Results
Physiological characteristics of broccoli
Figure 1 illustrates comparisons of antioxidant enzyme activities as well as the WP, EL, and Fv/Fm values under four temperatures at six different times in two genotypes of broccoli. The trend and rate of the increase in APX activity under heat stress over time differed among the genotypes.
For example, APX activity in GM plants increased at a different rate from 0 (0.56 lmol g-1 FW) to 48 h (0.86 lmol g-1 FW), and then dropped to 0.73 lmol g-1 FW after 72 h with 30C treatment, but increased from 0 (0.68 lmol g-1 FW) to 72 h (1.01 lmol g-1 FW) with 40C treatment in SF plants. Slight increases in CAT levels were noted in all plants with 30, 35, and 40C treatments as the heat-stressed time was extended. When genotypes were compared across heat-stressed times of 12–72 h, SF plants exhibited significantly higher CAT activities than GM plants with 30, 35, and 40C treatments. A maximal increase was found with 40C treatment over time for SF plants. SOD activities did not significantly differ among genotypes at different times with 35C treatment, with the exception of significantly higher values (1.64 and 1.92 lmol g-1 FW) in GM plants than values (1.45 and 1.69 lmol g-1 FW) in SF plants at 6 and 12 h, respectively. The trend of changes in SOD activities with 40C treatment was similar to that with 35C treatment.
As shown in Fig. 1, WP with 40C treatment was significantly lower than those with 25 and 30C treatments from 6 to 72 h for both genotypes, indicating that a high temperature may induce a decrease in the leaf water level, subsequently affecting the leaf WP. When different heatstress treatments across time were compared, SF exhibited a significantly higher WP (a less-negative value) than GM from 24 to 72 h. Thus, the WP of the different genotypes responded differently to heat stress. The lowest value of the WP (-19.7 bar) was detected at 72 h with exposure to 40C in GM plants. EL (%) was assessed over the time course under various temperature conditions, and its patterns and trends were similar to those of the WP. When plants were subjected to 40C treatment, all plants had significantly higher EL values over time than those of the 25 and 30C treatments. Overall, the study showed differential responses of both varieties towards different EL for different time durations. GM is a heat-sensitive variety and showed 24.9–47.4% EL, while heat-tolerant SF plants had EL percentages of 20.8–38.9% with 40C treatment from 6 to 72 h. The Fv/Fm values with 25 and 30C treatments showed no significant differences from 0 to 48 h. In general, high temperature reduced Fv/Fm values, and gradual decreases in Fv/Fm values were observed with both 35 and 40C treatments over time.
Physiological characteristics of Chinese cabbage
Figure 2 demonstrates the effect of temperature conditions on Chinese cabbage varieties monitored by measuring the changes in antioxidant enzyme activities, as well as the RWC, EL and MDA contents for various time periods. APX activity significantly differed in the two genotypes at different time points under heat stress. APX activities of AS plants over time with 35 and 40C treatments were signifi- cantly higher than those of RN plants. It is noteworthy that AS (12.8 lmol g-1 FW) displayed a tenfold greater increase over RN (1.2 lmol g-1 FW) with 40C treatment at 72 h.
This increase in APX activity was clear with 40C treatment, which suggests that it was a result of the oxidative stress induced by the high temperature. The trends of change in CAT activity did not significantly differ in any heat-stressed treatments (30–40C) from 0 to 72 h in any plants, with the exceptions of elevated values of 4.89 lmol g-1 FW at 48 h with 30C treatment, 7.94 lmol g-1 FW at 24 h with 35C treatment, and 6.24 lmol g-1 FW at 48 h with 40C treatment in RN plants. SOD activities also showed no significant differences for any stress treatments over time for either RN or AS, with the exceptions of 35C treatment at 6 h (14.2 lmol g-1 FW) and 40C treatment at 48 h (12.1 lmol g-1 FW) in RN plants. As shown in Fig. 2, levels of leaf RWC (%) in all plants decreased at different rates as heat-stress durations were extended. RWC in RN plants showed significantly lower values after 6 h with 35 and 40C treatments compared to AS plants, and the lowest value (60.2%) was found with 40C treatment at 72 h. The EL (%) in all plants with 35 and 40C treatments gradually increased over the time course of the experiment. In addition, RN plants under 35 and 40C conditions exhibited higher levels of EL compared to AS plants from 24 to 72 h. However, EL showed no significant difference for plants during the time course under non-stressed conditions. MDA levels responded differently under heat-stressed conditions for the two plant genotypes. A significantly lower MDA content with 30–40C treatments over time was observed in AS plants compared to RN plants, except for 30C treatment at 6 h.
The MDA level of RN remained high and stable from 6 to 72 h with 35 and 40C treatments. At different points in time of temperature treatment, both the RN and AS genotypes exhibited the same patterns where the plants showed significantly increased MDA amounts as the temperature increased.
Cloning of CAT and APX cDNAs by RACE Inner and outer fragments were used to amplify full-length cDNAs of CAT and APX, using the 50 - and 30 -RACE method. The 550/700 (CAT/APX) 50 -RACE and 500/350 (CAT/APX) 30 -RACE products were amplified (data not shown). These PCR products were purified, cloned, and sequenced, and the sequences were compared to sequences in the GenBank database of NCBI-blastn. The full-lengthclone contained a broccoli CAT cDNA 1,768 bp long, and had an ORF 1,476 bp long (Fig. 3; GenBank accession no. GQ 500124). The cDNA was initiated by an ATG, terminated by TAA, and contained an 88-bp 50 untranslated region (UTR) and a 204-bp 30 UTR. The GC content of the coding region was 50%. The broccoli CAT gene encoded a putative protein of 492 amino acid (aa) residues, and had a molecular mass of 56.7 kDa with an isoelectric point (pI) of 7.0, as determined by the pI/Mw computer programs at Swiss-Port/TrEMBL (http://au.expasy.org/tools).
The resulting full-length Chinese cabbage APX cDNA was 1,070 bp, including a 750-bp ORF, a 65-bp 50
-UTR, and a 255-bp 30
-UTR (Fig. 4; GenBank accession no. GQ 500125). The ORF of the Chinese cabbage APX gene encoded 250 aa with a predicted molecular weight of 27.7 kDa and a pI of 5.8. The GC content of the ORF was 51%. Figures 3 and 4 also show the locations of the degenerate primers of both CAT (CAT-F and CAT-R) and APX (APX-F and APX-R) used for the PCR Sequence homology and phylogenetic analysis of CAT and APX In total, 12 deduced CAT amino acid sequences from nine plant species were aligned and compared. Figure 5a shows a phylogenetic tree from the conserved region using the NJ method, and shows the division of sequences into two classes. Broccoli (B. oleracea) CAT was more closely related to B. juncea CAT1 and CAT3 than to Raphanus sativus CAT.
Brassica oleracea and B. juncea CAT1 and CAT3 showed 42% homology to CAT. Brassica juncea, B. olerace, R. sativus, and A. thalinana are members of the Cruciferae, and were clustered together. However, G. hirsutum was clustered with P. persica, H. perfuratum, and M. crystallmumwith a bootstrap value of 96, which formed a clade with Brassica species. Brassica juncea contained four CAT isoforms within the same group. As shown in Fig. 5b, the sequence of Chinese cabbage (B. campestris) APX cDNA revealed a significant level of similarity with B. oleracea BO-APX1, R. sativus, B. napus, B. juncea, A. thaliana, and B. oleracea BO-APX2. Two major groups were distinguished within the phylogenetic tree. Chinese cabbage (B. campestris) and B. olerace BO-APX1 clustered into the same subgroup, which had the farthest phylogenetic distance from other groups of APX of other species. Brassica oleracea contained two CAT isoforms from separate groups.
Changes in RNA levels of CAT and APX genes with different temperature treatments RNA levels of the CAT gene were quantified using RT-qPCR of reverse transcripts of RNA from both GM and SF that had been subjected to different temperatures for 0–72 h (Fig. 6). The data were normalized with respect to the RNA level of actin, a housekeeping gene that is consistently expressed in plants. CAT gene transcripts in all plants constitutively accumulated at different rates over time. The peak of CAT transcript levels increased in SF plants after 72 h at 40C. Expression levels of CAT RNA in SF plants were significantly higher than those in GM plants at 24 h with 35 and 40C treatments. Although the transcripts were detected at all times, the CAT gene was most strongly active in heat-stressed (35 and 40C) treatments. In contrast, this gene showed rather low activity in non-stressed (25C) treatment. Hence, higher RNA levels were induced by the elevated heat stress.
Average RNA levels of the APX gene in the leaves of Chinese cabbage are summarized in Fig. 7. In all treatments, the RNA abundance of APX was upregulated. The trends and levels of the increase in APX RNA expression over time differed between genotypes. The APX gene was significantly more highly expressed in AS plants than in RN plants after 12 h of heat-stress conditions. Its expression in all plants was significantly upregulated with 35 and 40C treatments at 24–72 h compared to 25C treatment.