Climate change consequences: Excess ozone causes major changes in the redox proteome of soybean plants

Ozone reduces crop yields

 

Increasing ozone levels in the atmosphere not only influence the climate and human health, but also have a direct effect on crops. Numerous studies have illustrated the detrimental effect of ozone on crop yields which will have an enormous effect in the future as ozone concentrations continue their inexorable rise.

Since the 19th Century, the amount of ozone at ground level has doubled. Current levels in industrialised countries are rising at an annual rate of 0.5-2.5%, which is dwarfed by that of China, India and the USA, which are facing increases of up to 10% per year. Currently, daytime levels are regularly above 40 ppb, which is the recognised threshold for crop losses.

One of the major food crops is soybean and it has the unfortunate distinction of being one of the most susceptible crops to increased atmospheric ozone levels. Trials have shown that many different soybean varieties suffer a drop in yield of about 20% when ozone levels increase from 56 to 69 ppb. The implications for the future are ominous.

Some of the effects of ozone on plants include leaf bronzing, chlorophyll loss, lowering of the rate of photosynthesis and reductions in seed mass and number. Growth chamber experiments have also illustrated changes in protein metabolism under very high ozone levels of more than 200 ppb for soybean, rice and wheat, but are these changes replicated in the field?

Joseph Jez from Washington University, St. Louis, MO, decided to check this out for soybean plants. With colleagues from the University of Illinois, Urbana-Champaign, the USDA-ARS Global Change and Photosynthesis Research Unit, Urbana, IL, and the Donald Danforth Plant Science Center, St. Louis, MO, he also wanted to investigate the effects of ozone on the redox proteins for a better understanding of the responses to oxidative stress.

Soybeans under ozone

Soybean plants were grown at the Free Air Concentration Enrichment facility under ambient, moderate and high ozone concentrations, which corresponded to 37, 58 and 116 ppb after seasonal averaging. They were planted on June 9th 2009 and harvested on 5th August of the same year, following typical meteorological conditions over the growing season.

The proteins were extracted from root and leaf tissue, with the content of total soluble protein roughly the same for each plant. They were treated with N-ethylmaleimide to label free thiol groups, but not any that had been oxidised by ozone, then reduced with dithiothreitol and reacted with iodoacetamidofluorescein, to label the oxidised thiols.

The protein mixture was then resolved by 2D gel electrophoresis and the labelled proteins were imaged by excitation with blue light at 488 nm and detection at 520 nm. Thereafter, all of the proteins were imaged with a standard dye. A total of 1455 spots were detected and those that were differentially expressed and/or oxidised across the ozone treatments were cut from the gel for identification by mass spectrometry.

In this way, 159 proteins had altered abundances and/or oxidation states, with 55, 27, 9 and 30 unique to the 116 ppb ozone leaf, 116 ppb ozone root, 58 ppb ozone leaf and 58 ppb ozone root, respectively. The remaining 38 proteins differed in multiple tissue-ozone combinations.

Oxidised proteins induced under ozone

The changes observed for moderate ozone exposure were comparable to those found in the earlier growth chamber studies. However, those for high ozone exposure were far more drastic, with every protein identified in leaves having increased abundance or increased oxidation. Jez recognised that there is a threshold between 58 and 116 ppb ozone above which the expression and oxidation of proteins sharply increased.

For these high ozone proteins, 35 were up to five-fold more abundant, 22 were up to five-fold more oxidised and 22 were both more abundant and had increased oxidation. Examination of the proteins involved revealed that they were involved in a wide range of metabolic processes. Photosynthesis, carbon metabolism, amino acid synthesis, flavonoid/isoflavonoid synthesis, signalling, homeostasis and antioxidant pathways were all affected by increasing ozone levels.

For the first time, ozone exposure was proven to affect the thiol oxidation state of proteins in several different pathways, with long-term exposure initiating several redox protection mechanisms. For instance, methionine sulfoxide reductase was increased three-fold, indicating that repair of oxidised methionine residues is one key process activated by ozone stress.

Despite the significant number of changes that were uncovered, Jez reckons that "these changes in the redox proteome of soybean leaf are likely only a small fraction of the total number of proteins that change in oxidative state." Studies with more sensitive isolation and detection will help to uncover them.

He recommends a more complete time-dependent and tissue-specific analysis to reveal the molecular basis behind the initial response to oxidative stress and its transformation during long-term growth under ozone. This will help in the design of genetically modified soybean that is better protected against the high levels of ozone that are inevitable under our continuing climate change.