded by <i>PNRS1</i> (46 kD), MBP (50 kD) and MBP fused 4CL code by <i>At4CL2</i> (103 kD) were indicated on the right. <b>B</b> HPLC identification of Res production in <i>PNRS1</i> and <i>At4CL2</i> co-expressed <i>E</i>. <i>coli</i>. Both the peaks of <i>p</i>-coumaric acid and <i>trans</i>-resveratrol were obtained in the chromatogram of extracts from the co-cultured medium, which were detected at 306 nm under the same chromatographic conditions.</p

Contents of trans-resveratrol in different parts of transgenic rice detected by HPLC.

<p>Each value represents the mean ± standard deviation (SD) from three independent experiments.</p><p>^<b>a</b> Significant variance existed among the groups of each kind when analyzed by one-way ANOVA (p<0.01).</p><p>^<b>b</b> The data showed the highest value from total averages in each kind of sample when analyzed by Student’s t-test (p<0.01). WT: wild-type rice; EV: empty vector transformed rice; L1–L8: different transgenic lines of rice.</p><p>Contents of trans-resveratrol in different parts of transgenic rice detected by HPLC.</p

Alignment of Hd17 amino acid sequences from HJX74 and W12-S4.

<p>The green triangles indicate the prematurely terminated coding site in W12-S4, whereas red triangles indicate amino acid substitutions between HJX74 and W12-S4.</p

Molecular identification of <i>PNRS1</i> transgenic rice.

<p>The weight of DNA marker was shown on the left. <b>A</b> PCR identification of the integration of <i>PNRS1</i> in the rice genome. The genomic DNA from <i>PNRS1</i> transgenic rice lines of L1–L8 (L1–L8), from empty-vector transformed rice (EV), from wild-type rice “Shengdao 13” (WT), and the plasmid pCA1300-Ubi-PNRS1 (CK), were used as the templates for PCR amplification. <b>B</b> Semi-quantitative RT-PCR of <i>PNRS1</i> in transgenic rice lines L1–L8, using the internal control of <i>actin</i>. <b>C</b> The relative expression levels of <i>PNRS1</i> in transgenic lines L1–L8. Bars represent the mean value ± SD from three independent experiments (10 seedlings each).<b>*</b> the value significantly higher than the average value analyzed by student t-test (p<0.01).</p

Domain Dissection of AvrRxo1 for Suppressor, Avirulence and Cytotoxicity Functions

<div><p>AvrRxo1, a type III effector from <i>Xanthomonas oryzae</i> pv. <i>oryzicola</i> (Xoc) which causes bacterial leaf streak (BLS) in rice, can be recognised by non-host resistance protein Rxo1. It triggers a hypersensitive response (HR) in maize. Little is known regarding the virulence function of AvrRxo1. In this study, we determined that AvrRxo1 is able to suppress the HR caused by the non-host resistance recognition of <i>Xanthomonas oryzae</i> pv. <i>oryzae</i> (Xoo) by <i>Nicotiana benthamiana</i>. It is toxic, inducing cell death from transient expression in <i>N. benthamiana</i>, as well as in yeast. Among the four AvrRxo1 alleles from different Xoc strains, we concluded that the toxicity is abolished by a single amino acid substitution at residue 344 in two AvrRxo1 alleles. A series of truncations from the carboxyl terminus (C-terminus) indicate that the complete C-terminus of AvrRxo1 plays an essential role as a suppressor or cytotoxic protein. The C-terminus was also required for the avirulence function, but the last two residues were not necessary. The first 52 amino acids of N-terminus are unessential for toxicity. Point mutagenesis experiments indicate that the ATP/GTP binding site motif A is required for all three functions of AvrRxo1, and NLS is required for both the avirulence and the suppression of non-host resistance. The putative thiol protease site is only required for the cytotoxicity function. These results determine that AvrRxo1 plays a role in the complex interaction with host proteins after delivery into plant cells.</p></div