Comparative genomics in salt tolerance between Arabidopsis and aRabidopsis-related halophyte salt cress using Arabidopsis microarray

Abstract

Salt cress (Thellungiella halophila), a halophyte, is a genetic model system with a small plant size, short life cycle, copious seed production, small genome size, and an efficient transformation. Its genes have a high sequence identity (90%-95% at cDNA level) to genes of its close relative, Arabidopsis. These qualities are advantageous not only in genetics but also in genomics, such as gene expression profiling using Arabidopsis cDNA microarrays. Although salt cress plants are salt tolerant and can grow in 500 mm NaCl medium, they do not have salt glands or other morphological alterations either before or after salt adaptation. This suggests that the salt tolerance in salt cress results from mechanisms that are similar to those operating in glycophytes. To elucidate the differences in the regulation of salt tolerance between salt cress and Arabidopsis, we analyzed the gene expression profiles in salt cress by using a full-length Arabidopsis cDNA microarray. In salt cress, only a few genes were induced by 250 mm NaCl stress in contrast to Arabidopsis. Notably a large number of known abiotic- and biotic-stress inducible genes, including Fe-SOD, P5CS, PDF1.2, AtNCED, P-protein, beta-glucosidase, and SOS1, were expressed in salt cress at high levels even in the absence of stress. Under normal growing conditions, salt cress accumulated Pro at much higher levels than did Arabidopsis, and this corresponded to a higher expression of AtP5CS in salt cress, a key enzyme of Pro biosynthesis. Furthermore, salt cress was more tolerant to oxidative stress than Arabidopsis. Stress tolerance of salt cress may be due to constitutive overexpression of many genes that function in stress tolerance and that are stress inducible in Arabidopsis.

Figure 1.

Survivability of salt cress and Arabidopsis under high-salt stress. A, High-salt stress tolerance of salt cress and Arabidopsis of the same size. Three-week-old Arabidopsis and 4-week-old salt cress plants that were the same in size were exposed to 500 mm NaCl solution for 3 weeks. B, High salt stress tolerance of salt cress and Arabidopsis of the same age. Three-week-old Arabidopsis and salt cress plants that were the same in size were exposed to 500 mm NaCl solution for 3 weeks.

Figure 2.

Evaluation of salt tolerance and Na⁺ uptake in hydroponic culture system. A, High salinity stress tolerance in hydroponic culture system. Three-week-old Arabidopsis and 4-week-old salt cress plants that were the same in size were exposed to 250 mm NaCl solution for 24, 48, and 96 h. B, Accumulation of NaCl in leaves of Arabidopsis and salt cress during high salt stress in hydroponic culture system. Three-week-old Arabidopsis and 4-week-old salt cress plants that were the same size were exposed to 250 mm NaCl solution for 2, 5, 10, and 24 h.

Figure 3.

Classification of salt stress-inducible genes in salt cress and Arabidopsis. The salt stress-inducible genes identified were categorized into three groups: (1) genes induced in Arabidopsis; (2) genes induced in salt cress; and (3) genes induced in both Arabidopsis and salt cress. The genes with expression log₂ ratios (salt stressed/unstressed) greater than 1.5 times the average of the three experimental sets were regarded as salt stress-inducible genes.

Figure 4.

Hierarchical clustering among the genes highly expressed in salt cress and the results of microarray analyses that included various abiotic stress treatments or various biotic stress and biotic-related treatments of Arabidopsis using this Arabidopsis cDNA microarray. A, Overview of the hierarchical cluster display. The fold change values for each sample, relative to untreated control samples, were log₂ transformed and subjected to complete linkage hierarchical clustering. Expression values higher and lower than those of the control are shown in red and green, respectively. As absolute value of fold difference increased, the color intensity increased. The bars at the right end indicate five rough groups: abiotic, biotic, both abiotic and biotic stress-inducible genes, abiotic and biotic stress-suppressible genes, and abiotic and biotic stress-noninducible genes. B, The expansion figure of A. The relationship among experiments across all of the genes included in the cluster analysis and the type of each experiment are indicated (for details, see Seki et al., 2002a, 2002b; Narusaka et al., 2003).

Figure 5.

RNA gel-blot analysis of AtP5CS, ThP5CS, SOS1, and PDF1.2 genes in salt cress and Arabidopsis. Total RNA was prepared from 3-week-old Arabidopsis and 4-week-old salt cress plants that were the same in size during nonstress or 250 mm NaCl stress using the microarray analyses. Each lane was loaded with 10 μg of total RNA. The RNA was fractionated on a 1% agarose gel, blotted onto a nylon membrane, and hybridized with ³²P-labeled AtP5CS, ThP5CS, SOS1, and PDF1.2 DNA fragments as probes.

Figure 6.

ABA and Pro contents in salt cress and Arabidopsis. A, Accumulation of ABA in the entire plant of salt cress and Arabidopsis during normal growth conditions in hydroponic culture. Three-week-old Arabidopsis and 4-week-old salt cress plants grown under normal conditions were used for the analysis. B, Pro content in the entire plant of salt cress and Arabidopsis during normal growth conditions in hydroponic culture. Three-week-old Arabidopsis and 4-week-old salt cress plants grown under normal conditions were used for the analysis.

Figure 7.

Oxidative stress tolerance of salt cress. A, Root elongation assay with paraquat. Salt cress and Arabidopsis seedlings were grown on vertical MS plates containing 1.2% (w/v) agar and 3% Suc. One week after germination, the seedlings were transferred onto either the control MS agar plates (left) or MS agar plates containing 1 μm paraquat (right). The root tips of the seedlings were arranged uniformly along the light-blue line to ensure accurate measurements. Red and yellow bars show root ends of salt cress and Arabidopsis. The pictures were taken 10 d after the seedlings were transferred. B, Root elongation ratio in salt cress (black bar) and Arabidopsis (gray bar) during the exogenous paraquat treatment. The sd of 10 replicated measurements is indicated.