The comparison of the anthocyanin accumulation tissues and the CBP60g expression pattern.

<p>(A), (C) and (E) The anthocyanin accumulation tissues. (B), (D) and (F) CBP60g expression tissues. (A) and (B) The abaxial side of 14 days seedlings. (C) and (D) The adaxial side of 14 days seedlings. (E) and (F) The flank side of 14 days seedlings.</p

CBP60g repressed the anthocyanin accumulation in a calcium independent manner.

<p>Wild-type, the <i>cbp60g</i> mutant and <i>CBP60g</i> overexpression lines grown in the solid 1/2 MS medium with 3% sucrose, 40 μM kinetin and various concentrations of EGTA were observed (A). The fresh weight and anthocyanin content were quantified (B) and (C). Three biological replicates were performed for every experiment. Each data point represents the average of three technical replicates ±SD.</p

CBP60g repressed the expression of anthocyanin biosynthetic and regulatory genes under AIC.

<p>Twelve-day-old wild-type, the <i>cbp60g</i> mutant and <i>CBP60g</i> overexpression lines grown in the liquid 1/2 MS medium were treated with 100 μM kinetin. The expression of anthocyanin biosynthetic genes <i>CHS</i>, <i>CHI</i> and <i>DFR</i>, along with the anthocyanin regulatory genes <i>PAP1</i> and <i>TT8</i> were detected. Three biological replicates were performed for every experiment. Each data point represents the average of three technical replicates ±SD.</p

The <i>acs6-2</i> mutant allele is a knockdown mutant.

<p>(A) <i>B. cinerea</i>-induced ethylene production in wild type, <i>acs6-2</i> (SALK_090423), and <i>acs6-1</i> (SALK_025672) plants. Twelve-day-old seedlings grown in GC vials were inoculated with <i>B. cinerea</i> spores. Ethylene accumulation in GC vials was monitored at indicated times, and seedlings were collected for gene expression analysis. Error bars indicate standard deviations (n = 3). (B) Induction of <i>ACS6</i> expression in wild type (Col-0), <i>acs6-2</i>, and <i>acs6-1</i> seedlings after <i>B. cinerea</i> inoculation. Total RNA was isolated from the seedlings collected in (A). Expression of the <i>ACS6</i> gene was quantified by real-time PCR. <i>ACS6</i> transcript levels were expressed as fold of induction relative to the zero time point (upper panel) and as a percentage of the <i>EF1α</i> transcript (lower panel). Error bars indicate standard deviations (n = 3).</p

WRKY33 transcription factor binds to the promoter of <i>ACS2</i> and <i>ACS6</i> genes <i>in vivo</i>.

<p>(A) The promoters of <i>ACS2</i> and <i>ACS6</i> genes are rich in W-boxes, the cis-element binding sites of the WRKY transcription factor. A diagram indicates the number and relative position of the W-boxes in the promoters of the <i>ACS2</i> and <i>ACS6</i> genes. Line arrows indicate the position of primers used for qPCR after chromatin immunoprecipitation (ChIP). Positions of the predicted transcriptional starting sites are indicated by arrows with turning lines and negative numbers. The T-DNA insertion site in the SALK_090423 <i>acs6-2</i> allele, which locates in the promoter region of <i>ACS6</i> gene, is also indicated. (B) ChIP-qPCR analysis was performed using <i>DD/4myc-WRKY33^WT</i> plants generated from the cross of <i>wrky33/4myc-WRKY33^WT</i> with <i>DD</i> lines. Input chromatin was isolated from two-week-old seedlings 12 hr after DEX treatment. Epitope-tagged WRKY33-chromatin complex was immunoprecipitated with an anti-myc antibody. A control reaction was processed side-by-side using mouse IgG. ChIP- and input-DNA samples were quantified by real-time qPCR using primers specific to the promoters of <i>ACS2</i> (left panel) and <i>ACS6</i> (right panel) genes. ChIP results are presented as percentage of input DNA. Error bars indicate standard deviations (n = 3).</p

<i>WRKY33</i> functions downstream of the MPK3/MPK6 cascade in inducing the expression of <i>ACS2</i> and <i>ACS6</i> genes in the gain-of-function <i>DD</i> seedlings.

<p>(A) Mutation of <i>WRKY33</i> compromises ethylene induction in <i>DD</i> seedlings. Twelve-day-old <i>DD</i> and <i>DD/wrky33</i> seedlings grown in GC vials were treated with 1-µM DEX. Ethylene accumulation in GC vials was monitored at indicated times, and then seedlings were collected for gene expression analysis. Error bars indicate standard deviations (n = 3). (B) MPK3/MPK6-induced <i>ACS2</i> and <i>ACS6</i> gene expression in <i>DD</i> plants is dependent on <i>WRKY33</i>. Total RNA was extracted from seedlings collected in (A). Expressions of <i>ACS2</i> (upper panel) and <i>ACS6</i> (lower panel) genes were quantified by real-time PCR. <i>ACS</i> transcript levels were calculated as a percentage of the <i>EF1α</i> transcript. Error bars indicate standard deviations (n = 3).</p

A model depicting the dual-level regulation of ACS activity by MPK3/MPK6-dependent and independent pathways during pathogen-induced ethylene production.

<p>Members of all three types of ACS isoforms are involved in pathogen-induced ethylene production. In <i>B. cinerea</i>-infected plants, Type I (ACS2/ACS6) isoforms contribute the most (∼85%). ACS2 and ACS6 are regulated by the MPK3/MPK6 cascade at both transcriptional and protein stability levels. The transcriptional up-regulation is mediated by WRKY33, a MPK3/MPK6 substrate. Type II (ACS8 and ACS11) and Type III (ACS7) isoforms are activated at the transcriptional level although the regulatory pathway(s) involved is not clear at present. Increase in total cellular ACS activity drives the elevated ethylene production, which triggers downstream responses.</p

<i>ACS7</i> also contributes to <i>B. cinerea</i>-induced ethylene production in Arabidopsis.

<p>Twelve-day-old wild type (Col-0), <i>acs2-1/acs6-2</i> double mutant, and <i>acs2-1/acs6-2/acs7-1</i> triple mutant Arabidopsis seedlings grown in GC vials were inoculated with <i>B. cinerea</i>. Ethylene levels in the headspace were determined at indicated times. Error bars indicate standard deviations (n = 3).</p

Additional file 2: of Two NAC transcription factors from Caragana intermedia altered salt tolerance of the transgenic Arabidopsis

Germination of the CiNAC3 transgenic seeds under ABA treatment. (A) The transgenic lines showed a higher germination rate on 3 μM ABA medium compared with wild-type. The picture was taken 7 d (3 d for control) after imbibition. The germination rate of wild-type and two overexpression lines on medium with (B) or without (C) 6 μM ABA. (D) Germination of transgenic seeds without stratification. Error bars are standard errors of the means from three replications. Three independent biological replicates have been performed. (TIFF 1072 kb

<i>B. cinerea</i> induced <i>ACS2</i> and <i>ACS6</i> gene activation is dependent on functional MPK3 and MPK6.

<p>Twelve-day-old wild type (Col-0), <i>mpk3</i>, <i>mpk6</i>, and rescued <i>mpk3/mpk6</i> double mutant seedlings grown in GC vials were inoculated with <i>B. cinerea</i> spores. Samples were collected at indicated times. Total RNAs were extracted and treated with DNase to remove trace genomic DNA contamination. After reverse transcription, expressions of <i>ACS2</i> (A) and <i>ACS6</i> (B) genes were quantified by real-time PCR. <i>ACS</i> transcript levels were calculated as a percentage of the <i>EF1α</i> transcript. Error bars indicate standard deviations (n = 3).</p