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A rice FATB insertional mutant exhibits improved growth and reduced photoinhibition at high temperatures

E.S. Whitelaw1,2, Q. Liu1, F. Chow3, Z. Li1, C.L. Blanchard2 and S. Rahman1

1 CSIRO Plant Industry, Canberra, ACT, 2602
2
Charles Sturt University, Wagga Wagga, NSW, 2650
3
Australian National University, Canberra, ACT, 2601

Introduction

Lipids play a critical role in plant function as they are the predominant component of membranes. In some plants, such as the oil seeds, lipids can make up to 60% of the weight of the grain (Ohlrogge and Jaworski, 1997). In cereals such as rice, the percentage is far lower (4%), but lipids can still make up 37% of the dry weight of the embryo (Choudhury and Juliano 1980). In the rice grain, lipids are concentrated in the outer bran layer as storage lipids in the form of triacylglycerols (TAGs). In addition lipids in all plants have important roles in other tissues, including leaves, where they are crucially involved in photosynthesis. In leaf, fatty acids are used to form membrane glycerolipids in cell membranes and chloroplast membranes. The saturation of fatty acid in leaf has been found to affect plant growth and photosynthesis at high and low temperatures (Ariizumi et al, 2002; Bonaventure et al, 2003; Gombos et al, 1994; and Murakami et al, 2000).

Fatty acids are initially synthesised in the plastid by two enzyme systems acetyl-CoA carboxylase (ACCase) and fatty acid synthase (FAS). ACCase catalyses the formation of malonyl-CoA from acetyl-CoA. FAS transfers the malonyl moiety to acyl carrier protein (ACP) then elongates the growing acyl chain by addition of malonyl units. The final products formed are predominantly palmitoyl (16:0)-ACP and stearoyl (18:0)-ACP. Thioesterase enzymes then hydrolyse the acyl chains from ACP and release free fatty acids. The released fatty acids enter the eukaryotic pathway where they are mostly used for glycerolipid synthesis in the endoplasmic reticulum (Ohlrogge and Jaworski, 1997). One of the thioesterase enzymes acyl-acyl carrier protein thioesterase B (FATB) has a high affinity for 16:0-ACP, and so effectively catalyses the formation of palmitic acid (16:0) (Salas and Ohlrogge, 2002). Four rice FATB isoforms have been identified through BLAST analysis of the NCBI, TIGR and Chinese rice databases (Whitelaw et al, 2004).

Retrotransposons are mobile genetic elements that propagate in the genome via reverse transcription of RNA intermediates. The rice retrotransposon Tos17 is activated by tissue culture propagation. In addition, Tos17 seems to prefer to insert into gene-rich regions of the rice genome. Insertional mutagenesis by the retrotransposon Tos17 can knock out gene expression (Yamazaki et al, 2001). A Tos17 insertional mutant has been identified in one FATB isoform in rice. Insertion of this ~4000 bp retrotransposon in exon 4 of the FATB isoform is likely to inactivate the gene. We report here preliminary investigation of this rice line.

results and discussion

Sequencing of cDNA produced by reverse transcribing RNA found that the four isoforms are expressed in both rice grain and leaf. Reverse transcription PCR of leaf RNA indicated greatly reduced RNA expression of the targeted FATB isoform in the mutant (Figure 1). Analysis of the fatty acid composition from rice leaf and grain was performed by Gas Chromatography (GC). It was found that palmitic acid content was reduced by around 10% in grain and leaf of mutant plants. In addition, there was an increase in oleic acid content in the grain of the Tos17 mutant. It has been found that the mutant grew better at high temperatures (Figure 2), especially in the first 10 to 20 days when the plants were transferred into the 36C cabinet.

Figure 1. Reverse transcription PCR indicates reduced FATB expression in the Tos17 mutant.
1= Tos17 FATB mutant, 2= Wild-type, 3= Negative control.

A

B

Figure 2. Tos17 FATB mutant has improved growth at 36C.
A. Change in number of leaves/plant during growth in the 36C cabinet.
B. Change in total leaf area in the 36C cabinet

The Tos17 mutants may grow better at high temperatures due to reduced photoinhibition at high temperatures. Photoinhibition is the inhibition of photosynthesis due to excessive visible light. The D1 thylakoid membrane protein has an enhanced rate of degradation and synthesis in response to light. D1 protein turnover is thought to be an important factor contributing to photoinhibition (Chow, 1994). D1 repair requires movement of the protein from the stacked to the unstacked thylakoid membrane. The FATB mutant may have improved thylakoid membrane fluidity due to the reduction in palmitic acid content. This may help to improve the efficiency of the D1 repair process at high temperatures. This may be an advantage at high temperatures when there is a high rate of D1 damage and repair. Figure 3 shows an analysis of leaf photoinhibition for both wild-type and mutant plants at high temperatures by looking at alterations in optimum quantum yield (Fv/Fm) at high temperatures. Fv/Fm is a measure of photosynthetic efficiency of Photosystem II (responsible for water splitting to evolve oxygen), determined as the ratio of the variable fluorescence to the maximum possible leaf fluorescence. Any reduction in Fv/Fm indicates a drop in photosynthetic efficiency under light-limiting conditions. It can be seen that the wild type plant appears to suffer greater photoinhibition at high temperatures.

A

B

Figure 3. Photoinhibition for Wild type vs. Tos17 FATB mutant rice.
A. Change in optimum quantum yield (Fv/Fm) values for wild and mutant plants over the second day of high temperature (36C) and high light stress.
B. Initial Fv/Fm values at different temperatures from plants grown at normal glass house conditions (22-32C).

Conclusion

This research indicates that the FATB Tos17 mutant has reduced palmitic acid content in grain and leaf. The mutant may also have improved growth at high temperatures through greater resistance to photoinhibition. Further work is needed to confirm enhancement of growth at high temperatures.

Acknowledgements

We would like to thank Dr. Akio Miyao from the National Institute of Agrobiological Sciences (NIAS), Japan, for providing Tos17 insertion mutant rice seeds. We would also like to thank Lorraine Mason for GC analysis.

References

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