Biochemical and Structural Diversification of C4 Photosynthesis in Tribe Zoysieae (Poaceae).

C4 photosynthesis has evolved independently multiple times in grass lineages with nine anatomical and three biochemical subtypes. Chloridoideae represents one of the separate events and contains species of two biochemical subtypes, NAD-ME and PEP-CK. Assessment of C4 photosynthesis diversification is limited by species sampling. In this study, the biochemical subtypes together with anatomical leaf traits were analyzed in 19 species to reveal the evolutionary scenario for diversification of C4 photosynthesis in tribe Zoysieae (Chloridoideae). The effect of habitat on anatomical and biochemical diversification was also evaluated. The results for the 19 species studied indicate that 11 species have only NAD-ME as a decarboxylating enzyme, while eight species belong to the PEP-CK subtype. Leaf anatomy corresponds to the biochemical subtype. Analysis of Zoysieae phylogeny indicates multiple switches between PEP-CK and NAD-ME photosynthetic subtypes, with PEP-CK most likely as the ancestral subtype, and with multiple independent PEP-CK decarboxylase losses and its secondary acquisition. A strong correlation was detected between C4 biochemical subtypes studied and habitat annual precipitation wherein NAD-ME species are confined to drier habitats, while PEP-CK species prefer humid areas. Structural adaptations to arid climate include increases in leaf thickness and interveinal distance. Our analysis suggests that multiple loss of PEP-CK decarboxylase could have been driven by climate aridization followed by continued adaptive changes in leaf anatomy.

Altered cell wall hydroxycinnamate composition impacts leaf- and canopy-level CO2 uptake and water use in rice.

Cell wall properties play a major role in determining photosynthetic carbon uptake and water use through their impact on mesophyll conductance (CO2 diffusion from substomatal cavities into photosynthetic mesophyll cells) and leaf hydraulic conductance (water movement from xylem, through leaf tissue, to stomata). Consequently, modification of cell wall (CW) properties might help improve photosynthesis and crop water use efficiency (WUE). We tested this using 2 independent transgenic rice (Oryza sativa) lines overexpressing the rice OsAT10 gene (encoding a "BAHD" CoA acyltransferase), which alters CW hydroxycinnamic acid content (more para-coumaric acid and less ferulic acid). Plants were grown under high and low water levels, and traits related to leaf anatomy, CW composition, gas exchange, hydraulics, plant biomass, and canopy-level water use were measured. Alteration of hydroxycinnamic acid content led to statistically significant decreases in mesophyll CW thickness (-14%) and increased mesophyll conductance (+120%) and photosynthesis (+22%). However, concomitant increases in stomatal conductance negated the increased photosynthesis, resulting in no change in intrinsic WUE (ratio of photosynthesis to stomatal conductance). Leaf hydraulic conductance was also unchanged; however, transgenic plants showed small but statistically significant increases in aboveground biomass (AGB) (+12.5%) and canopy-level WUE (+8.8%; ratio of AGB to water used) and performed better under low water levels than wild-type plants. Our results demonstrate that changes in CW composition, specifically hydroxycinnamic acid content, can increase mesophyll conductance and photosynthesis in C3 cereal crops such as rice. However, attempts to improve photosynthetic WUE will need to enhance mesophyll conductance and photosynthesis while maintaining or decreasing stomatal conductance.

Leaf cell wall properties and stomatal density influence oxygen isotope enrichment of leaf water.

Measurements of oxygen isotope enrichment of leaf water above source water (Δ18 OLW ) can improve our understanding of the interaction between leaf anatomy and physiology on leaf water transport. Models have been developed to predict Δ18 OLW such as the string-of-lakes model, which describes the mixing of leaf water pools, and the Péclet effect model, which incorporates transpiration rate and the mixing length between unenriched xylem and enriched mesophyll water in the mesophyll (Lm ) or veins (Lv ). Here we compare measurements and models of Δ18 OLW on two cell wall composition mutants grown under two light intensities and relative humidities to evaluate cell wall properties on leaf water transport. In maize (Zea mays), the compromised ultrastructure of the suberin lamellae in the bundle sheath of the ALIPHATIC SUBERIN FERULOYL TRANSFERASE mutant (Zmasft) reduced barriers to apoplastic water movement, resulting in higher E and, potentially, Lv and, consequently, lower Δ18 OLW . The difference in Δ18 OLW in cellulose synthase-like F6 (CslF6) mutants and wild-type of rice (Oryza sativa) grown under two light intensities co-varied with stomatal density. These results show that cell wall composition and stomatal density influence Δ18 OLW and that stable isotopes can facilitate the development of a physiologically and anatomically explicit water transport model.

Structural diversity in salt excreting glands and salinity tolerance in Oryza coarctata, Sporobolus anglicus and Urochondra setulosa.

MAIN CONCLUSION: Unlike the bicellular glands characteristic of all known excreting grasses, unique single-celled salt glands were discovered in the only salt tolerant species of the genus Oryza, Oryza coarctata. Salt tolerance has evolved frequently in a large number of grass lineages with distinct difference in mechanisms. Mechanisms of salt tolerance were studied in three species of grasses characterized by salt excretion: C3 wild rice species Oryza coarctata, and C4 species Sporobolus anglicus and Urochondra setulosa. The leaf anatomy and ultrastructure of salt glands, pattern of salt excretion, gas exchange, accumulation of key photosynthetic enzymes, leaf water content and osmolality, and levels of some osmolytes, were compared when grown without salt, with 200 mM NaCl versus 200 mM KCl. Under salt treatments, there was little effect on the capacity for CO2 assimilation, while stomatal conductance decreased with a reduction in water loss by transpiration and an increase in water use efficiency. All three species accumulate compatible solutes but with drastic differences in osmolyte composition. Having high capacity for salt excretion, they have distinct structural differences in the salt excreting machinery. S. anglicus and U. setulosa have bicellular glands while O. coarctata has unique single-celled salt glands with a partitioning membrane system that are responsible for salt excretion rather than multiple hairs as previously suggested. The features of physiological responses and salt excretion indicate similar mechanisms are involved in providing tolerance and excretion of Na+ and K+.

Mesophyll conductance response to short-term changes in pCO2 is related to leaf anatomy and biochemistry in diverse C4 grasses.

Mesophyll CO2 conductance (gm ) in C3 species responds to short-term (minutes) changes in environment potentially due to changes in leaf anatomical and biochemical properties and measurement artefacts. Compared with C3 species, there is less information on gm responses to short-term changes in environmental conditions such as partial pressure of CO2 (pCO2 ) across diverse C4 species and the potential determinants of these responses. Using 16 C4 grasses we investigated the response of gm to short-term changes in pCO2 and its relationship with leaf anatomy and biochemistry. In general, gm increased as pCO2 decreased (statistically significant increase in 12 species), with percentage increases in gm ranging from +13% to +250%. Greater increase in gm at low pCO2 was observed in species exhibiting relatively thinner mesophyll cell walls along with greater mesophyll surface area exposed to intercellular air spaces, leaf N, photosynthetic capacity and activities of phosphoenolpyruvate carboxylase and Rubisco. Species with greater CO2 responses of gm were also able to maintain their leaf water-use efficiencies (TEi ) under low CO2 . Our study advances understanding of CO2 response of gm in diverse C4 species, identifies the key leaf traits related to this response and has implications for improving C4 photosynthetic models and TEi through modification of gm .

Analysis of formin functions during cytokinesis using specific inhibitor SMIFH2.

The phragmoplast separates daughter cells during cytokinesis by constructing the cell plate, which depends on interaction between cytoskeleton and membrane compartments. Proteins responsible for these interactions remain unknown, but formins can link cytoskeleton with membranes and several members of formin protein family localize to the cell plate. Progress in functional characterization of formins in cytokinesis is hindered by functional redundancies within the large formin gene family. We addressed this limitation by employing Small Molecular Inhibitor of Formin Homology 2 (SMIFH2), a small-molecule inhibitor of formins. Treatment of tobacco (Nicotiana tabacum) tissue culture cells with SMIFH2 perturbed localization of actin at the cell plate; slowed down both microtubule polymerization and phragmoplast expansion; diminished association of dynamin-related proteins with the cell plate independently of actin and microtubules; and caused cell plate swelling. Another impact of SMIFH2 was shortening of the END BINDING1b (EB1b) and EB1c comets on the growing microtubule plus ends in N. tabacum tissue culture cells and Arabidopsis thaliana cotyledon epidermis cells. The shape of the EB1 comets in the SMIFH2-treated cells resembled that of the knockdown mutant of plant Xenopus Microtubule-Associated protein of 215 kDa (XMAP215) homolog MICROTUBULE ORGANIZATION 1/GEMINI 1 (MOR1/GEM1). This outcome suggests that formins promote elongation of tubulin flares on the growing plus ends. Formins AtFH1 (A. thaliana Formin Homology 1) and AtFH8 can also interact with EB1. Besides cytokinesis, formins function in the mitotic spindle assembly and metaphase to anaphase transition. Our data suggest that during cytokinesis formins function in: (1) promoting microtubule polymerization; (2) nucleating F-actin at the cell plate; (3) retaining dynamin-related proteins at the cell plate; and (4) remodeling of the cell plate membrane.

Differences in leaf anatomy determines temperature response of leaf hydraulic and mesophyll CO2 conductance in phylogenetically related C4 and C3 grass species.

Leaf hydraulic and mesophyll CO2 conductance are both influenced by leaf anatomical traits, however it is poorly understood how the temperature response of these conductances differs between C4 and C3 species with distinct leaf anatomy. This study investigated the temperature response of leaf hydraulic conductance (Kleaf ), stomatal (gs ) and mesophyll (gm ) conductance to CO2 , and leaf anatomical traits in phylogenetically related Panicum antidotale (C4 ) and P. bisulcatum (C3 ) grasses. The C4 species had lower hydraulic conductance outside xylem (Kox ) and Kleaf compared with the C3 species. However, the C4 species had higher gm compared with the C3 species. Traits associated with leaf water movement, Kleaf and Kox , increased with temperature more in the C3 than in the C4 species, whereas traits related to carbon uptake, Anet and gm , increased more with temperature in the C4 than the C3 species. Our findings demonstrate that, in addition to a CO2 concentrating mechanism, outside-xylem leaf anatomy in the C4 species P. antidotale favours lower water movement through the leaf and stomata that provides an additional advantage for greater leaf carbon uptake relative to water loss with increasing leaf temperature than in the C3 species P. bisulcatum.

Increased adaxial stomatal density is associated with greater mesophyll surface area exposed to intercellular air spaces and mesophyll conductance in diverse C4 grasses.

Mesophyll conductance (gm ) is the diffusion of CO2 from intercellular air spaces (IAS) to the first site of carboxylation in the mesophyll cells. In C3 species, gm is influenced by diverse leaf structural and anatomical traits; however, little is known about traits affecting gm in C4 species. To address this knowledge gap, we used online oxygen isotope discrimination measurements to estimate gm and microscopy techniques to measure leaf structural and anatomical traits potentially related to gm in 18 C4 grasses. In this study, gm scaled positively with photosynthesis and intrinsic water-use efficiency (TEi ), but not with stomatal conductance. Also, gm was not determined by a single trait but was positively correlated with adaxial stomatal densities (SDada ), stomatal ratio (SR), mesophyll surface area exposed to IAS (Smes ) and leaf thickness. However, gm was not related to abaxial stomatal densities (SDaba ) and mesophyll cell wall thickness (TCW ). Our study suggests that greater SDada and SR increased gm by increasing Smes and creating additional parallel pathways for CO2 diffusion inside mesophyll cells. Thus, SDada , SR and Smes are important determinants of C4 -gm and could be the target traits selected or modified for achieving greater gm and TEi in C4 species.

Transgenic maize phosphoenolpyruvate carboxylase alters leaf-atmosphere CO2 and 13CO2 exchanges in Oryza sativa.

The engineering process of C4 photosynthesis into C3 plants requires an increased activity of phosphoenolpyruvate carboxylase (PEPC) in the cytosol of leaf mesophyll cells. The literature varies on the physiological effect of transgenic maize (Zea mays) PEPC (ZmPEPC) leaf expression in Oryza sativa (rice). Therefore, to address this issue, leaf-atmosphere CO2 and 13CO2 exchanges were measured, both in the light (at atmospheric O2 partial pressure of 1.84 kPa and at different CO2 levels) and in the dark, in transgenic rice expressing ZmPEPC and wild-type (WT) plants. The in vitro PEPC activity was 25 times higher in the PEPC overexpressing (PEPC-OE) plants (~20% of maize) compared to the negligible activity in WT. In the PEPC-OE plants, the estimated fraction of carboxylation by PEPC (ß) was ~6% and leaf net biochemical discrimination against 13CO2[Formula: see text] was ~ 2‰ lower than in WT. However, there were no differences in leaf net CO2 assimilation rates (A) between genotypes, while the leaf dark respiration rates (Rd) over three hours after light-dark transition were enhanced (~ 30%) and with a higher 13C composition [Formula: see text] in the PEPC-OE plants compared to WT. These data indicate that ZmPEPC in the PEPC-OE rice plants contributes to leaf carbon metabolism in both the light and in the dark. However, there are some factors, potentially posttranslational regulation and PEP availability, which reduce ZmPEPC activity in vivo.

Knockdown of glycine decarboxylase complex alters photorespiratory carbon isotope fractionation in Oryza sativa leaves.

The influence of reduced glycine decarboxylase complex (GDC) activity on leaf atmosphere CO2 and 13CO2 exchange was tested in transgenic Oryza sativa with the GDC H-subunit knocked down in leaf mesophyll cells. Leaf measurements on transgenic gdch knockdown and wild-type plants were carried out in the light under photorespiratory and low photorespiratory conditions (i.e. 18.4 kPa and 1.84 kPa atmospheric O2 partial pressure, respectively), and in the dark. Under approximately current ambient O2 partial pressure (18.4 kPa pO2), the gdch knockdown plants showed an expected photorespiratory-deficient phenotype, with lower leaf net CO2 assimilation rates (A) than the wild-type. Additionally, under these conditions, the gdch knockdown plants had greater leaf net discrimination against 13CO2 (Δo) than the wild-type. This difference in Δo was in part due to lower 13C photorespiratory fractionation (f) ascribed to alternative decarboxylation of photorespiratory intermediates. Furthermore, the leaf dark respiration rate (Rd) was enhanced and the 13CO2 composition of respired CO2 (δ13CRd) showed a tendency to be more depleted in the gdch knockdown plants. These changes in Rd and δ13CRd were due to the amount and carbon isotopic composition of substrates available for dark respiration. These results demonstrate that impairment of the photorespiratory pathway affects leaf 13CO2 exchange, particularly the 13C decarboxylation fractionation associated with photorespiration.

Cell wall properties in Oryza sativa influence mesophyll CO2 conductance.

Diffusion of CO2 from the leaf intercellular air space to the site of carboxylation (gm ) is a potential trait for increasing net rates of CO2 assimilation (Anet ), photosynthetic efficiency, and crop productivity. Leaf anatomy plays a key role in this process; however, there are few investigations into how cell wall properties impact gm and Anet . Online carbon isotope discrimination was used to determine gm and Anet in Oryza sativa wild-type (WT) plants and mutants with disruptions in cell wall mixed-linkage glucan (MLG) production (CslF6 knockouts) under high- and low-light growth conditions. Cell wall thickness (Tcw ), surface area of chloroplast exposed to intercellular air spaces (Sc ), leaf dry mass per area (LMA), effective porosity, and other leaf anatomical traits were also analyzed. The gm of CslF6 mutants decreased by 83% relative to the WT, with c. 28% of the reduction in gm explained by Sc . Although Anet /LMA and Anet /Chl partially explained differences in Anet between genotypes, the change in cell wall properties influenced the diffusivity and availability of CO2 . The data presented here indicate that the loss of MLG in CslF6 plants had an impact on gm and demonstrate the importance of cell wall effective porosity and liquid path length on gm .

Diversity in structure and forms of carbon assimilation in photosynthetic organs in Cleome (Cleomaceae).

Photosynthesis in different organs of Cleome was analysed in four species known to have differences in leaf photosynthesis: Cleome africana Botsch. (C3), Cleome paradoxa R.Br. (C3-C4 intermediate), Cleome angustifolia Forssk. and Cleome gynandra L. (C4). The chlorophyll content, carbon isotope composition, stomatal densities, anatomy, levels and compartmentation of some key photosynthetic enzymes, and the form and function of photosynthesis were determined in different organs of these species. In the three xerophytes, C. africana, C. paradoxa, and C. angustifolia, multiple organs contribute to photosynthesis (cotyledons, leaves, petioles, stems and pods) which is considered important for their survival under arid conditions. In C. africana, all photosynthetic organs have C3 photosynthesis. In C. paradoxa, cotyledons, leaves, stems and petioles have C3-C4 type features. In C. angustifolia, the pods have C3 photosynthesis, whereas all other organs have C4 photosynthesis with Kranz anatomy formed by a continuous, dual layer of chlorenchyma cells. In the subtropical C4 species C. gynandra, cotyledons, leaves, and pods develop C4 photosynthesis, with Kranz anatomy around individual veins; but not in stems and petioles which have limited function of photosynthesis. The diversity in forms and the capacity of photosynthesis in organs of these species to contribute to their carbon economy is discussed.

Unique photosynthetic phenotypes in Portulaca (Portulacaceae): C3-C4 intermediates and NAD-ME C4 species with Pilosoid-type Kranz anatomy.

Portulacaceae is a family that has considerable diversity in photosynthetic phenotypes. It is one of 19 families of terrestrial plants where species having C4 photosynthesis have been found. Most species in Portulaca are in the alternate-leaved (AL) lineage, which includes one clade (Cryptopetala) with taxa lacking C4 photosynthesis and three clades having C4 species (Oleracea, Umbraticola and Pilosa). All three species in the Cryptopetala clade lack Kranz anatomy, the leaves have C3-like carbon isotope composition and they have low levels of C4 cycle enzymes. Anatomical, biochemical and physiological analyses show they are all C3-C4 intermediates. They have intermediate CO2 compensation points, enrichment of organelles in the centripetal position in bundle sheath (BS) cells, with selective localization of glycine decarboxylase in BS mitochondria. In the three C4 clades there are differences in Kranz anatomy types and form of malic enzyme (ME) reported to function in C4 (NAD-ME versus NADP-ME): Oleracea (Atriplicoid, NAD-ME), Umbraticola (Atriplicoid, NADP-ME) and Pilosa (Pilosoid, NADP-ME). Structural and biochemical analyses were performed on Pilosa clade representatives having Pilosoid-type leaf anatomy with Kranz tissue enclosing individual peripheral vascular bundles and water storage in the center of the leaf. In this clade, all species except P. elatior are NADP-ME-type C4 species with grana-deficient BS chloroplasts and grana-enriched M chloroplasts. Surprisingly, P. elatior has BS chloroplasts enriched in grana and NAD-ME-type photosynthesis. The results suggest photosynthetic phenotypes were probably derived from an ancestor with NADP-ME-type C4, with two independent switches to NAD-ME type.

Molecular phylogeny and forms of photosynthesis in tribe Salsoleae (Chenopodiaceae).

While many C4 lineages have Kranz anatomy around individual veins, Salsoleae have evolved the Salsoloid Kranz anatomy where a continuous dual layer of chlorenchyma cells encloses the vascular and water-storage tissue. With the aim of elucidating the evolution of C4 photosynthesis in Salsoleae, a broadly sampled molecular phylogeny and anatomical survey was conducted, together with biochemical, microscopic, and physiological analyses of selected photosynthetic types. From analyses of photosynthetic phenotypes, a model for evolution of this form of C4 was compared with models for evolution of Kranz anatomy around individual veins. A functionally C3 proto-Kranz phenotype (Proto-Kranz Sympegmoid) and intermediates with a photorespiratory pump (Kranz-like Sympegmoid and Kranz-like Salsoloid types) are considered crucial transitional steps towards C4 development. The molecular phylogeny provides evidence for C3 being the ancestral photosynthetic pathway but there is no phylogenetic evidence for the ancestry of C3-C4 intermediacy with respect to C4 in Salsoleae. Traits considered advantageous in arid conditions, such as annual life form, central sclerenchyma in leaves, and reduction of surface area, evolved repeatedly in Salsoleae. The recurrent evolution of a green stem cortex taking over photosynthesis in C4 clades of Salsoleae concurrent with leaf reduction was probably favoured by the higher productivity of the C4 cycle.

Influence of light and nitrogen on the photosynthetic efficiency in the C4 plant Miscanthus × giganteus.

There are numerous studies describing how growth conditions influence the efficiency of C4 photosynthesis. However, it remains unclear how changes in the biochemical capacity versus leaf anatomy drives this acclimation. Therefore, the aim of this study was to determine how growth light and nitrogen availability influence leaf anatomy, biochemistry and the efficiency of the CO2 concentrating mechanism in Miscanthus × giganteus. There was an increase in the mesophyll cell wall surface area but not cell well thickness in the high-light (HL) compared to the low-light (LL) grown plants suggesting a higher mesophyll conductance in the HL plants, which also had greater photosynthetic capacity. Additionally, the HL plants had greater surface area and thickness of bundle-sheath cell walls compared to LL plants, suggesting limited differences in bundle-sheath CO2 conductance because the increased area was offset by thicker cell walls. The gas exchange estimates of phosphoenolpyruvate carboxylase (PEPc) activity were significantly less than the in vitro PEPc activity, suggesting limited substrate availability in the leaf due to low mesophyll CO2 conductance. Finally, leakiness was similar across all growth conditions and generally did not change under the different measurement light conditions. However, differences in the stable isotope composition of leaf material did not correlate with leakiness indicating that dry matter isotope measurements are not a good proxy for leakiness. Taken together, these data suggest that the CO2 concentrating mechanism in Miscanthus is robust under low-light and limited nitrogen growth conditions, and that the observed changes in leaf anatomy and biochemistry likely help to maintain this efficiency.

The unique structural and biochemical development of single cell C4 photosynthesis along longitudinal leaf gradients in Bienertia sinuspersici and Suaeda aralocaspica (Chenopodiaceae).

Temporal and spatial patterns of photosynthetic enzyme expression and structural maturation of chlorenchyma cells along longitudinal developmental gradients were characterized in young leaves of two single cell C4 species, Bienertia sinuspersici and Suaeda aralocaspica Both species partition photosynthetic functions between distinct intracellular domains. In the C4-C domain, C4 acids are formed in the C4 cycle during capture of atmospheric CO2 by phosphoenolpyruvate carboxylase. In the C4-D domain, CO2 released in the C4 cycle via mitochondrial NAD-malic enzyme is refixed by Rubisco. Despite striking differences in origin and intracellular positioning of domains, these species show strong convergence in C4 developmental patterns. Both progress through a gradual developmental transition towards full C4 photosynthesis, with an associated increase in levels of photosynthetic enzymes. Analysis of longitudinal sections showed undeveloped domains at the leaf base, with Rubisco rbcL mRNA and protein contained within all chloroplasts. The two domains were first distinguishable in chlorenchyma cells at the leaf mid-regions, but still contained structurally similar chloroplasts with equivalent amounts of rbcL mRNA and protein; while mitochondria had become confined to just one domain (proto-C4-D). The C4 state was fully formed towards the leaf tips, Rubisco transcripts and protein were compartmentalized specifically to structurally distinct chloroplasts in the C4-D domains indicating selective regulation of Rubisco expression may occur by control of transcription or stability of rbcL mRNA. Determination of CO2 compensation points showed young leaves were not functionally C4, consistent with cytological observations of the developmental progression from C3 default to intermediate to C4 photosynthesis.

An assessment of the capacity for phosphoenolpyruvate carboxykinase to contribute to C4 photosynthesis.

Three C4 acid decarboxylases, phosphoenolpyruvate carboxykinase (PEPCK), NADP-malic enzyme (NADP-ME), and NAD-malic enzyme (NAD-ME) were recruited from C3 plants to support C4 photosynthesis. In Poaceae, there are established lineages having PEPCK type species, and some NADP-ME lineages in which PEPCK contributes to C4. Besides family Poaceae, recently PEPCK has been reported to function in C4 photosynthesis in eudicot species including Cleome gynandra (Cleomaceae), Trianthema portulacastrum and Zaleya pentandra (Aizoaceae). We evaluated PEPCK by enzyme assay and western blots in representatives of Poaceae, Aizoaceae, Cleomaceae, and Chenopodiaceae compared to that in the PEPCK type C4 grass Spartina anglica. Eragrostis nutans was identified as the first NAD-ME type C4 grass having substantial amounts of PEPCK. In the eudicots, including C. gynandra, Cleome angustifolia, T. portulacastrum, Z. pentandra, and nine C4 members of family Chenopodiaceae (which has the most C4 species and diversity in forms among eudicot families), amounts of PEPCK were generally very low (barely detectable up to 4% of that in S. anglica). Based on these results, C4 species can be classified biochemically according to the dominant decarboxylase recruited for C4 function; and, Poaceae remains the only family in which PEPCK is known to have a significant role in C4 photosynthesis.

Pollen development in Rhododendron in relation to winter dormancy and bloom time.

Microsporogenesis and microgametogenesis of Rhododendron ledebourii (semi-deciduous), Rhododendron luteum (deciduous), and Rhododendron catawbiense (evergreen) were studied by light and electron microscopies in order to determine the stages of pollen development in relation to period of winter dormancy and bloom time throughout an annual growth cycle. Development of generative organs starts in June in R. ledebourii and in July in R. luteum and R. catawbiense and reaches completion about 11 months later. R. luteum and R. catawbiense microspores undergo meiosis at the end of the August and spend winter at the vacuolization stage. Mitosis with the formation of bicellular pollen grain occurs shortly before flowering at the beginning of June. R. ledebourii develops two types of flowers which differ in the timing of microgametogenesis. The first type is characterized by early microspore meiosis and mitosis leading to development of bicellular pollen grains by the end of August, and is prone to fall blooming during warm autumn temperatures. Microspores of the second flower type have a more prolonged vacuolization stage with mitosis and subsequent bicellular pollen grains occurring in November. By winter, flower buds in R. ledebourii are more advanced developmentally than in R. catawbiense and R. luteum, and bloom about 1 month earlier. The different strategies of pollen development identified both within and between these three Rhododendron species were recognized which are not associated with leaf drop during winter but appear to be related to the time of spring flowering and the frequency of autumn flowering.

The acclimation of photosynthesis and respiration to temperature in the C3 -C4 intermediate Salsola divaricata: induction of high respiratory CO2 release under low temperature.

Photosynthesis in C(3) -C(4) intermediates reduces carbon loss by photorespiration through refixing photorespired CO(2) within bundle sheath cells. This is beneficial under warm temperatures where rates of photorespiration are high; however, it is unknown how photosynthesis in C(3) -C(4) plants acclimates to growth under cold conditions. Therefore, the cold tolerance of the C(3) -C(4) Salsola divaricata was tested to determine whether it reverts to C(3) photosynthesis when grown under low temperatures. Plants were grown under cold (15/10 °C), moderate (25/18 °C) or hot (35/25 °C) day/night temperatures and analysed to determine how photosynthesis, respiration and C(3) -C(4) features acclimate to these growth conditions. The CO(2) compensation point and net rates of CO(2) assimilation in cold-grown plants changed dramatically when measured in response to temperature. However, this was not due to the loss of C(3) -C(4) intermediacy, but rather to a large increase in mitochondrial respiration supported primarily by the non-phosphorylating alternative oxidative pathway (AOP) and, to a lesser degree, the cytochrome oxidative pathway (COP). The increase in respiration and AOP capacity in cold-grown plants likely protects against reactive oxygen species (ROS) in mitochondria and photodamage in chloroplasts by consuming excess reductant via the alternative mitochondrial respiratory electron transport chain.

Differentiation of C4 photosynthesis along a leaf developmental gradient in two Cleome species having different forms of Kranz anatomy.

In family Cleomaceae there are NAD-malic enzyme-type C4 species having different forms of leaf anatomy. Leaves of Cleome angustifolia have Glossocardioid-type anatomy with a single complex Kranz unit which surrounds all the veins, while C. gynandra has Atriplicoid anatomy with multiple Kranz units, each surrounding an individual vein. Biochemical and ultrastructural differentiation of mesophyll (M) and bundle sheath (BS) cells were studied along a developmental gradient, from the leaf base (youngest) to the tip (mature). Initially, there is cell-specific expression of certain photosynthetic enzymes, which subsequently increase along with structural differentiation. At the base of the leaf, following division of ground tissue to form M and BS cells which are structurally similar, there is selective localization of Rubisco and glycine decarboxylase to BS cells. Thus, a biochemical C3 default stage, with Rubisco expression in both cell types, does not occur. Additionally, phosphoenolpyruvate carboxylase (PEPC) is selectively expressed in M cells near the base. Surprisingly, in both species, an additional layer of spongy M cells on the abaxial side of the leaf has the same differentiation with PEPC, even though it is not in contact with BS cells. During development along the longitudinal gradient there is structural differentiation of the cells, chloroplasts, and mitochondria, resulting in complete formation of Kranz anatomy. In both species, development of the C4 system occurs similarly, irrespective of having very different types of Kranz anatomy, different ontogenetic origins of BS and M, and independent evolutionary origins of C4 photosynthesis.