Progress 09/01/02 to 08/31/05
Outputs
While most C4 species are adapted to warm temperatures, a few species can be found in cold climates. Among these cold-tolerant species, the grass Miscanthus x giganteus is exceptional in being one of the most productive. In replicated trials in SE Britain (52 degrees N) it achieved annual net primary productivities >27 t/ha dry matter, the highest plant productivity recorded in Britain. Additionally, when grown continually at low temperature (14 degrees C), M. x giganteus produces leaves which can photosynthesize across a range of temperatures at rates almost identical to 25 degrees C-grown leaves. Thus, this species has solved the problem of C4 photosynthesis at low temperatures. Zea mays also utilizes the NADP-ME C4 pathway, yet, as in other C4 crops, cannot produce leaves capable of photosynthesis below 20 degrees C, which limits both early season growth and its environmental range. If global climate change results in increased temperature fluctuations, as
predicted, cold temperature interludes early in the growing season could further limit productivity of maize in future environments. Our early work suggested that, at low temperature, M. x giganteus may have increased amounts of ribulose bisphosphate carboxylase/oxygenase (Rubisco), pyruvate orthophosphate dikinase (PPDK), and phosphoenolpyruvate carboxylase (PEPc), the three key enzymes of the C4 photosynthetic pathway, in comparison to maize. Molecular analysis further indicated that steady-state mRNA levels did not differ with growth temperature for either PPDK or PEPc in either species, while steady-state levels of the small sub-unit of Rubisco (rbcS) increased in the cold in maize. These results suggest that differences seen in protein amounts are post-transcriptional. We cloned both PPDK and rbcS from M. x giganteus and compared their translated protein sequences with published maize sequences: they were ~89% homologous. There were no consistent patterns that might suggest major
structural or functional differences; however, other researchers have shown that minor sequence differences can confer cold-tolerance to Flaveria PPDK. We have since created in vivo expression vectors for M. x giganteus and maize PPDK and will soon be comparing the in vivo-expressed enzyme activities. Recently, we measured the temperature response (5-40 degrees C) of in vivo enzyme function using gas exchange to analyze the response of photosynthesis to internal CO2 concentration and of in vitro enzyme function using crude enzyme extracts. There was little difference in in vitro enzyme function of PPDK at low temperature between M. x giganteus and maize, while both PEPc and Rubisco from M. x giganteus had lower activation energies (AE). For in vivo enzyme function, the AE of maximum PEP regeneration capacity, influenced by both Rubisco and PPDK, was lower in M. x giganteus. Under normal field conditions of growth, functional limitations to photosynthetic rate at high light and low
temperature are likely influenced by Rubisco and/or PPDK rather than PEPc. Therefore, we conclude that the lower AE of Rubisco is more important in low temperature tolerance of C4 photosynthesis than differences in other enzymes.
Impacts
Miscanthus x giganteus photosynthesizes and grows with great efficiency in low temperature in contrast to close taxonomic relatives (sugarcane, sorghum, maize). Our work has increased understanding of mechanisms for cold-tolerant C4 photosynthesis, and provides a novel source of genes for improving cold tolerance in corn and sorghum, or may indicate specific genetic markers for conventional breeding.
Publications
- Naidu, S.L., Szarejko, J.M., Ramig, K.M., Portis, A.R. and Long, S.P. 2005. The exceptional low temperature tolerance of C4 photosynthesis in Miscanthus x giganteus corresponds to a lower activation energy of Rubisco. Abstract #41004, Minisymposium 28: Photosynthesis II, American Society of Plant Biologists Annual Meeting, Seattle, WA.
- Naidu, S.L. and Long, S.P. 2004. Potential mechanisms of low-temperature tolerance of C4 photosynthesis in Miscanthus x giganteus: An in vivo analysis. Planta 220:145-155.
- Naidu, S.L., Moose, S.P., Al-Shoabi, K.A., Raines, C.A. and Long, S.P. 2003. Cold-tolerance of C4 photosynthesis in Miscanthus x giganteus effects of cold on photosynthetic enzymes. Plant Physiology 132: 1688-1697.
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Progress 01/01/04 to 12/31/04
Outputs
While the C4 mechanism of photosynthesis provides a theoretical advantage to productivity potential, C4 species are conspicuous for their rarity in cool or cold climates. As in other C4 crops, maize grown at 14C and below cannot produce photosynthetically competent leaves; this limits the length of the growing season and range of economically viable cultivation. While future scenarios of global climate change predict rising temperature means, increased temperature fluctuations are also expected. More cold temperature interludes early in the growing season would further limit productivity of maize in future environments. The grass Miscanthus x giganteus is from the same taxonomic group as sugar cane and maize, and uses the same form of C4 photosynthesis. Yet, it performs efficiently at temperatures down to 5 degrees C and develops photosynthetically competent leaves at 8 degrees C, thus having solved the problem of C4 photosynthesis at low temperatures. Our research
has shown that M. x giganteus leaves developed at low temperature have photosynthetic rates similar to leaves developed at warm temperature, while low-temperature-grown maize leaves show an 80% reduction in photosynthesis. Three key photosynthetic enzymes are considered to limit C4 photosynthesis at low temperatures, and we have investigated their expression, gene sequences and kinetic properties. This has shown that at low temperature, ribulose bisphosphate carboxylase/oxygenase (Rubisco) from M x giganteus is more efficient than the equivalent enzyme from maize. Pyruvate orthophosphate dikinase (PPDK) from M x giganteus shows little kinetic or sequence difference from that of maize, but is produced in much larger quantities at low temperature. These two changes may be critical to the success of M x giganteus at low temperature. Our analyses suggest that the third enzyme, Phosphoenol Pyruvate Carboxylase (PEPc) does not explain the difference between the two species. Under normal
field conditions, in vivo analysis of limitations to photosynthetic rate at high light and low temperature also suggest that Rubisco and/or PPDK rather than PEPc are limiting and responsible for the species differences. Other data suggest that reduced susceptibility to photoinhibition, and the ability to maintain high levels of leaf absorptance may also contribute to the better low-temperature performance of M. x giganteus. Molecular analysis has indicated that steady-state mRNA levels did not differ with growth temperature for either PPDK or PEPc in either species, while steady-state levels of the small sub-unit of Rubisco (rbcS) increased in the cold in maize. These results suggest that differences seen in protein amounts are post-transcriptional. Sequence analysis of PPDK cDNA from the two species revealed no differences at the active site; however minor sequence differences existed which could be responsible for conferring cold-tolerance to PPDK in M. x giganteus. Initial analysis
of small sub-unit Rubisco cDNA (rbcS) indicates that some sequence differences do exist between M. x giganteus and maize rbcS, which may explain the different temperature kinetics, although further investigation is needed.
Impacts
Miscanthus x giganteus photosynthesizes and grows with great efficiency in low temperature in contrast to close taxonomic relatives such as sugarcane, sorghum, corn. Our work will increase understanding of mechanisms for cold-tolerant C4 photosynthesis, and may provide a novel source of genes for improving cold tolerance in corn and sorghum, or indicate specific genetic markers for conventional breeding.
Publications
- Naidu, S.L. and Long, S.P. 2004. Potential mechanisms of low-temperature tolerance of C4 photosynthesis in Miscanthus x giganteus: An in vivo analysis. Planta 220:145-155.
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Progress 01/01/03 to 12/31/03
Outputs
The C4 photosynthetic pathway has the highest efficiency of CO2 fixation known. As in other C4 crops, this potential in corn is lost at temperatures below 20 degrees C. The grass Miscanthus x giganteus is from the same taxonomic group as sugar cane, sorghum and corn, and uses the same form of C4 photosynthesis. In contrast to these other closely-related C4 species, M. x giganteus is capable of developing photosynthetically competent leaves under chilling temperatures and achieves productivities in excess of those of C3 grasses at 52 degrees N latitude. Thus, this species has solved the problem of C4 photosynthesis at low temperatures. A possible mechanism for this phenotype is the continued accumulation and/or improved function of one or more major photosynthetic enzymes of the C4 pathway-ribulose bisphosphate carboxylase/oxygenase (Rubisco), pyruvate orthophosphate dikinase (PPDK), and phosphoenolpyruvate carboxylase (PEPc). Our previous results indicate that an
accumulation of PPDK in M. x giganteus may play a key role in its cold tolerance. Data indicate that this increase may result from differences in the protein itself, such as increased stability in the cold. Sequence analysis of PPDK cDNA revealed no differences at the active site. However, minor sequence differences existed which could be responsible for conferring cold-tolerance to PPDK in M. x giganteus. We also cloned and sequenced small sub-unit Rubisco cDNA (rbcS) from M. x giganteus. Initial analysis indicates that some sequence differences exist between M. x giganteus and maize rbcS, which will be investigated further in the next year of funding. We also investigated whether alternative electron sinks play a role in the cold-tolerant C4 photosynthesis of M. x giganteus. Efficient conversion of radiation into net carbon fixation requires minimum diversion of absorbed energy to non-photosynthetic processes, e.g., photorespiration or the Mehler reaction. Low temperature-grown corn
exhibits large alternative electron sinks which may protect against photodamage under chilling conditions. Simultaneous measurements of chlorophyll fluorescence and gas exchange on an attached leaf segment were used to determine photosynthetic and fluorescent parameters. For both species at both temperatures, the linear relationship between operating efficiency of whole chain electron transport through PSII and the efficiency of CO2 assimilation had a zero intercept, indicating the absence of non-photosynthetic electron sinks. Photorespiration was inferred only in warm temperature corn at trace levels. Additionally, leaf absorbance was 16% lower in low temperature corn but unchanged in M. x giganteus. These data indicate that alternative electron sinks are negligible in both warm and low temperature-grown M. x giganteus and corn. The maintenance of high photosynthetic rates in M. x giganteus may be controlled by the properties of Rubisco and/or PPDK, reduced susceptibility to
photoinhibition, and the ability to maintain high levels of leaf absorptance during growth at low temperature, in contrast to corn.
Impacts
Miscanthus x giganteus photosynthesizes and grows with great efficiency in low temperature in contrast to close taxonomic relatives such as sugarcane, sorghum, corn. Our work will increase understanding of mechanisms for cold-tolerant C4 photosynthesis, and may provide a novel source of genes for improving cold tolerance in corn and sorghum, or indicate specific genetic markers for conventional breeding.
Publications
- Naidu, S.L., Moose S.P., Al-Shoabi, K.A., Raines, C.A. and Long, S.P. 2003. Cold-tolerance of C4 photosynthesis in Miscanthus x giganteus-effects of cold on photosynthetic enzymes. Plant Physiology 132: 1688-1697.
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Progress 01/01/02 to 12/31/02
Outputs
The objective of this research is to determine the physiological and molecular bases of effective C4 photosynthesis at low temperatures. The C4 photosynthetic pathway has the highest efficiency of CO2 fixation known. As in other C4 crops, this potential in corn is lost at temperatures below 20 degrees Celsius. In addition, photoinhibition and lesions within the C4 photosynthetic apparatus of leaves developed at low temperature further limit both early season growth and the environmental range of corn, as well as other important C4 crops. The grass Miscanthus x giganteus is from the same taxonomic group as sugar cane, sorghum and corn, and uses the same form of C4 photosynthesis. However, in contrast to the vast majority of C4 species, it achieves productivities in excess of those of C3 grasses at 52 degrees N latitude and has high photosynthetic rates at low temperatures. Thus, this species has solved the problem of C4 photosynthesis at low temperatures. A possible
mechanism for this phenotype is the continued accumulation of pyruvate orthophosphate dikinase (PPDK) in Miscanthus leaves under chilling conditions. PPDK activity is both cold-sensitive and potentially limiting for C4 photosynthesis in corn. In the first 2 years of our research, we showed that cold-grown corn exhibits large decreases in amounts of PPDK, while amounts of ribulose bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPc) are not significantly affected by growth temperature in either corn or M. x giganteus. In contrast, cold-grown M. x giganteus exhibits a marked accumulation of PPDK. Cold did not affect steady-state amounts of PPDK mRNA in either species. Furthermore, we showed that the difference in amount of PPDK is insufficient to account for the difference in photosynthetic rate at low temperature. These data indicate that the increase in PPDK protein in M. x giganteus may result from differences in the protein itself, such as
increased stability in the cold. We have cloned and sequenced M. x giganteus C4-PPDK cDNA and found at least three transcripts coding for C4-PPDK in M. x giganteus. In comparison to corn, there were no differences at the active site; however minor sequence differences existed and could be responsible for conferring cold-tolerance to PPDK in M. x giganteus. In further research we will use three approaches to determine the physiological basis for effective C4 photosynthesis at low temperatures in M. x giganteus and to identify the molecular components responsible: 1) comparative analysis of differences in C4 photosynthesis and C4 photosynthetic enzyme activities between corn and M. x giganteus grown in controlled optimal and low temperature environments; 2) clone genes encoding enzymes specific to the C4 pathway from M. x giganteus and compare their sequences and expression patterns with the orthologous corn enzymes; and 3) express functional M. x giganteus PPDK enzyme in E.coli to
investigate the potential role of increased enzyme stability in conferring cold-tolerance to C4 photosynthesis.
Impacts
Miscanthus x giganteus photosynthesizes and grows with great efficiency in low temperature in contrast to corn. Our research will increase understanding of mechanisms for cold-tolerant C4 photosynthesis, and may provide a novel source of genes for improving cold tolerance in corn, or indicate specific genetic markers for conventional breeding to improve productivity of corn.
Publications
- No publications reported this period
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