Differential Responses of <i>NHX1</i> and <i>SOS1</i> Gene Expressions to Salinity in two <i>Miscanthus sinensis</i> Anderss. Accessions with Different Salt Tolerance

The lignocellulosic crop Miscanthus spp. has been identified as a good candidate for biomass production. The responses of Miscanthus sinensis Anderss. to salinity were studied to satisfy the needs for high yields in marginal areas and to avoid competition with food production. The results indicated that the relative advantages of the tolerant accession over the sensitive one under saline conditions were associated with restricted Na⁺ accumulation in shoots. Seedlings of two accessions (salt-tolerant ‘JM0119’ and salt-sensitive ‘JM0099’) were subjected to 0 (control), 100, 200, and 300 mM NaCl stress to better understand the salt-induced biochemical responses of genes involved in Na⁺ accumulation in M. sinensis. The adaptation responses of genes encoding for Na⁺ /H⁺ antiporters, NHX1 and SOS1 to NaCl stress were examined in JM0119 and JM0099.The cDNA sequences of genes examined were highly conserved among the relatives of M. sinensis based on the sequencing on approximate 600 bp-long cDNA fragments obtained from degenerate PCR. These salt-induced variations of gene expression investigated by quantitative real-time PCR provided evidences for insights of the molecular mechanisms of salt tolerance in M. sinensis. The expression of NHX1 was up-regulated by salt stress in JM0119 shoot and root tissues. However, it was hardly affected in JM0099 shoot tissue except for a significant increase at the 100 mM salt treatment, and it was salt-suppressed in the JM0099 root tissue. In the root tissue, the expression of SOS1 was induced by the high salt treatment in JM0119 but repressed by all salt treatments in JM0099. Thus, the remarkably higher expression of NHX1 and SOS1 were associated with the resistance to Na⁺ toxicity by regulation of the Na⁺ influx, efflux, and sequestration under different salt conditions.     

Maintenance of photosynthesis and the antioxidant defence systems have key roles for survival of Halopeplis perfoliata (Amaranthaceae) in a saline environment

• Coastal salt marsh plants employ various combinations of morphological and physiological adaptations to survive under saline conditions. Little information is available on salinity tolerance mechanisms of Halopeplis perfoliata, a C3 stem succulent halophyte.
• We investigated the growth, photosynthesis and antioxidant defence mechanisms of H. perfoliata under saline conditions (0, 150, 300 and 600 mM NaCl) in an open greenhouse.
• Optimal shoot succulence, projected shoot area and relative growth rate were obtained in the low (150 mm NaCl) salinity treatment, while growth was inhibited at the highest salinity (600 mm NaCl). The CO₂ compensation point and carbon isotope composition of biomass confirmed C₃ photosynthesis. Increases in salinity did not affect the photosynthetic pigment content or maximum quantum efficiency of PSII of H. perfoliata. Assimilation of CO₂ (A) also remained unaffected by salinity. A modest effect on some gas exchange and photochemistry parameters was observed at 600 mm NaCl. With increasing salinity, there was a continual increase in respiration, suggesting utilisation of energy to cope with saline conditions. Under 300 and 600 mm NaCl, there was an increase in H₂O₂ and MDA with a concomitant rise in AsA, GR content and CAT activity.
• Hence, H. perfoliata appears to be an obligate halophyte that can grow up to seawater salinities by modulating photosynthetic gas exchange, photochemistry and the antioxidant defence systems.

     

Carbon assimilation by tree stems: potential involvement of phosphoenolpyruvate carboxylase

In woody species, the photosynthesis of stems, especially young branches, occurs by refixing part of the internal respiratory CO₂. The present study aims to improve the physiological characterization of stem photosynthesis by examining enzymatic characteristics. During an entire growing season, three enzymatic activities that are linked to C₃ and C₄ metabolism were investigated in relation to the CO₂ efflux and chlorophyll content of current year stems of European beech and were compared to the corresponding characteristics of leaves. High activities of phosphoenolpyruvate carboxylase (PEPC) and NADP malic enzyme were detected in stems (up to 13 times and 30 times higher in stems than in leaves, respectively), whereas Rubisco activity remained low in comparison with leaves. Stem maximal Rubisco and PEPC activities occurred at the beginning of the season when the total chlorophyll content and the CO₂ assimilation rate were also maximal. Stems were characterized by a PEPC:Rubisco ratio that was equal to 2.5 [an intermediate value between that of C₃-plants (about 0.1) and that of C₄-plants (about 10)], whereas it was equal to 0.1 in leaves. Eight other tree species were also measured and the PEPC:Rubisco ratio was, on average, equal to 3.6. The potential role of PEPC in stem carbon assimilation is discussed in relation to its known involvement in the anaplerotic function of C₃ plants and in the carbon metabolism of the C₄ pathway.     

Photosynthesis, immediate export and carbon partitioning in source leaves of C3, C3-C4 intermediate, and C4Panicum and Flaveria species at ambient and elevated CO2 levels

Immediate export in leaves of C₃‐C₄ intermediates were compared with their C₃ and C₄ relatives within the Panicum and Flaveria genera. At 35 Pa CO₂, photosynthesis and export were highest in C₄ species in each genera. Within the Panicum, photosynthesis and export in ‘type I’ C₃‐C₄ intermediates were greater than those in C₃ species. However, ‘type I’ C₃‐C₄ intermediates exported a similar proportion of newly fixed ¹⁴C as did C₄ species. Within the Flaveria, ‘type II’ C₃‐C₄ intermediate species had the lowest export rather than the C₃ species. At ambient CO₂, immediate export was strongly correlated with photosynthesis. However, at 90 Pa CO₂, when photosynthesis and immediate export increased in all C₃ and C₃‐C₄ intermediate species, proportionally less C was exported in all photosynthetic types than that at ambient CO₂. All species accumulated starch and sugars at both CO₂ levels. There was no correlation between immediate export and the pattern of ¹⁴C‐labelling into sugars and starch among the photosynthetic types within each genus. However, during CO₂ enrichment, C₄Panicum species accumulated sugars above the level of sugars and starch normally made at ambient CO₂, whereas the C₄Flaveria species accumulated only additional starch.     

Beneficial role of acetylcholine in chlorophyll metabolism and photosynthetic gas exchange in Nicotiana benthamiana seedlings under salinity stress

• Acetylcholine (ACh) is believed to improve plant growth. However, regulation at biochemical and molecular levels is largely unknown.
• The present study investigated the impact of exogenously applied ACh (10 µm ) on growth and chlorophyll metabolism in hydroponically grown Nicotiana benthamiana under salt stress (150 mm NaCl).
• Salinity reduced root hydraulic conductivity while ACh‐treated seedlings exhibited a significant increase, resulting in increased relative water content. Salinity induced a reduction in chlorophyll biosynthetic intermediates, such as protoporphyrin‐IX, Mg‐photoporphyrin‐IX and protochlorophyllide, which were significantly ameliorated in the presence of ACh. This influence of ACh on chlorophyll synthesis was confirmed by up‐regulation of HEMA1, CHLH, CAO and POR genes. Gas exchange parameters, i.e. stomatal conductance, internal CO₂ concentration and transpiration rate, increased with ACh, thereby alleviating the salinity effects on photosynthesis. In addition, the salinity‐induced enhancement of lipid peroxidation declined after ACh treatment through modulation of the activity of the assayed antioxidant enzymes (superoxide dismutase and peroxidase). Importantly, ACh significantly reduced the uptake of Na and increased uptake of K, resulting in a decline in the Na/K ratio.
• Results of the present study indicate that ACh can be effective in ameliorating NaCl‐induced osmotic stress, altering chlorophyll metabolism and thus photosynthesis by maintaining ion homeostasis, hydraulic conductivity and water balance.

     

Guard cell photosynthesis is critical for stomatal turgor production,yet does not directly mediate CO2‐ and ABA‐induced stomatal closing

Stomata mediate gas exchange between the inter‐cellular spaces of leaves and the atmosphere. CO₂ levels in leaves (Ci) are determined by respiration, photosynthesis, stomatal conductance and atmospheric [CO₂]. [CO₂] in leaves mediates stomatal movements. The role of guard cell photosynthesis in stomatal conductance responses is a matter of debate, and genetic approaches are needed. We have generated transgenic Arabidopsis plants that are chlorophyll‐deficient in guard cells only, expressing a constitutively active chlorophyllase in a guard cell specific enhancer trap line. Our data show that more than 90% of guard cells were chlorophyll‐deficient. Interestingly, approximately 45% of stomata had an unusual, previously not‐described, morphology of thin‐shaped chlorophyll‐less stomata. Nevertheless, stomatal size, stomatal index, plant morphology, and whole‐leaf photosynthetic parameters (PSII, qP, qN, F_V′/F_M′) were comparable with wild‐type plants. Time‐resolved intact leaf gas‐exchange analyses showed a reduction in stomatal conductance and CO₂‐assimilation rates of the transgenic plants. Normalization of CO₂ responses showed that stomata of transgenic plants respond to [CO₂] shifts. Detailed stomatal aperture measurements of normal kidney‐shaped stomata, which lack chlorophyll, showed stomatal closing responses to [CO₂] elevation and abscisic acid (ABA), while thin‐shaped stomata were continuously closed. Our present findings show that stomatal movement responses to [CO₂] and ABA are functional in guard cells that lack chlorophyll. These data suggest that guard cell CO₂ and ABA signal transduction are not directly modulated by guard cell photosynthesis/electron transport. Moreover, the finding that chlorophyll‐less stomata cause a ‘deflated’ thin‐shaped phenotype, suggests that photosynthesis in guard cells is critical for energization and guard cell turgor production.     

Kinetic variation in grass phosphoenolpyruvate carboxylases provides opportunity to enhance C4 photosynthetic efficiency

The high rates of photosynthesis and the carbon-concentrating mechanism (CCM) in C₄ plants are initiated by the enzyme phosphoenolpyruvate (PEP) carboxylase (PEPC). The flow of inorganic carbon into the CCM of C₄ plants is driven by PEPC’s affinity for bicarbonate (K_HCO3), which can be rate limiting when atmospheric CO₂ availability is restricted due to low stomatal conductance. We hypothesize that natural variation in K_HCO3 across C₄ plants is driven by specific amino acid substitutions to impact rates of C₄ photosynthesis under environments such as drought that restrict stomatal conductance. To test this hypothesis, we measured K_HCO3 from 20 C₄ grasses to compare kinetic properties with specific amino acid substitutions. There was nearly a twofold range in K_HCO3 across these C₄ grasses (24.3 ± 1.5 to 46.3 ± 2.4 μm ), which significantly impacts modeled rates of C₄ photosynthesis. Additionally, molecular engineering of a low-HCO₃⁻ affinity PEPC identified key domains that confer variation in K_HCO3. This study advances our understanding of PEPC kinetics and builds the foundation for engineering increased-HCO₃⁻ affinity and C₄ photosynthetic efficiency in important C₄ crops.     

Photosynthetic activity and proteomic analysis highlights the utilization of atmospheric CO2 by Ulva prolifera (Chlorophyta) for rapid growth

Free‐floating Ulva prolifera is one of the causative species of green tides. When green tides occur, massive mats of floating U. prolifera thalli accumulate rapidly in surface waters with daily growth rates as high as 56%. The upper thalli of the mats experience environmental changes such as the change in carbon source, high salinity, and desiccation. In this study, the photosynthetic performances of PSI and PSII in U. prolifera thalli exposed to different atmospheric carbon dioxide (CO₂) levels were measured. Changes in photosynthesis within salinity treatments and dehydration under different CO₂ concentrations were also analyzed. The results showed that PSII activity was enhanced as CO₂ increased, suggesting that CO₂ assimilation was enhanced and U. prolifera thalli can utilize CO₂ in the atmosphere directly, even when under moderate stress. In addition, changes in the proteome of U. prolifera in response to salt stress were investigated. Stress‐tolerance proteins appeared to have an important role in the response to salinity stress, whereas the abundance of proteins related to metabolism showed no significant change under low salinity treatments. These findings may be one of the main reasons for the extremely high growth rate of free‐floating U. prolifera when green tides occur.     

Crassulacean acid metabolism in the Basellaceae (Caryophyllales)

• C₄ and crassulacean acid metabolism (CAM) have evolved in the order Caryophyllales many times but neither C₄ nor CAM have been recorded for the Basellaceae, a small family in the CAM‐rich sub‐order Portulacineae.
• 24 h gas exchange and day–night changes in titratable acidity were measured in leaves of Anredera baselloides exposed to wet–dry–wet cycles.
• While net CO₂ uptake was restricted to the light period in well‐watered plants, net CO₂ fixation in the dark, accompanied by significant nocturnal increases in leaf acidity, developed in droughted plants. Plants reverted to solely C₃ photosynthesis upon rewatering.
• The reversible induction of nocturnal net CO₂ uptake by drought stress indicates that this species is able to exhibit CAM in a facultative manner. This is the first report of CAM in a member of the Basellaceae.

     

Effect of salt stress on photosynthesis and chlorophyll fluorescence in Medicago truncatula

In the present study, photosynthetic parameters including gas exchanges, pigment contents, and chlorophyll fluorescence, were compared in two contrasting local Medicago truncatula lines TN6.18 and TN8.20, in response to salt added to the nutrient solution. Plants were cultivated under symbiotic nitrogen fixation (SNF) after inoculation with a reference strain Sinorhizobium meliloti 2011, a very tolerant strain to salinity (700 mM NaCl), and grown in a controlled glasshouse. On one month old plants (with active SNF), salt treatment (75 mM NaCl) was gradually applied. Photosynthesis, assimilating pigments and chlorophyll fluorescence were monitored throughout the experiment during both short and long terms, compared to control (non-saline) conditions. A genotypic variation in salt tolerance was found; TN6.18 was the more sensitive to salinity. The relative tolerance of TN8.20 was concomitant with the highest photochemical quenching coefficient (q_P) affecting the maximum quantum yield of PSII (Y); the real quantum yield (?_exc) was the most affected in the sensitive line. Moreover, stomatal and PSII reaction centers activities differed clearly between the studied lines. We found that the effect of salinity on photosynthesis of M. truncatula was related to PSII activity reduction rather than to stomatal conductance limitation. Photosynthesis was reduced by the inhibition of CO₂ assimilation caused by PSII damage. This was clearly estimated by the Y, ?_exc and especially by the quantum yield of electron transport of PSII (Φ_PSII). Thus, on the basis of our results on the two local M. truncatula lines, we recommend the use of chlorophyll fluorescence as non-destructive screening method to discriminate susceptible and resistant legumes to salt stress.     

Light‐induced systemic signalling down‐regulates photosynthetic performance of soybean leaves with different directional effects

• When plants are exposed to a heterogeneous environment, photosynthesis of leaves is not only determined by their local condition, but also by certain signals from other parts of the same plant, termed systemic regulation. Our present study was conducted to investigate the effects of light‐dependent systemic regulation on the photosynthetic performance of soybean (Glycine max L. Merr.) under heterogeneous light conditions.
• Soybean plants were treated with heterogeneous light. Then gas exchange characteristics were measured to evaluate the photosynthetic performance of leaves. Parameters related to photosynthetic pigments, chlorophyll fluorescence, Rubisco and photosynthates were examined to study the mechanisms of light‐dependent systemic regulation on photosynthesis.
• Light‐induced systemic signalling by illuminated leaves reduced the P_n of both upper and lower non‐illuminated leaves on the same soybean plant. The decrease in g_s and increase in C_i in these non‐illuminated leaves indicated restriction of carbon assimilation, which was further verified by the decline in content and activity of Rubisco. However, the activation state of Rubisco decreased only in upper non‐illuminated leaves. Quantum efficiency of PSII (ΦPSII) and ETR also decreased only in upper non‐illuminated leaves. Moreover, the effects of light‐induced systemic signalling on carbohydrate content were also detectable only in upper non‐illuminated leaves.
• Light‐induced systemic signalling by illuminated leaves restricts carbon assimilation and down‐regulates photosynthetic performance of non‐illuminated leaves within a soybean plant. However, effects of such systemic regulation differed when regulated in upward or downward direction.

     

Drought limitation of photosynthesis differs between C3 and C4 grass species in a comparative experiment

Phylogenetic analyses show that C₄ grasses typically occupy drier habitats than their C₃ relatives, but recent experiments comparing the physiology of closely related C₃ and C₄ species have shown that advantages of C₄ photosynthesis can be lost under drought. We tested the generality of these paradoxical findings in grass species representing the known evolutionary diversity of C₄ NADP‐me and C₃ photosynthetic types. Our experiment investigated the effects of drought on leaf photosynthesis, water potential, nitrogen, chlorophyll content and mortality. C₄ grasses in control treatments were characterized by higher CO₂ assimilation rates and water potential, but lower stomatal conductance and nitrogen content. Under drought, stomatal conductance declined more dramatically in C₃ than C₄ species, and photosynthetic water‐use and nitrogen‐use efficiency advantages held by C₄ species under control conditions were each diminished by 40%. Leaf mortality was slightly higher in C₄ than C₃ grasses, but leaf condition under drought otherwise showed no dependence on photosynthetic‐type. This phylogenetically controlled experiment suggested that a drought‐induced reduction in the photosynthetic performance advantages of C₄ NADP‐me relative to C₃ grasses is a general phenomenon.     

Upregulation of bundle sheath electron transport capacity under limiting light in C4 Setaria viridis

C₄ photosynthesis is a biochemical pathway that operates across mesophyll and bundle sheath (BS) cells to increase CO₂ concentration at the site of CO₂ fixation. C₄ plants benefit from high irradiance but their efficiency decreases under shade, causing a loss of productivity in crop canopies. We investigated shade acclimation responses of Setaria viridis, a model monocot of NADP-dependent malic enzyme subtype, focussing on cell-specific electron transport capacity. Plants grown under low light (LL) maintained CO₂ assimilation rates similar to high light plants but had an increased chlorophyll and light-harvesting-protein content, predominantly in BS cells. Photosystem II (PSII) protein abundance, oxygen-evolving activity and the PSII/PSI ratio were enhanced in LL BS cells, indicating a higher capacity for linear electron flow. Abundances of PSI, ATP synthase, Cytochrome b₆f and the chloroplast NAD(P)H dehydrogenase complex, which constitute the BS cyclic electron flow machinery, were also increased in LL plants. A decline in PEP carboxylase activity in mesophyll cells and a consequent shortage of reducing power in BS chloroplasts were associated with a more oxidised plastoquinone pool in LL plants and the formation of PSII – light-harvesting complex II supercomplexes with an increased oxygen evolution rate. Our results suggest that the supramolecular composition of PSII in BS cells is adjusted according to the redox state of the plastoquinone pool. This discovery contributes to the understanding of the acclimation of PSII activity in C₄ plants and will support the development of strategies for crop improvement, including the engineering of C₄ photosynthesis into C₃ plants.     

Enhanced tolerance of photosynthesis against high temperature damage in salt-adapted halophyte Atriplex centralasiatica plants

Thermotolerance of photosynthesis in salt‐adapted Atriplex centralasiatica plants (100–400 mm NaCl) was evaluated in this study after detached leaves and whole plants were exposed to high temperature stress (30–48 °C) either in the dark or under high light (1200 mol m^?2 s^?1). In parallel with the decrease in stomatal conductance, intercellular CO₂ concentration and CO₂ assimilation rate decreased significantly with increasing salt concentration. There was no change in the maximal efficiency of PSII photochemistry (F_v/F_m) with increasing salt concentration, suggesting that there was no damage to PSII in salt‐adapted plants. On the other hand, there was a striking difference in the response of PSII and CO₂ assimilation capacity to heat stress in non‐salt‐adapted and salt‐adapted leaves. Leaves from salt‐adapted plants maintained significantly higher F_v/F_m values than those from non‐salt‐adapted leaves at temperatures higher than 42 °C. The F_v/F_m differences between non‐salt‐adapted and salt‐adapted plants persisted for at least 24 h following heat stress. Leaves from salt‐adapted plants also maintained a higher net CO₂ assimilation rate than those in non‐salt‐adapted plants at temperatures higher than 42 °C. This increased thermotolerance was independent of the degree of salinity since no significant changes in F_v/F_m and net CO₂ assimilation rate were observed among the plants treated with different concentrations of NaCl. The increased thermotolerance of PSII induced by salinity was still evident when heat treatments were carried out under high light. Given that photosynthesis is considered to be the physiological process most sensitive to high temperature damage, increased thermotolerance of photosynthesis may be of significance since A. centralasiatica, a typical halophyte, grows in the high salinity regions in the north of China, where the temperature in the summer is often as high as 45 °C.     

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.