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First published online March 7, 2008; 10.1104/pp.108.116889 Plant Physiology 147:228-238 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Isolation and Characterization of Mutants of Common Ice Plant Deficient in Crassulacean Acid Metabolism1,[W],[OA]Department of Biochemistry and Molecular Biology, University of Nevada, Reno, Nevada 89557–0200 (J.C.C., R.L.A.); Faculty of Agriculture, Saga University, Saga 840–8502, Japan (S.A.); and Institute for Research on Environment and Sustainability, School of Biology, Newcastle University, Newcastle upon Tyne NE1 7RU, United Kingdom (S.M.E., T.T., A.M.B.)
Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that improves water use efficiency by shifting part or all of net atmospheric CO2 uptake to the night. Genetic dissection of regulatory and metabolic attributes of CAM has been limited by the difficulty of identifying a reliable phenotype for mutant screening. We developed a novel and simple colorimetric assay to measure leaf pH to screen fast neutron-mutagenized populations of common ice plant (Mesembryanthemum crystallinum), a facultative CAM species, to detect CAM-deficient mutants with limited nocturnal acidification. The isolated CAM-deficient mutants showed negligible net dark CO2 uptake compared with wild-type plants following the imposition of salinity stress. The mutants and wild-type plants accumulated nearly comparable levels of sodium in leaves, but the mutants grew more slowly than the wild-type plants. The mutants also had substantially reduced seed set and seed weight relative to wild type under salinity stress. Carbon-isotope ratios of seed collected from 4-month-old plants indicated that C3 photosynthesis made a greater contribution to seed production in mutants compared to wild type. The CAM-deficient mutants were deficient in leaf starch and lacked plastidic phosphoglucomutase, an enzyme critical for gluconeogenesis and starch formation, resulting in substrate limitation of nocturnal C4 acid formation. The restoration of nocturnal acidification by feeding detached leaves of salt-stressed mutants with glucose or sucrose supported this defect and served to illustrate the flexibility of CAM. The CAM-deficient mutants described here constitute important models for exploring regulatory features and metabolic consequences of CAM.
Crassulacean acid metabolism (CAM), one of three modes of photosynthetic assimilation of atmospheric CO2, has evolved multiple times in approximately 7% of vascular plant species. The high number of independent origins of CAM in over 30 taxonomically diverse plant families is indicative of convergent evolution (Crayn et al., 2004  ; Silvera et al., 2005) and, as such, serves as a useful model for probing how complex traits evolve in response to a changing environment. CAM is typically characterized by nocturnal CO2 uptake via the enzyme phosphoenolpyruvate (PEP) carboxylase (PEPC), which is activated via phosphorylation by a dedicated protein kinase, PEPC kinase (PPCK; Nimmo, 2000). The 3-C substrate for nocturnal carboxylation, PEP, is produced via the glycolytic breakdown of carbohydrates at night. The organic acids produced via the dark carboxylation of PEP are stored overnight in a large central vacuole (Epimashko et al., 2004) and are subsequently decarboxylated during the day to release CO2 for fixation by Rubisco behind closed stomata. CAM facilitates CO2 acquisition in water-limited environments and is typically found in species that inhabit semiarid regions (Cushman and Borland, 2002), including tropical epiphytes that must endure intermittent or seasonal water availability (Zotz, 2004). In addition, CAM can lead to greater carbon acquisition in aquatic environments where daytime CO2 availability can be limiting (Keeley, 1998).
In recent years, efforts to elucidate the metabolic, regulatory, and signaling elements that define CAM have focused largely on the facultative CAM halophyte common ice plant (Mesembryanthemum crystallinum). When grown under well-watered, nonstressed conditions, common ice plant performs C3 photosynthesis from the seedling stage through seed set (Winter and Holtum, 2007
To date, genetic dissection of the regulatory and metabolic attributes of CAM in common ice plant has been limited by the difficulty of identifying a reliable phenotype for mutant screening. Here, we report the establishment of large-scale mutant collections of any CAM species and the development of a novel screening strategy for the isolation of CAM-deficient mutants in common ice plant. Fast neutron (Nf) mutagenesis was selected over chemical mutagenesis because mutants generated in this way have kilobase pair-size deletions or rearrangements, rather than point mutations as in chemically mutagenized plants (Bruggemann et al., 1996
Isolation of CAM-Deficient Mutants
We investigated whether a simple visual screen using a colorimetric pH indicator dye could be used for high-throughput screening of ice plant mutants with a reduced ability to conduct nocturnal acidification, a key feature of CAM. Traditionally, nocturnal acidification is measured by the change in titratable acidity in leaf sample extracts collected at the end of the dark period (dawn) and at the end of the light period (dusk). However, this approach is too labor intensive for high-throughput screening. To simplify the screening process, leaf discs were sampled from plants at dawn and dusk in a microtiter plate assay containing the pH indicator, chlorophenol red (see Supplemental Fig. S1), rather than by measuring the absolute leaf pH of each individual plant independently (Fig. 1
). To confirm putative CAM mutants, a secondary, complementary iodine staining assay (Caspar et al., 1985
From an estimated total of 11,666 M2 plants (derived from 779 M1 plants irradiated with 50 Gy) and 5,954 M2 plants screened (derived from 292 M1 plants irradiated with 40 Gy), 167 candidates were selected during the primary screen on the basis of a failure to undergo nocturnal acidification following salinity stress treatment. Within these populations, 50 lines contained morphological aberrations (e.g. alterations in pigmentation or morphology of leaves and stems, etc.), including an epidermal bladder cell-less mutant with reduced salt tolerance (Agarie et al., 2007 ). The frequency of putative CAM mutants within the M2 generation irradiated at 40 and 50 Gy were 0.3% and 0.42%, respectively. In the subsequent M3 generation, the frequency of putative CAM-deficient mutants increased to 0.7%. Two lines were isolated and designated 351 and 371, which showed 18 and 22, respectively, of 24 plants assayed that were CAM deficient. These two mutant lines were subjected to further physiological and biochemical characterization.
Patterns of net CO2 assimilation in leaves over a day/night cycle were monitored over a 2-week time course of salinity treatment and confirmed that M4 generation lines 351 and 371 were deficient in CAM (Fig. 2 ). Net dark CO2 uptake was apparent in wild-type plants after 7 d of salinity treatment and increased further by 14 d of salinity (Fig. 2). In contrast, there was negligible net dark CO2 uptake in either of the mutant lines after 14-d treatment with 0.3 M NaCl. Both mutant lines also showed lower rates of daytime net CO2 assimilation compared to wild type in well-watered conditions (day 0; Fig. 2), and this was also reflected in lower stomatal conductance and transpiration rates in the mutant lines (data not shown).
Growth rate measurements of wild type and M4 generation mutant lines under well-watered (unstressed) conditions up to 4 weeks of age showed that both mutant lines displayed significant retardation in growth (Fig. 3, A and B ). To determine whether the lower transpiration and growth rates in the mutants, as compared to wild type, might be due to sodium toxicity, sodium accumulation was measured after 2 weeks of exposure to salinity. The mutant lines contained slightly less sodium than wild-type plants. Although the sodium content differences observed were statistically significant, the growth differences were unlikely to be due to sodium toxicity (Fig. 3C).
Reproductive Output
Wild-type plants and M4 generations of both mutant lines were taken through to seed. Both mutant lines produced substantially fewer and lighter seeds compared to wild type (Fig. 4, A and B
). Line 351 showed the most significant reduction in reproductive output as compared to wild type, with seed production only 10% of that shown by wild type and average seed weight less than one-half of that noted in wild type. Carbon-isotope ratios of harvested seed from mutant line 351 were depleted in 13C by up to 3
Metabolite Levels and CAM Expression Following treatment with 0.3 M NaCl for 14 d, wild-type plants showed a more than 4.5-fold day/night change in the content of leaf titratable acids, whereas both mutant lines showed less than a 1.5-fold change in leaf titratable acids (Fig. 5A ). Salt-stressed wild-type plants also showed more than a 4-fold day/night change in leaf starch content, whereas the leaves of both mutant lines showed very low levels of starch (3-fold lower than wild type at dawn) that failed to exhibit any diel fluxes (Fig. 5B). Control (unstressed) plants of both mutant lines showed similarly depleted levels of leaf starch relative to wild-type plants (data not shown). Assuming that two H+ are equivalent to one Mal and one Glc equivalent can generate two PEP, there was a close stoichiometry between starch degradation and titratable acid accumulation at night in the salt-stressed wild type. The day/night changes in leaf soluble sugar content were more pronounced and greater in absolute abundance during the afternoon period in both of the mutant lines compared to wild type (Fig. 5C). In both of the mutant lines, the small amounts of acid accumulated overnight could have been furnished by PEP produced from soluble sugar degradation using the same stoichiometric relationship as described above.
Restoration of CAM by Sugar Feeding To test the hypothesis that the CAM deficiency in the mutants was due to a lack of substrate for nocturnal malate synthesis, detached salted leaves of line 351 were incubated with either 0.2 M Glc or Suc. Suc feeding restored the overnight accumulation of titratable acidity in detached leaves of mutant lines 351 and 371 (Fig. 6 ) to levels comparable to those found in salted wild-type plants (Fig. 5A). Feeding mutant leaves with 0.2 M Glc also resulted in a nearly identical restoration of titratable acidity in detached leaves of the mutant lines (data not shown). Such results imply that Suc was degraded to Fru and Glc, which were then processed to produce PEP for organic acid synthesis. Bulk analysis and HPLC analyses of the sugar-fed leaves confirmed that the mutant was capable of metabolizing exogenous Suc because there was an increase in total soluble sugars and Glc and Fru content in the leaf in parallel with the increase in Suc while starch content remained unchanged (E. Antony and T. Taybi, unpublished data). Detached leaves were also fed with 0.2 M mannitol to confirm that alterations in titratable acidity were not due to osmotic effects. Mannitol treatment had no significant impact on titratable acidity changes in either wild type or mutant lines (Fig. 6).
Plastidic PGM Enzyme Activity and mRNA Expression
To test the hypothesis that a defect in starch biosynthesis and/or breakdown was responsible for the observed CAM-deficient phenotype, key gluconeogenic enzymes leading to starch biosynthesis were surveyed by in-gel activity staining assays. Phosphoglucoisomerase (PGI), which catalyzes the interconversion of Fru-6-P and Glc-6-P, is present as three distinct isozymes in the ice plant, one of which is localized to the plastid (Fig. 7A
). All three isozymes of PGI showed similar activity in the wild type and mutant lines 351 and 371 under control conditions, whereas salinity elicited an increase in the activity of the highest Mr isozyme in all plants (Fig. 7A). Two isozymes of PGM, which catalyzes the interconversion of Glc-6-P
To verify that plastidic PGM expression was impaired in the CAM-deficient mutant lines, transcripts for a variety of genes important for CAM, including Ppck1 (Taybi et al., 2000 ), CAM-specific PEPC (Ppc1; Cushman et al., 1989), CAM-specific Glc-6-P/Pi translocator (Gpt2; Kore-eda et al., 2005), and plastidic Pgm2 were monitored by semiquantitative reverse transcription (RT)-PCR. The transcript abundances of all genes were similar in wild type and CAM-deficient mutant lines (Fig. 8
), with the exception of plastidic PGM, which lacked any detectable mRNA in both mutant lines 351 and 371. Uniform transcript abundance of constitutively expressed Ubq1 indicated that the total RNA input was similar for all reactions (Fig. 8A). Quantitative analyses, however, revealed approximately 4-h shifts in the day/night patterns of transcript abundance of the CAM-related genes in the mutant lines in comparison to wild type (Fig. 8, C–E). This was particularly marked for both Ppc1 and Gpt2, which exhibited reduced transcript abundance during afternoon and dusk (2 PM and 6 PM) and elevated abundance during the night (10 PM and 2 AM) in both 351 and 371 relative to wild type. Ppck1 also exhibited approximately a 4-h shift in relative transcript abundance over the course of the day/night period (Fig. 8). Such observations indicate a shift in circadian and/or metabolite-mediated control of transcription in the CAM-deficient lines.
Isolation and Characterization of CAM-Deficient Mutants
We have shown that a novel, yet simple, visual screen using a colorimetric pH indicator dye can be used for high-throughput screening and isolation of ice plant mutants with a reduced ability to conduct nocturnal acidification, a key feature of CAM (Fig. 5A). Two mutant lines, designated 351 and 371, were isolated using this screening protocol and were confirmed to be CAM deficient by their failure to conduct nocturnal net CO2 uptake after 2 weeks of treatment with 0.3 M NaCl (Fig. 2). In wild-type plants, the same salinity regime induced net dark CO2 uptake within 7 d. After 2 weeks of salinity stress, approximately 50% of net 24-h carbon gain occurred at night in wild-type plants. However, lines 351 and 371 could not be designated as CAM-null mutants because a slight, but significant, change in titratable acidity was measured over 24 h in plants from both mutant lines exposed to salinity for 2 weeks. Such nocturnal acidification can be attributed to the refixation of respiratory CO2 by PEPC (Griffiths, 1988
Both of the mutant lines isolated in this study showed a substantial reduction in leaf growth, seed number, and seed weight compared to the wild-type plants (Fig. 4). At least part of the reduced growth and reproductive output in mutant lines 351 and 371 can be attributed to starch deficiency. The slow-growth phenotype is similar to that observed for the starchless mutant of Arabidopsis deficient in chloroplast PGM activity also grown under a 12-h photoperiod (Caspar et al., 1985
As a complementary screen, and to confirm the putative CAM defect indicated via the colorimetric pH screen, salt-stressed mutants were also tested for defects in day/night starch accumulation/depletion using a high-throughput iodine-staining protocol (Fig. 1). A diagnostic feature of the CAM cycle in common ice plant is reciprocal cycling of organic acids and starch, so a failure to accumulate starch over the day might be anticipated of a CAM-deficient mutant after the imposition of salt stress. Both of the isolated mutant lines showed this starch-deficient phenotype (Fig. 5B), but, because well-watered plants of both lines also had very low levels of leaf starch, it appears that starch deficiency may be the cause, rather than a consequence, of CAM deficiency. Interestingly, both CAM-deficient mutant lines also exhibited elevated soluble sugar accumulation (Fig. 5B). Starch deficiency and increased soluble sugar accumulation is typically observed in the leaves of Arabidopsis and N. sylvestris plastidic PGM mutants (Caspar et al., 1985
Previous work has indicated that starch turnover and content are key regulatory factors for the expression of CAM in common ice plant (Borland and Dodd, 2002
In CAM plants, the expression, abundance, and activity of PEPC serve as a diagnostic marker for the potential capacity of nocturnal carboxylation and magnitude of CAM (Winter, 1982
In conclusion, as a consequence of Nf mutagenesis and high-throughput screening methods, a large collection of morphological and salt-sensitive (Agarie et al., 2007
Establishment of Mutant Seed Banks Common ice plant (Mesembryanthemum crystallinum) seeds were irradiated by Nf bombardment at a range of doses from 20 to 120 Gy using a cobalt 60 source at the reactor of the Atomic Energy Agency (courtesy of Dr. H. Brunner, Vienna). Wild-type Nf-irradiated seeds (M0) were surface sterilized and germinated in 150-mm disposable plastic petri dishes containing Murashige and Skoog agar (0.43% Murashige and Skoog salts, 1x B5 vitamins, 3% Suc, and 0.5% agar). The seedlings (M1) were grown in a growth chamber under 100 µmol m–2 s–1 of cool-white fluorescent light on a 12-h (26°C) light/12-h (18°C) dark photoperiod. After 1 month, the survival of the seedlings that grew to an expanded second leaf pair and beyond was scored as positive for survival. Based on survival rates, wild-type seeds that were irradiated with Nf doses of 40 and 50 Gy (55%–60% survival) were taken forward for mutant screening and were germinated on agar plates as described above. The plants were selfed to obtain an M2 population.
The M2 seeds were germinated on agar as described above and 1-week-old seedlings were transplanted into commercial soil mix (MetroMix 200; Scotts Sierra Horticultural Products) and irrigated daily with 0.5x Hoagland solution number 2 (Hoagland and Arnon, 1950
Leaf pH assays were conducted on 24 individuals from each M2 line before and after the imposition of salinity by punching out a 0.23-cm2 leaf disc with a paper punch from the fourth or fifth leaves both at the end of the dark period (dawn) and at the end of the light period (dusk), and immersing the leaf discs in a solution of 0.025% (w/v) chlorophenol red (Sigma-Aldrich; indicator grade no. 199524), a colorimetric pH indicator dye. Chlorophenol red has a visual transition interval from pH = 4.8 (yellow) to pH = 6.4 (red), which matches the pH difference observed between morning and evening measurements made in CAM-performing ice plant (see Supplemental Fig. S1). The goal was to identify individual mutants amid a population of wild-type plants by simply visualizing the color differences between leaf samples in a microtiter plate assay, rather than by measuring the absolute leaf pH of each individual plant independently. After 4 h, the plates were examined for color development. Plants that failed to undergo nocturnal acidification as indicated by the colorimetric pH assay were classified as putative mutants. These plants were retested after 1 and 2 weeks of additional salt stress to rule out false positives. Seed (M3) was collected from each plant of a putative mutant line, planted, and M3 plants reassayed as above. To confirm putative mutants, positive and apparent false positives that were identified using the pH screen were subjected to a leaf starch content assay as previously described (Caspar et al., 1985
M4 seeds from wild type and two putative CAM-deficient mutant lines (351 and 371) were germinated and propagated as described above. Plants were grown in a controlled environment chamber with a 12-h photoperiod, a day/night temperature regime of 25°C/18°C, and photon flux density of 350 µmol m–2 s–1 at plant height. Five-week-old plants (primary leaf 4 fully emerged) were irrigated daily with 0.5x Hoagland solution containing 0.3 M NaCl for up to 14 d. Gas exchange measurements were made at day 0 (control), day 7, and day 14 of the salinity treatment. Net CO2 uptake was monitored continuously on a single leaf (primary leaf 4) over 24-h periods with three separate runs made for each treatment. The leaf was enclosed in a gas exchange cuvette, which tracked the environmental conditions in the growth chamber with gas exchange parameters measured using an open infrared gas analyzer (H. Walz). Rates of net CO2 exchange were calculated using DIAGAS software provided by Walz.
The sodium content of leaves was determined by flame photometry. Dried plant tissue was combusted overnight at 550°C. The ashed samples were placed in ceramic crucibles and moistened with a few drops of distilled water and extracted three times with concentrated HCl while incubated in a steam bath. The sodium was determined in diluted extracts via flame photometry using 0, 0.8, 1.6, 2.4, 3.2, and 4.0 µg mL–1 sodium as standards. Statistical significance was determined using Student's t test, assuming unequal variances (as indicated by a simple test for the homogeneity of variances) using the R statistical software package (http://cran.r-project.org).
Wild-type and M4 seeds from two putative CAM-deficient mutant lines (351 and 371) were germinated and propagated as described above under greenhouse conditions except that individual plants were grown in 1-L styrofoam pots containing commercial soil mix (MetroMix 200) and irrigated every other day with 0.5x Hoagland solution number 2. Five-week-old plants were irrigated every other day with 0.5x Hoagland solution containing 0.3 M NaCl for up to 3 months until flowering was completed. Plants were then allowed to dry completely. Seed pods were harvested and seeds were cleaned, counted, and weighed from individual plants.
Samples of 1 ± 0.1 mg dry weight were taken from each batch of seeds from wild type and lines 351 and 371 (10 samples from each line) and were placed in tin capsules (D1006; Elemental Microanalysis) before positioning in an autosampler. Samples were then passed through a combustion furnace, reduction furnace, water traps, and gas chromatograph before being transferred to the mass spectrometer. Enrichment in 13C was determined using continuous flow isotope ratio mass spectrometry (ANCA SL preparation unit interfaced with a 20/20 mass spectrometer; PDZ Europa). All samples were analyzed in duplicate and referenced to an internal flour standard. The flour standard had been previously calibrated against NBS22 IAEA standard. Results are given as
Leaves (primary leaf 4) were harvested from wild type, lines 351 and 371, at 4-h intervals over a 24-h light/dark cycle after the plants had been treated with 0.3 M NaCl for 2 weeks. The sampled leaves were snap frozen, powdered in liquid N2, and stored at –80°C until analyzed. Frozen tissue (0.3 g) was ground completely using a mortar and pestle and placed in 3 mL of 80% ethanol at 80°C for 1 h. Aliquots of the methanol extracts were titrated against 1 mol m–3 NaOH to a neutral endpoint, as indicated by phenolphthalein or direct measure with a pH meter, and leaf titratable acidities were expressed as µmol H+ g–1 fresh weight.
The soluble sugar content of methanol extracts was determined using the colorimetric phenol-sulfuric acid test as described (Dubois et al., 1956
Four-week-old plants were irrigated every other day with 0.5x Hoagland solution (control) or with 0.5x Hoagland solution containing 0.3 M NaCl (salt stressed) for 10 d. Replicates of primary leaf 4 were detached, weighed, and the petiole immersed in water for the controls and 0.2 M Glc, Suc, or mannitol solutions for the treatments. Sugar feeding commenced 2 h into the photoperiod and lasted until the leaves were sampled at the end of the photoperiod (10 h in sugar solution) and at the end of the dark period (22 h in sugar solution). Leaves were frozen in liquid N2 and stored at –80°C until analyzed for titratable acids and soluble sugar content as described above.
Five-week-old plants were irrigated every other day with 0.5x Hoagland solution (control) or with 0.5x Hoagland solution containing 0.3 M NaCl (salt stressed) for 14 d. In-gel enzyme activity assays for PGI (EC 5.3.1.9), PGM (EC 5.4.2.2), and AGP (EC 2.7.7.27) were conducted according to previously described methods using 7% (w/v) PAGE with PGI and PGM activity detected as the formation of formazan in enzyme-coupled reactions, whereas AGP activity was detected by calcium pyrophosphate precipitation (Wang et al., 1998
Total RNA was purified from 150 mg of powdered leaf tissue bulked from leaves of four individual plants using TRIzol reagent (Invitrogen) as described (Taybi and Cushman, 1999 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AF158091 (Ppck1), X13660 (Ppc1), BG269256 (Pgm2), AB190772 (Gpt2), and AF053563 (Ubq1).
The following materials are available in the online version of this article.
We thank Mary Ann Cushman and Susan Patterson for technical assistance, Edna Antony and Richard French for help with the carbohydrate analyses, and Karen A. Schlauch for assistance with statistical data analysis. We gratefully acknowledge Davina Bufford, Laura Clay, Monica Dennis, Dennis Shoun, Ryan Snow, Katherine Baumann, Joshua Branco, Monica Orten, and João P. Maroco for technical assistance with plant care, seed cleaning, and routine screening of ice plant mutant collections. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation or the National Institutes of Health. Received January 27, 2008; accepted March 2, 2008; published March 7, 2008.
1 This work was supported in part by the National Science Foundation (grant nos. IBN–9722285 and IBN–0196070 to J.C.C.), as well as the Nevada Agricultural Experiment Station (publication no. 03087098), the Natural Environment Research Council UK (grant no. NER/A/S/2001/01163 to A.M.B.), and Newcastle University. This publication was also made possible by National Institutes of Health Grant Number P20 RR–016464 from the INBRE Program of the National Center for Research Resources through its support of the Nevada Genomics, Proteomics, and Bioinformatics centers.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: John C. Cushman (jcushman{at}unr.edu).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.116889 * Corresponding author; e-mail jcushman{at}unr.edu.
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ABSTRACT
RESULTS
Glc-1-P and is critical to subsequent gluconeogenesis and starch formation. Validation of this genetic lesion was demonstrated by the restoration of the CAM phenotype by feeding detached leaves with Glc or Suc. We discuss how the availability of such CAM-deficient mutants could improve our understanding of the complex day/night metabolic and circadian regulatory processes that govern CAM. 
(i.e. more C3-like) compared to wild-type seed, whereas seed from mutant line 371 was depleted in 13C by up to 1
) in leaf titratable acidity at the start and end of the subsequent dark period were determined and plotted as the difference between the two measurements. Each point is the mean of five replicates ± SE. Student's t test P value for control between WT and 351: P = 5.4 x 10–4** and WT and 371: P = 3.3 x 10–4**. Student's t test P value for Suc feeding between WT and 351: P = 1.4 x 10–2* and WT and 371: P = 1.8 x 10–2*. Student's t test P value for mannitol feeding between WT and 351: P = 2.0 x 10–4** and WT and 371: P = 1.2 x 10–6**.
ADP-Glc + pyrophosphate (PPi; Lee et al., 2007
13C (
-amylase (Sigma-Aldrich) overnight at 40°C. After centrifugation at 12,000g for 10 min, the supernatant was assayed for Glc equivalents as described (Dubois et al., 1956
