Fig 1.
Diagram of pE1801-ocs/mas ‘superpromoter’-HSP101 plasmid.
PAg7 = Transcription termination and poly-Adenylation signal sequence from Octopine Ti-Plasmid T-DNA (gene for transcript #7) [57]; NptII = Neomycin phosphotransferase II coding region [58]; Pnos = Nopaline synthase promoter from Nopaline Ti-Plasmid T-DNA [59]; Aocs X 3 = Octopine synthase enhancer element (3 copies) from Octopine T-Plasmid T-DNA [60]; AmasPmas = Manopine synthase promoter from Octopine Ti-Plasmid T-DNA [61]; HSP101 = Heat shock protein (101 kdalton molecular weight) from A. thaliana [32]; Ags-ter = Transcription termination and poly-Adenylation signal sequence from Octopine Ti-Plasmid T-DNA (Agropine synthase gene) [62].
Fig 2.
Tobacco seedling responses to a high temperature challenge.
(A) Photograph of segregating SR1-GS24 F2 tobacco seedlings that had been heat treated for 5 min at 50°C and returned to the dark at 22°C for 16 hours. The seedlings with the asterisk above them were negative when analyzed for AtHSP101 by PCR. (B) PCR analysis of the segregating SR1-GS24 F2 tobacco seedlings shown in (A) using P-AGS-> HS101-5’<- primers. (C) Photograph of the characterization of tobacco heat tolerance using the hypocotyl elongation assay on SR1 (AtHSP101 minus) and SR1-GS2 (AtHSP101 plus) seedlings following a 5 min 50°C heat challenge and 16 h recovery. These assays were used to check for line homogeneity.
Fig 3.
Genomic PCR analysis of cellulose synthase and AtHSP101 in segregating transgenic cotton lines and a commercial check (Phytogen 72).
Fig 4.
Photographs of RT-PCR and Western Blot analysis showing the expression of AtHSP101 and presence of HSP101 proteins in transgenic cotton lines.
RT-PCR products from pollen (A) and leaf (B) tissues of transgenic line 40 plants grown in 31°C/27°C greenhouse; Western blot analysis of HSP101 for: mature pollen of transgenic line 40 (C) and null line 52 (F, AtHSP101 minus) plants grown in a greenhouse set to 31°C/27°C day/night; leaf tissues of transgenic line 40 (D) and null line 52 (G) plants grown in a greenhouse set to 31C°/27°C day/night; leaf tissues of transgenic line 40 (E) and null line 52 (H) plants grown in a hot greenhouse set to 43°C/28°C day/night temperatures.
Fig 5.
Photomicrographs of tobacco pollen following a high temperature challenge.
SR1 (A), SR1-GS2 (B), and SR1-GS3 (C) tobacco pollen germination when incubated at 30°C for 3 hours; and SR1 (D), SR1-GS2 (E), SR1-GS3 (F) pollen germination when incubated at 46°C for 3 hours.
Fig 6.
Tobacco pollen germination following a high temperature challenge.
Graph comparing the relative pollen tube lengths of SR1, SR1-GS3, SR1-GS2, SR1-GS7, and SR1-GS17 tobacco pollen following a 60-min incubation at 30°C (control—white bars) or 7 min at 50°C followed by 53 min at 30°C (heat treated—black bars).
Fig 7.
Photomicrographs of tobacco pollen following a high temperature challenge.
SR1 (A), SR1-GS2 (B), SR1-GS7 (C), and SR1-GS17 (D) tobacco pollen incubated for 7 minutes at 50°C and then 53 minutes at 30°C.
Fig 8.
Cotton pollen germination following a high temperature challenge.
Graph showing the percent germination of control (AtHSP101 minus, plant 39) and transgenic (AtHSP101 plus, plant 40) pollen from greenhouse grown cotton germinated for one hour at either 23°C or 39°C on the solid media described by Burke et al. [17]. Error bars represent Standard Deviation and the sample size was n = 20, p value is 0.048388.
Fig 9.
Photomicrographs of cotton pollen following a high temperature challenge.
(A) pollen from plant 39 (AtHSP101 minus) germination at 28°C after treating flowers for five hours at 23°C, (B) pollen from plant 39 (AtHSP101 minus) germination at 28°C after treating flowers for five hours at 37°C, (C) pollen from plant 40 (AtHSP101 plus) germination at 28°C after treating flowers for five hours at 23°C, and (D) pollen from plant 40 (AtHSP101 plus) germination at 28°C after treating flowers for five hours at 37°C.
Fig 10.
Photomicrographs of cotton pollen following a high temperature challenge.
Representative photomicrographs showing pollen germination at 28°C according to the procedure described by Burke et al. [17] from plants grown under control temperatures (31°C/27°C day/night temperatures) and elevated temperatures (43°C/28°C day/night temperatures). (A) Plant 52 Control, (B) Plant 40 Control, (C) Plant 52 from a greenhouse with elevated day/night temperatures, (D) Plant 40 from a greenhouse with elevated day/night temperatures.
Table 1.
Characterization of the heat tolerance of AtHSP101 plus and AtHSP101 minus cotton plants from different cell lines.
Fig 11.
Boll accumulation on control and heat-treated cotton plants.
Photograph of cotton bolls harvested from individual plants of plant line 24 (AtHSP101 plus) and plant line 39 (AtHSP101 minus) that were grown in a greenhouse with a 43°C/28°C day/night temperature regime. Paired plants (line 24 and 39) were randomly distributed throughout the greenhouse because of a measured uneven temperature distribution (set point ± 3°C) during the winter months.
Fig 12.
Photographs of anthers from high temperature grown cotton plants.
(A) Line 39 [AtHSP101 minus], (B) Line 40 [AtHSP101 plus], (C) Line 52 [AtHSP101 minus], (D) Line 24 [AtHSP101 plus] flowers from plants grown under high temperatures (43°C/28°C day/night) in a greenhouse. Pollen dehiscence is apparent in all lines evaluated.
Table 2.
Characterization of the relative heat tolerance of AtHSP101 plus and AtHSP101 minus cotton plants from the 1–8 cell line.
Fig 13.
Field air temperatures during evaluation of boll accumulation.
This graph shows the maximum and minimum air temperatures in Maricopa, Arizona during the 2007-growing season. Temperatures over 38°C were common throughout the flowering and boll development period. The Bar line indicates the time period where the opened bolls were set.
Table 3.
Field performance of AtHSP101 minus (seed from plant #39) and AtHSP101 plus (seed from plant #40) cotton in Maricopa, Arizona during the 2007 growing season.