Evidence for the expression of morphological and biochemical characteristics of C3-photosynthesis in chlorohyllous callus cultures of Zea mays

A Zea mays callus culture containing chlorophyll was established and grown photomixotrophically. Cell chloroplast structure, and pigment and soluble protein contents were examined. Expression of some key enzymes of C₄ carbon metabolism was compared with that of etiolated (heterotrophic) and green photoautotrophic leaves. Chlorophyll content of the callus was 15–20% that of green leaves. Soluble protein content of callus was half that of leaf cells. Electron microscopic observations showed that green callus cells contained only typical granal chloroplasts. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.38) activities in green callus were ca 30% those of green leaves but 2–3 times higher than in etiolated leaves. Quantitative enzyme protein determination, using antibodies specific to maize leaf Rubisco showed that the chloroplastic carboxylase represented about 7% of total soluble protein in green callus, in parallel to its low chlorophyll content. The specific activity of Rubisco in callus and leaves was unchanged. Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) activity in green callus was about 20% that of green leaves and similar to that measured in etiolated leaves. Apparent K_m (PEP) values (0.08 mM) for PEPC isolated from green callus and etiolated leaves were very different from values (0.5 mM) obtained with PEPC from green leaves. These kinetic characteristics together with the absence of inhibition by malate and activation by glucose-6-phosphate suggest that the properties of PEPC isolated from green callus and etiolated maize leaves are very similar to those of PEPPC from C₃ plants. Using PEPC antibodies specific to green maize leaf enzyme, immunotitration of PEPC preparations containing identical enzyme units allowed complete precipitation of the green leaf enzyme with increasing antibody volumes. In contrast, 60–70% of the activity of PEPC from etiolated and green callus was inhibited, suggesting low affinity for the maize green leaf PEPC antiserum (typical C₄ form). Ouchterlony double diffusion tests revealed only partial recognition of PEPC in green callus and etiolated leaves. NAD-malate dehydrogenase (NAD-MDH, EC 1.1.1.37) activity in callus was 2 and 3 times higher, respectively, than in etiolated and green leaves. NADP-malic enzyme (NADP-ME, EC 1.1.1.40) activity in callus cultures was much lower than in green leaves. All our data support the hypothesis that cultures of fully dedifferentiated chlorophyllous tissues of Zea mays possess a C₃-like metabolism.     

High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize

A high throughput genetic transformation system in maize has been developed with Agrobacterium tumefaciens mediated T-DNA delivery. With optimized conditions, stable callus transformation frequencies for Hi-II immature embryos averaged approximately 40%, with results in some experiments as high as 50%. The optimized conditions include N6 medium system for Agrobacterium inoculation, co-cultivation, resting and selection steps; no AgNo₃ in the infection medium and adding AgNo₃ in co-cultivation, resting and selection medium; Agrobacterium concentration at 0.5×10⁹ c.f.u. ml^–1 for bacterium inoculation; 100 mg l^–1 carbenicillin used in the medium to eliminate Agrobacterium after inoculation; and 3 days for co-cultivation and 4 days for resting. A combination of all of these conditions resulted in establishing a high throughput transformation system. Over 500 T₀ plants were regenerated and these plants were assayed by transgene expression and some of them were also analyzed by Southern hybridization. T₁ plants were analyzed and transmission of transgenes to the T₁ generation was verified. This represents a highly reproducible and reliable system for genetic transformation of maize Hi-II.     

Silicon Carbide Whisker-Mediated Embryogenic Callus Transformation of Cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) and Regeneration of Salt Tolerant Plants

A silicon carbide whisker-mediated gene transfer system with recovery of fertile and stable transformants was developed for cotton (Gossypium hirsutum L.) cv. Coker-312. Two-month-old hypocotyl-derived embryogenic/non-embryogenic calli at different days after subculture were treated with silicon carbide whiskers for 2 min in order to deliver pGreen0029 encoding GUS gene and pRG229 AVP1 gene, encoding Arabidopsis vacuolar pyrophosphatase, having neomycin phosphotransferaseII (nptII) genes as plant-selectable markers. Three crucial transformation parameters, i.e., callus type, days after subculture and selection marker concentration for transformation of cotton calli were evaluated for optimum efficiency of cotton embryogenic callus transformation giving upto 94% transformation efficiency. Within six weeks, emergence of kanamycin-resistant (km^r) callus colonies was noted on selection medium. GUS and Southern blot analysis showed expression of intact and multiple transgene copies in the transformed tissues. Kanamycin wiping of leaves from T₁, T₂, and T₃ progeny plants revealed that transgenes were inherited in a Mendelian fashion. Salt treatment of T₁ AVP1 transgenic cotton plants showed significant enhancement in salt tolerance as compared to control plants. Thus far, this is first viable physical procedure after particle bombardment available for cotton that successfully can be used to generate fertile cotton transformants.     

Transgenic zoysiagrass (Zoysia japonica) plants obtained by Agrobacterium-mediated transformation

Zoysiagrass (Zoysia japonica Steud.) is an important turfgrass that spreads by stolons and rhizomes. By exploring the potential of direct shoot formation from stolons, we developed a straightforward and efficient transformation protocol without callus induction and propagation. Sterilized stolon nodes were infected and co-cultivated with Agrobacterium tumefaciens harboring pCAMBIA vectors. Hygromycin phosphotransferase gene (hph) was used as the selectable marker and hygromycin was used as the selection agent. Both green and albino shoots were directly regenerated from the infected stolon nodes 4–5 weeks after hygromycin selection. Greenhouse-grown plants were obtained 10–12 weeks after Agrobacterium-mediated transformation. Based on the number of transgenic plants obtained and the number of stolon nodes infected, a transformation frequency of 6.8% was achieved. Stable integration of the transgenes in the plant genome was demonstrated by PCR and Southern blot hybridization analyses. Expression of the transgenes was confirmed by RT-PCR analysis and GUS staining. The new transformation system opens up new opportunities for the functional characterization of genes and promoters and the development of novel germplasm in zoysiagrass.     

Generation of transgenic Lolium temulentum plants by Agrobacterium tumefaciens-mediated transformation

Lolium temulentum L. (Darnel ryegrass) has been proposed to be used as a model species for functional genomics studies in forage and turf grasses, because it is a self-fertile, diploid species with a short life cycle and is closely related to other grasses. Embryogenic calluses were induced from mature embryos of a double haploid line developed through anther culture. The calluses were broken up into small pieces and used for Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harboring pCAMBIA1301 and pCAMBIA1305.2 vectors were used to infect embryogenic callus pieces. Hygromycin was used as a selection agent in stable transformation experiments. Hygromycin resistant calluses were obtained after 4–6 weeks of selection and transgenic plants were produced in 10–13 weeks after Agrobacterium-mediated transformation. Fertile plants were readily obtained after transferring the transgenics to the greenhouse. Transgenic nature of the regenerated plants was demonstrated by Polymerase chain reaction (PCR), Southern hybridization analysis, and GUS staining. Progeny analysis showed Mendelian inheritance of the transgenes. The transformation system provides a valuable tool for functionality tests of candidate genes in forage and turf grasses.     

Self-fertile transgenic wheat plants regenerated from isolated scutellar tissues following microprojectile bombardment with two distinct gene constructs†

A system for enhanced induction of somatic embryo-genesis and regeneration of plants from isolated scutellar tissue of wheat has been developed. This system has been successfully used in the development of a simple and reproducible protocol for the production of self-fertile transgenic wheat plants. The procedure is rapid resulting in the production of transgenic plantlets within 12 weeks from initiation of cultures and it avoids the need for establishing long-term callus, cell suspension or protoplast cultures. Somatic embryos regenerated from scutella bombarded with plasmid pBARGUS were selected on L-phosphinothricin (L-PPT) to obtain herbicide-resistant self-fertile transgenic plants. Phosphinothricin acetyltransferase (PAT) activity was observed at varying levels in 50% of the plants selected on L-PPT whereas none of the plants showed β-glucuronidase (GUS) activity. Molecular analysis of PAT-positive plants confirmed stable integration of both bar and gus genes in R₀ and R₁ progeny plants. Segregation of the PAT activity and herbicide resistance in R₁ progeny plants confirmed the Mendelian inheritance of the bar gene. Additionally, isolated scutella bombarded with plasmid DNA containing a gus::nptII fusion gene driven by a rice actin promoter and its first intron were selected in the presence of geneticin to obtain fully fertile transgenic plants. Functional expression of the fusion gene was demonstrated in transgenic plants by GUS and neomycin phospho-transferase (NPTII) enzyme assays. Southern blot analysis confirmed the integration of transgenes into the wheat genome. Histochemical GUS staining showed transmission of the fusion gene to floral organs of primary transformants and confirmed Mendelian segregation of the transgene in R₁ progeny.     

Molecular weights of glycollate oxidase from C3 and C4 plants determined during early stages of purification

The M_rs of glycollate oxidase (EC 1.1.3.1) (GAO) determined soon after extraction from the leaves of several C₃ and C₄ plants are reported. The enzyme isolated from the C₃ plants wheat, barley, spinach, pea and tobacco has M_r in the range 160–180 000 and is probably a homotetramer. GAO purified from pea was previously reported as a dimer and as an octamer from spinach leaves. Therefore the quaternary structure of these GAOs soon after extraction differs from that of the purified proteins. The enzymes from the C₄ plants maize and sugar cane have M_rs ca twice this value in the range 290–310 000, whilst that of the C₄ grass Panicum maximum has an M_r of 162 000. An improved spectrophotometric assay for GAO, using a non-carcinogenic dye, is described.     

Accelerated production and identification of fertile,homozygous transgenic wheat lines by anther culture

Anther culture has been developed in the winter wheat cultivar Florida to achieve accelerated production and identification of homozygous transgenic lines. With untransformed, seed-derived plants to develop the culture system, it was shown that cold pre-treatment of spikes excised from donor plants and addition of 2,4-dichlorophenoxyacetic acid together with either kinetin or 6-benzylaminopurine in the callus induction medium improves the anther culture response. The procedure developed allowed production of fertile homozygous lines within 8–9 months, which includes an 8-week vernalisation period. With transgenic wheat plants produced by particle bombardment as donors, we show that the system can be used to produce homozygous transgenics, requiring one generation cycle. Both T₀ tissue culture-derived plants and their T₁ seed-derived descendents serve as suitable donors. We show that an anther culture response comparable to that of untransformed, seed-derived plants can be achieved with T₀ tissue culture-derived plants. PCR and Southern molecular analyses of anther culture-derived transgenics show that the transgenes are stably inherited; there are no perturbations at the chromosomal level around the sites of transgene integration as a result of in vitro chromosome manipulation during anther culture.     

Photosynthetic responses to temperature of phosphoenolpyruvate carboxykinase type C4 species differing in cold sensitivity

Photosynthetic rates, the activities of key enzymes associated with the C₄ cycle and ribulose-1,5-bisphosphate carboxylase (RuBPCase), and the levels of metabolites involved in the C₄ cycle were compared between the two phosphoenolpyruvate carboxykinase (PCK) type C₄ species Spartina anglica, which is cold-tolerant, and Zoysia japonica, which is cold-sensitive, during exposure to low temperature. Plants of both species grown outside in summer were placed in a growth chamber at 27/20 °C day/night temperatures. After 1 week, plants were exposed to 20/17 °C for 1 week and then to 10/7 °C for 2 weeks. Photosynthetic rates in Z. japonica decreased progressively to about 25% during the chilling treatments. In contrast, S. anglica exhibited a 43% increase in photosynthetic rates after exposure to 20 °C for 1 week, which remained relatively constant thereafter. Consistent with these observations, most of the C₄ enzymes and RuBPCase in Z. japonica declined. Phosphoenolpyruvate carboxylase (PEPC) and PCK activities declined particularly drastically during the treatments. However, the activities of these enzymes in S. anglica showed either a slight increase or decrease upon a mild cold treatment, and remained relatively constant during further chilling treatments. There was a sharp decline in phosphoenolpyruvate in Z. japonica after exposure to 10 °C. On the other hand, metabolite levels in S. anglica were largely unaffected by the chilling treatments. These results suggest that the drastic declines of both PEPC and PCK activities may be important limiting factors responsible for cold sensitivity in C₄ photosynthesis of Z. japonica.     

Effects of growth temperature and nitrogen nutrition on expression of C3–C4 intermediate traits in Chenopodium album

Proto-Kranz plants represent an initial phase in the evolution from C₃ to C₃–C₄ intermediate to C₄ plants. The ecological and adaptive aspects of C₃–C₄ plants would provide an important clue to understand the evolution of C₃–C₄ plants. We investigated whether growth temperature and nitrogen (N) nutrition influence the expression of C₃–C₄ traits in Chenopodium album (proto-Kranz) in comparison with Chenopodium quinoa (C₃). Plants were grown during 5 weeks at 20 or 30 °C under standard or low N supply levels (referred to as 20SN, 20LN, 30SN, and 30LN). Net photosynthetic rate and leaf N content were higher in 20SN and 30SN plants than in 20LN and 30LN plants of C. album but did not differ among growth conditions in C. quinoa. The CO₂ compensation point (Γ) of C. album was lowest in 30LN plants (36 µmol mol^–1), highest in 20SN plants (51 µmol mol^–1), and intermediate in 20LN and 30SN plants, whereas Γ of C. quinoa did not differ among the growth conditions (51–52 µmol mol^–1). The anatomical structure of leaves was not considerably affected by growth conditions in either species. However, ultrastructural observations in C. album showed that the number of mitochondria per mesophyll or bundle sheath (BS) cell was lower in 20LN and 30LN plants than in 20SN and 30SN plants. Immunohistochemical observations revealed that lower accumulation level of P-protein of glycine decarboxylase (GDC-P) in mesophyll mitochondria than in BS mitochondria is the major factor causing the decrease in Γ values in C. album plants grown under low N supply and high temperature. These results suggest that high growth temperature and low N supply lead to the expression of C₃–C₄ traits (the reduction of Γ) in the proto-Kranz plants of C. album through the regulation of GDC-P expression.

     

Naturally low carbonic anhydrase activity in C4 and C3 plants limits discrimination against C18OO during photosynthesis

The ¹⁸O content of CO₂ is a powerful tracer of photosynthetic activity at the ecosystem and global scale. Due to oxygen exchange between CO₂ and ¹⁸O-enriched leaf water and retrodiffusion of most of this CO₂ back to the atmosphere, leaves effectively discriminate against ¹⁸O during photosynthesis. Discrimination against ¹⁸O ( Δ ¹⁸O) is expected to be lower in C₄ plants because of low c_i and hence low retrodiffusing CO₂ flux. C₄ plants also generally show lower levels of carbonic anhydrase (CA) activities than C₃ plants. Low CA may limit the extent of ¹⁸O exchange and further reduce Δ ¹⁸O. We investigated CO₂–H₂O isotopic equilibrium in plants with naturally low CA activity, including two C₄ (Zea mays, Sorghum bicolor) and one C₃ (Phragmites australis) species. The results confirmed experimentally the occurrence of low Δ ¹⁸O in C₄, as well as in some C₃, plants. Variations in CA activity and in the extent of CO₂–H₂O isotopic equilibrium ( θ _eq) estimated from on-line measurements of Δ ¹⁸O showed large range of 0–100% isotopic equilibrium ( θ _eq = 0–1). This was consistent with direct estimates based on assays of CA activity and measurements of CO₂ concentrations and residence times in the leaves. The results demonstrate the potential usefulness of Δ ¹⁸O as indicator of CA activity in vivo. Sensitivity tests indicated also that the impact of θ _eq < 1 (incomplete isotopic equilibrium) on ¹⁸O of atmospheric CO₂ can be similar for C₃ and C₄ plants and in both cases it increases with natural enrichment of ¹⁸O in leaf water.     

Molecular evolution of phosphoeno/pyruvate carboxylase

Phosphoenolpyruvate carboxylase is an enzyme involved in a wide variety of important metabolic pathways of plants such as anaplerotic reactions and C4 and CAM photosynthetic pathways. The accumulation of molecular sequence data of phosphoenolpyruvate carboxylases has enabled us to investigate the function and molecular evolution of the enzymes by computer-assisted sequence comparison. Here we report the results of sequence comparison of phosphoenolpyruvate carboxylases: (1) Phosphoenofpyruvate carboxylases were classified into four groups; a subgroup of bacterial enzymes and three subgroups of plants enzymes. (2) The divergence time of the monocot enzymes involved in the C₄ pathways was roughly estimated to be 150—300 million years. On the other hand, the phylogenetic tree of the enzymes suggested that those for the dicot enzymes involved in the C₄ and CAM pathways might be close to the divergence time between the monocots and the dicots. (3) The evolutionary positions of the enzymes prevalent in roots or root nodules were identified. (4) Although sorghum and maize contained at least three genes for the enzymes in their genomes, the rates of amino acid substitution of the enzymes were different from gene to gene. The difference could not be explained by either lineage effects nor bias in base contents.     

<Emphasis Type="Italic">Organogenic callus</Emphasis> as the target for plant regeneration and transformation via <Emphasis Type="Italic">Agrobacterium</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.)

A regeneration and transformation system has been developed using organogenic calluses derived from soybean axillary nodes as the starting explants. Leaf-node or cotyledonary-node explants were prepared from 7 to 8-d-old seedlings. Callus was induced on medium containing either Murashige and Skoog (MS) salts or modified Finer and Nagasawa (FNL) salts and B5 vitamins with various concentrations of benzylamino purine (BA) and thidiazuron (TDZ). The combination of BA and TDZ had a synergistic effect on callus induction. Shoot differentiation from the callus occurred once the callus was transferred to medium containing a low concentration of BA. Subsequently, shoots were elongated on medium containing indole-3-acetic acid (IAA), zeatin riboside, and gibberellic acid (GA). Plant regeneration from callus occurred 90 ∼ 120 d after the callus was cultured on shoot induction medium. Both the primary callus and the proliferated callus were used as explants for Agrobacterium-mediated transformation. The calluses were inoculated with A. tumefaciens harboring a binary vector with the bar gene as the selectable marker gene and the gusINT gene for GUS expression. Usually 60–100% of the callus showed transient GUS expression 5 d after inoculation. Infected calluses were then selected on media amended with various concentrations of glufosinate. Transgenic soybean plants have been regenerated and established in the greenhouse. GUS expression was exhibited in various tissues and plant organs, including leaf, stem, and roots. Southern and T₁ plant segregation analysis of transgenic events showed that transgenes were integrated into the soybean genome with a copy number ranging from 1–5 copies.     

Acclimation of photosynthesis in relation to Rubisco and non-structural carbohydrate contents and in situ carboxylase activity in Scirpus olneyi grown at elevated CO2 in the field

Stands of Scirpus olneyi, a native saltmarsh sedge with C₃ photosynthesis, had been exposed to normal ambient and elevated atmospheric CO₂ concentrations (C_a) in their native habitat since 1987. The objective of this investigation was to characterize the acclimation of photosynthesis of Scirpus olneyi stems, the photosynthesizing organs of this species, to long-term elevated C_a treatment in relation to the concentrations of Rubisco and non-structural carbohydrates. Measurements were made on intact stems in the Held under existing natural conditions and in the laboratory under controlled conditions on stems excised in the field early in the morning. Plants grown at elevated C_a had a significantly higher (30–59%) net CO₂ assimilation rate (A) than those grown at ambient C_a when measurements were performed on excised stems at the respective growth C_a. However, when measurements were made at normal ambient C_a, A was smaller (45–53%) in plants grown at elevated C_a than in those grown at ambient C_a. The reductions in A at normal ambient C_a, carboxylation efficiency and in situ carboxylase activity were caused by a decreased Rubisco concentration (30–58%) in plants grown at elevated C_a; these plants also contained less soluble protein (39–52%). The Rubisco content was 43 to 58% of soluble protein, and this relationship was not significantly altered by the growth CO₂ concentrations. The Rubisco activation state increased slightly, but the in situ carboxylase activity decreased substantially in plants grown at elevated C_a. When measurements were made on intact stems in the field, the elevated C_a treatment caused a greater stimulation of,A (100%) and a smaller reduction in carboxylation efficiency (which was not statistically significant) than when measurements were made on excised stems in the laboratory. The possible reasons for this arc discussed. Plants grown at elevated C_a contained more non-structural carbohydrates (25–53%) than those grown at ambient C_a. Plants grown at elevated C_a appear to have sufficient sink capacity to utilize the additional carbohydrates formed during photosynthesis. Overall, our results are in agreement with the hypothesis that elevated C_a leads to an increased carbohydrate concentration and the ensuing acclimation of the photo-synthetic apparatus in C₃ plants results in a reduction in the protein complement, especially Rubisco, which reduces the photosynthetic capacity in plants grown at elevated C_a, relative to plants grown at normal ambient C_a. Nevertheless, when compared at their respective growth C_a, Scirpus olneyi plants grown at elevated C_a in their native habitat maintained a substantially higher rate of photosynthesis than those grown at normal ambient C_a even after 8 years of growth at elevated C_a.     

Stability and inheritance of endosperm-specific expression of two transgenes in progeny from crossing independently transformed barley plants

To study stability and inheritance of two different transgenes in barley, we crossed a homozygous T₈ plant, having uidA (or gus) driven by the barley endosperm-specific B₁-hordein promoter (localized in the near centromeric region of chromosome 7H) with a second homozygous T₄ plant, having sgfp(S65T) driven by the barley endosperm-specific D-hordein promoter (localized on the subtelomeric region of chromosome 2H). Both lines stably expressed the two transgenes in the generations prior to the cross. Three independently crossed F₁ progeny were analyzed by PCR for both uidA and sgfp(S65T) in each plant and functional expression of GUS and GFP in F₂ seeds followed a 3:1 Mendelian segregation ratio and transgenes were localized by FISH to the same location as in the parental plants. FISH was used to screen F₂ plants for homozygosity of both transgenes; four homozygous plants were identified from the two crossed lines tested. FISH results showing presence of transgenes were consistent with segregation ratios of expression of both transgenes, indicating that the two transgenes were expressed without transgene silencing in homozygous progeny advanced to the F₃ and F₄ generations. Thus, even after crossing independently transformed, homozygous parental plants containing a single, stably expressed transgene, progeny were obtained that continued to express multiple transgenes through generation advance. Such stability of transgenes, following outcrossing, is an important attribute for trait modification and for gene flow studies.     

The promoters of two carboxylases in a C4 plant (maize) direct cell-specific, light-regulated expression in a C3 plant (rice)

C₄ plants have two carboxylases which function in photosynthesis. One, phosphoenolpyruvate carboxylase (PEPC) is localized in mesophyll cells, and the other, ribulose bisphosphate carboxylase (RuBPC) is found in bundle sheath cells. In contrast, C₃ plants have only one photosynthetic carboxylase, RuBPC, which is localized in mesophyll cells. The expression of PEPC in C₃ mesophyll cells is quite low relative to PEPC expression in C₄ mesophyll cells. Two chimeric genes have been constructed consisting of the structural gene encoding β-glucuronidase (GUS) controlled by two promoters from C₄ (maize) photosynthetic genes: (i) the PEPC gene (pepc) and (ii) the small subunit of RuBPC (rbcS). These constructs were introduced into a C₃ cereal, rice. Both chimeric genes were expressed almost exclusively in mesophyll cells in the leaf blades and leaf sheaths at high levels, and no or very little activity was observed in other cells. The expression of both genes was also regulated by light. These observations indicate that the regulation systems which direct cell-specific and light-inducible expression of pepc and rbcS in C₄ plants are also present in C₃ plants. Nevertheless, expression of endogenous pepc in C₃ plants is very low in C₃ mesophyll cells, and the cell specificity of rbcS expression in C₃ plants differs from that in C₄ plants. Rice nuclear extracts were assayed for DNA-binding protein(s) which interact with a cis-regulatory element in the pepc promoter. Gel-retardation assays indicate that a nuclear protein with similar DNA-binding specificity to a maize nuclear protein is present in rice. The possibility that differences in pepc expression in a C₃ plant (rice) and C₄ plant (maize) may be the result of changes in cis-acting elements between pepc in rice and maize is discussed. It also appears that differences in the cellular localization of rbcS expression are probably due to changes in a trans-acting factor(s) required for rbcS expression.     

Production of transgenic maize plants and progeny by bombardment of hi-II immature embryos

Summary Production of transgenic maize (Zea mays L.) callus, plants, and progeny from microprojectile bombardment of 2–5-d cultured Hi-II immature embryos is described. Histological evidence indicates that these tissues are amenable to transformation due to surface layer cell division of the scutellum. Two out of every 100 bombarded embryos produced transgenic callus and R₀ transgenic plants were both male and female fertile. Expected segregation of transgenes was observed in progeny. The primary advantage of bombarding these tissues is increased male and female fertility of transgenic plants compared with those produced using long-term callus or suspension cultures.     

Insects and fungi on a C3 sedge and a C4 grass exposed to elevated atmospheric CO2 concentrations in open-top chambers in the field

The effects of elevated atmospheric CO₂ concentration on plant-fungi and plant-insect interactions were studied in an emergent marsh in the Chesapeake Bay. Stands of the C₃ sedge Scirpus olneyi Grey, and the C₄ grass Spartina patens (Ait.) Muhl. have been exposed to elevated atmospheric CO₂ concentrations during each growing season since 1987. In August 1991 the severities of fungal infections and insect infestations were quantified. Shoot nitrogen concentration ([N]) and water content (WC) were determined. In elevated concentrations of atmospheric CO₂, 32% fewer S. olneyi plants were infested by insects, and there was a 37% reduction in the severity of a pathogenic fungal infection, compared with plants grown in ambient CO₂ concentrations. S. olneyi also had reduced [N], which correlated positively with the severities of fungal infections and insect infestations. Conversely, S. patens had increased WC but unchanged [N] in elevated concentrations of atmospheric CO₂ and the severity of fungal infection increased. Elevated atmospheric CO₂ concentration increased or decreased the severity of fungal infection depending on at least two interacting factors, [N] and WC; but it did not change the number of plants that were infected with fungi. In contrast, the major results for insects were that the number of plants infected with insects decreased, and that the amount of tissue that each insect ate also decreased.     

Does elevated atmospheric CO2 concentration inhibit mitochondrial respiration in green plants?

ATP, adenosine triphosphate
K_m, Michaelis-Menton coefficient
C_a, concentration of CO₂ in the air (μmol mol^–1)
NAD, oxidized nicotin adenine dinucleotide
NADH, reduced nicotin adenine dinucleotide
NADP, oxidized nicotin adenine phosphate dinucleotide
NADPH, reduced nicotine adenine phosphate dinucleotide
R, rate of respiration per unit DW [μmol g
DW^–1], Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase
V_c,_max, maximum in vivo rate of carboxylation at Rubisco (μmol m^–2 s^–1)

There is abundant evidence that a reduction in mitochondrial respiration of plants occurs when atmospheric CO₂ (C_a) is increased. Recent reviews suggest that doubling the present C_a will reduce the respiration rate [per unit dry weight (DW)] by 15 to 18%. The effect has two components: an immediate, reversible effect observed in leaves, stems, and roots of plants as well as soil microbes, and an irreversible effect which occurs as a consequence of growth in elevated C_a and appears to be specific to C₃ species. The direct effect has been correlated with inhibition of certain respiratory enzymes, namely cytochrome-c-oxidase and succinate dehydrogenase, and the indirect or acclimation effect may be related to changes in tissue composition. Although no satisfactory mechanisms to explain these effects have been demonstrated, plausible mechanisms have been proposed and await experimental testing. These are carbamylation of proteins and direct inhibition of enzymes of respiration. A reduction of foliar respiration of 15% by doubling present ambient C_a would represent 3 Gt of carbon per annum in the global carbon budget.     

C4 photosynthesis in a single C3 cell is theoretically inefficient but may ameliorate internal CO2 diffusion limitations of C3 leaves

Attempts are being made to introduce C₄ photosynthetic characteristics into C₃ crop plants by genetic manipulation. This research has focused on engineering single‐celled C₄‐type CO₂ concentrating mechanisms into C₃ plants such as rice. Herein the pros and cons of such approaches are discussed with a focus on CO₂ diffusion, utilizing a mathematical model of single‐cell C₄ photosynthesis. It is shown that a high bundle sheath resistance to CO₂ diffusion is an essential feature of energy‐efficient C₄ photosynthesis. The large chloroplast surface area appressed to the intercellular airspace in C₃ leaves generates low internal resistance to CO₂ diffusion, thereby limiting the energy efficiency of a single‐cell C₄ concentrating mechanism, which relies on concentrating CO₂ within chloroplasts of C₃ leaves. Nevertheless the model demonstrates that the drop in CO₂ partial pressure, pCO₂, that exists between intercellular airspace and chloroplasts in C₃ leaves at high photosynthetic rates, can be reversed under high irradiance when energy is not limiting. The model shows that this is particularly effective at lower intercellular pCO₂. Such a system may therefore be of benefit in water‐limited conditions when stomata are closed and low intercellular pCO₂ increases photorespiration.