The physiological response of marine diatoms to ocean acidification: differential roles of seawater pCO2 and pH

Although increasing the pCO₂ for diatoms will presumably down‐regulate the CO₂‐concentrating mechanism (CCM) to save energy for growth, different species have been reported to respond differently to ocean acidification (OA). To better understand their growth responses to OA, we acclimated the diatoms Thalassiosira pseudonana, Phaeodactylum tricornutum, and Chaetoceros muelleri to ambient (pCO₂ 400 μatm, pH 8.1), carbonated (pCO₂ 800 μatm, pH 8.1), acidified (pCO₂ 400 μatm, pH 7.8), and OA (pCO₂ 800 μatm, pH 7.8) conditions and investigated how seawater pCO₂ and pH affect their CCMs, photosynthesis, and respiration both individually and jointly. In all three diatoms, carbonation down‐regulated the CCMs, while acidification increased both the photosynthetic carbon fixation rate and the fraction of CO₂ as the inorganic carbon source. The positive OA effect on photosynthetic carbon fixation was more pronounced in C. muelleri, which had a relatively lower photosynthetic affinity for CO₂, than in either T. pseudonana or P. tricornutum. In response to OA, T. pseudonana increased respiration for active disposal of H⁺ to maintain its intracellular pH, whereas P. tricornutum and C. muelleri retained their respiration rate but lowered the intracellular pH to maintain the cross‐membrane electrochemical gradient for H⁺ efflux. As the net result of changes in photosynthesis and respiration, growth enhancement to OA of the three diatoms followed the order of C. muelleri > P. tricornutum > T. pseudonana. This study demonstrates that elucidating the separate and joint impacts of increased pCO₂ and decreased pH aids the mechanistic understanding of OA effects on diatoms in the future, acidified oceans.     

Effects of non-steady-state iron limitation on nitrogen assimilatory enzymes in the marine diatom thalassiosira weissflogii (BACILLARIOPHYCEAE)

Since the recognition of iron‐limited high nitrate (or nutrient) low chlorophyll (HNLC) regions of the ocean, low iron availability has been hypothesized to limit the assimilation of nitrate by diatoms. To determine the influence of non‐steady‐state iron availability on nitrogen assimilatory enzymes, cultures of Thalassiosira weissflogii (Grunow) Fryxell et Hasle were grown under iron‐limited and iron‐replete conditions using artificial seawater medium. Iron‐limited cultures suffered from decreased efficiency of PSII as indicated by the DCMU‐induced variable fluorescence signal (F_v/F_m). Under iron‐replete conditions, in vitro nitrate reductase (NR) activity was rate limiting to nitrogen assimilation and in vitro nitrite reductase (NiR) activity was 50‐fold higher. Under iron limitation, cultures excreted up to 100 fmol NO₂^?·cell^?1·d^?1 (about 10% of incorporated N) and NiR activities declined by 50‐fold while internal NO₂^? pools remained relatively constant. Activities of both NR and NiR remained in excess of nitrogen incorporation rates throughout iron‐limited growth. One possible explanation is that the supply of photosynthetically derived reductant to NiR may be responsible for the limitation of nitrogen assimilation at the NO₂^? reduction step. Urease activity showed no response to iron limitation. Carbon:nitrogen ratios were equivalent in both iron conditions, indicating that, relative to carbon, nitrogen was assimilated at similar rates whether iron was limiting growth or not. We hypothesize that, diatoms in HNLC regions are not deficient in their ability to assimilate nitrate when they are iron limited. Rather, it appears that diatoms are limited in their ability to process photons within the photosynthetic electron transport chain which results in nitrite reduction becoming the rate‐limiting step in nitrogenassimilation.     

Metabolic and photosynthetic consequences of blocking starch biosynthesis in the green alga Chlamydomonas reinhardtii sta6 mutant

Upon nutrient deprivation, microalgae partition photosynthate into starch and lipids at the expense of protein synthesis and growth. We investigated the role of starch biosynthesis with respect to photosynthetic growth and carbon partitioning in the Chlamydomonas reinhardtii starchless mutant, sta6, which lacks ADP‐glucose pyrophosphorylase. This mutant is unable to convert glucose‐1–phosphate to ADP‐glucose, the precursor of starch biosynthesis. During nutrient‐replete culturing, sta6 does not re‐direct metabolism to make more proteins or lipids, and accumulates 20% less biomass. The underlying molecular basis for the decreased biomass phenotype was identified using LC–MS metabolomics studies and flux methods. Above a threshold light intensity, photosynthetic electron transport rates (water → CO₂) decrease in sta6 due to attenuated rates of NADPH re‐oxidation, without affecting photosystems I or II (no change in isolated photosynthetic electron transport). We observed large accumulations of carbon metabolites that are precursors for the biosynthesis of lipids, amino acids and sugars/starch, indicating system‐wide consequences of slower NADPH re‐oxidation. Attenuated carbon fixation resulted in imbalances in both redox and adenylate energy. The pool sizes of both pyridine and adenylate nucleotides in sta6 increased substantially to compensate for the slower rate of turnover. Mitochondrial respiration partially relieved the reductant stress; however, prolonged high‐light exposure caused accelerated photoinhibition. Thus, starch biosynthesis in Chlamydomonas plays a critical role as a principal carbon sink influencing cellular energy balance however, disrupting starch biosynthesis does not redirect resources to other bioproducts (lipids or proteins) during nutrient‐replete culturing, resulting in cells that are susceptible to photochemical damage caused by redox stress.     

Acclimation of wild-type cells and CO2-insensitive mutants of the green alga Chlorella ellipsoidea to elevated [CO2

CO(2)-insensitive mutants of the green alga Chlorella ellipsoidea were previously shown to be unable to repress an inorganic carbon-concentrating mechanism (CCM) when grown under 5% CO(2). When air-grown, wild-type (WT) cells were transferred to 5% CO(2), an abrupt drop of P(max) to 43% the original level of air-grown cells was observed within the initial 12 h. Photosynthetic affinities of WT cells to dissolved inorganic carbon (DIC) were maintained at high levels for the initial 4 d of acclimation, and then decreased gradually to lower levels over the next 6 d. In contrast to WT cells, the CO(2)-insensitive mutant, ENU16, exhibited a constant P(max) at maximum levels and a low K(1/2)[DIC] throughout the acclimation period. The rapid P(max) drop within 12 h of acclimation in WT cells was significantly reduced by treatment with 0.5 mm of 6-ethoxybenzothiazole-2-sulphonamide (EZA), a specific membrane-permeable inhibitor of carbonic anhydrase (CA), suggesting the participation of internal CAs in the temporary drop in P(max) in WT cells. WT and ENU16 cells were grown in controlled equilibrium [CO(2)], and the photosynthetic rate of each acclimated cell type was measured under equilibrated growth [DIC] conditions. In WT cells acclimated to 0.14-0.4% [CO(2)], K(1/2)[DIC] values increased as [CO(2)] increased, and the photosynthetic rates at growth DIC conditions were shown to decrease to about 70% the P(max) level in this intermediate [CO(2)] range. Such decreases in the net photosynthetic rates were not observed in ENU16. These results suggest that algal primary production could be depressed significantly under moderately enriched CO(2) conditions as a result of acquiring intermediate affinities for DIC because of their sensitive responses to changes in the ambient [CO(2)].     

Effect of iron on the growth of Phaeodactylum tricornutum via photosynthesis

Iron is a limiting factor that controls the phytoplankton biomass in the modern ocean, and iron fertilization of the ocean could lead to blooms dominated by diatoms. Thus, iron plays an important role in controlling the distribution of diatoms. In this study, we measured the growth rate and photosynthetic activity of the model diatom Phaeodactylum tricornutum cultured under different iron concentrations and found that it grew more rapidly and had a much higher photosynthetic efficiency under higher iron concentrations. In order to explore the unique mechanism of the response of diatoms to iron, a proteomic analysis was carried out, and the results indicated that iron promotes the Calvin cycle of P. tricornutum. Diatoms can tolerate the pressure of iron limitation by replacing iron‐rich proteins with flavodoxin, and so on. Moreover, we found that the photosystem I (PSI) activity of iron‐limited algae that were treated by N’,N’,N’,N’‐tetramethyl‐p‐phenylenediamine (TMPD) was increased significantly. As TMPD plays the role of a cytochrome b₆/f complex that transfers electrons from photosystem II to PSI, the cytochrome b₆/f complex is the key to photosynthesis regulation. Iron could influence the growth of P. tricornutum by regulating its biosynthesis. All of the results suggest that iron might affect the growth of diatoms through the Calvin cycle and the cytochrome b₆/f complex.     

Arbuscular mycorrhizal fungi effect growth and photosynthesis of Phragmites australis (Cav.) Trin ex. Steudel under copper stress

• Arbuscular mycorrhizal fungi (AMF) is an effective way to remove heavy metals’ inhibition on plants, however, few relevant research attempts have been made to determine the contribution of AMF to the physiological and biochemical changes related to the enhanced copper tolerance of Phragmites australis under metal‐stressed conditions.
• In this study, the effects of AMF inoculation on P. australis under different concentrations of copper stress were investigated according to the changes in the parameters related to growth and development, and photosynthetic charateristics. Then, differentially expressed proteins (DEPs) were evaluated by the Isobaric Tag for Relative and Absolute Quantification (iTRAQ) system, which could accurately quantify the DEPs by measuring peak intensities of reporter ions in tandem mass spectrometry (MS/MS) spectra.
• It was found that AMF inoculation may relieve the photosynthesis inhibition caused by copper stress on P. australis and thus promote growth. Proteomic analysis results showed that under copper stress, the inoculation of R. irregularis resulted in a total of 459 differently‐expressed proteins (200 up‐regulated and 259 down‐regulated) in root buds. In addition, the photosynthetic changes caused by AMF inoculation mainly involve the up‐regulated expression of transmembrane protein–pigment complexes CP43 (photosystem II) and FNR (ferredoxin‐NADP+ oxidoreductase related to photosynthetic electron transport).
• These results indicate that AMF could effectively improve the growth and physiological activity of P. australis under copper stress, and thus provides a new direction and instructive evidence for determining the mechanisms by which AMF inoculation enhances the copper tolerance of plants.

     

CHARACTERIZATION OF DIATOM (BACILLARIOPHYCEAE) NITRATE REDUCTASE GENES AND THEIR DETECTION IN MARINE PHYTOPLANKTON COMMUNITIES1

The complete assimilatory nitrate reductase (NR) gene from the pennate diatom Phaeodactylum triconutum Bohlin was sequenced from cDNA and compared with NR sequences from fungi, green algae, vascular plants, and the recently sequenced genome of the centric diatom Thalassiosira pseudonana Hasle and Heimdal CCMP1335. In all the major eukaryotic nitrate reductase (Euk‐NR) functional domains, diatom NR gene sequences are generally 50%–60% identical to plant and alga sequences at the amino acid level. In the less conserved N‐terminal, hinge 1, and hinge 2 regions, homology to other NR sequences is weak, generally<30%. Two PCR primer sets capable of amplifying Euk‐NR from plants, algae, and diatoms were designed. One primer set was used to amplify a 750‐base pair (bp) NR fragment from the cDNA of five additional diatom strains. The PCR amplicon spans part of the well‐conserved dimer interface region, the more variable hinge 1 region, and part of the conserved cytochrome b heme binding region. The second primer set, targeted to the dimer region, was used to amplify an approximately 400‐bp fragment of the NR gene from DNA samples collected in Monterey Bay, California and in central New Jersey inner continental shelf (LEO‐15 site) waters. Only diatom‐like NR sequences were recovered from Monterey Bay samples, whereas LEO‐15 samples yielded NR sequences from a range of photosynthetic eukaryotes. The prospect of using DNA‐ and RNA‐based methods to target the NR genes of diatoms specifically is a promising approach for future physiological and ecological experiments.     

FGF21 deletion exacerbates diabetic cardiomyopathy by aggravating cardiac lipid accumulation

Fibroblast growth factor 21 (FGF21) plays an important role in energy homoeostasis. The unaddressed question of FGF21's effect on the development and progression of diabetic cardiomyopathy (DCM) is investigated here with FGF21 knockout (FGF21KO) diabetic mice. Type 1 diabetes was induced in both FGF21KO and C57BL/6J wild‐type (WT) mice via streptozotocin. At 1, 2 and 4 months after diabetes onset, the plasma FGF21 levels were significantly decreased in WT diabetic mice compared to controls. There was no significant difference between FGF21KO and WT diabetic mice in blood glucose and triglyceride levels. FGF21KO diabetic mice showed earlier and more severe cardiac dysfunction, remodelling and oxidative stress, as well as greater increase in cardiac lipid accumulation than WT diabetic mice. Western blots showed that increased cardiac lipid accumulation was accompanied by further increases in the expression of nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) and its target protein CD36, along with decreases in the phosphorylation of AMP‐activated protein kinase and the expression of hexokinase II and peroxisome proliferator‐activated receptor gamma co‐activator 1α in the heart of FGF21KO diabetic mice compared to WT diabetic mice. Our results demonstrate that FGF21 deletion‐aggravated cardiac lipid accumulation is likely mediated by cardiac Nrf2‐driven CD36 up‐regulation, which may contribute to the increased cardiac oxidative stress and remodelling, and the eventual development of DCM. These findings suggest that FGF21 may be a therapeutic target for the treatment of DCM.     

Expression of a constitutively active nitrate reductase variant in tobacco reduces tobacco‐specific nitrosamine accumulation in cured leaves and cigarette smoke

Burley tobaccos (Nicotiana tabacum) display a nitrogen‐use‐deficiency phenotype that is associated with the accumulation of high levels of nitrate within the leaf, a trait correlated with production of a class of compounds referred to as tobacco‐specific nitrosamines (TSNAs). Two TSNA species, 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) and N‐nitrosonornicotine (NNN), have been shown to be strong carcinogens in numerous animal studies. We investigated the potential of molecular genetic strategies to lower nitrate levels in burley tobaccos by overexpressing genes encoding key enzymes of the nitrogen‐assimilation pathway. Of the various constructs tested, only the expression of a constitutively active nitrate reductase (NR) dramatically decreased free nitrate levels in the leaves. Field‐grown tobacco plants expressing this NR variant exhibited greatly reduced levels of TSNAs in both cured leaves and mainstream smoke of cigarettes made from these materials. Decreasing leaf nitrate levels via expression of a constitutively active NR enzyme represents an exceptionally promising means for reducing the production of NNN and NNK, two of the most well‐documented animal carcinogens found in tobacco products.     

Fitness effects of a photosynthetic mutation across contrasting environments

To test the hypothesis that variation in photosynthesis can cause differences in fitness, we compared wild‐type (WT) Amaranthus hybridus genotypes to those having a single‐gene mutation (R) that affects photosynthetic rate. By using light and water treatments, we generated a range of differences between WT and R genotypes in photosynthetic rate, growth and reproduction at three developmental stages. In two cases photosynthetic differences were in the expected direction (WT > R), they did not differ in others, and in one case the R genotype had a higher rate than the WT. Across light and water treatments, higher rates of photosynthesis were related to increases in specific leaf area, leaf nitrogen content and stomatal conductance relative to the other genotype. Differences between genotypes in growth and allocation paralleled those in photosynthesis; in treatments where photosynthetic rate differed between the genotypes (high light), growth and reproduction did as well. In high light, the effects of genotype on fitness were indirect with high‐water availability, but were direct with low‐water availability. When photosynthetic rate did not differ between genotypes (low light), neither did growth and reproduction. These results demonstrate that variation in photosynthesis can cause differences in growth and reproduction. Furthermore, resource availability can moderate the ways in which selection operates on photosynthetic traits.     

Agronomic nitrogen‐use efficiency of rice can be increased by driving OsNRT2.1 expression with the OsNAR2.1 promoter

The importance of the nitrate () transporter for yield and nitrogen‐use efficiency (NUE) in rice was previously demonstrated using map‐based cloning. In this study, we enhanced the expression of the OsNRT2.1 gene, which encodes a high‐affinity transporter, using a ubiquitin (Ubi) promoter and the ‐inducible promoter of the OsNAR2.1 gene to drive OsNRT2.1 expression in transgenic rice plants. Transgenic lines expressing pUbi:OsNRT2.1 or pOsNAR2.1:OsNRT2.1 constructs exhibited the increased total biomass including yields of approximately 21% and 38% compared with wild‐type (WT) plants. The agricultural NUE (ANUE) of the pUbi:OsNRT2.1 lines decreased to 83% of that of the WT plants, while the ANUE of the pOsNAR2.1:OsNRT2.1 lines increased to 128% of that of the WT plants. The dry matter transfer into grain decreased by 68% in the pUbi:OsNRT2.1 lines and increased by 46% in the pOsNAR2.1:OsNRT2.1 lines relative to the WT. The expression of OsNRT2.1 in shoot and grain showed that Ubi enhanced OsNRT2.1 expression by 7.5‐fold averagely and OsNAR2.1 promoters increased by about 80% higher than the WT. Interestingly, we found that the OsNAR2.1 was expressed higher in all the organs of pUbi:OsNRT2.1 lines; however, for pOsNAR2.1:OsNRT2.1 lines, OsNAR2.1 expression was only increased in root, leaf sheaths and internodes. We show that increased expression of OsNRT2.1, especially driven by OsNAR2.1 promoter, can improve the yield and NUE in rice.     

Arabidopsis thaliana ggt1 photorespiratory mutants maintain leaf carbon/nitrogen balance by reducing RuBisCO content and plant growth

Metabolic and physiological analyses of glutamate:glyoxylate aminotransferase 1 (GGT1) mutants were performed at the global leaf scale to elucidate the mechanisms involved in their photorespiratory growth phenotype. Air‐grown ggt1 mutants showed retarded growth and development, that was not observed at high CO₂ (3000 μL L^?1). When compared to wild‐type (WT) plants, air‐grown ggt1 plants exhibited glyoxylate accumulation, global changes in amino acid amounts including a decrease in serine content, lower organic acid levels, and modified ATP/ADP and NADP⁺/NADPH ratios. When compared to WT plants, their net CO₂ assimilation rates (A_n) were 50% lower and this mirrored decreases in ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) contents. High CO₂‐grown ggt1 plants transferred to air revealed a rapid decrease of A_n and photosynthetic electron transfer rate while maintaining a high energetic state. Short‐term (a night period and 4 h of light) transferred ggt1 leaves accumulated glyoxylate and exhibited low serine contents, while other amino acid levels were not modified. RuBisCO content, activity and activation state were not altered after a short‐term transfer while the ATP/ADP ratio was lowered in ggt1 rosettes. However, plant growth and RuBisCO levels were both reduced in ggt1 leaves after a long‐term (12 days) acclimation to air from high CO₂ when compared to WT plants. The data are discussed with respect to a reduced photorespiratory carbon recycling in the mutants. It is proposed that the low A_n limits nitrogen‐assimilation, this decreases leaf RuBisCO content until plants attain a new homeostatic state that maintains a constant C/N balance and leads to smaller, slower growing plants.     

Auxin biosynthetic gene TAR2 is involved in low nitrogen‐mediated reprogramming of root architecture in Arabidopsis

In plants, the plasticity of root architecture in response to nitrogen availability largely determines nitrogen acquisition efficiency. One poorly understood root growth response to low nitrogen availability is an observed increase in the number and length of lateral roots (LRs). Here, we show that low nitrogen‐induced Arabidopsis LR growth depends on the function of the auxin biosynthesis gene TAR2 (tryptophan aminotransferase related 2). TAR2 was expressed in the pericycle and the vasculature of the mature root zone near the root tip, and was induced under low nitrogen conditions. In wild type plants, low nitrogen stimulated auxin accumulation in the non‐emerged LR primordia with more than three cell layers and LR emergence. Conversely, these low nitrogen‐mediated auxin accumulation and root growth responses were impaired in the tar2‐c null mutant. Overexpression of TAR2 increased LR numbers under both high and low nitrogen conditions. Our results suggested that TAR2 is required for reprogramming root architecture in response to low nitrogen conditions. This finding suggests a new strategy for improving nitrogen use efficiency through the engineering of TAR2 expression in roots.