Olavi Kiirats

Response of mannitol-producing Arabidopsis thaliana to abiotic stress

In celery, mannitol is a primary photosynthetic product that is associated with celerys exceptional salt tolerance. Arabidopsis plants transformed with celerys mannose-6-phosphate reductase (M6PR) gene produce mannitol and grow normally in the absence of stress. Daily analysis of the increase in growth (fresh and dry weight, leaf number, leaf area per plant and specific leaf weight) over a 12-day period showed less effect of salt (100 mm NaCl) on the M2 transformant than wild type (WT). Following a 12-day treatment of WT, M2 and M5 plants with 100 or 200 mm NaCl the total shoot fresh weight, leaf number, and leaf area were significantly greater in transformants than in WT plants. The efficiency of use of energy for photochemistry by PSII was measured daily under growth conditions. In WT plants treated with 100 mm NaCl, the PSII yield begin decreasing after 6 days with a 50% loss in yield after 12 days, indicating a severe loss in PSII efficiency; whereas, there was no effect on the transformants. Under atmospheric levels of CO2, growth with 200 mm NaCl caused an increase in the substomatal levels of CO2 in WT plants but not in transformants. It also caused a marked decrease in carboxylation efficiency under limiting levels of CO2 in WT compared with transformants. When stress was imposed and growth reduced by withholding water for 12 days, which resulted in a similar decrease in relative water content to salt-treated plants, there were no differences among the genotypes in PSII yields or growth. The results suggest mannitol, which is known to be a compatible solute and antioxidant, protects photosynthesis against salt-related damage to chloroplasts.

Interactions of Nitrate and CO2 Enrichment on Growth, Carbohydrates, and Rubisco in Arabidopsis Starch Mutants. Significance of Starch and Hexose

Wild-type (wt) Arabidopsis plants, the starch-deficient mutant TL46, and the near-starchless mutant TL25 were grown in hydroponics under two levels of nitrate, 0.2 versus 6 mm, and two levels of CO2, 35 versus 100 Pa. Growth (fresh weight and leaf area basis) was highest in wt plants, lower in TL46, and much lower in TL25 plants under a given treatment. It is surprising that the inability to synthesize starch restricted leaf area development under both low N (N L ) and high N (NH). For each genotype, the order of greatest growth among the four treatments was high CO2/NH > low CO2/NH, > high CO2/N L , which was similar to low CO2/N L . Under high CO2/N L , wt and TL46 plants retained considerable starch in leaves at the end of the night period, and TL25 accumulated large amounts of soluble sugars, indicative of N-limited restraints on utilization of photosynthates. The lowest ribulose-1,5-bisphosphate carboxylase/oxygenase per leaf area was in plants grown under high CO2/N L . When N supply is limited, the increase in soluble sugars, particularly in the starch mutants, apparently accentuates the feedback and down-regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase, resulting in greater reduction of growth. With an adequate supply of N, growth is limited in the starch mutants due to insufficient carbohydrate reserves during the dark period. A combination of limited N and a limited capacity to synthesize starch, which restrict the capacity to use photosynthate, and high CO2, which increases the potential to produce photosynthate, provides conditions for strong down-regulation of photosynthesis.

Bundle Sheath Diffusive Resistance to CO2 and Effectiveness of C4 Photosynthesis and Refixation of Photorespired CO2 in a C4 Cycle Mutant and Wild-Type Amaranthus edulis

A mutant of the NAD-malic enzyme-type C4 plant,Amaranthus edulis, which lacks phosphoenolpyruvate carboxylase (PEPC) in the mesophyll cells was studied. Analysis of CO2 response curves of photosynthesis of the mutant, which has normal Kranz anatomy but lacks a functional C4 cycle, provided a direct means of determining the liquid phase-diffusive resistance of atmospheric CO2 to sites of ribulose 1,5-bisphosphate carboxylation inside bundle sheath (BS) chloroplasts (r bs) within intact plants. Comparisons were made with excised shoots of wild-type plants fed 3,3-dichloro-2-(dihydroxyphosphinoyl-methyl)-propenoate, an inhibitor of PEPC. Values of r bs in A. edulis were 70 to 180 m2 s−1mol−1, increasing as the leaf matured. This is about 70-fold higher than the liquid phase resistance for diffusion of CO2 to Rubisco in mesophyll cells of C3 plants. The values of r bs in A. edulis are sufficient for C4 photosynthesis to elevate CO2 in BS cells and to minimize photorespiration. The calculated CO2 concentration in BS cells, which is dependent on input of r bs, was about 2,000 μbar under maximum rates of CO2 fixation, which is about six times the ambient level of CO2. High re-assimilation of photorespired CO2 was demonstrated in both mutant and wild-type plants at limiting CO2 concentrations, which can be explained by high r bs. Increasing O2 from near zero up to ambient levels under low CO2, resulted in an increase in the gross rate of O2 evolution measured by chlorophyll fluorescence analysis in the PEPC mutant; this increase was simulated from a Rubisco kinetic model, which indicates effective refixation of photorespired CO2 in BS cells.

Development of biochemical specialization and organelle partitioning in the single-cell C4 system in leaves of Borszczowia aralocaspica (Chenopodiaceae)

The terrestrial plant Borszczowia aralocaspica (Chenopodiaceae) has recently been shown to contain the entire C(4) photosynthesis mechanism within individual, structurally and biochemically polarized chlorenchyma cells rather than in a dual cell system, as has been the paradigm for this type of carbon fixation (Nature 414: 543-546, 2001). Analysis of carbon isotope composition and (14)CO(2) fixation shows that photosynthesis and growth of B. aralocaspica occurs through carbon acquired by C(4) photosynthesis. The development of this unique single-cell C(4) system in chlorenchyma cells was studied by analysis of young (0.2-0.3 cm length), intermediate (ca. 0.5-0.6 cm length), and mature leaves (ca. 3 cm length). The length of chlorenchyma cells approximately doubles from young to intermediate and again from intermediate to the mature leaf stage. In young chlorenchyma cells, there is a single type of chloroplast; the chloroplasts are evenly distributed throughout the cytosol, and all contain starch and rubisco. During leaf development, the activities of phosphoenolpyruvate carboxylase (PEPC; which is cytosolic), rubisco, and pyruvate,Pi dikinase (PPDK) increase on a chlorophyll basis. As leaves mature, chloroplasts differentiate into two distinct structural and biochemical types that are spatially separated into the proximal and distal parts of the cell (the proximal end being closest to the center of the leaf). The early stages of this polarization are observed in intermediate leaves, and the polarization is fully developed in mature leaves. The chloroplasts in the distal ends of the cell have reduced grana and little starch, while those at the proximal ends have well-developed grana and abundant starch. In mature leaves, PPDK is expressed in chloroplasts at the distal end of the cells, while rubisco and adenosine diphosphate glucose (ADPG) pyrophosphorylase are selectively expressed in chloroplasts at the proximal end of the cell. Mitochondrial polarization also occurs during development as nicotinamide-adenine dinucleotide phosphate-malic enzyme (NAD-ME) and the photorespiratory enzyme glycine decarboxylase are expressed in mature but not young leaves and are localized in mitochondria at the proximal end of the cells. The data show that single-cell C(4) develops from a single pool of identical organelles that develop differential biochemical functions and spatial partitioning in the cell during maturation.

Effects of salinity on chlorophyll fluorescence and CO2 fixation in C4 estuarine grasses

The effects of salinity (sea water at 0 ‰ versus 30 ‰) on gross rates of O2 evolution (JO2) and net rates of CO2 uptake (PN) were measured in the halotolerant estuarine C4 grasses Spartina patens, S. alterniflora, S. densiflora, and Distichlis spicata in controlled growth environments. Under high irradiance, salinity had no significant effect on the intercellular to ambient CO2 concentration ratio (Ci/Ca). However, during photosynthesis under limiting irradiance, the maximum quantum efficiency of CO2 fixation decreased under salinity across species, suggesting there is increased leakage of the CO2 delivered to the bundle sheath cells by the C4 pump. Growth under salinity did not affect the maximum intrinsic efficiency of photosystem 2, PS2 (FV/FM) in these species, suggesting salinity had no effect on photosynthesis by inactivation of PS2 reaction centers. Under saline conditions and high irradiance, PN was reduced by 75 % in Spartina patens and S. alterniflora, whereas salinity had no effect on PN in S. densiflora or D. spicata. This inhibition of PN in S. patens and S. alterniflora was not due to an effect on stomatal conductance since the ratio of Ci/Ca did not decrease under saline conditions. In growth with and without salt, PN was saturated at ∼500 µmol(quantum) m−2 s−1 while JO2 continued to increase up to full sunlight, indicating that carbon assimilation was not tightly coupled to photochemistry in these halophytic species. This increase in alternative electron flow under high irradiance might be an inherent function in these halophytes for dissipating excess energy.

Feedback limitation of photosynthesis at high CO2 acts by modulating the activity of the chloroplast ATP synthase

It was previously shown that photosynthetic electron transfer is controlled under low CO2 via regulation of the chloroplast ATP synthase. In the current work, we studied the regulation of photosynthesis under feedback limiting conditions, where photosynthesis is limited by the capacity to utilise triose-phosphate for synthesis of end products (starch or sucrose), in a starch-deficient mutant of Nicotiana sylvestris Speg. & Comes. At high CO2, we observed feedback control that was progressively reversed by increasing O2 levels from 2 to 40%. The activity of the ATP synthase, probed in vivo by the dark-interval relaxation kinetics of the electrochromic shift, was proportional to the O2-induced increases in O2 evolution from PSII (JO2), as well as the sum of Rubisco oxygenation (vo) and carboxylation (vc) rates. The altered ATP synthase activity led to changes in the light-driven proton motive force, resulting in regulation of the rate of plastoquinol oxidation at the cytochrome b6f complex, quantitatively accounting for the observed control of photosynthetic electron transfer. The ATP content of the cell decreases under feedback limitation, suggesting that the ATP synthesis was downregulated to a larger extent than ATP consumption. This likely resulted in slowing of ribulose bisphosphate regeneration and JO2). Overall, our results indicate that, just as at low CO2, feedback limitations control the light reactions of photosynthesis via regulation of the ATP synthase, and can be reconciled with regulation via stromal Pi, or an unknown allosteric affector.

Structural, biochemical, and physiological characterization of photosynthesis in two C4 subspecies of Tecticornia indica and the C3 species Tecticornia pergranulata (Chenopodiaceae)

Among dicotyledon families, Chenopodiaceae has the most C(4) species and the greatest diversity in structural forms of C(4). In subfamily Salicornioideae, C(4) photosynthesis has, so far, only been found in the genus Halosarcia which is now included in the broadly circumscribed Tecticornia. Comparative anatomical, cytochemical, and physiological studies on these taxa, which have near-aphyllous photosynthetic shoots, show that T. pergranulata is C(3), and that two subspecies of T. indica (bidens and indica) are C(4) (Kranz-tecticornoid type). In T. pergranulata, the stems have two layers of chlorenchyma cells surrounding the centrally located water storage tissue. The two subspecies of T. indica have Kranz anatomy in reduced leaves and in the fleshy stem cortex. They are NAD-malic enzyme-type C(4) species, with mesophyll chloroplasts having reduced grana, characteristic of this subtype. The Kranz-tecticornoid-type anatomy is unique among C(4) types in the family in having groups of chlorenchymatous cells separated by a network of large colourless cells (which may provide mechanical support or optimize the distribution of radiation in the tissue), and in having peripheral vascular bundles with the phloem side facing the bundle sheath cells. Also, the bundle sheath cells have chloroplasts in a centrifugal position, which is atypical for C(4) dicots. Fluorescence analyses in fresh sections indicate that all non-lignified cell walls have ferulic acid, a cell wall cross-linker. Structural-functional relationships of C(4) photosynthesis in T. indica are discussed. Recent molecular studies show that the C(4) taxa in Tecticornia form a monophyletic group, with incorporation of the Australian endemic genera of Salicornioideae, including Halosarcia, Pachycornia, Sclerostegia, and Tegicornia, into Tecticornia.

Flowers of Bienertia cycloptera and Suaeda aralocaspica (Chenopodiaceae) complete the life cycle performing single-cell C4 photosynthesis

Leaves and cotyledons of the terrestrial C4 plants, Bienertia cycloptera Bunge ex Boiss. and Suaeda aralocaspica (Bunge) Freitag & Schütze (Chenopodiaceae), accomplish C4 photosynthesis within individual chlorenchyma cells: each species having a unique means of intracellular spatial partitioning of biochemistry and organelles. In this study the chlorenchyma tissue in flowers and stems of these species was investigated. Flowers have an outer whorl of green tepals with a layer of chlorenchyma cells, which are located on the abaxial side, exposed to the atmosphere. Anatomical, immunocytochemical, western blots and starch analyses show that the chlorenchyma cells in tepals are specialised for performance of single-cell C4 photosynthesis like that in leaves. In the tepals of B. cycloptera, chlorenchyma cells have a distinctive central cytoplasmic compartment, with chloroplasts which contain Rubisco, separated by cytoplasmic channels from a peripheral chloroplast-containing compartment, with phosphoenolpyruvate carboxylase (PEPC) distributed throughout the cytoplasm. In the tepals of S. aralocaspica, chlorenchyma cells have chloroplasts polarised towards opposite ends of the cells. Rubisco is found in chloroplasts towards the proximal end of the cell and PEPC is found throughout the cytoplasm. Also, green stems of B. cycloptera have a single layer of the specialised C4 type chlorenchyma cells beneath the epidermis, and in stems of S. aralocaspica, chlorenchyma cells are scattered throughout the cortical tissue with chloroplasts around their periphery, typical of C3 type chlorenchyma. During reproductive development, green flowers become very conspicuous, and their photosynthesis is suggested to be important in completion of the life cycle of these single-cell C4 functioning species.

Requirements for the CO2-concentrating mechanism in C4 plants relative to limitations on carbon assimilation in rice.

CO 2 -concentrating mechanisms in photosynthetic organisms have in common a requirement for energy (ATP) and investment in proteins (enzymes/transporters). This investment can benefit the plant with a sufficient increase in the supply of CO 2 to Rubisco under CO 2 -limited photosynthesis. The effectiveness of the CO 2 -concentrating mechanism in a C 4 plant is discussed with respect to overcycling by the C 4 pathway, the energetics of photorespiration, and CO 2 diffusive resistance between Rubisco and phospho enol pyruvate carboxylase. Comparisons of the minimum calculated quantum requirement for CO 2 fixation at different intercellular levels of CO 2 for a C 4 plant (with variable overcycling and bundle sheath resistance) versus C 3 photosynthesis illustrate when C 4 photosynthesis may be of benefit as well as constraints in engineering C 4 photosynthesis into C 3 plants. Conditions in which photosynthesis is CO 2 -limited and conditions where rice may benefit from a CO 2 -concentrating mechanism are discussed.

CO2 diffusion resistance of bundle sheath cells in Amaranthus edulis, as revealed from the study of a PEP carboxylase deficient mutant

A mutant of the NAD-ME type C4 plant Amaranthus edulis, that lacks PEP carboxylase in the mesophyll cells was studied. Analysis of CO2 response curves of the mutant in the absence of a functional C4 cycle, provided a direct means of determining the liquid phase diffusive resistance of atmospheric CO2 to the sites of RuBP carboxylation inside the bundle sheath (BS) chloroplasts (rbs) within intact plants. The magnitude of rbs has been difficult to determine and previous estimates using different methods have ranged from 15 -1400 m2 s mol-1. The values of rbs in A. edulis were 70-180 m2 s mol-1 and they increased as the leaf matured. This is about 70-fold higher than the liquid phase resistance or diffusion of CO2 to Rubisco in the mesophyll cells of C3 plants. The values of rbs in A. edulis are sufficient for C4 photosynthesis to elevate the CO2 concentration inside the BS cells and thus reduce photorespiration. High reassimilation of photorespired CO2 was demonstrated in both mutant and wild type plants at limiting CO2 concentrations, which can be explained by high rbs. Based on O2 sensitivity analysis of wild type plants, the calculated CO2 concentration in BS cells under maximum rates of CO2 fixation in young leaves (lower rbs) and in expanded leaves (higher rbs) was about the same (1000-1200 m bar), which is sufficient to minimise photorespiration. This indicates that overcycling of the pathway and the rate of leakage of CO2 from the BS cells was higher in the younger leaves.