Simplified hormone biosynthetic pathways.

<p>The hormone biosynthetic pathways of Arabidopsis for gibberellins <b><i>A.</i></b>, ABA <b><i>B.</i></b>, and, brassinolide <b><i>C.</i></b>. The biosynthesis mutants used in this study and sites of their lesions are shown. Also, the biosynthetic genes over-expressed to increase the levels of respective hormones are indicated. <b><i>A.</i></b> The <i>ga1</i> mutant is impaired in the first stage of GA-biosynthesis: the cyclization of geranylgeranyl diphosphate (GGPP) to copalyl diphosphate (CPP). <b><i>B.</i></b> The <i>aba2</i> mutant is blocked at the cis-xanthoxin to ABA-aldehyde conversion. <b><i>C.</i></b> The conversion of 6-Deoxocathasterone/Cathasterone to 6- Deoxoteasterone/teasterone does not occur in the <i>cpd</i> mutant. <b><i>A.</i></b> The <i>GA5</i> gene encodes a GA 20-oxidase that catalyzes the formation of the GA20 and GA9, the final precursors of the bioactive GAs. <b><i>B.</i></b> The <i>NCED3</i> encodes 9-<i>cis</i>-epoxycarotenoid dioxygenase that catalyzes the oxidative cleavage of a 9-<i>cis</i> isomer of epoxycarotenoid (9-<i>cis</i>-violaxanthin or 9’-<i>cis</i>-neoxanthin) to form xanthoxin. <b><i>C.</i></b> The <i>DWF4</i> gene encodes a 22-a hydroxylase (CYP90B1) that catalyzes the conversion of 6- Oxocampestanol/Campestanol to 6-Deoxocathasterone/Cathasterone. IPP, Isopentenyl pyrophosphate. ABA, abscisic acid. Adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014012#pone.0014012-Srivastava1" target="_blank">[49]</a>.</p

Overexpression lines for rate-limiting enzymes in various phytohormone pathways.

<p>Transgenic lines harboring <i>35S::DWF4</i>, <i>35S::GA5</i> and <i>35S::NCED3</i> constructs. <b><i>A.</i></b> Over-expression was confirmed by RT-PCR with primers specific for <i>DWF4</i>, <i>GA5</i> and <i>NCED3</i>. Primers specific for the elongation factor 1-alpha gene were used as a control. Representative lines are shown. All lines tested showed over-expression of the gene of interest >3 fold. <b><i>B.</i></b> Images of 3-weeks-old plants grown under long days (16 h light/8 h darkness) in the greenhouse. The white bar indicates 1 cm.</p

Student's t-test for flowering-time differences between mutant genotypes.

<p>Listed are pairs of compared genotypes. P values for each pair are provided.</p><p>ø No significant difference P>0.05;</p><p>statistically significant differences:</p><p>***P<0.0001,</p><p>**P<0.001,</p><p>*P<0.05.</p

Root and shoot production in non‐transformed wildtype (60444) and the four independent transformants at harvest.

<p>(a) Storage-root dry weight; (b) total plant dry weight; (c) harvest index (HI), calculated as HI = (storage-root dry mass)/ [(storage-root dry mass) + (above-ground dry mass)]; (d) number of storage-roots. Shown are the means ± SEM.</p

Transformed and non-transformed plants at various stages of floral development.

<p>(a): FT-17 transgenic plant at 2 months <i>in vitro</i>. (b and c): FT-17 transgenic plantlet at one month after transfer from <i>in vitro</i> to culture box and soil respectively. (d): Advanced stage transgenic plants flowering at 3 months. (e): Non-transformed (left) vs. transformed (right) plants at 5 months old. (f and g): Close up view of the apical region of 5-month old non-transformed (f) and transformed (g) plants, respectively. Arrows indicate flowers.</p

Schematic representation of the transformation vector.

<p><i>Arabidopsis FT</i> cDNA was inserted into the construct through Gateway cloning. pAnos, nopaline synthase polyadenylation signal; pat, phosphinothricin acetyltransferase; Tnos, terminator of nopaline synthase; pAlcA, promoter of alcohol dehydrogenase I (Adh-I) encoded by the <i>alc</i>A gene; <i>FT</i> cDNA, cDNA of Flowering Locus (FT) gene; pA35S, polyadenylation sequence of Cauliflower mosaic virus 35S gene; nos, nopaline synthase terminator; ALCR, transcriptional factor which binds to <i>AlcA promoter</i>; p35S, Cauliflower Mosaic Virus 35S promoter; LB, left border; RB, right border.</p

Expression of Arabidopsis <i>FT</i> gene in cassava.

<p>The qRT-PCR results were obtained from four biological replicates and two technical replicates for each sample. 60444 represents the non‐transformed wildtype line and FT-02, FT-11, FT-13 and FT-17 represent the four independent transformants. The levels of detected amplification were normalized using 18S and Ubiquitin as reference genes. The expression cassette had an ethanol‐inducible promoter. In each case, potted cassava transgenic plants were either watered normally (H₂O), or the soil was drenched with 1% (v/v) ethanol for two weeks before leaves were harvested and analyzed.</p

Flowering traits in non‐transformed wildtype line (60444) and in the four independent transformants.

<p>(a) Flowering time in days from establishment in soil to flowering at the 1st, 2nd, and 3rd tier of flowering, as defined by fork-type branching at the apical meristems. (b) Number of shoot nodes to forking events where inflorescences develop. The number of nodes between the soil surface and the first fork, between the first-tier and second-tier forks, and between the second- and third-tier forks. (c) Number of flowers per tier, per plant. (d) Time to start of floral and/or inflorescence senescence. Floral traits were recorded weekly to determine the date of inflorescence appearance, and initial date of floral senescence. The total number of days from flower appearance to start of inflorescence and/or flower senescence was calculated from these weekly records. Shown are the means ± SEM.</p

GA responses of the <i>brm-1</i> mutant.

<p>(A, B), Elongation of <i>brm-1</i> hypocotyls and roots in response to 1 µM GA₄. Plants were grown on ½ MS medium for 8 days under long-days conditions in the presence or absence of 1 µM GA₄. GA application caused considerable elongation of the hypocotyls, but had little effect on <i>brm-1</i> root growth. Bar = 5 mm. (B), Hypocotyl length of plants grown as in A. Presented data are the means of 12 measurements ± s.d. (C), Flowering of <i>brm-1</i> plants in response to exogenous gibberellins. Plants were grown in soil under short-day conditions and treated with 10 µM GA₃. At least 15 plants of each line/condition were scored. Data are the means ± s.d. Asterisks indicate significant differences from the wild type plants (p<0.01).</p

<i>brm</i> mutants show GA-related phenotypic traits and increased sensitivity to paclobutrazol.

<p>(A), Comparison of <i>brm-1</i> and <i>ga1-3</i> mutants grown on ½ MS medium for 18 days under long-day conditions. (B), Germination of the <i>brm-1</i> mutant is abolished in the presence of 10 µM PAC and rescued upon addition of exogenous gibberellin. The progeny of <i>brm-1/BRM</i> plants were analyzed 14 days after sowing. (C), Phenotype of <i>brm-1</i> plants grown for 25 days on 10 µM PAC after incubation of seeds with exogenous GA. (D), Germination assay of wild type, <i>brm-3</i> and <i>3xdella</i> (<i>rga/rgl1/rgl2</i>) lines. Seed coat rupture after 14 days was scored as germination. (E), Root elongation assay of wild type and <i>brm-3</i> plants grown for 12 days on PAC-containing medium. Bars in A, C and E = 5 mm.</p