Stress tolerance to stress escape in plants: role of the OXS2 zinc-finger transcription factor family

Abstract

During dire conditions, the channelling of resources into reproduction ensures species preservation. This strategy of survival through the next generation is particularly important for plants that are unable to escape their environment but can produce hardy seeds. Here, we describe the multiple roles of OXIDATIVE STRESS 2 (OXS2) in maintaining vegetative growth, activating stress tolerance, or entering into stress-induced reproduction. In the absence of stress, OXS2 is cytoplasmic and is needed for vegetative growth; in its absence, the plant flowers earlier. Upon stress, OXS2 is nuclear and is needed for stress tolerance; in its absence, the plant is stress sensitive. OXS2 can activate its own gene and those of floral integrator genes, with direct binding to the floral integrator promoter SOC1. Stress-induced SOC1 expression and stress-induced flowering are impaired in mutants with defects in OXS2 and three of the four OXS2-like paralogues. The autoactivation of OXS2 may be a commensurate response to the stress intensity, stepping up from a strategy based on tolerating the effects of stress to one of escaping the stress via reproduction.

Figure 1

OXS2 is needed to delay floral transition. (A) Abbreviated model for the regulation of floral transition. (B) Structure of OXS2 gene (At2g41900) and partial OXS2 cDNA (AT3, black rectangle). Blue boxes: ANKYRIN repeats; red box: ZF domain; green boxes: polyglutamine stretches; grey boxes: untranslated regions; line: intron; red lollipop: T-DNA insertion in oxs2 alleles. (C, D) Flowering time of growth chamber plants measured by total number of primary leaves at bolting. (C) Homozygous plants subjected to long days at 21°C without or with a drought stress (water withheld after 7 days of growth); s.e. from >12 plants. Lower panel: RT–PCR analysis on 12-day-old seedlings grown on plates; g, genomic DNA. (D) Mutant rescue of oxs2-1 with a 35S-OXS2 construct. A, G, and H represent three independent lines homozygous for the transgene; Lower panel: northern blot analysis of OXS2 expression.

Figure 2

OXS2 paralogous genes display differences and similarities in their regulation and function relative to stress. (A) Chromosome map of OXS2 and O2L paralogues; genetic linkage of OXS2 and O2L4 was broken once in 400 F2 siblings of a cross between oxs2-1 and o2l4-1. (B) Gene structure and T-DNA insertions as in Figure 1B. Polyglutamine stretches are specific features of OXS2 but not other members of the family, and hence not indicated. RT–PCR data show transcripts not detected in homozygotes of mutant alleles. (C, D) Root length of Arabidopsis seedlings grown vertically for 10 days on plates containing growth medium without or with diamide (1 mM result chosen for illustration) or a range of t-BOOH concentrations; error bars=s.d. from ⩾10 root measurements; ^* indicates statistical difference between wild type and the mean value of the four oxs2 alleles combined (α<5%). (E). Subcellular localization of OXS2 and paralogous proteins tagged with GFP and transiently expressed in onion epidermal cells before and after 1 h exposure at 4°C.

Figure 3

OXS2 localization affects flowering time. (A) Subcellular localization in onion epidermal cells of OXS2 or derivative proteins tagged with GFP or dsRed expressed from 35S promoter; numbers indicate amino-acid positions; ankyrin repeats (ANK), ZF domain (ZF), and polyglutamine stretches (Q) as Figure 1B; L708P is point mutation in OXS2 NES; inverted triangles show NES from OXS2, O2L1, or HIV-1 Rev (rev); NLS is from SV40 protein. Bar=20 μm. (B, C) Confocal microscopy of (B) protoplasts from plants transgenic for OXS2–GFP (RB39) treated 3 h without or with ABA (100 μM) or leptomycin B (LMB, 50 ng/ml). Bar=1 μm; or (C) adaxial side of Arabidopsis leaves from RB39 (full-length OXS2), RB43 (N-terminus deletion), RB44 (NES-rev in place of one ankyrin repeat), RB40 (C-terminus deletion); c: cytosol, chl: chloroplast, gc: guard cell, n: nucleus. (D) Localization determined by transient expression in onion cells and confirmed in transgenic Arabidopsis (except for RB206 (L708P aa substitution) as none of the tested transgenic plants (n>12) yielded sufficient GFP signal for imaging, although transgene expression was confirmed by RT–PCR); + or − indicate the relative presence or absence, respectively, of OXS2 or derivative proteins tagged with GFP in (c) cytoplasm, (n) nucleus, or (n^S) nucleus of cells subjected to stress; ND, not determined. Flowering time recorded for primary leaves at bolting on two separate lines expressing similar levels of detectable OXS2–GFP; grown in greenhouse corresponding to mild stress conditions; s.e. from 12 plants; Ler, wild type. (E) OXS2 transactivation of Gal4 promoter–luciferase gene fusion. Each activator construct encodes a fusion to the Gal4 DNA binding domain (G4DBD). G4AD, Gal4 activation domain; ΔZF, deletion of ZF domain (aa 251–446, as in RB41); ΔC1 and ΔC2, 186 aa (as in RB40) and 112 aa (as in RB50) C-terminal deletions of the putative activation domain, respectively; ΔQ: deletion of the polyglutamine rich domain (aa 508–634). (F) RT–PCR analysis of flowering genes in plants transgenic for RB43, RB206, RB39 and control RB40 that exhibit wild-type flowering time; 7-day-old seedlings grown on agar plates 21°C, continuous light. ^*Expression of SOC1 and LFY on 4-day-old seedlings; EF1α, internal control. Unlike other OXS2 variants, inert RB40 is expressed at higher level, hence higher OXS2 mRNA due also to the RB40 transgene. (G) Relative expression of SOC1 and LFY determined by quantitative RT–PCR. Measurements of the mean±s.d. of triplicates extrapolated on standard curves of each tested gene.

Figure 4

OXS2 activation of floral integration genes associated with BOXS2 elements. (A) Selection and amplification binding (SAAB) assay using OXS2 recombinant protein. Sequence logo: letter sizes proportional to relative occurrence of the nucleotide in the SAAB population. P^(e): estimated occurrence of the motif in A. thaliana promoter regions (C, G=0.15; T, A=0.35). (B) SOC1 promoter and EMSA (electromobility shift assay) with E. coli-produced OXS2 protein using probe x, y, or z. Left panels, 4% acrylamide gel; right panel, 1.5% agarose vertical gel. FP, free probe: band x, y, or z; b, bound probe. ^*Low-molecular weight complexes. Cold probe is unlabelled z competitor; dIdC, non-specific competitor. (C) Immunoblot using anti-GFP antibody on cytoplasmic (C) or nuclear (N) fraction from 10-day-old seedlings transgenic for RB39, RB40, or histone H4–GFP (H4). (D) Chromatin immunoprecipitation (ChIP) on SOC1 promoter (−446 to +23) from plants transgenic for GFP or OXS2–GFP (RB39); i, input; b, beads; αG, anti-GFP serum used for IP; ACT2, ACTIN2 primers used as control; Enrichment established as (αG/i)SOC1/(αG/i)ACT2 after qPCR. qPCR results presented as SOC1 enrichment in OXS2–GFP plants versus GFP plants normalized to the ACT2 DNA enrichment set to 1. (E) Transient expression assays. Firefly luciferase gene fused to promoter from SOC1 (P_SOC1) (Hepworth et al, 2002), LFY (P_LFY), AP1 (P_AP1), FUL (P_FUL), TFL1 (P_TFL1) or OXS2 (P_OXS2) assayed for expression with (+) or without (−) co-transformed 35S-OXS2. Grey box: CT-rich region; black boxes: putative BOXS2 motifs with sequence and position shown relative to transcript start (arrowheads). Error bar=s.e. of triplicate experiments.

Figure 5

OXS2 and O2L genes regulate stress-induced flowering. (A) Flowering time of greenhouse grown plants measured as total leaf number; mean±s.d. (n=12–16); filled square, mutant allele; open square, wild-type allele. (B–E) Plants grown in environmental chambers. (B) Stress-induced flowering in wild type and the most delayed mutant combination o2l⁴ (oxs2-1 o2l1-1 o2l3-1 o2l4-1); drought stress applied 7 days after germination by withholding water until bolting. (C–E) SOC1 expression assessed by RT–qPCR. (C) Wild-type seedlings, the quadruple mutant o2l⁴ and the quintuple mutant o2l⁵ grown 10 days in short days—to prevent photoperiodic induction of SOC1—and treated with or without t-BOOH for 3 h. (D) Wild type and o2l⁵ from days 4 to 11 under continuous light. (E) SOC1 induction in wild type and o2l⁵ by shifting to long-day growth following 10 short days.

Figure 6

Model of OXS2 regulation of stress tolerance or stress escape. OXS2^C: cytoplasmic OXS2; OXS2^N: nuclear OXS2; bottom arrow: vegetative to reproductive transition. T-bars and arrows indicate repression and induction, respectively. In the absence of stress (green lines), OXS2^C is due to XPO1-mediated nuclear exclusion and is needed to delay flowering. During low stress intensity (orange lines), nuclear accumulation of OXS2 activates stress tolerance and the alleviation of stress replenishes OXS2^C for delayed flowering. Under high stress intensity (red lines), higher accumulation of OXS2^N results from autoactivation and translocation from the cytoplasm for a stress escape response, and in conjunction with family members O2L1, 3, and 4, activate the reproductive phase via the floral integrator genes.