Article Figures & Data
Figures
- Figure 1.
Production of Ubi1:CBF3 and Ubi1:ABF3 plants. A, Ubi1:CBF3 and Ubi1:ABF3 consist of the maize ubiquitin promoter (Ubi1) linked to the CBF3 and ABF3 coding regions, respectively, and the 3′-region of the potato proteinase inhibitor II gene (3′pinII), and a gene expression cassette that contains the 35S promoter, the bar coding region, and the 3′-region of the nopaline synthase gene (3′nos). The entire expression cassette is flanked by the 5′-matrix attachment region (MAR) of the chicken lysozyme gene (Phi-Van and Strätling, 1996). B, RNA gel-blot analysis was performed using total RNAs from young leaves of 6 Ubi1:CBF3 (top) and 5 Ubi1:ABF3 (bottom) lines and from NC plants. The blots were hybridized with the CBF3 and ABF3 probes (described in Supplemental Fig. 1) and reprobed with the rice RbcS gene (Kyozuka et al., 1993). Ethidium bromide (EtBr) staining of total RNA was for equal loading of RNAs.
- Figure 2.
Growth characteristics of Ubi1:CBF3 and Ubi1:ABF3 plants. A, Growth phenotypes of 3 independent T4 homozygous lines for Ubi1:CBF3, Ubi1:ABF3, and NC plants at indicated days after germination (DAG). B, Dry weight and fresh weight accumulation of Ubi1:CBF3, Ubi1:ABF3, and NC plants. Plants grown in the greenhouse during the same time course shown in A were harvested and fresh and dry weight/10 plants measured. Each data point represents the mean ± sd of triplicate experiments with three different transgenic lines.
- Figure 3.
Appearance of plants and changes in chlorophyll fluorescence during drought stress. A, Four and three independent T4 homozygous lines for Ubi1:CBF3 and Ubi1:ABF3, respectively, and NC seedlings were grown in the greenhouse for 4 weeks and then subjected to 4 d of drought stress followed by 5 to 7 d of watering. Eighteen plants per each line were tested. Photos were taken at 1- or 2-d intervals; + followed by number denotes days of watering. B, Fv/Fm of the transgenic and NC plants in the same time course of drought stress shown in A was measured using a pulse modulation fluorometer (mini-PAM). Fv/Fm is a measure of accumulated photooxidative damage to PSII. Each data point represents the mean ± se of triplicate experiments (n = 6).
- Figure 4.
Changes in chlorophyll fluorescence during drought, high-salinity, and low-temperature stresses. Three independent T4 homozygous lines for Ubi1:CBF3, Ubi1:ABF3, and NC seedlings grown in the greenhouse for 14 d were subjected to various stress conditions as described: for drought stress, the seedlings were air-dried for 2 h at 28°C and for high-salinity stress seedlings were exposed to 400 mm NaCl for 2 h at 28°C. For low-temperature stress, they were exposed to 4°C for 6 h. All of the experiments were carried out under continuous light of 150 μmol m−2 s−1. Each data point represents the mean ± se of triplicate experiments (n = 6).
- Figure 5.
Induction of stress-related genes in Ubi1:CBF3 and Ubi1:ABF3 plants. Three independent T4 homozygous lines for Ubi1:CBF3, Ubi1:ABF3, and NC seedlings were grown in the greenhouse for 14 d. Transgenic and NC plants were then treated for 2 h with drought (the seedlings excised before being air-dried for 2 h), high salinity (400 mm NaCl) at the greenhouse, and with low-temperature stress (4°C) at the cold chamber under continuous light of 150 μmol m−2 s−1. For ABA treatments, 100 μm ABA was applied to each 14-d-old seedling for 2 h. RNA gel blots of total RNAs from transgenic and NC plants grown either under normal growth conditions (left) and under stress conditions (right) are indicated. RNA gel blots of NC plants grown under normal growth conditions were included on the left-hand side of each section for clarity of comparison. The blots were hybridized with probes for CBF3, ABF3, Lip5 (AB011368), Dip1 (AY587109), Jacalin1 (AK066682), Jacalin2 (AK101991), LOX (AJ270938), PSLS (AK072958), Hsp70 (CF280418), PP2Ca (CF304401), Wsi18 (D26536), Rab21 (Y00842), LEA4 (AK107930), PP2Cb (AK069274), and RbcS (Kyozuka et al., 1993). EtBr staining of total RNA was used to ensure equal RNA loading.
- Figure 6.
Transactivation of the Wsi18:GUS fusion by ABF3. The Wsi18:GUS construct was cotransformed with the effector constructs, either Ubi1:CBF3 or Ubi1:ABF3, or with the expression vector alone; 4 μg of each construct with 2 μg of Ubi1:LUC as an internal standard was used in all cases. Each bar represents the mean value of the relative GUS/LUC activities from four independent experiments.
Tables
Plantsa Totalb Survivalc Survival Rated Ubi1:CBF3 NC 36 0 0% 1-4-1 36 34 94% 2-1-1 36 36 100% 4-8-1 36 36 100% 6-5-1 36 14 39% Ubi1:ABF3 NC 36 3 8% 1-4-1 36 21 58% 2-1-1 36 24 67% 5-8-1 36 18 50% ↵a Four-week-old soil-grown plants withheld water for 4 d followed by watering for 7 d and results scored. Plants were considered dead if there was no regrowth 7 d of rewatering. Water loss during the drought periods was similar for all pots.
↵b Total number of plants used in each assay.
↵c Number of survival plants.
↵d Percent of survived plants (survival/total × 100).
- Table II.
List of genes that are up-regulated in Ubi1:CBF3 and Ubi1:ABF3 plants under normal and drought stress
Numbers appearing in bold are the ones that are induced 1.6-fold or more in Ubi1:CBF3 or Ubi1:ABF3 plants.
Gene Name Accession No.a Normalb Droughtd Ubi1:CBF3c Ubi1:ABF3c Ubi1:CBF3c Ubi1:ABF3c Jacalin1 AK066682 3.56 −1.11 3.04 2.92 Jacalin2 AK101991 3.32 −1.17 2.42 2.10 Dip1 AY587109 1.66 1.13 1.10 1.39 Lip5 AB011368 2.32 1.29 1.20 −1.10 Lipoxygenase (LOX) AJ270938 3.10 1.43 −1.59 1.11 Glutelin AK107238 2.18 −1.10 −1.22 1.13 Bowman Birk trypsin inhibitor1 AK065846 1.83 1.08 −1.19 1.27 Bowman Birk trypsin inhibitor2 AK105455 1.65 −1.22 −1.19 1.10 Receptor kinase containing LRR repeats AK119823 1.67 1.64 1.37 1.99 Unknown protein AK059202 1.62 −2.07 1.12 1.26 Cyt P450 AK069394 1.34 1.11 5.21 3.30 Seed imbibition protein (Sip1) AK065100 1.03 −1.03 2.20 1.12 Sicrotubule-associated protein MAP65-1a AK102553 −1.08 1.06 1.91 1.22 Unknown protein NM_188534 −1.07 1.09 1.93 1.06 Unknown protein AK107624 1.10 −1.07 1.86 1.17 Unknown protein AK061456 −1.03 −1.00 1.68 1.25 Polygalacturonase (PG2) AK108477 1.02 1.09 1.70 1.47 FtsJ cell division protein AK070075 1.10 1.10 1.78 −1.02 mRNA cleavage factor I subunit AK061260 1.03 1.13 1.79 1.04 Beclin AK101033 1.19 1.02 1.73 1.05 Cyclophilin (CyP) AK111654 1.04 −1.01 1.70 1.21 Phospho sulfolactate synthase (PSLS) AK072958 −1.01 −1.04 1.61 1.02 Gag-pol polyprotein AK063408 −1.08 −1.18 1.82 −1.01 α-Expansin OsEXPA5 AF394546 1.16 1.14 1.41 1.94 Hsp70 AK072830 1.99 1.94 1.39 1.28 Protein phosphatase 2Ca (PP2Ca) XM_463364 1.65 1.67 1.21 1.48 Wsi18 D26536 1.00 2.11 1.06 2.27 RAB21 Y00842 −1.03 2.60 1.22 1.54 Phosphate-induced protein 1(phi1) AK070419 −1.11 1.67 1.10 −1.39 Antioxidant protein AK066452 1.17 1.68 1.25 1.15 LEA4 AK107930 −1.01 1.02 1.08 4.33 Protein phosphatase 2Cb (PP2Cb) AK069274 1.12 1.05 1.05 3.45 PHD-type zinc finger protein AK059311 −1.17 1.09 1.07 3.11 Unknown protein AK063747 −1.16 1.09 1.17 2.83 Little protein (LP1) AK063634 1.09 1.09 −1.22 3.01 Unknown protein AK072034 1.08 1.06 −1.11 2.66 Unknown protein AK063680 −1.00 −1.05 −1.08 2.63 CCCH-type zinc finger protein AK106392 1.37 1.05 1.40 2.56 26S proteasome AAA-ATPase subunit AK103936 1.05 1.22 −1.00 2.26 Unknown protein NM_197832 −1.34 1.18 −1.00 2.24 Unknown protein XM_480395 1.01 1.09 1.05 2.34 Unknown protein AK058851 1.03 −1.00 −1.12 2.38 Aquaporin (TIP4) AK121671 1.08 −1.06 1.07 2.62 Disease resistance protein (RPH8A) AK072531 1.13 1.18 −1.07 2.10 Unknown protein AK104155 1.05 1.00 −1.01 2.20 1,4-β-d xylan xylanohydrolase NM_184104 1.09 1.04 1.03 1.97 Spore coat protein AK108917 1.02 −1.02 1.05 1.93 Cellulase XM_469540 −1.06 1.07 1.21 1.98 LTI6B-like AK104060 −1.12 1.09 −1.07 1.88 Fusarium resistance protein AK058343 1.05 1.11 −1.03 2.20 ABC transporter AK105712 −1.08 1.17 −1.19 1.91 LEA AK067556 1.13 1.10 1.22 1.87 LEA3 AK102039 −1.02 1.17 −1.17 2.14 ↵a GenBank accession numbers for full-length cDNA sequences of corresponding genes.
↵b Microarrays were hybridized with Cy3- and Cy5-labeled probe pairs of either Ubi1:CBF3 and nontransgenic plants or Ubi1:ABF3 and nontransgenic plants grown in normal growth conditions.
↵c The microarray-data sets can be found at the www.http://www.ncbi.nlm.nih.gov/geo/ (Gene Expression Omnibus, GEO). GEO accession number of microarray-data set is GSE2211.
↵d Microarrays were hybridized with Cy3- and Cy5-labeled probe pairs of either Ubi1:CBF3 and nontransgenic plants or Ubi1:ABF3 and nontransgenic plants grown in drought-stress conditions.
Supplemental Data
Supplemental Figures, Tables, and Material
Files in this Data Supplement:
- Supplemental Data - Supplemental Figure 1A
- Supplemental Data - Supplemental Figure 1B,C
- Supplemental Data - Supplemental Figure Legend
- Supplemental Data - Supplemental Table I
- Supplemental Data - Supplemental Table II
- Supplemental Data - Supplemental Material Legend
- Supplemental Data - Supplemental Material 1A
- Supplemental Data - Supplemental Material 1B,C
- Supplemental Data - Supplemental Material 2
- Supplemental Data - Supplemental Material 3
