The Solanum lycopersicum Zinc Finger2 cysteine-2/histidine-2 repressor-like transcription factor regulates development and tolerance to salinity in tomato and Arabidopsis

Abstract

The zinc finger superfamily includes transcription factors that regulate multiple aspects of plant development and were recently shown to regulate abiotic stress tolerance. Cultivated tomato (Solanum lycopersicum Zinc Finger2 [SIZF2]) is a cysteine-2/histidine-2-type zinc finger transcription factor bearing an ERF-associated amphiphilic repression domain and binding to the ACGTCAGTG sequence containing two AGT core motifs. SlZF2 is ubiquitously expressed during plant development, and is rapidly induced by sodium chloride, drought, and potassium chloride treatments. Its ectopic expression in Arabidopsis (Arabidopsis thaliana) and tomato impaired development and influenced leaf and flower shape, while causing a general stress visible by anthocyanin and malonyldialdehyde accumulation. SlZF2 enhanced salt sensitivity in Arabidopsis, whereas SlZF2 delayed senescence and improved tomato salt tolerance, particularly by maintaining photosynthesis and increasing polyamine biosynthesis, in salt-treated hydroponic cultures (125 mm sodium chloride, 20 d). SlZF2 may be involved in abscisic acid (ABA) biosynthesis/signaling, because SlZF2 is rapidly induced by ABA treatment and 35S::SlZF2 tomatoes accumulate more ABA than wild-type plants. Transcriptome analysis of 35S::SlZF2 revealed that SlZF2 both increased and reduced expression of a comparable number of genes involved in various physiological processes such as photosynthesis, polyamine biosynthesis, and hormone (notably ABA) biosynthesis/signaling. Involvement of these different metabolic pathways in salt stress tolerance is discussed.

Figure 1.

Identification of SlZF2. A, Amino acid sequence comparison of SlZF2 and StZFP1, SlZF1, CaZFP1, AtZAT10, and AtAZF1. Identical residues are shown in black and conserved residues are in dark gray. The C2H2 domains, as well as the nuclear localization site (B box), L box, and EAR motif, are labeled. B, Phylogenetic relationships between SlZF2 (ADZ15317) and zinc finger proteins from different species: tomato SlZF1 (ACG50000), Arabidopsis ZAT10 (Q96289), AZF1 (BAA85108), ZAT7 (Q42453), ZAT12 (Q42410), tobacco NtZFP (AAC06243), potato StZFP1 (ABK78777), pepper CaZFP1 (AAQ10954), and rice OsZFP252 (AAO46041).

Figure 2.

qRT-PCR analysis of the SlZF2 spatiotemporal expression profile during tomato plant development in vegetative organs (A) and reproductive organs (B). Actin and GAPDH were used as internal controls. Data represent means and sd of three replicates. Bre, Breaker stage; Flo, flower; Gre, green stage; OL, old leaf; Red, red stage of tomato development; Ro, root; St, stem; YL, young leaf. Letters indicate values of SlZF2 expression that differ significantly between tomato organs according to the Student-Newman-Keuls test at P < 0.05.

Figure 3.

SlZF2 transcriptional properties. A, Transient expression of YFP-SlZF2 fusion protein in tomato leaf protoplasts: YFP-SlZF2 (Aa and Aa′), YFP control fluorescence (Ab and Ab′), and bright field/chlorophyll/YFP fluorescence respectively. B, SlZF2 transactivation ability. The SlZF2 coding region was fused to GAL4 DNA binding domain (DBD) into the pGBKT7 vector carrying the nutritional marker TRYPTOPHAN1 as the reporter gene. Yeasts were transformed with pGBKT7 empty vector (Ba and Ba′) or with the GAL4-DBD-SlZF2 construct (Bb and Bb′). Both constructs were transformed into the yeast strain Y8930 harboring the ß-galactosidase L, ADE2, and HIS3 reporter genes. Yeasts were separately grown on synthetic dropout medium lacking either Trp (Ba and Bb), or A and His (Ba′ and Bb′). C, Position weight matrix representation of the three top-scoring 8 mers obtained in a seed-and-wobble algorithm. D, Box plot representation of signal intensities of the probes containing the elements indicated. Different combinations of the AGT modules in bipartite motifs are shown in blue, whereas their corresponding mutant versions are shown in red. Letters above the boxes represent different groups of statistical significance, relative to the motif ACTnnnAGT with highest median intensity, as follows: a, P > 0.05 (no significant differences); b, P < 0.01; c, P < 0.001; and d, P < 0.0001 (Wilcoxon exact test). All of the mutant derivatives grouped together at the same significance group (P < 2.2e-16). The number of probes containing the elements indicated ranges from 243 to 303.

Figure 4.

A and B, qRT-PCR analysis of SlZF2 expression pattern in response to salinity (150 mm NaCl) in roots (A) or leaves (B; young and old leaves for 50-d-old plants). C, Expression of SlZF2 in roots of young tomato plants (3 weeks old) submitted to 150 mm NaCl, 150 mm KCl, or drought. Actin and GAPDH were used as internal controls. Data represent means and sd of three replicates. Letters indicate values of SlZF2 expression that differ significantly between treatments according to the Student-Newman-Keuls test at P < 0.05.

Figure 5.

Overexpression of SlZF2 enhances salinity sensitivity in Arabidopsis. A, Transfer DNA (T-DNA) insertion in the Arabidopsis genome and expression of SlZF2 in L5, L6, and L9 transgenic lines and a control line transformed with empty vector (V) were analyzed by PCR. B, Phenotype of transgenic Arabidopsis plants grown on one-half-strength MS medium and then transferred to soil, and ectopically expressing SlZF2 (Ba, Bc, Be, Bg, and Bh) compared with control plants (Bd and Bf). C, Phenotype of Arabidopsis V (Ca), L5 (Cb), L6 (Cc), and L9 (Cd) lines 5 d after transfer on 125 mM NaCl medium. D, Pro accumulation in control (V) and SlZF2 transgenic lines grown for 5 d on 80 mm NaCl. E, MDA accumulation in control (V) and SlZF2 transgenic lines grown 5 d on 80 mm NaCl. Data represent means and se of three replicates. Letters indicate values that differ significantly between SlZF2-transgenic Arabidopsis and control V lines according to the Student-Newman-Keuls test at P < 0.05.

Figure 6.

Effects of SlZF2 ectopic expression in tomato. A, Transfer DNA (T-DNA) insertion in tomato genome and expression of SlZF2 in Z4, Z5, and Z16 transgenic lines and wild-type (WT) plants were analyzed by PCR. B, Defaults of germination of SlZF2 transgenic seeds on MS medium supplemented with 100 mg/L kanamycin. C, Dwarfism of 35S::SlZF2 tomato seedlings grown in vitro compared with wild-type tomatoes. D, Fully expanded leaf of transgenic (Da) and wild-type (Db) plants. E, Flowers of SlZF2-transgenic (Ea, Eb, and Ec) or wild-type (Ed and Ee) plants. F, Details of 35S::SlZF2 and wild-type tomato fruits (Fa and Fb, respectively) and according to a longitudinal section (Fc and Fd, respectively), and seeds of transgenic (Fe) and wild-type (Ff) tomatoes.

Figure 7.

Characterization of hydroponic cultures of SlZF2 transgenic and wild-type tomatoes exposed to salinity (125 mm NaCl) during 3 weeks. A, Phenotype of wild-type (WT; Aa) and transgenic (Ab) tomatoes after 10 d. Accelerated senescence of the wild type (Ac and Ae) compared with transgenic tomatoes (Ad and Ae) is revealed by leaf chlorosis and flower abortion. Transfer of transgenic and wild-type tomatoes from hydroponics to soil results in death of wild-type plants (Af). B, MDA accumulation in transgenic (Z4, Z5, and Z16) and wild-type tomato lines exposed to salinity. C, Water content of transgenic and wild-type leaf 3. D, Osmotic potential of transgenic and wild-type leaf 5. E, Sodium accumulation in leaf 3 of Z4, Z5, Z16, and wild-type plants. Data represent means and se of at least two replicates. Letters indicate values that significantly differ between SlZF2 transgenic tomatoes and the wild type according to the Student-Newman-Keuls test at P < 0.05.

Figure 8.

Photosynthesis parameters of SlZF2 transgenic and wild-type (WT) tomatoes exposed to salt (125 mm NaCl) for 3 weeks. A, F_v/F_m, ΦPSII, and NPQ of transgenic and wild-type leaf 3. B and C, Leaf stomatal conductance (g_s; B) and net assimilation of CO₂ (A) and net transpiration rate (E) of transgenic and wild-type tomato lines (C). Data represent means and se of at least two replicates. Letters indicate values that significantly differ between SlZF2 transgenic tomatoes and the wild type according to the Student-Newman-Keuls test at P < 0.05.

Figure 9.

Free PA (spermine, spermidine, and putrescine) content in tomato seedlings grown in hydroponics under control conditions or 5 d of salt (125 mm NaCl) stress. Data represent means and se of six replicates. Letters indicate values that significantly differ between SlZF2 transgenic tomatoes and wild-type (WT) lines according to the Student-Newman-Keuls test at P < 0.05.

Figure 10.

SlZF2 hormonal regulation. A, qRT-PCR analysis of SlZF2 expression in response to different hormonal treatments. EF1α and GAPDH were used as internal controls for ABA, CK, and IAA treatments. Data represent means and sds of three replicates. B, Viviparous phenotype of SlZF2 seeds in Z4 transgenic fruits. Germinating seeds developing in transgenic tomato fruits at harvest (Ba). Details of emerging radicals and hypocotyls (Bb). C, Insensitivity of Arabidopsis L5 (Cb), L6 (Cc), and L9 (Cd) transgenic seeds to ABA compared with a control line (Ca). Plants were grown on one-half-strength MS medium supplemented with 0.2 μM ABA. D and E, Phytohormone content in Z4, Z5, and Z16 compared with wild-type tomato seedlings grown in vitro on regular MS medium. Data represent means and se of two readings of the same extract. Each extract concerns at least four plants, and the experiment was repeated twice. Different letters indicate significant differences between treatments or tomato lines according to the Student-Newman-Keuls test at P < 0.05. cZ, cis-Zeatin; tZ, trans-Zeatin; WT, Wild type.

Figure 11.

Microarray analysis of differentially regulated genes between wild-type and Z4 transgenic lines: number and categories of genes up- and down-regulated in tomato Z4 transgenic line compared with the wild type.

Figure 12.

qRT-PCR analysis of SlZF2 putative target genes expression in transgenic tomato lines Z4, Z5, and Z16 compared with wild-type (WT) tomatoes, under control conditions (A) or after salt stress (B; 125 mm NaCl, 48 h). Actin and GAPDH were used as internal controls. Data represent means and sds of three replicates.

Figure 12.

qRT-PCR analysis of SlZF2 putative target genes expression in transgenic tomato lines Z4, Z5, and Z16 compared with wild-type (WT) tomatoes, under control conditions (A) or after salt stress (B; 125 mm NaCl, 48 h). Actin and GAPDH were used as internal controls. Data represent means and sds of three replicates.