Engineering topics
Sharkey, Thomas D.
Publications and source records attributed to Sharkey, Thomas D..
The metabolic origins of non-photorespiratory CO 2 release during photosynthesis: a metabolic flux analysis
Respiration in the light (R L ) releases CO 2 in photosynthesizing leaves and is a phenomenon that occurs independently from photorespiration. Since R L lowers net carbon fixation, understanding R L could help improve plant carbon-use efficiency and models of crop photosynthesis. Although R L was identified more than 75 years ago, its biochemical mechanisms remain unclear. To identify reactions contributing to R L , we mapped metabolic fluxes in photosynthesizing source leaves of the oilseed crop and model plant camelina (Camelina sativa). We performed a flux analysis using isotopic labeling patterns of central metabolites during 13 CO 2 labeling time course, gas exchange, and carbohydrate production rate experiments. To quantify the contributions of multiple potential CO 2 sources with statistical and biological confidence, we increased the number of metabolites measured and reduced biological and technical heterogeneity by using single mature source leaves and quickly quenching metabolism by directly injecting liquid N 2 ; we then compared the goodness-of-fit between these data and data from models with alternative metabolic network structures and constraints. Our analysis predicted that R L releases 5.2 μmol CO2 g –1 FW h –1 of CO 2 , which is relatively consistent with a value of 9.3 μmol CO 2 g –1 FW h –1 measured by CO 2 gas exchange. The results indicated that ≤10% of R L results from TCA cycle reactions, which are widely considered to dominate R L . Further analysis of the results indicated that oxidation of glucose-6-phosphate to pentose phosphate via 6-phosphogluconate (the G6P/OPP shunt) can account for >% of CO 2 released by R L .
Phosphoglucoisomerase Is an Important Regulatory Enzyme in Partitioning Carbon out of the Calvin-Benson Cycle
Phosphoglucoisomerase (PGI) isomerizes fructose 6-phosphate (F6P) and glucose 6-phosphate (G6P) in starch and sucrose biosynthesis. Both plastidic and cytosolic isoforms are found in plant leaves. Using recombinant enzymes and isolated chloroplasts, we have characterized the plastidic and cytosolic isoforms of PGI. We have found that the Arabidopsis plastidic PGI K m for G6P is three-fold greater compared to that for F6P and that erythrose 4-phosphate is a key regulator of PGI activity. Additionally, the K m of spinach plastidic PGI can be dynamically regulated in the dark compared to the light and increases by 200% in the dark. We also found that targeting Arabidopsis cytosolic PGI into plastids of Nicotiana tabacum disrupts starch accumulation and degradation. Our results, in combination with the observation that plastidic PGI is not in equilibrium, indicates that PGI is an important regulatory enzyme that restricts flow and acts as a one-way valve preventing backflow of G6P into the Calvin-Benson cycle. We propose the PGI may be manipulated to improve flow of carbon to desired targets of biotechnology.
Source of 12 C in Calvin–Benson cycle intermediates and isoprene emitted from plant leaves fed with 13 CO 2
Feeding 14 CO 2 was crucial to uncovering the path of carbon in photosynthesis. Feeding 13 CO 2 to photosynthesizing leaves emitting isoprene has been used to develop hypotheses about the sources of carbon for the methylerythritol 4-phosphate pathway, which makes the precursors for terpene synthesis in chloroplasts and bacteria. Both photosynthesis and isoprene studies found that products label very quickly (<10 min) up to 80–90% but the last 10–20% of labeling requires hours indicating a source of 12 C during photosynthesis and isoprene emission. Furthermore, studies with isoprene showed that the proportion of slow label could vary significantly. This was interpreted as a variable contribution of carbon from sources other than the Calvin–Benson cycle (CBC) feeding the methylerythritol 4-phosphate pathway. Here, we measured the degree of label in isoprene and photosynthetic metabolites 20 min after beginning to feed 13 CO 2 . Isoprene labeling was the same as labeling of photosynthesis intermediates. High temperature reduced the label in isoprene and photosynthesis intermediates by the same amount indicating no role for alternative carbon sources for isoprene. A model assuming glucose, fructose, and/or sucrose reenters the CBC as ribulose 5-phosphate through a cytosolic shunt involving glucose 6-phosphate dehydrogenase was consistent with the observations.
Phaseolus vulgaris SUT1.1 is a high affinity sucrose–proton co–transporter
Plant sucrose transporters are required for phloem loading, and therefore are essential for plant growth and development. In common beans (Phaseolus vulgaris) there are only two sucrose transporters functionally characterized. Through a previous RNA-seq study, we identified a putative sucrose transporter in common bean, which we hypothesize to function in import of sucrose into plant cells. In silico analysis revealed that PvSUT1.1 is a putative sucrose-proton co-transporter distinct from other characterized sucrose transporters in common bean indicating that this is a previously undescribed transporter protein in beans. Further analysis revealed that PvSUT1.1 shares high protein sequence homology to the phloem loader Arabidopsis SUC2; both have 12 transmembrane domains, a typical characteristic of plant sucrose transporters. Heterologous expression in yeast further showed PvSUT1.1 to be functional and it imported sucrose into yeast cells with a K m of 0.7 mM sucrose. Import of sucrose through PvSUT1.1 is also pH-dependent with highest uptake at pH 4.0, and activity is lost in the presence of the uncoupler carbonyl cyanide 3-chlorophenylhydrazone. Consistent with identification of PvSUT1.1 as a Type I transporter, PvSUT1.1 also transports esculin. Finally, PvSUT1.1 showed expression in multiple tissues and the protein was localized to the plasma membrane. The results show that PvSUT1.1 is a sucrose transporter that is probably involved in the uptake of sucrose into source and sink cells. The potential role of PvSUT1.1 in leaf phloem loading of sucrose in common beans and its importance in heat tolerance of reproductive tissues are further discussed.