Development of novel tools for plant bioenergetics studies
Adenosine triphosphate (ATP), nicotinamide adenine dinucleotide phosphate (NADPH), and nicotinamide adenine dinucleotide (NADH) are crucial energy molecules in living systems. Monitoring in planta dynamic changes of ATP, NADPH, and NADH/NAD+ratio at the subcellular level is a major hurdle in plant bioenergetics studies. Previous methods measure these metabolites in vitro, using bioluminescence, HPLC, mass spectrometry, enzymatic and radioactive methods. However, these in vitro methods require the extraction of ATP, NADPH, and NADH from tissues before measurements can be carried out and therefore cannot determine the instant levels of these metabolites in different subcellular compartments of different cells in a tissue. We solve these problems by introducing several fluorescent protein sensors into C3 plant Arabidopsis thaliana.
Now, we can observe real-time in planta dynamic changes of these energy molecules (ATP, NADPH, NADH/NAD+ratio) in several subcellular compartments of various tissues in plants. We are the first group to introduce these three novel energy sensors in plants. By developing innovative methods to visualize energy changes in subcellular compartments in live plants, we have clarified some important questions about photosynthesis.
By studying in vivo changes of ATP levels in the plastids and cytosol of Arabidopsis thaliana using an novel ATP sensor, we have shown that (1) while the chloroplast is the major energy-harvesting organelle in photosynthetic cells, the stromal ATP concentration (0.2 mM) is significantly lower than the cytosolic ATP concentration (>1.4 mM) (2) chloroplasts consume ATP rapidly; (3) the import of ATP into mature chloroplasts is impeded by the low density of NTT transporter; (4) unlike in diatoms (Nature 524:366-369), where ATP is imported into chloroplasts to support the CBB cycle, in photosynthetic cells of higher plants, cytosolic ATP does not enter chloroplasts efficiently and is, therefore, unlikely to support the CBB cycle to any significant extent; (5) instead of ATP import, in higher plant it is the export of reducing equivalents from chloroplasts to balance the NADPH/ATP demand of the CBB cycle and NADPH/ATP supply from the linear electron flow and (6) in higher plants, reducing equivalents from chloroplasts are the major fuel for the mitochondrion to supply ATP to the cytosol.
Do mature plant chloroplasts import cytosolic ATP?
Since the publication of the article “ADENINE NUCLEOTIDE TRANSLOCATION IN SPINACH CHLOROPLASTS” in 1969, it is generally believed that ATP can be freely translocated across chloroplast membrane. Since then, it is believed that mature chloroplasts import ATP from cytosol to aid carbon fixation and to provide energy to chloroplasts at night.
After almost half a century, Boon Leong Lim and his research group introduced a fluorescent ATP sensor into the plant cytosol and chloroplast. ‘We found that the ATP concentrations in the chloroplast and cytosol were similar in young seedlings. But the ATP concentration in the chloroplast dropped significantly when the seedlings grew older.’. ‘Given that previous studies had demonstrated the ATP can move freely across the chloroplast membrane, our observation was quite a surprise.’ The question is why chloroplast ATP drops when it matures?
To validate if the chloroplasts can import ATP, chloroplasts containing functional ATP sensors were isolated from plants of different ages. “We found that only chloroplasts isolated from young seedlings, but not mature plants were able to uptake exogenous ATP’, said Dr Chiapao Voon. This finding contradicts with the previous reports. In these studies, radioactive quantitative method was used to show that exogenous ATP was able to enter mature chloroplasts isolated from spinach and Digitaria sanguinalis. Why are our observations contradictory to these reports?
‘By reading these earlier publications carefully, I found that these data actually demonstrated that ATP was not being imported by the mature chloroplasts. The import rate of ATP into chloroplasts was measured by using radioactive ATP. First, isolated chloroplasts were incubated with radioactive ATP. Afterward, non-radioactive ATP was used to exchange any radioactive ATP which has been ‘imported’ into the chloroplasts. At the end, the chloroplasts were removed and the radioactivity in the exchange buffer was measured. Since the ‘exchange efficiency’ of non-radioactive ATP was higher than GTP/CTP/TTP, it was concluded that the free movement of ATP across chloroplast membrane was possible. However, in 2004, a German group identified two ATP transporters on the chloroplast membrane which were responsible for the exchange of ATP with ADP in opposite directions. In the radioactive experiments, non-radioactive ADP was also applied. Intriguingly, the exchange efficiency of non-radioactive ADP is merely 12% compared to ATP. This is not reasonable because ADP is supposed to exchange with ATP through the ATP transporters. I believe that the truth is the radioactive ATP did not enter the chloroplasts but instead adhering on the surface of the chloroplasts. Therefore, the radioactivity in the exchange buffer was due to displacement of radioactive ATP adhered on the chloroplast surface, but not due to translocation of radioactive ATP”, explained Lim, ‘Furthermore, the publication in 2004 showed that the ATP transporters were only expressed in young seedlings but not in mature leaves, which concurred with our findings.’. The novel findings were reported in the Proceedings of the National Academy of Sciences of the United States of America (PNAS), and how these findings update the understanding of chloroplast bioenergetics was reported in an article published in the National Science Review.
Figure 1.Three different scenarios of chloroplast bioenergetics in Arabidopsis thaliana. (A) During the transition of proplastid (grey) to chloroplast; (B) Energy flow in mature chloroplasts during photosynthesis; (C) How mature chloroplasts obtain ATP at night.