GFP expression segregation in seeds of tobacco T1 progeny overexpressing <i>EaZIP</i>.

<p>Tobacco plants were grown to maturity. Seeds were collected from each line and subject to GFP expression observation. Representative images from examined lines were presented. Segregation ratios between GFP positive and negative seeds were indicated in parentheses. A, non-transformed tobacco plant; B and C: <i>EaZIPwocTP</i> plant lines; and D and E: <i>EaZIPwcTP</i> plant lines.</p

Data_Sheet_1_Transcriptome analysis reveals molecular mechanisms underlying salt tolerance in halophyte Sesuvium portulacastrum.xlsx

Soil salinity is an important environmental problem that seriously affects plant growth and crop productivity. Phytoremediation is a cost-effective solution for reducing soil salinity and potentially converting the soils for crop production. Sesuvium portulacastrum is a typical halophyte which can grow at high salt concentrations. In order to explore the salt tolerance mechanism of S. portulacastrum, rooted cuttings were grown in a hydroponic culture containing ½ Hoagland solution with or without addition of 400 mM Na for 21 days. Root and leaf samples were taken 1 h and 21 days after Na treatment, and RNA-Seq was used to analyze transcript differences in roots and leaves of the Na-treated and control plants. A large number of differentially expressed genes (DEGs) were identified in the roots and leaves of plants grown under salt stress. Several key pathways related to salt tolerance were identified through KEGG analysis. Combined with physiological data and expression analysis, it appeared that cyclic nucleotide gated channels (CNGCs) were implicated in Na uptake and Na+/H+ exchangers (NHXs) were responsible for the extrusion and sequestration of Na, which facilitated a balance between Na+ and K+ in S. portulacastrum under salt stress. Soluble sugar and proline were identified as important osmoprotectant in salt-stressed S. portulacastrum plants. Glutathione metabolism played an important role in scavenging reactive oxygen species. Results from this study show that S. portulacastrum as a halophytic species possesses a suite of mechanisms for accumulating and tolerating a high level of Na; thus, it could be a valuable plant species used for phytoremediation of saline soils.</p

T-DNA and PCR-detection of <i>EaZIP</i> and <i>NPTII</i> sequences from transgenic tobacco plants.

<p>A. Schematic presentation of T-DNA region containing marker gene and <i>EaZIP</i> genes (<i>EaZIPwocTP</i> or <i>EaZIPwcTP</i>) for overexpression analysis. R and L: Right and left borders of T-DNA; EGFP/NPTII: EGFP and NPTII translational fusion marker; BDPC: Bidirectional dual promoter complex derived from double enhancer CaMV35S and CsVMV promoters; Ter: 35S transcript terminator. PCR primers including NRT53, NRT34, ENEW-53 and ENC-32 along with expected amplicons are marked. B. PCR products to show presence of <i>EaZIP</i> cDNA fragment in analyzed tobacco lines. M indicates marker DNA, CEJ225: plasmid DNA containing the <i>EaZIP</i> short version; nontransgenic control plant; <i>EaZIPwocTP</i> lines 1–4; and <i>EaZIPwcTP</i> lines 1–4. C. PCR products detecting presence of <i>NPTII</i> gene sequence in <i>EaZIPwocTP</i> and <i>EaZIPwcTP</i> tobacco lines as well as nontransgenic control plant.</p

Overexpression of an <i>EaZIP</i> gene devoid of transit peptide sequence induced leaf variegation in tobacco

<div><p>Leaf variegation is an ornamental trait that is not only biologically but also economically important. In our previous study, a Mg-protoporphyrin IX monomethyl ester cyclase homologue, <i>EaZIP</i> (<i>Epipremnum aureum</i> leucine zipper) was found to be associated with leaf variegation in <i>Epipremnum aureum</i> (Linden & Andre) G.S. Bunting. The protein product of this nuclear-encoded gene is targeted back to chloroplast involving in chlorophyll biosynthesis. Based on a web-based homology analysis, the <i>EaZIP</i> was found to lack a chloroplast transit peptide (cTP) sequence. In the present study, we tested if overexpression of the <i>EaZIP</i> cDNA with or without the cTP sequence could affect leaf variegation. Transgenic tobacco plants overexpressing <i>EaZIP</i> genes with (<i>EaZIPwcTP</i>) and without (<i>EaZIPwocTP</i>) cTP sequence were generated. Many plant lines harboring <i>EaZIPwocTP</i> showed variegated leaves, while none of the plant lines with <i>EaZIPwcTP</i> produced such a phenotype. Molecular analysis of T0 plants and selfed T1 progeny, as well as observations of tagged marker GFP (green fluorescent protein) did not show any other difference in patterns of gene integrity and expression. Results from this study indicate that transgenic approach for expressing <i>EaZIPwocTP</i> could be a novel method of generating variegated plants even through the underlying mechanisms remain to be elucidated.</p></div

Extended <i>EaZIP</i> cDNA sequence of <i>Epipremnum aureum</i> ‘Golden Pothos’.

<p>Genomic DNA was used for PCR amplification to extend 5’ region of <i>EaZIP</i> cDNA. Putative cTP region is highlighted.</p

Detection of <i>EaZIP</i> transcripts from tobacco and pothos plants.

<p>Total RNA was isolated from leaf tissues of indicated tobacco and pothos plants and subject to reverse transcription reaction followed by amplification of target fragments using regular PCR. M indicates marker DNA. Kanamycin gene amplicon is 323 bp in length, and <i>EaZIP</i> gene amplicon is 553 bp in length.</p

Determination of transgene <i>EGFP</i> copy numbers using quantitative real-time PCR based on external plasmid controls.

<p>Determination of transgene <i>EGFP</i> copy numbers using quantitative real-time PCR based on external plasmid controls.</p

Transgenic tobacco plants grown in a greenhouse.

<p>A. independent lines expressing <i>EaZIPwcTP</i> (no variegated leaves); B. an <i>EGFP</i> line (no variegated leaves); and C. an <i>EaZIPwocTP</i> line with variegated leaves.</p

Transgenic tobacco leaves.

<p>A. EGFP-CK, plants harboring EGFP gene as a control, plants did not exhibit leaf variegation; B-F. Plants harboring <i>EaZIPwocTP</i> gene, which showed different degrees of leaf variegation.</p

Comparison of chloroplast density of leaf tissues among transgenic tobacco plant lines overexpressing <i>EGFP</i> (EGFP-CK), <i>EaZIPwocTP</i>, and <i>EaZIPwcTP</i>.

<p>Samples from three each of <i>EGFP</i>, <i>EaZIPwocTP</i>, and <i>EaZIPwcTP</i> plant lines were examined. Bar represent average 1000 x chloroplasts per mg tissue. Standard errors were indicated (n = 3). Different letters above bars indicate significant difference (<i>P</i> < 0.05) among examined leaves according to Fisher’s LSD test.</p