Figure 1.
Expression analysis of wheat TaHsfA2d.
(A) Northern blot expression analysis of EST representing wheat HSF in shoot, root tissues of two weeks old seedling and developing seed tissues. Potted plants were given heat shock at indicated temperature for two hrs and RNA from control (C) and heat shock treated (HS) plants was probed with EST as the probe (B) Expression analysis by semi quantitative RT-PCR, TaHsf (upper) and wheat actin (lower) in developing seed tissues of wheat (DAA-days after anthesis) at indicated time points. Heat stress was provided to potted plants at different time points during various stages of seed development after anthesis. The experiment was repeated two times.
Figure 2.
Phylogenetic relationship of TaHsfA2d with rice HSF gene family.
The tree was constructed in ClustalX by neighbor joining method with boot strap values and by using deduced amino acid sequence of newly identified wheat Hsf and amino acid sequences of all known rice Hsf proteins as reported earlier in Chauhan et al. (5)
Figure 3.
Schematic representation of wheat HsfA2d domains.
(A) numbers on top represents the positions of first and last amino acid of the particular domain. Relative length and identification of different domains was done by aligning deduced amino acid sequence of TaHsfA2d with rice OsHsfA2d (Os03g06630, http://www.uniprot.org/uniprot/Q8H7Y6). (B) Transcriptional activation assay of TaHsfA2d protein in yeast (AH109). ORF of TaHSFA2d was fused with GAL4 DNA-binding domain in plasmid pGBKT7 and transformed in AH109 yeast strain harboring the LacZ and HIS3 reporter genes as mentioned and selected on nutrient medium lacking His and Trp (right panel). Transformed yeast cells with the pGBKT7 vector alone were used as a control (Left panel). Transcriptional activation was also checked by β-galactosidase activity of LacZ reporter gene. The experiment was repeated three times.
Figure 4.
Expression analysis of AtHsfA2 and TaHsfA2d.
(A) Semi quantitative RT-PCR in two-weeks-old different Arabidopsis plants (WT, mutant and transgenics) under control and heat stress conditions, Arabidopsis Actin was used as internal control and the experiment was repeated three times. (B) Effect of high temperature stress of 42°C on PSII activity in terms of maximum photosynthetic efficiency (Fv/Fm) in rosette leaves of two-weeks-old different Arabidopsis plants (WT, mutant and transgenics). Measurements were taken by PAM fluorometer at different time points during stress and after two days of recovery. The values represent mean of at least ten individual plants of each line and the experiment was repeated three times. Error bar represents standard error (** represents p value = <0.01).
Figure 5.
Effect of constant heat stress on growth and development of different Arabidopsis plants.
(A) Growth and biomass accumulation in four weeks old different Arabidopsis plants (WT, mutant and transgenics) grown under moderate heat stress of 30°C. The seeds were first germinated at 20°C and grown for two weeks before transferring them to high temperature conditions for another two weeks and then photographed and other data was recorded. The values under fresh weight represent mean of at least ten individual plants of each line and error bar represents standard error based on three experiments (p = <0.01) (B) Steady state PSII activity in four-weeks-old Arabidopsis plants in terms of ETR and effective photosynthetic efficiency (YII) under continuous moderate heat stress of 30°C. The values represent mean percent reduction of at least ten individual plants of each line and the experiment was done three times and error bar represents standard error (** represents p value = <0.01).
Figure 6.
Growth and yield of Arabidopsis plants in terms of number of productive siliques and seed weight per plant.
The plants were grown in Soilrite under constant heat stress of 30°C in a growth chamber. The data was recorded after physiological maturity and represent means of at least ten individual plants per line and error bar represents standard error (** represents p value = <0.01). The scale bar represents 1 cm.
Figure 7.
Effect of salt stress on Arabidopsis plants.
(A) Effect of different concentrations of salt stress (NaCl) on percent germination of different Arabidopsis plants (WT, mutant and transgenics). Arabidopsis seeds were germinated on ½ MS for control condition and ½ MS supplemented with different concentrations of NaCl in Petri plates with a density of 50 seeds per plate. The data was recorded after two weeks of germination as seen in control plates and represents mean of three different plates per line. The error bar represents standard error. (B) Effect of 4 days salt stress (NaCl) during vegetative stage of two-weeks-old seedlings of different Arabidopsis plants. For seedling response under salinity stress, salt stress was provided by immersing two-week-old seedlings of different Arabidopsis plants (WT, mutant and transgenics) in ½ MS medium supplemented with 150 mM and 300 mM NaCl. (C) Effect of salt stress on total chlorophyll content. Total chlorophyll content was measured in 50 mg of plant material for each line and the data represents mean percent reduction of five replicates. The error bar represents standard error (p = 0.05(*) and 0.01(**).
Figure 8.
Effect of water stress on different Arabidopsis plants.
(A) Growth and biomass accumulation of two-weeks-old different Arabidopsis plants (WT, mutant and transgenics) grown under drought stress caused by 300 mM mannitol (scale bar = 1 cm). (B) Effect of simulated drought stress on some developmental parameters. The seeds were germinated on ½ MS for control condition and ½ MS supplemented with 300 mM mannitol in Petri plates. The data represents mean of ten individual plants and error bar represents standard error (** represents p = <0.01). The experiment was repeated three times.
Figure 9.
Expression analysis of some target genes of AtHSFA2.
Quantitative RT-PCR was done in different tissues of Arabidopsis plants (two-weeks-old plants for seedling and 8-weeks-old plants for flower and siliques tissues) of WT, 35S-TaHsfA2d (OE), knock out mutant AtHsfA2 (KO) and complemented mutants with TaHsfA2d (Comp-KO). Values on Y-axis represent relative transcript abundance, and non-treated tissues were used as control ( = 1)