Salt cress. A halophyte and cryophyte Arabidopsis relative model system and its applicability to molecular genetic analyses of growth and development of extremophiles

Abstract

Salt cress (Thellungiella halophila) is a small winter annual crucifer with a short life cycle. It has a small genome (about 2 x Arabidopsis) with high sequence identity (average 92%) with Arabidopsis, and can be genetically transformed by the simple floral dip procedure. It is capable of copious seed production. Salt cress is an extremophile native to harsh environments and can reproduce after exposure to extreme salinity (500 mm NaCl) or cold to -15 degrees C. It is a typical halophyte that accumulates NaCl at controlled rates and also dramatic levels of Pro (>150 mm) during exposure to high salinity. Stomata of salt cress are distributed on the leaf surface at higher density, but are less open than the stomata of Arabidopsis and respond to salt stress by closing more tightly. Leaves of salt cress are more succulent-like, have a second layer of palisade mesophyll cells, and are frequently shed during extreme salt stress. Roots of salt cress develop both an extra endodermis and cortex cell layer compared to Arabidopsis. Salt cress, although salt and cold tolerant, is not exceptionally tolerant of soil desiccation. We have isolated several ethyl methanesulfonate mutants of salt cress that have reduced salinity tolerance, which provide evidence that salt tolerance in this halophyte can be significantly affected by individual genetic loci. Analysis of salt cress expressed sequence tags provides evidence for the presence of paralogs, missing in the Arabidopsis genome, and for genes with abiotic stress-relevant functions. Hybridizations of salt cress RNA targets to an Arabidopsis whole-genome oligonucleotide array indicate that commonly stress-associated transcripts are expressed at a noticeably higher level in unstressed salt cress plants and are induced rapidly under stress. Efficient transformation of salt cress allows for simple gene exchange between Arabidopsis and salt cress. In addition, the generation of T-DNA-tagged mutant collections of salt cress, already in progress, will open the door to a new era of forward and reverse genetic studies of extremophile plant biology.

Figure 1.

Effect of NaCl on shoot FW of salt cress and Arabidopsis (Col gl1). Plants were grown from seeds in Turface, and NaCl was increased incrementally in the irrigation water every 7 d to final concentrations of 100, 150, and 200 mm for Arabidopsis and 100, 200, 300, and 500 mm for salt cress. Shoots were harvested every 7 d through day 42, after which time the sampling interval was reduced to 4 to 6 d. A to C, Comparisons of the two species at 0, 100, and 200 mm, respectively. D and E, Effects of unequal treatment levels (higher in salt cress and lower in Arabidopsis) because concentrations above 200 mm were lethal in Arabidopsis. White circles, salt cress (T.h.); black circles, Arabidopsis (A.t.). Values are means ± se; n = 8.

Figure 2.

Effect of NaCl on root FW of salt cress and Arabidopsis (Col gl1). Plants were grown from seeds in Turface, and NaCl was increased incrementally in the irrigation water every 7 d to final concentrations of 100, 150, and 200 mm for Arabidopsis and 100, 200, 300, and 500 mm for salt cress. Roots were harvested every 7 d through day 42, after which time the sampling interval was reduced to 4 to 6 d. A to C, Comparisons of the two species at 0, 100, and 200 mm, respectively. D and E, Effects of unequal treatment levels (higher in salt cress and lower in Arabidopsis) because concentrations above 200 mm were lethal in Arabidopsis. White circles, salt cress (T.h.); black circles, Arabidopsis (A.t.). Values are means ± se; n = 8.

Figure 3.

Effect of increasing concentration of NaCl on shoot and root FW of salt cress and Arabidopsis (Col gl1). Three-week-old seedlings grown in Turface were irrigated with 75 mm NaCl for 5 d, and salt concentration was then gradually increased to 150, 200, 300, 400, and 500 mm on days 5, 9, 16, 22, and 26, respectively, as denoted by vertical arrows on the x axis. Salt cress plants were irrigated every other day with 500 mm NaCl from day 30 to 58. After sampling on day 58, the remaining plants were rewatered with either 0 or 100 mm, and growth was assessed again after 9 d. Arabidopsis did not survive beyond day 30. White circles, salt cress; black circles, Arabidopsis. Values are means ± se; n = 8.

Figure 4.

Effect of NaCl on Na⁺ and K⁺ content in leaves of salt cress and Arabidopsis. Three-week-old plants were grown in Turface and salinized incrementally, every 7 d, to final concentrations of 0, 100, 200, 300, and 500 mm NaCl. For salt cress, concentration was incremented at 100 mm intervals, while 50 mm increments were used for Arabidopsis. Arabidopsis did not survive NaCl concentrations higher than 200 mm. Na⁺ and K⁺ content were measured 28 d after the onset of salt treatment. White bars, salt cress; black bars, Arabidopsis. Values are means ± se; n = 9.

Figure 5.

Pro and total soluble sugar accumulation of salt cress plants subjected to NaCl stress. A, Three-week-old plants were grown in Turface and salinized incrementally, every 7 d, to final concentrations of 0, 100, 200, 300, and 500 mm NaCl. Leaf tissue was sampled for Pro determination on days 36, 47, 60, and 70.Values are means ± se, n=3. B, Four-week-old plants were grown in soil and treated with four levels of NaCl for 3 weeks. Total soluble sugar accumulation was measured by the anthrone method. Values are means ± se, n = 3.

Figure 6.

The effect of NaCl on germination of salt cress and Arabidopsis (Col gl1). A, Seeds were sown on agar plates containing MS medium or MS medium supplemented with NaCl. Germination was recorded 7 d after sowing. B, Seeds were sown on agar plates containing MS medium supplemented with NaCl and rescued to MS medium without salt after 8 d. Germination was measured following an additional 3 d and calculated as a percentage relative to that on MS medium. C, Seeds were sown on agar plates containing MS medium supplemented with ABA. Germination was recorded 10 d after sowing. White bars, salt cress; black bars, Arabidopsis. Values are means ± se; n = 3.

Figure 7.

Tissue water relations of salt cress and Arabidopsis (Col gl1) plants subjected to NaCl stress. Plants were grown in Turface and irrigated with four levels of NaCl for 18 d. Measurements were made every 6 d and data were combined over all sample dates. Responses of salt cress to 100 mm NaCl were not determined. A, Leaf osmotic potential measured on expressed sap of frozen and thawed leaf samples of plants irrigated with 200 mm NaCl for 18 d. B, Leaf turgor pressure was estimated as the difference between water potential and osmotic potential. C, Leaf osmotic potential measured as in A. D, Leaf water potential was measured in single leaves with a Scholander-type pressure chamber. White bars, salt cress; black bars, Arabidopsis. Values are means ± se; n = 9.

Figure 8.

Stomatal conductance and diurnal whole-plant transpiration in salt cress and Arabidopsis (Col gl1). A, Plants were grown in Turface and irrigated with NaCl for 18 d. Stomatal conductance was measured with a PP Systems CIRAS-1 portable photosynthesis system every 6 d, and data are combined over all sample dates. Responses of salt cress to 100 mm NaCl were not determined. White bars, salt cress; black bars, Arabidopsis. Values are means ± se; n = 9. B, Four-week-old Arabidopsis (Col-0) and equivalently sized salt cress seedlings grown under long-day conditions with cool-white fluorescent lighting were used for measurements of whole-plant water loss. Plants were grown singly in 9-cm pots, which were sealed in plastic wrap and placed on electronic balances. Weight was determined every 30 min for 7 d. Values are means of 8 and 16 plants for Arabidopsis and salt cress, respectively. White circles, salt cress; black circles, Arabidopsis.

Figure 9.

Cross-sectional leaf and root anatomy of salt cress. A, First fully expanded leaf from a 3-month-old plant. B, Mature fully expanded leaf, sampled from a 3-month-old plant that had been treated with 500 mm NaCl for 40 d. C, Root cross-section taken approximately 1 mm from the root tip. ep, Epidermis; c, cortex; e, endodermis; p, pericycle.

Figure 10.

Effect of NaCl on shoot succulence of salt cress and Arabidopsis (Col gl1). NaCl was increased in the irrigation water in 50 mm increments every 7 d to final concentrations of 100 and 200 mm. NaCl treatment continued up to day 70, at which time plants were rewatered with 0 mm NaCl for an additional 19 d. FW to DW ratios were measured on day 42 in Arabidopsis (black bars) and salt cress (white bars) and on day 89 in salt cress (hatched bars); no Arabidopsis plants survived beyond 58 d. Values are mean ± se; n = 8.

Figure 11.

Bright-field light microscopic images of adaxial leaf surfaces of Arabidopsis and salt cress. Images are from leaf surface imprints obtained using cyanoacrylate adhesive. Scale bar is 100 μm. A, Arabidopsis, rosette leaf; B, salt cress, cauline leaf; C, salt cress, rosette leaf.

Figure 12.

Cold tolerance of salt cress plants compared to Arabidopsis. Arabidopsis (A) and salt cress (B) plants after freezing treatment (−15°C for 24 h). Plants were acclimated for 7 d at 4°C prior to freezing treatment. The photograph was taken 1 week after freezing treatment.

Figure 13.

A, The chromosome complement of salt cress root cells comprises 14 chromosomes. B, DNA content of salt cress compared to Arabidopsis.

Figure 14.

Salt cress gene expression may be recorded using Arabidopsis oligonucleotide-based microarrays. Compared are sequences of two Arabidopsis genes (At2g41430—ERD15; At1g01720—similar to NAC domain protein No Apical Meristem (NAM) GB:AAD17313) with salt cress EST sequences (BM985810, BQ060374). Their signals on the microarray slides are boxed. Also, false-color intensities for three control genes are included. For ERD15, the position of the oligonucleotide printed on microarrays is identified by the single-letter amino acid code, indicating the C terminus of the protein. The comparison with salt cress shows a deletion of two amino acids and several single-nucleotide exchanges in the 3′ UTR.

Figure 15.

Root-bending assay of wild-type salt cress and the let-1 mutant. Surface-sterilized seeds were sown on cellophane membrane overlaying an agar surface. After stratification for 3 d at 4°C, plates were incubated at 22°C for 6 d in a vertical position. The membranes with seedlings were then transferred to plates with agar medium containing 0 or 200 mm NaCl and rotated 180°; the photograph was taken 10 d later.

Figure 16.

Effect of NaCl on Pro content and ion accumulation in leaves of salt cress wild type and EMS mutant let1. Plants were grown in Turface calcined clay and irrigated with 0, 200, and 400 mm NaCl for 18 d. White bars, salt cress wild type; black bars, salt cress mutant let1. Levels of Pro and Na⁺ and K⁺ were determined as described in “Materials and Methods.” Values are means ± se; n = 3. Leaf Na⁺ content of salt-treated salt cress wild type and EMS mutant let1 are significantly different from each other (P < 0.05), while leaf K⁺ content and leaf Pro content are not significantly different at P = 0.05; Student's t test analysis was done by using grand means.