Understanding the physiological and molecular basis of chilling tolerance across species of the C4 genera Miscanthus and Spartina

Spence, Ashley

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Description

Title

Understanding the physiological and molecular basis of chilling tolerance across species of the C4 genera Miscanthus and Spartina

Author(s)

Spence, Ashley

Issue Date

2012-06-27T21:21:37Z

Director of Research (if dissertation) or Advisor (if thesis)

Long, Stephen P.

Doctoral Committee Chair(s)

Long, Stephen P.

Committee Member(s)

• Moose, Stephen P.
• Ming, Ray R.
• Lee, DoKyoung

Department of Study

Plant Biology

Discipline

Plant Biology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• C4 Photosynthesis
• Chilling Tolerance
• Miscanthus
• Spartina
• pyruvate phosphate dikinase (PPDK)
• Rubisco
• PEP carboxykinase (PEP-CK)
• NADP-dependent ‘malic enzyme’ (NADP-ME)
• Zea mays
• transcriptome
• microarray
• cold stress
• photosystem II (PSII)
• light reactions
• biofuels

Abstract

Increasing demand and decreasing reserves of oil together with uncertainties in external supply and global atmospheric change has created a need for alternative and renewable fuel sources. Plant based fuels are expected to help close the gap between supply and demand and create a pathway to energy security and independence. Among the potential biofuel crop species, C4 plants are the top candidates, and include maize, sorghum, switchgrass, and sugarcane. C4 plants are capable of producing higher biomass yields and have greater water and nitrogen use efficiency than C3 plants, largely due to the elimination of photorespiration, but this greater yield and efficiency potential is only realized at optimal environmental conditions. C4 species are classically photosynthetically limited in cooler climates and are typically outcompeted by C3 species in regions where the mean summer temperature is below 8-10 °C. The lower frequency of C4 species in these regions has been hypothesized to be caused by either 1) an unavoidable breakdown of, or limitation by, a component of their carbon assimilation pathway, including ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), phosphoenolypyruvate carboxylase (PEPc), pyruvate phosphate dikinase (PPDK) or one of the three decarboxylating enzymes specific to the C4 sub-type, or 2) the fact that C4 photosynthesis evolved relatively recently from C3 species of tropical and sub-tropical origin, suggesting that there has not been sufficient evolutionary time for species possessing C4 photosynthesis to have fully adapted to cooler regions. C4 species of the genera Miscanthus and Spartina have successfully colonized cool temperate and cold climatic regions, with Spartina having the most northerly distribution of any known C4 genus. Within these genera, Miscanthus × giganteus, Miscanthus sinensis and Spartina pectinata have already been evaluated for their potential as biofuel crops, and M. × giganteus has been found to possess significant chilling tolerance relative to other C4 species. This dissertation aims to understand this apparently unique tolerance of C4 photosynthesis with three investigations (Chapters 2-4). Chapter 2 analyses the transcriptome of M. × giganteus on acclimation to chilling temperatures in comparison to that of chilling-intolerant Zea mays using microarray and qPCR. Notably, in response to 14 days of chilling (14 °C) M. × giganteus up-regulated 30 transcripts encoding chloroplast and light-reaction proteins. Validation and comparison of a portion of the key transcripts to Z. mays confirmed these results and found that the opposite response in transcription is seen in Z. mays during chilling. Western blot analysis of two key proteins encoded by the up-regulated transcripts in M. × giganteus, the D1 protein and LHCII type II chlorophyll a/b binding protein, showed significant increases in M. × giganteus during chilling and significant decreases in Z. mays. Chapter 3 examines whether chilling tolerance in Miscanthus is constitutive or has evolved progressively as species migrated northward through Japan. M. sinensis, one of the parent species of the M. × giganteus hybrid, is found throughout Japan. Three accessions of M. sinensis spanning from the sub-tropical southern-most tip to the cold northern-most tip of the major islands of Japan were grown in a common controlled environment and were then acclimated to chilling (14 °C) over 14 days. Following acclimation, the population from the northern-most location showed significantly higher photosynthetic rate of leaf CO2 uptake (A) and quantum yield of photosystem II (ΦPSII), and elevated contents of the rate-limiting enzymes PPDK and Rubisco (western blot analysis). By contrast, the chilling-intolerant southern population showed significant losses of these proteins. This suggests that genetic capacity for chilling acclimation has evolved as M. sinensis has dispersed to northward in Japan. Although M. × giganteus probably originated from the mid-latitudes of Japan, it showed even greater photosynthetic capacity following chilling acclimation, suggesting that its chilling tolerance is either a result of hybrid heterosis, or inheritance from the second parent, M. sacchariflorus. The findings add further credence to observations of previous studies that maintenance or increase in PPDK content is critical to the ability of C4 plants to photosynthesize efficiently in chilling conditions. Chilling tolerance in Spartina species has been suggested to result from its use of PEP carboxykinase (PEP-CK) as its C4 decarboxylating enzyme. PEP-CK forms PEP in the bundle sheath which is then translocated to the mesophyll, bypassing the need for PPDK, and in turn circumventing low temperature limitation by PPDK. Indeed, previous studies of Spartina from a cool climate have failed to detect PPDK. Chapter 4 compares the chilling response of Spartina pectinata cv. ‘Red River’, a C4-PEP-CK sub-type, to two species of the C4-NAPD-ME sub-type, one chilling tolerant, M. × giganteus, and one chilling intolerant, Z. mays. After acclimation with 14 days of chilling at 14 °C, S. pectinata maintained significantly higher A than M. × giganteus and Z. mays during chilling, and there was also a significant decrease in whole chain electron transport rate and photochemical quenching in M. × giganteus but not in S. pectinata. Surprisingly, S. pectinata and M. × giganteus had similar amounts of PPDK per unit leaf area at 25 °C, and both exhibited large and significant increases in PPDK during chilling. M. × giganteus showed a significant increase in Rubisco (33%), while Rubisco content was maintained in S. pectinata, and declined significantly (30%) in Z. mays. PEP-CK was also maintained during chilling in S. pectinata. These results show for the first time that PPDK is not only present in Spartina, but that ability to acclimate photosynthetic capacity to chilling conditions also corresponds to an increase in photosynthesis. The results also suggest that S. pectinata may provide an even greater capacity for photosynthetic productivity in cold climates than M. × giganteus. Collectively, the results of this dissertation show that chilling tolerance in C4 photosynthesis is not species or sub-type specific and that chilling tolerance within C4 photosynthesis may have adapted through similar means in two very separate clades and biochemical types of C4 grasses. In M. × giganteus, key transcriptional responses in chloroplast-specific genes correspond to the maintenance of higher rates of A and ΦPSII. The three species exhibiting the greatest chilling tolerance, measured as higher rates of A and ΦPSII, all showed the same response to chilling of an increase in PPDK content with no reduction in Rubisco content.

Graduation Semester

2012-05

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Copyright 2012 Ashley K. Spence

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