Genome of the pitcher plant <i>Cephalotus </i>reveals genetic changes associated with carnivory

Carnivorous plants exploit animals as a nutritional source and have inspired long-standing questions about the origin and evolution of carnivory-related traits. To investigate the molecular bases of carnivory, we sequenced the genome of the heterophyllous pitcher plant Cephalotus follicularis, in which we succeeded in regulating the developmental switch between carnivorous and non-carnivorous leaves. Transcriptome comparison of the two leaf types and gene repertoire analysis identified genetic changes associated with prey attraction, capture, digestion and nutrient absorption. Analysis of digestive fluid proteins from C. follicularis and three other carnivorous plants with independent carnivorous origins revealed repeated co-options of stress-responsive protein lineages coupled with convergent amino acid substitutions to acquire digestive physiology. These results imply constraints on the available routes to evolve plant carnivory

Development of an Agrobacterium-Mediated Stable Transformation Method for the Sensitive Plant <i>Mimosa pudica</i>

<div><p>The sensitive plant <i>Mimosa pudica</i> has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for <i>M. pudica</i> mediated by <i>Agrobacterium tumefaciens</i> (Agrobacterium). We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP). Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(<i>N</i>-morpholino)ethanesulfonic acid (MES) buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (<i>Glycine max</i>) and pea (<i>Pisum sativum</i>). The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in <i>M. pudica</i> movements.</p></div

Genomic Southern blot analysis of T₀ plants.

<p>Genomic DNAs of 13 independent T₀ plants (lines #1-13) and a non-transformed plant (C) were analyzed by <i>Eco</i>RI digestion and detection of the <i>sGFP</i> sequence. The size of several bands is shorter than the minimal length expected from the intact T-DNA sequence (3.0 kb; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088611#pone-0088611-g002" target="_blank">Figure 2A</a>), suggesting that the T-DNA sequence had undergone truncation and/or rearrangement.</p

Optimization of root induction conditions.

<p>A–D. Bright-field (A, C) and green fluorescent (B, D) images of a transformed shoot before (A, B) and after (C, D) 14 days of cultivation in irrigated vermiculite. Scale bar, 5 mm. E. Comparison of root induction conditions (n = 27). A statistical analysis was conducted using the frequencies at 14 days of cultivation. Significant differences (P<0.05 by Fisher's exact test with Holm's P-value adjustment) were observed between two groups that do not share the same lowercase letter. 1/2 MS, half-strength MS salts; SV, 2% sucrose and 1× Gamborg's vitamins.</p

Establishment of transgenic plants.

<p>A. Time course of establishment of transgenic T₀ plants from cotyledonary node explants (n = 160). Each line indicates the frequency of Agrobacterium-infected explants that reached the process indicated in (B). B. Schematic representation of the entire transformation procedure. C. Green fluorescent images of T₁ seedlings of a single T-DNA insertion line (#1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088611#pone-0088611-g006" target="_blank">Figure 6</a>). The zygosity of T₁ progeny [non-transformant (−/−), hemizygote (Tg/−) or homozygote (Tg/Tg)] produced by self-crossing of a T₀ plant could be determined based on fluorescence intensity of the plantlet. D. Green fluorescent image of homozygous T₂ seedling produced by self-crossing of a homozygous T₁ plant. Scale bar, 2 mm.</p

Shoot formation from <i>M. pudica</i> explants.

<p>A–E. Preparation of explants. A 2-day-old seedling cultured in the dark (A) was divided into the root, the cotyledons with petiole (B), and the remaining part (C). The epicotyl containing the shoot apex was then removed from the remaining part (C) to prepare the cotyledonary node explant (D) as illustrated in (E). Dashed lines in (A), (C), and (E) indicate the cutting positions. The circle in (D) indicates the position of the cotyledonary node. SA, shoot apex; Hc, hypocotyl. F, G. Shoot formation from the cotyledonary node (F) and petiolate cotyledon (G) explants after 4 and 6 weeks of cultivation in the presence of 0.5 µg/ml BAP, respectively. H. Comparison of the frequency of explants forming shoots after 4 weeks of cultivation with 0.5 µg/ml of BAP (n = 32). I, J. Effects of BAP and NAA on shoot formation from cotyledonary node (I) and petiolate cotyledon (J) explants after 4 and 6 weeks of cultivation, respectively. The distribution of the number of shoots formed per explant is shown as box-and-whisker plots (n = 32). Lower and upper whiskers indicate the range of values within 1.5 times the interquartile range from the box and circles indicate outliers. Significant differences were observed between two groups that do not share the same lowercase letter [P<0.05 by Fisher's exact test with Holm's P-value adjustment (H) or Steel-Dwass test (I, J)]. Scale bars, 1 cm (A, F, G), 1 mm (B–D).</p

Changes in medium pH during co-cultivation.

<p>A. Effects of Agrobacterium strain LBA4404 harboring pSB111-GFP (LBA), cotyledonary node explants (Exp), and MES buffer (MES) on medium pH (n = 4). The co-cultivation media were initially adjusted to pH 5.8 and sterilized by filtration to circumvent the pH decrease caused by autoclaving. In the absence of MES buffer, pH values had already declined in the time it took to prepare the Agrobacterium suspension in co-cultivation medium (∼30 minutes). B. Changes in pH of MES-buffered medium initially adjusted to pH 6.1 (n = 5). The medium was sterilized by autoclaving and then used for co-cultivation of Agrobacterium and explants. The effect of Silwet L-77 treatment prior to co-cultivation was also examined. Data are the means ± SD. The pH of the medium was measured at 0, 3, 6, 9, 12, 18, 24, 48, and 72 hours after the initiation of cultivation.</p

Data from: Genome of the pitcher plant Cephalotus reveals genetic changes associated with carnivory

Carnivorous plants exploit animals as a nutritional source and have inspired long-standing questions about the origin and evolution of carnivory-related traits. To investigate the molecular bases of carnivory, we sequenced the genome of the heterophyllous pitcher plant Cephalotus follicularis, in which we succeeded in regulating the developmental switch between carnivorous and non-carnivorous leaves. Transcriptome comparison of the two leaf types and gene repertoire analysis identified genetic changes associated with prey attraction, capture, digestion and nutrient absorption. Analysis of digestive fluid proteins from C. follicularis and three other carnivorous plants with independent carnivorous origins revealed repeated co-options of stress-responsive protein lineages coupled with convergent amino acid substitutions to acquire digestive physiology. These results imply constraints on the available routes to evolve plant carnivory

convergence_detection

convergence_detectio

transcriptome_comparison

transcriptome_compariso