An insertional mutagenesis programme with an enhancer trap for the identification and tagging of genes involved in abiotic stress tolerance in the tomato wild-related species Solanum pennellii

Abstract

Salinity and drought have a huge impact on agriculture since there are few areas free of these abiotic stresses and the problem continues to increase. In tomato, the most important horticultural crop worldwide, there are accessions of wild-related species with a high degree of tolerance to salinity and drought. Thus, the finding of insertional mutants with other tolerance levels could lead to the identification and tagging of key genes responsible for abiotic stress tolerance. To this end, we are performing an insertional mutagenesis programme with an enhancer trap in the tomato wild-related species Solanum pennellii. First, we developed an efficient transformation method which has allowed us to generate more than 2,000 T-DNA lines. Next, the collection of S. pennelli T(0) lines has been screened in saline or drought conditions and several presumptive mutants have been selected for their salt and drought sensitivity. Moreover, T-DNA lines with expression of the reporter uidA gene in specific organs, such as vascular bundles, trichomes and stomata, which may play key roles in processes related to abiotic stress tolerance, have been identified. Finally, the growth of T-DNA lines in control conditions allowed us the identification of different development mutants. Taking into account that progenies from the lines are being obtained and that the collection of T-DNA lines is going to enlarge progressively due to the high transformation efficiency achieved, there are great possibilities for identifying key genes involved in different tolerance mechanisms to salinity and drought.

Fig. 1

Summary of the screening process of the collections of S. pennellii enhancer trap lines carried out in non-stress and abiotic stress conditions. ¹Long-term and mid-term experiments are described in “Materials and methods”

Fig. 2

Salt tolerance in the accession ‘20164’ of S. pennellii. a, b No difference was observed between plants grown in control (a) and stress conditions (b) after irrigation with 200 mM NaCl for 4 months. c No symptoms of wilting, senescence or necrotic spots were seen in adult leaves of plants grown under salt stress (right) as compared with those grown in control conditions (left)

Fig. 3

A high-throughput transformation method for the generation of S. pennellii T-DNA lines. a Leaf explants developing multiple shoot-buds on kanamycin-free organogenic culture medium. b Absence of growth and organogenesis in non-inoculated explants sown in kanamycin containing culture medium. c–e Inoculated explants developing shoot-buds in selective conditions. Despite the great number of transformation events per inoculated explant only those sufficiently far away to one another were selected to avoid the possibility to recover plants coming from the same transformation event. f Shoot elongation process on kanamycin-containing culture medium. g Collection of T-DNA lines in the growth chamber. h, i Acclimatization and cultivation of T-DNA lines in the greenhouse

Fig. 4

Reporter gene expression in specific plant organs of different S. pennellii T-DNA lines. a Root hairs. b Pollen grains and anther tissue. c Stigma. Vascular bundles of stem (d) and leaf (e) under stress conditions. f, g Trichomes. Close-ups show reporter expression in all cells of these specialized structures (f) or just in the terminal cell (g). Stomata of leaves (h) and pedicel (i) Bar 1 mm

Fig. 5

S. pennellii mutants showing sensitivity to salinity and drought. a, b Plants grown in control conditions (a) and 200 mM NaCl for 15 days (b) of one selected line from ac. PE47, named SP salt sensitive-2; WT plants were unaffected by salt treatment at this time (close-ups), whereas mutant showed necrosis in leaves spreading from the bottom to the upper part of the plant. c, d Drought sensitivity of one selected line from ac. 20164, named SP drought sensitive-1; control plants were well-watered (c) and drought plants were watered with 30% of the nutrient solution used in the control plants for 60 days (d). The hypersensitivity symptoms were manifested by the wilting and senescence of basal leaves, which spread from the basal to the upper part of the plant, whereas WT plants only showed reduced leaf area but not symptoms of senescence (close-ups). Moreover, the reporter gene expression (GUS) was increased by stress; expression in stem vascular bundles and stomata of non-stressed plants (e, f) and drought stressed plants (g, h). Bar 1 mm

Fig. 6

S. pennellii mutants altered in developmental traits. a Leaves from wild-type plants. b Mutant with chlorophyll-deficient leaves. c Comparison between root development in vitro of a mutant (right) with respect to wild-type plants (left). d Two clonal replicates of an mutant (right) altered in growth habit with respect to that of wild-type plants (left). e Inflorescence architecture of an mutant (right) and that of a wild-type plant (left). f Homeotic conversion of floral whorls in a T-DNA line (down) as compared to normal floral development in wild type plants (up). g, h Fruits of WT (g) and a mutant with larger size fruit (h). Crosses between WT and mutant were carried out using the mutant as donor parental. i On a WT plant, one fruit of WT (left) and one of the hybrid WT × mutant (right), with a fruit size around three times greater than that of the WT

Fig. 7

Phenotype of the S. pennellii succulent leaves-1 mutant (ac. PE47). The higher chlorophyll content and the succulence of the leaves were clearly observed both in vitro and in vivo. Under salinity (17 days of 200 mM NaCl treatment), the mutant shows lower Na⁺ accumulation and Na⁺/K⁺ ratio in its leaves than WT (M = SPsl-1 mutant)