IC4R005-Proteomic-2010-19858118

Project Title

• Physiological and proteomic approaches to address heat tolerance during anthesis in rice (Oryza sativa L.)

The Background of This Project

• Episodes of high temperature at anthesis, which in rice is the most sensitive stage to temperature, are expected to occur more frequently in future climates. In this project, the morphology of the reproductive organs and pollen number, and changes in anther protein expression, were studied in response to high temperature at anthesis in three rice (Oryza sativa L.) genotypes.

Plant Culture & Treatment

• Plants were grown in a temperature controlled greenhouse maintained at 29/21 ℃ day/night temperature [Actual: 27.7 ℃ (SD¼0.80)/19.9 ℃ (SD¼0.11)] and RH of 75% [Actual 82.7% (SD¼2.83)] under natural sunlight conditions at the International Rice Research Institute (IRRI), Philippines.

• Three rice varieties, japonica type Moroberekan (highly susceptible to high temperature during anthesis), indica type IR64 (moderately tolerant), and an Aus type N22 (highly tolerant) were chosen for this study. Pregerminated seeds were sown into 2l trays. After 15 d, three seedlings each were transplanted into 10 l pots. For each of the three genotypes, 34 pots were sown. The plants were grown under flooded conditions throughout the crop cycle. There were no other pest or disease problems.

• On the first day of anthesis (i.e. the appearance of anthers), plants were transferred at 08.00 h into growth chambers (Thermoline, Australia) with temperatures gradually increasing from 29 ℃ to 38 ℃ by 09.00 h (2.5 h after dawn) and maintained at 38 ℃ (SD¼0.13) until 15.00 h, with an RH of 75% (SD¼1.10). Immediately after the heat treatment, plants were moved back to the control conditions (29/21 ℃) before being returned to the growth cabinets and exposed to the same conditions the following morning at 09.00 h, i.e. plants were exposed to 2 d of high temperature.

• Seventeen pots of each genotype were exposed to high (38 ℃) or ambient (29 ℃) temperature for 6 h. Two pots (six plants) were used for scoring spikelet fertility both at high and control temperatures while the remaining 15 pots were used to sample spikelets for the morphological and proteomic analyses. Spikelet fertility was measured on spikelets opening between 09.00 h and 15.00 h on the first day of anthesis and marked with acrylic paint for identification (Jagadish et al., 2007, 2008). Ten to 12 days later, the marked spikelets were scored for fertility by pressing the marked spikelets individually. 121 to 184 and 110 to 174 spikelets were scored under control and high temperature treatments, respectively. Ten unopened spikelets, each positioned third on the top most rachis branch of the panicle predicted to open the next day (Matsui and Kagata, 2003) were sampled at the end of the treatment period (at 15.00 h) to measure anther length and width. Following 1 h of high temperature exposure on the first day of high temperature treatment, spikelets just beginning to open were carefully marked and collected in order to record: the pistil length (20 spikelets); total number of pollen, number of germinated pollen on the stigma, and stigma length (15 spikelets); and pollen tube length (10 spikelets). All spikelets were transferred immediately into fixative containing 1:3 glacial acetic acid: absolute alcohol (v/v) in glass vials.

• Samples for proteomic analysis were collected from the top four rachis branches simultaneously from both high temperature and control treatments and stored in falcon tubes suspended in liquid N at –80 ℃ for further use.

Protein Extraction and 2-D PAGE

• Proteins from the anthers of three different genotypes collected under control and heat-stressed conditions were extracted by trichloroacetic acid (TCA) precipitation with minor modifications. Three biological replicate anther tissue samples of 1 g each were ground in liquid nitrogen and suspended in 10% w/v TCA in acetone with 0.07% w/v DTT at –20 ℃ for 1 h, followed by centrifugation for 25 min at 10 000 rpm. The pellets were washed with ice-cold acetone containing 0.07% DTT, incubated at –20 for 1 h, and centrifuged again at 4 ℃. The above step was repeated three times and the pellets were lyophilized. The lyophilized sample was solubilized in lysis buffer (9 M urea, 4% w/v CHAPS, 2.5% w/v Pharmalyte pH 3–10, 1% w/v DTT, 35mM TRIS) and the protein concentration was determined using a Bradford assay with BSA (bovine serum albumin) as the standard.

• 2D-PAGE separation of proteins was carried out with minor modifications according to Gorg et al.. Equal amount of proteins were rehydrated into 17 cm IPG strips (pH 4–7) for analytical (100 lg) and preparative gels (500 lg). IEF was carried out using a Pharmacia Multiphore II kit (Amersham Pharmacia Biotech) at 20 ℃ under high voltage (500 V for 1 h, 1000 V for 1 h, and finally 2950 V for 14 h). Second dimension protein separation was performed using 12% SDS-PAGE gels. The protein spots in analytical gels were visualized by staining with silver nitrate according to Blum et al. (1987), with some modifications as published at http://www.weihenstephan.de/bim/deg. Preparative gels were stained with colloidal Coomassie Brilliant Blue G-250.

Research Findings

• Photographs were taken with a Nikon D70 camera (Nikon Corp., Japan) when the anther pores were completely open (Fig. 1).

Figure 1 Images of Moroberekan (a–c), IR64 (d–f), and N22 (g–i) showing the apical and basal pore sizes under heat stress, and stigmas with germinated pollen under control and high temperature conditions. Bars¼100 lm.

• The three contrasting rice genotypes selected were exposed to temperatures of 38 ℃ and 29 ℃ at anthesis. Spikelet fertility was between 95% and 96% under control (29 ℃) conditions (Fig. 2). The 6 h high temperature treatment (38 ℃) during anthesis had a significant impact on spikelet fertility. Moroberekan was highly sensitive (18% fertility), IR64 intermediate (48%), and N22 tolerant (71%) to high temperature.

Figure 2 Spikelet fertility (%Odds Ratio), total and germinated pollen number on the stigma, and pollen tube length (mm) under control and high temperature stress in the three rice genotypes Bars indicate 6SE.

• Anther size (length and width) varied significantly between genotypes (P <0.001) but was not affected by temperature. There was no temperature3genotype interaction for pore area, length or width (P >0.05). IR64 had the longest and widest anthers, resulting in a cross-sectional area of 2.44 mm2, nearly double that of Morobekan and N22 which had similar sized anthers. However, larger anther size did not contribute to larger apical (r >–0.78; n¼6) or basal (r¼0.07 to –0.3) pore size or length, which were greater in N22 than other genotypes (Table 1). Apical pore size and length was greater than basal pore size or length, particularly in N22 and Moroberekan. High temperature increased apical and basal pore areas (P <0.05) and lengths (P <0.05). Apical and basal pore areas and lengths were not correlated in any genotype.

• Genotypic differences in stigma and pistil length (P <0.001) were observed, but these differences were unaffected by temperature. Stigmas and pistils were significantly shorter in N22 than IR64 or Moroberekan (Table 1). Stigma length, and more so pistil length, was positively correlated with anther size (r¼0.60 and 0.86, n¼6, respectively) and negatively correlated with apical pore size (r >–0.90).

Tabel 1 Effect of genotype and temperature during anthesis on anther dehiscence characteristics, anther length and width, stigma length and pistil length

• The number of pollen on the stigma was affected by genotype (P <0.001), temperature (P <0.001), and their interaction (P <0.001). There was also a significant effect of temperature on the number of germinated pollen in the three genotypes (Fig. 2), and germinated pollen number was highly correlated with spikelet fertility (r¼0.94; n¼6). Spikelet fertility was also strongly correlated (r¼0.97, n¼6) with the proportion of spikelets with >20 germinated pollen grains. The rate of pollen tube growth was also significantly affected by genotype, high temperature and their interaction (all at P <0.001), with almost no pollen germination in Moroberekan at high temperature (Fig. 2).

• A 2D-gel electrophoresis showed 46 protein spots changing in abundance(Fig. 3). Spots shown to be differentially accumulated at the 0.05 level of significance with >2-fold changes were excised from 2D gels and considered for further analysis. Hence, 13 out of 46 protein spots fitting these criteria were analysed by mass spectrometry. Proteins were annotated based on the NCBI and TIGR databases using BLASTp analyses (Table 2; Figs 4, 5). For seven proteins, sequence similarities to annotated proteins were found (Fig. 4).

Table 2 Spikelet fertility (%Odds Ratio), total and germinated pollen number on the stigma, and pollen tube length (mm) under control and high temperature stress in the three rice genotypes Bars indicate 6SE.

Figure 4 Annotated proteins identified by 2D gel electrophoresis and the sequence similarity to the annotated proteins was obtained from the TIGR database using protein mass fingerprinting data from mass spectrometry.

Figure 5 Protein spots identified by 2D gel electrophoresis which had no significant matches with the annotated protein sequences (proteins of unknown functions following Luhua et al., 2008) from the database search using protein mass fingerprinting data from mass spectrometry.

• A cold and a heat shock protein were found significantly up-regulated in N22, and this may have contributed to the greater heat tolerance of N22.

Labs working on this Project

• Plant Environment Laboratory, University of Reading, Cutbush Lane, Shinfield, Reading RG2 9AF, UK
• Plant Breeding, Genetics and Biochemistry Division, International Rice Research Institute, DAPO BOX 7777, Metro Manila, Philippines

Corresponding Author

• p.q.craufurd@reading.ac.uk