Change of function of the wheat stress-responsive transcriptional repressor TaRAP2.1L by repressor motif modification

Abstract

Plants respond to abiotic stresses by changes in gene regulation, including stress-inducible expression of transcriptional activators and repressors. One of the best characterized families of drought-related transcription factors are dehydration-responsive element binding (DREB) proteins, known as C-repeat binding factors (CBF). The wheat DREB/CBF gene TaRAP2.1L was isolated from drought-affected tissues using a dehydration-responsive element (DRE) as bait in a yeast one-hybrid screen. TaRAP2.1L is induced by elevated abscisic acid, drought and cold. A C-terminal ethylene responsive factor-associated amphiphilic repression (EAR) motif, known to be responsible for active repression of target genes, was identified in the TaRAP2.1L protein. It was found that TaRAP2.1L has a unique selectivity of DNA-binding, which differs from that of DREB activators. This binding selectivity remains unchanged in a TaRAP2.1L variant with an inactivated EAR motif (TaRAP2.1Lmut). To study the role of the TaRAP2.1L repressor activity associated with the EAR motif in planta, transgenic wheat overexpressing native or mutated TaRAP2.1L was generated. Overexpression of TaRAP2.1L under constitutive and stress-inducible promoters in transgenic wheat and barley led to dwarfism and decreased frost tolerance. By contrast, constitutive overexpression of the TaRAP2.1Lmut gene had little or no negative influence on wheat development or grain yield. Transgenic lines with the TaRAP2.1Lmut transgene had an enhanced ability to survive frost and drought. The improved stress tolerance is attributed to up-regulation of several stress-related genes known to be downstream genes of DREB/CBF activators.

Keywords: EAR motif; dehydration-responsive element binding proteins; drought; frost; transcriptional repressor; transgenic wheat.

Figure 1

Expression of TaRAP2.1L in a variety of wheat tissues (cv. RAC875) in the absence of stress and under different stresses. Levels of expression were detected by quantitative RT‐PCR and are shown as normalized transcript levels in arbitrary units. a, Expression in different tissue types under nonstressed conditions; b, expression in leaves in hydroponically grown seedlings treated with 200 μm ABA; c, expression in leaves of plants grown in soil during slowly developing drought; d, expression in leaves of 3‐week‐old seedlings under cold stress (4 °C); e, expression in mechanically wounded (with a fine metal brash) leaves of 3‐week‐old seedlings. Error bars represent standard errors.

Figure 2

Expression of DREB activator (TaDREB3) and repressor (TaRAP2.1L) genes in rapidly dehydrated leaves. Levels of expression were detected by quantitative RT‐PCR and are shown as normalized transcript levels in arbitrary units. TaCor39 was used to control the quality of cDNA series as a known dehydration‐responsive gene. Error bars represent standard errors.

Figure 3

A sequence alignment and molecular folds of AP2 domains of TaRAP2.1L and AtERF1 in complex with cis‐elements. (a) A sequence alignment of the AP2 domains of TaRAP2.1L (target) and AtERF1 (template). Residues in the template sequence which are identical to the residue in the target sequence are coloured. Colouring scheme is based on the property of amino acids, where Arg or Lys and Glu or Asp are coloured in blue and red, respectively, while other conserved residues are coloured in other colours. Secondary structure elements are indicated above the sequences (blue C‐coil, black S‐sheet, red H‐α‐helix). The positions of highly conserved Arg Gly and Trp residues involved in binding of cis‐elements are boxed. (b) C‐terminal sequences of the RAP/DREB clade (Figure S1, blue circles) with a DLNxxP EAR motif (highlighted in a cyan box); GenBank accession numbers of TFs are included. (c) Ribbon representations show the disposition of secondary structures, where antiparallel strands carry residues that contact cis‐elements. The ribbons are coloured in magenta (TaRAP2.1L/DRE), cyan (TaRAP2.1L/CRT), blue (TaRAP2.1L/GCC‐box) and yellow (AtERF1/GCC‐box). The black arrows point to the NH ₂‐termini of AP2 domains. The anticoding strands of cis‐elements GTCGGT, GTCGGC and GGCGGC are shown in sticks and are in atomic colours, except of the first (A or G) or penultimate (A or C) bases shown in cpk green and cpk yellow, respectively. Selected bases of coding strands (A, G and C) are shown in regular type, where bound to TaRAP2.1L. The interacting residues are shown in sticks and are coloured in cpk magenta (interacting with bases that differ between three elements), cpk cyan (interacting with bases that do not differ between three elements) and atomic cpk (noninteracting residues). Distances of 2.6 Å to 3.5 Å between contacting residues and cis‐elements are indicated by dotted lines.

Figure 4

DNA‐binding and trans‐activation properties of TaRAP2.1L and TaRAP2.1Lmut determined in yeast. (a) Activation was determined via activation assays in yeast using GAL4 BD fusions of the TaDREB3, TaRAP2.1L and TaRAP2.1Lmut proteins. BD‐TaDREB3 was used as a positive and the empty pGBKT7 vector as negative controls. (b) Binding of TaRAP2.1L and TaRAP2.1Lmut, fused with the GAL4 activation domains to DRE, CRT and GCC‐box cis‐elements, was determined via Y1H activation assays. A cis‐element HDZ1 specific for HD‐Zip I TFs and the original nonmodified y187 strain were used as negative controls.

Figure 5

TaRAP2.1L and TaRAP2.1Lmut overexpression in transgenic wheat and barley. (a) Phenotypes of representative plants of control wild‐type (WT) and two independent T₁ transgenic TaRAP2.1L barley lines (L6, upper panels; L15, lower panels) at 3 weeks (left panels) and 3 months (right panels) after germination. (b, c) Frost survival rates of (b) control (WT) and three transgenic lines of barley transformed with pUbi‐TaRAP2.1L, and (c) WT and four transgenic lines of wheat (cv. Bobwhite) transformed with pDhn8‐TaRAP2.1L. (d) Phenotypes of representative plants of WT and T₁ transgenic TaRAP2.1Lmut wheat (cv. Gladius) at grain filling. (e, f) Frost survival rates of WT and transgenic wheat transformed with pUbi‐TaRAP2Lmut. Frost survival rates of (e) T₁ lines and (f) T₃ homozygous progeny of the same lines. Statistical data were calculated from 12 plants. In panels b, c and f, standard error bars are indicated. In panel e, asterisks represent significant differences compared to control plants at the 5% (P < 0.05) significance level and were calculated by one‐way ANOVA (GenStat 9.0 VSN International, Hemel Hempstead, UK).

Figure 6

Evaluation of grain yields of transgenic wheat plants under two different water regimes using large containers. (a) Water tension parameters in soil and other conditions of the yield under drought experiment. (b) Images of wheat plants grown in large containers under well‐watered and drought conditions, for which growth characteristics and other analyses were performed.

Figure 7

Yield components and drought survival rates of control and transgenic wheat transformed with pUbi‐TaRAP2Lmut. (a) Growth characteristics and yield components of T₂ transgenic sublines grown in large containers under well‐watered conditions or moderate drought conditions. (b) Survival rates for T₃ transgenic sublines grown in pots, after rewatering from a severe drought event, were recorded after 6 weeks. Statistical data were calculated from 12 to 16 plants. Values represent means ± SE at P < 0.05 that were calculated by one‐way ANOVA (GenStat 9.0).

Figure 8

Expression levels of TaRAP2.1Lmut (transgene driven by the ubiquitin promoter), TaRAP2.1L (endogene) and several genes known to be downstream genes of DREB/CBF activators in leaves of nontransformed wild‐type (WT) (two control plants Control 1 and Control 2) and T₂ transgenic wheat lines. The levels of expression are shown as normalized transcript levels in arbitrary units. Error bars represent standard errors.