MOESM3 of Virus-induced plasma membrane aquaporin PsPIP2;1 silencing inhibits plant water transport of Pisum sativum

Additional file 3: Figure S1. Virus-induced gene silencing of P. sativum phytoene desaturase (PsPDS). (A) Leaves of a control plant inoculated with PEBV carrying a fragment of Bean yellow mosaic virus (pCAPE2-Con) remained green; (B) leaves of a plant inoculated with PEBV carrying a fragment of PsPDS (pCAPE2-PDS) showed a characteristic bleaching phenotype

Salvia glabrescens Makino

原著和名: アキギリ科名: シソ科 = Labiatae採集地: 新潟県 妙高山麓 (越後 妙高山麓)採集日: 1976/9/19採集者: 萩庭丈壽整理番号: JH016536国立科学博物館整理番号: TNS-VS-96653

Flower morphology of <i>A. hainanensis</i>, and expression analyses of <i>AhFUL, AhSEP3b, AhAGL6–like</i>, and<i>AhSEP4</i>.

<p>(A) Expression analyses of four genes by quantitative RT–PCR in different organs of the 2 cm length flowers (right), and in flowers at different developmental stages (left). The relative expression of the four genes was normalized to the expression level of rRNA <i>18S</i> with biological repeats in triplicate. (B)An early developing <i>A. hainanensis</i> in florescence with flower buds. (C) Anatomical structure of an <i>A. hainanensis</i> flower, consisting of one bract, one tubular sepal, three petals, one labellum with two lateral staminodes, one stamen and carpel. (D) A mature flower in the <i>A. hainanensis</i> inflorescence. (E–M) <i>In situ</i> hybridization patterns of <i>AhFUL</i> (E–J) and <i>AhAGL6–like</i> (K–M) transcripts in longitudinal sections of <i>A. hainanensis</i> flowers. Leave (l), bract (b), sepal (se), petal (pe), common primordium of stamens and petals (st–pe), common primordium of the labellum and petals (la–pe), stamen (st), labellum (la), carpel (ca). Bar  = 1 cm in (B) and (C), 100 µm in (E–M).</p

Floral organ homeotic conversion in the transgenic <i>Arabidopsis</i> plants by ectopic expression of <i>AhFUL</i>.

<p>(A) Early flowering and fewer rosettes of the 21–day–old <i>35S</i>::<i>AhFUL</i> transgenic <i>Arabidopsis</i> plant (white arrow bars) compared with the wild–type plant, when grown under long day photoperiod condition (16 h light/8 h dark). (B) The wild–type plant with round rosette leaves. (C) The <i>35S::AhFUL</i> transgenic <i>Arabidopsis</i> plants with four small curled rosettes. (D) The <i>35S::AhFUL</i> transgenic <i>Arabidopsis</i> plants were short and weak (right). (E) The wild–type flower. (F–O) Floral organ phenotypic analysis of the transgenic <i>Arabidopsis</i> ectopically expressing <i>AhFUL</i>, displaying mild (F, G, H), intermediate (I, J, K), and severe (L, M, N, O) abnormal phenotypes. White arrow bars indicate an extra flower raised from the axils of another flower. (P) Style of the wild–type plant. (Q) Style of the <i>35S::AhFUL</i> transgenic plant. (R) Siliques of the wild–type.(S) Siliques of the <i>35S::AhFUL</i> transgenic <i>Arabidopsis</i>. (T) Detection of <i>AhFUL</i> expression in transgenic <i>Arabidopsis</i> plants. Total RNA isolated from one wild–type <i>Arabidopsis</i> plant and two different 21–day–old <i>35S::AhFUL</i> transgenic plants was used as templates.</p

Evolutionary reconstruction of protein–protein interaction (PPI) pattern between <i>AP1</i>/<i>AGL9</i> lineage proteins.

<p>Ancestral character–state reconstructions of PPIs between proteins of the AP1, AGL2, AGL6, and AGL9 lineage members. Filled and open circles in (A–G) indicate presence and absence of interactions, respectively, with the probability of the interaction in ancestral taxa indicated at each interior node. Among the 10protein interaction combinations (H), five had conserved (in black) and three had variable (in grey) PPIs. In the remaining 2 cases, no interaction was observed or accumulated data so far is unavailable. Information on the proteins is listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114134#pone.0114134.s008" target="_blank">Table S3</a>. (I) Evolutionary historical model of the PPI network formed by AP1/AGL9 proteins, filled and open circles stand for the AP1 (blue), AGL2 (purple), AGL9 (red), and AGL6 (green) lineage members that can and cannot form homodimers.</p

Endogenous flowering–related gene expression changes in the three <i>35S::AhFUL</i> transgenic <i>Arabidopsis</i> lines, compared with the wild–type (WT).

<p>Data represent the mean±SE from three replicate. <i>AP1</i> (NM_105581), <i>AGL24</i> (AF005158), <i>SEP3</i>, (NM_180622), <i>LFY</i> (NM_125579), <i>FT</i> (AB027505), and <i>SOC1</i> (NM_130128).</p

Subcellular localization of four ginger MADS–box proteins.

<p>AhFUL–GFP, AhAGL6–like–GFP, AhSEP4–GFP, and AhSEP3b–GFP fusion proteins under control of the CaMV<i>35S</i> promoter were transiently expressed in <i>Arabidopsis</i> mesophyll protoplasts. Images were taken in the dark field for green fluorescence, while the outline of the cell and the combination were photographed in a bright field. mCherry–VirD2NLS was induced in each transfection to serve as a control for successful transfection as well as for nuclear localization. The length of the bar is indicated in the photographs.</p

Phylogenetic tree of AP1/AGL9–like MADS–box proteins.

<p>AP1/AGL9 lineage of the MADS–box genes could be further divided into four clades (AP1/AGL6/LOFSEP/SEP3). On the basis of amino acid sequence of the full–length protein, some differences with canonical <i>AGL6</i> genes may explain the position of AhAGL6–like close to <i>AGL6</i> lineage but in the external branch of this clade. MEGA 5 software was used with the neighbor–joining method using the parameters of p–distance, complete deletion, and bootstrap (1000 replicates). The four genes from <i>A.hainanensis</i> are labeled in yellow. The term “out” indicates the outgroup (D class genes in <i>Arabidopsis</i> and <i>Oryza sativa</i>) used for the analysis. Information of these genes is listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114134#pone.0114134.s007" target="_blank">Table S2</a>.</p

Physical protein–protein interaction of AhFUL, AhSEP4, AhSEP3b, and AhAGL6–like proteins by bimolecular fluorescence complementation (BiFC).

<p>Results of BiFC analyses in <i>Arabidopsis</i> mesophyll protoplast transient expression system showed that AhFUL–AhSEP4, AhFUL–AhAGL6–like, AhFUL–AhSEP3b, AhSEP4–AhAGL6–like, AhSEP4–AhSEP3b, AhAGL6–like–AhSEP3b, and AhSEP3b–AhSEP3b could form dimers. The full–length coding sequences of AhFUL, AhSEP4, AhAGL6–like, and AhSEP3b were fused with the N–terminal fragment of YFP in the pSAT1–nEYFP–C1 (YN–P) and the pSAT1 (A)–nEYFP–N1 (P–YN) vectors, while the four coding sequences were also cloned into pSAT1–cEYFP–C1(B) (YC–P) and pSAT1(A)–cEYFP–N1 (P–YC) as a fusion with the C–terminal fragment of YFP. Amino– and carboxyl– terminal fusions are represented by YC–P and YN–P, P–YC and P–YN respectively. All empty vectors were used as negative controls. mCherry–VirD2NLS was induced in each transfection to serve as a control for successful transfection as well as for nuclear localization. Symbols of cross–lined circles: negative interactions; N: no test, because fluorescence can be detected in the protein with empty vector.</p