Feature: Proteomics and plant respiration

This feature appeared in the July/August 2011 issue of Australian Life Scientist. To subscribe to the magazine, go here.

Read part I: Proteomics on the dark side.

Professor Harvey Millar’s team at the University of Western Australia, including PhD student Richard Jacoby and a Research Fellow Nicolas Taylor, are studying a range of popular wheat cultivars grown in WA where saline soils are a particular problem.

They are specifically comparing the performance of cultivars preferred for salty areas with those of cultivars grown in low-salinity soils. “We are comparing growth rates, respiration rates and then going right down to compare their proteomes , to see if we can correlate protein changes with respiratory responses,” he says.

In their first paper they compared today’s main salt-tolerant variety with a variety it had displaced. “Farmers grow the new variety because there’s an economic benefit in its greater salt tolerance, and we can show that difference under laboratory conditions.”

In ongoing research they are comparing these findings with data from many different wheat cultivars grown in WA, seeking correlations between protein expression patterns and salinity tolerance.

“We are trying to identify novel targets to explain why particular cultivars are tolerant, and following on from those findings, how salt tolerance is inherited when you cross tolerant with non-tolerant plants.

“Basically, the mitochondrial proteomic differences in salt tolerance between varieties come down to differences in the expression of two respiratory proteins. In the first two varieties we have compared, the over-expression of these proteins appears to be linked to their salt-tolerance.”

Millar says wheat cultivars employ both the classical mechanism of salt exclusion from the roots, which comes at least in part from a high respiration requirement, and the alternative mechanism of salt storage in internal compartments, away from vital tissues – so called ‘tissue tolerance.’

“Quite a few varieties use tissue tolerance, yet they still seem to be able to grow in saline soils, so we think part of the explanation lies in differences in metabolic pathways like respiration between cultivars.”

Millar says the WA Department of Agriculture and Food has an invaluable resource for comparative proteomics and proteome influences on phenotype: a 100-year archive of wheat-breeding data, complemented by a well organised germplasm connection, which is ready for exploration at the level of the proteome.

“There has been a lot of variety selection for local conditions. WA farmers and agronomists have very good information about the performance of these varieties in a range of growth conditions, including growth patterns and yield responses under different climatic and environmental influences, including salinity, resistance to disease, response to cold and flowering and maturation time.

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“A really interesting aspect of plant breeding, and where proteomics fits into the picture, is that historically, most breeding has used genetic markers. We now need to think about new opportunities for biomarkers at the protein level and bring these into plant breeding strategies, especially when breeding for tolerance to stress.”

Stress responses tend to be genetically complex, and Professor Millar says genomics alone will only provide part of the information about phenotypic responses to stress.

Rice and maize breeders are making steady progress towards linking microarray patterns of gene-expression to proteome patterns and making the further link to phenotypic variation. “Generally, the genetic information we have from wheat, for proteomic pattern-matching, is still not as good as for rice or maize.”

Millar says much of the problem is the much larger size of the tetraploid durum wheat genome, and the hexaploid bread wheat genome. Rice has two copies of its 20,000-odd genes in its diploid genome giving it roughly 40,000 alleles; maize about 80,000 alleles, while hexaploid wheat has somewhere over 200,000 different alleles!

It’s not just the number of alleles in the wheat genome, but the potential for a much greater repertoire of interactions between the four or six alleles of equivalent genes in the A, B and D genomes that challenges wheat breeders, interactions that are also responsive to environmental stresses.

Closing the gap

However, Millar says new super-fast next-generation sequencers and proteomics tools should soon begin to close the gap on linking proteins and gene alleles in wheat.

As well as synthesising ATP, plant mitochondria produce reactive oxygen species (ROS), but the actual role of these potent oxidising agents in plants has been something of a mystery.

In a paper published in Proceedings of the National Academy of Science in June, Dr Shaobai Huang from Millar’s group collaborated with researchers from CSIRO Plant Industry to provided proof that mitochondrial ROS (mROS) play a key role in regulating stress and defensive responses, including defending plants against bacterial and fungal pathogens.

They screened thousands of Arabidopsis mutants for plants carrying a mutation in a gene that rendered it unresponsive to the phytohormone salicylic acid. Plants produce salicylic acid when they come under attack from herbivores and bacterial or fungal pathogens or abiotic stressors such as heat, desiccation or salinity.

Millar says microarray analysis of the disrupted in stress response 1 (dsr1) mutant confirmed it had down-regulated expression of specific salicylic acid-activated stress response genes.

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The dsr1 mutant also showed significantly impaired activity of the respiration enzyme succinate dehydrogenase. The mutation in the dsr1 plants occurs in a conserved region of the succinate dehydrogenase 1-1 (SDH1-1) gene and produces mutant phenotypes that produce reduced levels of protective ROS.

Using an in situ fluorescence assay, they showed that ROS production in root cells occurs primarily in mitochondria, and that the salicylic acid response in the dsr1 mutant produced fewer ROS.

The mutant was also significantly impaired when it was exposed to bacterial and fungal pathogens. “It didn’t see the infections coming like wildtype plants and thus did not have time to respond and defend itself,” says Millar.

According to the PNAS paper, the dsr1 mutant provides critical insights into the function of mitochondrial respiration Complex II, which is highly conserved between most organisms. “These new findings may be relevant to understanding mitochondrial diseases of complex II in animals – including humans – as well as in plants,” says Millar.

“Respiration is an ancient evolutionary link we have with plants and with the environment. We all contribute to atmospheric CO2 as we breathe, and we all benefit from respiration as the most efficient process in nature to get cellular energy out of sugar.

“The ancient proteome of mitochondria can thus hold the keys to understanding both human disease and aging, as well as plant productivity and environmental tolerance.”