*Present address: Department of Biology, University of Texas – Pan American, Edinburg, TX 78539, USA.
Influence of atmospheric oxygen on leaf structure and starch deposition in Arabidopsis thaliana
Article first published online: 21 DEC 2001
Blackwell Science Ltd
Plant, Cell & Environment
Volume 24, Issue 4, pages 419–428, April 2001
How to Cite
Ramonell, K. M., Kuang, A., Porterfield, D. M., Crispi, M. L., Xiao, Y., Mcclure, G. and Musgrave, M. E. (2001), Influence of atmospheric oxygen on leaf structure and starch deposition in Arabidopsis thaliana. Plant, Cell & Environment, 24: 419–428. doi: 10.1046/j.1365-3040.2001.00691.x
- Issue published online: 21 DEC 2001
- Article first published online: 21 DEC 2001
- cell size;
- leaf ultrastructure
Plant culture in oxygen concentrations below ambient is known to stimulate vegetative growth, but apart from reports on increased leaf number and weight, little is known about development at subambient oxygen concentrations. Arabidopsis thaliana (L.) Heynh. (cv. Columbia) plants were grown full term in pre-mixed atmospheres with oxygen partial pressures of 2·5, 5·1, 10·1, 16·2, and 21·3 kPa O2, 0·035 kPa CO2 and the balance nitrogen under continuous light. Fully expanded leaves were harvested and processed for light and transmission electron microscopy or for starch quantification. Growth in subambient oxygen concentrations caused changes in leaf anatomy (increased thickness, stomatal density and starch content) that have also been described for plants grown under carbon dioxide enrichment. However, at the lowest oxygen treatment (2·5 kPa), developmental changes occurred that could not be explained by changes in carbon budget caused by suppressed photorespiration, resulting in very thick leaves and a dwarf morphology. This study establishes the leaf parameters that change during growth under low O2, and identifies the lower concentration at which O2 limitation on transport and biosynthetic pathways detrimentally affects leaf development.
In the 1970s, experiments with representative C3 and C4 plants were conducted to understand the consequences of long-term inhibition of the oxygenase activity of Rubisco for plant growth. As expected, a great stimulation of vegetative growth occurred for C3, but not for C4 species when plants were grown at 5·1 kPa O2. Reproductive development (scored as amount of seed produced) in both types of plants was completely inhibited by growth at 5·1 kPa O2, leading Quebedeaux & Hardy (1973, 1975, 1976) to speculate that low oxygen had effects on plant growth and development apart from the easily demonstrable inhibition of photorespiration.
Photorespiration is decreased in C3 species by lowering O2 concentrations or by raising CO2 concentrations during measurement. Although the response of gas exchange to O2 and CO2 concentrations has been well characterized, there is little information on the effects of long-term exposure to different CO2 and O2 levels on leaf development. Long-term exposure to elevated CO2 has been shown to change leaf development in some species by increasing leaf thickness. Extra palisade layers and/or increased spongy mesophyll have been implicated in this response.
In short-term (4–12 d) experiments, Byrd & Brown (1989) tested the effects of O2 and CO2 concentrations by growing C3, C4, and C3–C4 intermediate species under different gas concentrations. Leaves developed under these conditions were tested for gas exchange and anatomical characteristics. Byrd & Brown (1989) concluded that short-term changes in CO2 concentration (0·075 kPa) during growth produced no direct effects on leaf anatomy. However, growth in low O2 (2 kPa) strongly influenced leaf development, especially that of the C3–C4 intermediate, Panicum milioides, suggesting a more critical role of O2 on development that is not related to changes in photorespiratory activity.
We recently reported details on the control of seed development by atmospheric oxygen in Arabidopsis thaliana (Kuang, Crispi & Musgrave 1998). Here we report on oxygen control of development of leaf structure, including a detailed analysis of starch deposition over atmospheric oxygen partial pressures ranging from 2·5 to 21·3 (ambient) kPa.
The National Aeronautics and Space Administration (NASA) is interested in using plants to regenerate air, cleanse water, and produce food as part of a life-support system for long-duration space missions (Sager & Drysdale 1997). As plant productivity is higher at low oxygen tensions and less oxygen would be required to maintain the growth environment, a low oxygen/low pressure growth scenario is being considered for this specialized application. However, more information is needed about how oxygen concentrations control plant development. Using the model plant Arabidopsis thaliana we have conducted a detailed microscopy study of leaf development during long-term plant growth in subambient oxygen.
MATERIALS AND METHODS
Arabidopsis thaliana (L.) Heynh var. ‘Columbia’ plants were grown on Terralite (peat-vermiculite artificial soil medium, Grace Sierra Horticultural Products Co., Milpitas, CA, USA) supplemented with Osmocote slow-release fertilizer. Six days after seeding, plants were transferred to Sun transparent bags (Sigma, St Louis, MO, USA) under 250 μmol m−2 s−1 PAR continuous illumination at 25 °C. The bags were continuously purged with air or with premixed gases containing different oxygen levels (2·5, 5·1, 10·1, 16·2 kPa), 0·035 kPa CO2, and the balance N2 (Crispi, Porterfield & Musgrave 1996b). The gas composition in the premixed tanks and in the bags was confirmed by periodic sampling and gas chromatography.
Processing for microscopy and starch quantification
After 25 d in the bags, plants were harvested for analysis. Fully expanded leaves from the basal rosettes and flowers prior to anthesis were dissected and fixed with 1% formaldehyde and 2·5% glutaraldehyde in 0·1 M phosphate buffer, pH 7·0, and post-fixed in 1% osmium tetroxide. After being washed with the same buffer, leaf tissues were dehydrated in an ethanol series, then infiltrated with and embedded in Spurr's resin (Spurr 1969). Embedded leaf tissues were sectioned with a DuPont Sorvall microtome (DuPont, Newton, CN, USA). Sections 1 μm thick were stained with 1% toluidine blue O (TBO) in 2% sodium borate for general tissue staining, and periodic-Schiff's reagent (PAS) for polysaccharides. Sections were observed and photographed using a Nikon light microscope (Nikon, Melville, NY, USA). Thin sections were stained with uranyl acetate and lead citrate, and observed and photographed under a JEOL JEM 100CX transmission electron microscope (JEOL, Ltd, Tokyo, Japan).
For photometric quantification, a grid with 2 mm sections was laid on photographs and points of cell area were counted. Leaf blade thicknesses were determined by measuring the thickness of leaf cross-sections at four locations midway between the minor veins immediately flanking the midvein of the lamina. The results were analysed by analysis of variance (ANOVA) using Number Cruncher Statistical Software (Hintze 1987).
Leaf samples for starch quantification were lyophilized overnight and ground using a mortar and pestle. An aliquot (25 mg) of each of these samples was then placed into a lock top microcentrifuge tube with 1·25 mL of 80% ethanol and heated at 90 °C for 5 min with intermittent vortexing. The solutions were then centrifuged at 400 g in a microcentrifuge for 10 min. This ethanol extraction step was repeated three times. The supernatant solutions were removed and evaporated to dryness using a vacuum evaporator and subjected to soluble sugar analysis (Jones, Outlaw & Lowry 1977). The pellet was saved for starch analysis according to the procedures outlined by Sasek, Delucia & Strain (1984). The resulting data were analysed using ANOVA.
Experiments with det2, a brassinolide-deficient mutant
Seeds of the Arabidopsis mutant det2 were obtained from the Arabidopsis Biological Resource Center (ABRC, Ohio State University, Columbus, OH, USA). For comparison of leaf development, gas and brassinolide response of this mutant with var. Columbia, surface-sterilized seeds were sown in Petri plates containing nutrient agar (0·6% Hoagland's solution amended with 0·5% sucrose) and vernalized at 4°C for 2 d. The plants were grown under continuous fluorescent light (250 μmol m−2 s−1) for 10 d until the first leaves had emerged and expanded. Plants were then aseptically transferred to autoclaved, coupled Magenta vessels containing nine 7 mL polycarbonate centrifuge tubes. The tubes contained either nutrient agar or nutrient agar amended with 10−8, 10−7 or 10−6M brassinolide (Cidtech Research, Ontario, Canada). Fixation, microscopy, measurement of leaf thickness, and data analysis were accomplished as described above. To examine the interaction between low O2 and brassinolide supplementation in var. Columbia, the plants grown in the coupled Magenta vessels were then connected to the compressed gas mixtures described previously. Gas flow into each Magenta unit was set to 35 mL min−1, creating a slight positive pressure inside each unit. After 10 d in the gas treatment, the plants were harvested for analysis.
In order to determine quantitatively the effect of low oxygen on brassinolide biosynthesis, plants (var. Columbia) were grown in the five oxygen treatments using the Sunbag growth system and Terralite-filled flats as described before, and the entire plant population produced in each treatment was frozen and analysed for brassinolide content. Plant material was ground in liquid nitrogen, extracted in methanol and left to stir overnight. It was then filtered and the aqueous residue partitioned three times against chloroform. The chloroform phases were combined and evaporated to dryness. The residue was partitioned between hexane and 80% methanol. The 80% methanol phases were combined and evaporated to dryness. The residue was resolubilized in ethanol, derivatized with methylboronic acid, and examined by gas chromatography-mass spectroscopy (GC-MS).
Arabidopsis thaliana plants grown in the environment containing 2·5 kPa O2 are small in comparison with plants grown in the environments containing higher oxygen concentrations (Fig. 1). The leaf blades are smaller and are tinged reddish-purple due to accumulation of anthocyanins. Measurement of leaf thickness indicates that these leaves are also thicker in comparison with leaves from plants grown in higher oxygen concentrations (Table 1). Observation of leaf structure shows that these leaves are very different from the leaves from the higher oxygen environments (Fig. 2). The most obvious characteristic is that a thick cuticle layer covers the leaf surface, and the cell wall of the epidermal and mesophyll cells of the leaf blades are thicker (Fig. 3). These leaves have a small fraction of intercellular space and therefore the leaf structure is compacted (Fig. 1, Table 1). However, there were no significant differences in leaf thickness, cell wall thickness, and the fraction of intercellular space at any of the higher oxygen concentrations (Table 1).
|Cell size (μm2)||Fractions in the leaf (%)|
|O2 (kPa)||Thickness (μm)||epidermis||mesophyll||space||epidermis||mesophyll|
Scanning electron microscope analysis of the leaf surface revealed striking differences in stomatal density, epidermal cell shape, and stomatal distribution with decreasing oxygen. As shown in Fig. 4, stomata occur with increasing frequency per unit area as O2 partial pressure in the environment declines. Similar trends of increased stomatal frequency with decreased oxygen occurred in both the adaxial and abaxial surfaces of the leaves.
Mesophyll cells are highly differentiated. A mesophyll cell has a large vacuole in the centre of the cell and many chloroplasts align along the cell membrane. At 2·5 kPa O2, grana in the chloroplasts were observed, although they had less distinct stacking compared with those from the 21·3 kPa oxygen environment (Fig. 5). Chloroplasts that contain large starch grains are expanded, thus their shape is more rounded (Fig. 6). Large and elongated mitochondria with well-developed cristae were observed in mesophyll cells (Fig. 7).
At 2·5 kPa O2, large starch grains accumulate in plastids (Fig. 2). The size of starch grains was significantly larger in the plastids from plants grown in an oxygen environments below 21·3 kPa (Table 2, Fig. 6). Biochemical analysis of starch content in the leaves indicates that leaves from all oxygen treatments below 15 kPa had significantly higher starch (expressed as glucose equivalents) than the ambient controls (Table 2). Furthermore, leaf starch was significantly higher in the 2·5 kPa treatment than in all other treatments.
|O2 (kPa)||n/plastid||size (μm2)||n/cell||size (μm2)||Starch (μg mg−1)|
Numerous large starch grains were also observed in the anther tissues from plants grown in the 2·5 kPa treatment (Fig. 8). In those anthers, pollen grains did not develop well or aborted before anthesis. In contrast, anthers from plants grown in higher atmospheric oxygen did not have obvious starch grains located in the anther tissues but many starch grains accumulated in the developing pollen grains (Fig. 8).
Experiments with det2, a brassinolide-deficient mutant
Leaf blade thickness was compared in two lines of Arabidopsis thaliana, Columbia (wild-type), and the det2 mutant, which is blocked in the biosynthetic pathway of brassinosteroids (Li et al. 1996). The det2 mutant had significantly thicker leaves than the wild-type when grown under normal oxygen conditions (Fig. 9), and addition of brassinolide to the growth medium resulted in leaves that were more similar in morphology to those of Columbia (Fig. 9). Interestingly, the det2 mutant was also found to be insensitive to the dwarfing effects of very low O2 (2·5 kPa) and furthermore did not show the purpling stress response of Columbia (Fig. 10). This led us to hypothesize that the unique oxygen response of Columbia at the lowest O2 concentration might be due to a loss of brassinolide biosynthesis. Addition of brassinolide (10−7M) to the growth medium of Columbia could prevent the expected increase in leaf thickness when plants were grown under low O2 (Fig. 11), resulting in partial restoration of the ambient O2 morphology. Brassinolide content of Columbia plants grown under the five oxygen treatments for 28 d was below the limit of detection by GC-MS.
Leaf anatomy changes – similarities with CO2 enrichment responses
Elevated carbon dioxide is known to increase leaf thickness and deposition of starch in mesophyll cells. The gradual increase of mesophyll cell area and decrease of intercellular space shown by Arabidopsis with decreasing O2 concentration (Table 1) mirrors the changes in leaf anatomy shown by Lolium perenne with CO2 enrichment (Ferris et al. 1996). Low oxygen treatments of 2·5 kPa significantly increased leaf and mesophyll thickness (Table 1) and stomatal density (Fig. 4) in Arabidopsis. Leaf thickness, area of mesophyll cells, and thickness of epidermal cell layer increased with a doubling of CO2 in Lolium perenne L. (Ferris et al. 1996). Significant increases in leaf thickness were found for three C3 species (soybean, loblolly pine, sweet gum) under enriched CO2 conditions (520– 910 p.p.m.), but not for corn, a C4 plant (Thomas & Hardy 1983). Variation occurred among species concerning the location of the thickness increase: palisade or mesophyll cells.
The size of starch grains and plastids increased in Arabidopsis leaves as the oxygen partial pressure of their growth environment decreased. A concomitant rise in the amount of leaf starch occurred with decreasing oxygen (Table 2). The literature on carbon dioxide enrichment also describes an increase in leaf starch due to assimilation rates in excess of sink consumption of carbohydrate (Wulff & Strain 1982; Delucia, Sasek & Strain 1985), and in Desmodium, starch build-up under CO2 enrichment was sufficient to disrupt plastid structure (Wulff & Strain 1982). In Arabidopsis, extreme (three-fold increase) starch accumulation occurred in the 2·5 kPa oxygen treatment (Fig. 2; Table 2). At this O2 concentration, individual starch grains were more than twice as large in cross-section as in the ambient O2 controls (Table 2).
Ultrastructural changes – indications for physiological function
Although plant metabolic adaptations to hypoxia in the root-zone have been most extensively studied, Dolferus et al. (1997) have shown that genes involved in the alcoholic fermentation pathway (adh1 and pdc1) are also inducible in leaves of Arabidopsis, albeit less strongly than in the roots. Although we observed no evidence of ultrastructural changes associated with hypoxia in the higher subambient oxygen treatments, elongated mitochondria (Fig. 7b) were found in transmission electron micrographs of leaf tissue from plants grown in the 2·5 kPa oxygen environment. Vartapetian (1991) has described similar ultrastructural features in mitochondria from plant tissue that was exposed to sudden anoxia through nitrogen purging.
The striking build-up of starch in Arabidopsis leaves and anther tissues during pollen development in the 2·5 kPa oxygen treatment suggests that transport and/or utilization of carbohydrate is impaired during plant growth at very low oxygen concentrations. In addition to starch, the foliar concentration of soluble sugars increases dramatically at 2·5 kPa O2 in Arabidopsis (Porterfield, Crispi & Musgrave 1997). Low O2 effects on the carbon budget are also manifest during reproductive development. Although Arabidopsis pollen can be viable when plants are grown at 2·5 kPa oxygen, the viability is reduced in comparison with well-oxygenated conditions (Crispi et al. 1996a). Porterfield et al. (1999) have demonstrated hypoxic microsites within developing reproductive structures of Brassicaceae inside unopened flower buds and siliques. Thorne (1982) showed that import of 14C photosynthate by soybean embryos through phloem in vivo was completely blocked at 0 kPa oxygen, and suggested that this explains the reproductive sterility of soybean plants grown at 5 kPa O2 (Quebedeaux & Hardy 1975). However, Thorpe, Minchin & Dye (1979) showed that phloem loading, tracked using 11CO2, was only reduced by 70% following leaf anoxia in selected C3 species, whereas phloem loading in C4 species was completely unaffected by leaf anoxia. Furthermore, treatment of wheat leaves with 1 kPa O2 hardly affected phloem loading at all; full anoxia was needed to achieve a substantial inhibition. Our results support Byrd & Brown's (1989) conclusion that the effects of low O2 may be on the rate of photosynthate consumption rather than on phloem loading or unloading.
In the few previous studies of changes in leaf anatomy induced by extended growth under reduced oxygen, an increased deposition of leaf starch has also been observed, and has been explained by an increase in net carbon gain due to suppressed photorespiration. To understand the impact of photorespiration on growth, the gas exchange behaviour of species in the genus Flaveria, which contains both C3 and C4 plants, was studied under reduced (1·5 kPa) oxygen in order to find naturally occurring C3–C4 intermediate plants (Holaday, Lee & Chollet 1984; Ku et al. 1983). A later study found no change in leaf anatomy when Flaveria C3–C4 intermediates were grown under elevated (0·075 kPa) CO2, although a C3–C4 intermediate Panicum species (as well as two C3 species) grown under reduced O2 (2 kPa) showed great increases in the amount of starch visible in leaf cross-sections. Total available carbohydrates were about 50% higher in the 2 kPa O2 plants (Byrd & Brown 1989). Both C3 (Panicum laxum;Festuca arundinacea) and a C3–C4 intermediate (P. milioides) showed strong accumulation of starch in mesophyll cells (and bundle sheath cells when applicable) when grown under 2 kPa oxygen conditions (Byrd & Brown 1989). Reduction in apparent net photosynthesis in P. milioides under these conditions was attributed to acute starch build up.
A role for brassinolides in low O2 response?
Arabidopsis plants growing in 16·2, 10·1, and 5·1 kPa O2 did not differ substantially in appearance from plants grown under ambient conditions, despite subtle changes that were occurring at the cellular level (Tables 1 & 2). However, the lowest oxygen growth environment, 2·5 kPa, resulted in dwarf plants (Fig. 1). The appearance of Arabidopsis plants grown at 2·5 kPa O2 resembles that of two recently described Arabidopsis mutants that are deficient in the brassinosteroid biosynthetic pathway, det2 and cpd. Brassinosteroids are implicated in the normal development and elongation of cells and require molecular oxygen at several steps in their biosynthesis (Mandava 1988; Fujioka et al. 1995; Clouse, Langford & McMorris 1996; Szekeres et al. 1996; Hooley 1996). Both the det2 and cpd mutants display overall dwarfism and male sterility (Szekeres et al. 1996; Li et al. 1996), analogous to traits shown by Columbia when grown under 2·5 kPa oxygen. Although the cpd mutant was not available for this study, greatly increased leaf thickness in the det2 mutant is restored to normal with addition of brassinolide (Fig. 9). Growth of det2 under low O2 showed its growth to be unchanged in the 2·5 kPa O2 treatment, making its growth form insensitive to the effects of very low O2 (Fig. 10). These findings are consistent with a hypothesis that very low O2 prevents brassinolide biosynthesis in Arabidopsis, and indeed, addition of brassinolide to low O2-grown Columbia plants could prevent the expected low O2-induced increase in leaf thickness. Nonetheless, GC-MS failed to detect brassinolide in any of the treatments. Correlative data on brassinolide effects on low O2 changes in leaf length and stomatal density were reported by Ramonell (1999).
Because of the similarity of leaf anatomy changes (increased cell density, starch content) at lower atmospheric O2 concentrations to those caused by carbon dioxide enrichment, they are probably mediated through a change evoked by the favoured ratio of photosynthesis to photorespiration that both treatments cause. For Arabidopsis, growth at 2·5 kPa O2 results in leaf starch contents that are three times those of ambient-grown plants, and carbohydrate transport into developing pollen grains is seriously impaired. Because leaf thickness shows a very large increase between 5 and 2·5 kPa O2, we suggest that low O2 impairment of the brassinosteroid biosynthetic pathway contributes to this phenomenon, as well as to the overall dwarf appearance of plants growing in 2·5 kPa O2. The consequences of reduced photorespiration over the full life cycle on plant growth and development will show a species dependency, since source–sink relationship will vary according to the developmental programme. However, this study should serve to establish the leaf parameters that will change during growth under low O2, with special emphasis on identifying the lower limit, where O2 limitation of carbohydrate transport and perhaps brassinolide synthesis detrimentally affect leaf development.
This study was supported by NASA grants to M.E.M. (NAG5–3756; NAG2–1020; NAG2–1375), and by a fellowship to K.M.R. from the Louisiana Space Consortium.
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