Genome-wide analysis of the diatom cell cycle unveils a novel type of cyclins involved in environmental signaling

Background

Despite the enormous importance of diatoms in aquatic ecosystems and their broad industrial potential, little is known about their life cycle control. Diatoms typically inhabit rapidly changing and unstable environments, suggesting that cell cycle regulation in diatoms must have evolved to adequately integrate various environmental signals. The recent genome sequencing of Thalassiosira pseudonana and Phaeodactylum tricornutum allows us to explore the molecular conservation of cell cycle regulation in diatoms.

Results

By profile-based annotation of cell cycle genes, counterparts of conserved as well as new regulators were identified in T. pseudonana and P. tricornutum. In particular, the cyclin gene family was found to be expanded extensively compared to that of other eukaryotes and a novel type of cyclins was discovered, the diatom-specific cyclins. We established a synchronization method for P. tricornutum that enabled assignment of the different annotated genes to specific cell cycle phase transitions. The diatom-specific cyclins are predominantly expressed at the G1-to-S transition and some respond to phosphate availability, hinting at a role in connecting cell division to environmental stimuli.

Conclusion

The discovery of highly conserved and new cell cycle regulators suggests the evolution of unique control mechanisms for diatom cell division, probably contributing to their ability to adapt and survive under highly fluctuating environmental conditions.     

Differential regulatory role of nitric oxide in mediating nitrate reductase activity in roots of tomato (Solanum lycocarpum)

Background and Aims

Nitric oxide (NO) has been demonstrated to stimulate the activity of nitrate reductase (NR) in plant roots supplied with a low level of nitrate, and to affect proteins differently, depending on the ratio of NO to the level of protein. Nitrate has been suggested to regulate the level of NO in plants. This present study examined interactive effects of NO and nitrate level on NR activity in roots of tomato (Solanum lycocarpum).

Methods

NR activity, mRNA level of NR gene and concentration of NR protein in roots fed with 0·5 mm or 5 mm nitrate and treated with the NO donors, sodium nitroprusside (SNP) and diethylamine NONOate sodium (NONOate), and the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO), were measured in 25-d-old seedlings.

Key Results

Addition of SNP and NONOate enhanced but cPTIO decreased NR activity in the roots fed with 0·5 mm nitrate. The opposite was true for the roots fed with 5 mm nitrate. However, the mRNA level of the NR gene and the protein concentration of NR enzyme in the roots were not affected by SNP treatment, irrespective of nitrate pre-treatment. Nevertheless, a low rate of NO gas increased while cPTIO decreased the NR activities of the enzyme extracts from the roots at both nitrate levels. Increasing the rate of NO gas further increased NR activity in the enzyme extracts of the roots fed with 0·5 mm nitrate but decreased it when 5 mm nitrate was supplied. Interestingly, the stimulative effect of NO gas on NR activity could be reversed by NO removal through N₂ flushing in the enzyme extracts from the roots fed with 0·5 mm nitrate but not from those with 5 mm nitrate.

Conclusions

The effects of NO on NR activity in tomato roots depend on levels of nitrate supply, and probably result from direct interactions between NO and NR protein.Key words: Nitric oxide, nitrate, nitrate reductase, post-translational regulation, tomato, Solanum lycocarpum     

Involvement of putrescine and nitric oxide in aluminum tolerance by modulating citrate secretion from roots of red kidney bean

Background and Aims

Polyamines and nitric oxide (NO) are two important molecules modulating numerous environment stresses in plants. This study was to investigate the roles of polyamines and NO in aluminum (Al) tolerance in red kidney bean.

Methods

The interaction between putrescine (Put) and NO under Al stress was examined. NO donor and scavenger were used to further examine the role of NO in Al-induced citrate secretion from roots by high performance liquid chromatography.

Results

Al stress caused increase of endogenous free Put, and exogenous Put alleviated Al-induced inhibition of root elongation and Al accumulation. In addition, Put induced NO production and nitrate reductase (NR) activity under Al stress. Al- and Put-induced NO production could be reversed by NR inhibitor. Furthermore, Al stress stimulated citrate secretion from roots, and this response was stimulated by NO donor, whereas NO scavenger inhibited Al-induced citrate secretion from roots. Concomitantly, NO donor reduced Al accumulation in root apexes, while NO scavenger further enhanced Al accumulation. Al-induced inhibition of root growth was significantly improved by exogenous citrate treatment.

Conclusions

Put and NO enhanced Al tolerance by modulating citrate secretion from roots, and NO may act downstream of Put in red kidney bean under Al stress.     

Gene Expression of Lupeol Synthase and Biosynthesis of Nitric Oxide in Cell Suspension Cultures of Betula platyphylla in Response to a Phomopsis Elicitor

The current work aimed to characterize the generation of nitric oxide (NO) and gene expression of lupeol synthase (LUS) in Betula platyphylla cells exposed to a Phomopsis elicitor. The effects of nitrate reductase (NR) and NO synthase (NOS), the two key enzymes responsible for endogenous NO biosynthesis in plants, were also investigated. NO production in B. platyphylla cell cultures exhibited a biphasic pattern, reaching the Wrst plateau within 1.0–10 h of exposure to the Phomopsis elicitor. LUS gene expression was found to increase abruptly 10 h after Phomopsis induction, reaching its highest level (18.08) at 24 h. The maximum levels of NOS and NR activities in elicitor-treated cells were found to be 1.7-fold and 6.9-fold those of untreated cells, respectively. Pharmacological experiments showed that Phomopsis elicitor-induced NO production and LUS gene expression level were significantly suppressed by the NOS inhibitor N^G-nitro-l-Arg methyl ester (l-NAME), the NR inhibitor sodium azide (NaN₃), and the NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). NaNO₂ and l-arginine (the substrates that produce NO via NR and NOS) and NO donor sodium nitroprusside (SNP) were found to increase both NO production and LUS gene expression. These results suggest that the increase in LUS gene expression due to fungal elicitor-induced NO may involve the NR and NOS biosynthetic pathways.     

24-Epibrassinolide regulates carbohydrate metabolism and increases polyamine content in cucumber exposed to Ca(NO3)2 stress

Key message

This study focuses on the impact of carbohydrate metabolism and endogenous polyamines levels in leaves of cucumber seedlings under salt stress by exogenous BRs.

Abstract

The effects of 24-epibrassinolide (EBL) on carbohydrate metabolism and endogenous content of polyamines were investigated in cucumber seedlings (Cucumis sativus L. cv. Jinyou No. 4) exposed to salinity stress [80 mM Ca(NO₃)₂]. Spraying of exogenous EBL partially enhanced the enzyme activities of sucrose phosphate synthase, sucrose synthase and acid invertase; thus, raising the level of sucrose, fructose and total soluble sugars. The amylase activity was also increased by EBL, companied by the rising of sucrose level. These results indicated that EBL improved the carbohydrate metabolism of cucumber under Ca(NO₃)₂ stress. Moreover, EBL raised the levels of soluble conjugated and insoluble bound polyamines while lowered the free polyamines content, particularly putrescine. Our experiment demonstrated that exogenous EBL elevated stability of cellular membrane and positively improve the carbohydrate metabolism in cucumber growing under Ca(NO₃)₂ stress.     

Dark induction of nitrate reductase in the halophilic alga Dunaliella salina

The effect of nitrogen starvation on the NO₃-dependent induction of nitrate reductase (NR) and nitrite reductases (NIR) has been investigated in the halophilic alga Dunaliella salina. When D. salina cells previously grown in a medium with NH ₄ ⁺ as the only nitrogen source (NH ₄ ⁺ -cells) were transferred into NO ₃ ^? medium, NR was induced in the light. In contrast, when cells previously grown in N-free medium were transferred into a medium containing NO ₃ ^? , NR was induced in light or in darkness. Nitrate-dependent NR induction, in darkness, in D. salina cells previously grown at a photon flux density of 500 umol · m^?2 s^?1 was observed after 4 h preculture in N-free medium, whilst in cells grown at 100 umol · m^?2 s^?1 NR induction was observed after 7–8 h. An inhibitor of mRNA synthesis (6-methylpurine) did not inhibit NO ₃ ^? -induced NR synthesis when the cells, previously grown in NH ₄ ⁺ medium, were transferred into NO ₃ ^? medium (at time 0 h) after 4-h-N starvation. However, when 6-methylpurine was added simultaneously with the transfer of the cells from NH ₄ ⁺ to NO ₃ ^? medium (at time 0 h), NO ₃ ^? induced NR synthesis was completely inhibited. The activity of NIR decreased in N-starved cells and the addition of NO ₃ ^? to those cells greatly stimulated NIR activity in the light. The ability to induce NR in darkness was observed when glutamine synthetase activity reached its maximal level during N starvation. Although cells grown in NO ₃ ^? medium exhibited high NR activity, only 0.33% of the total NR was found in intact chloroplasts. We suggest that the ability, to induce NR in darkness is dependent on the level of N starvation, and that NR in D. salina is located in the cytosol. Light seems to play an indirect regulatory role on NO ₃ ^? uptake and NR induction due to the expression of NR and NO ₃ ^? -transporter mRNAs.     

Comparison of complete nuclear receptor sets from the human, Caenorhabditis elegans and Drosophila genomes

Background

The availability of complete genome sequences enables all the members of a gene family to be identified without limitations imposed by temporal, spatial or quantitative aspects of mRNA expression. Using the nearly completed human genome sequence, we combined in silico and experimental approaches to define the complete human nuclear receptor (NR) set. This information was used to carry out a comparative genomic study of the NR superfamily.

Results

Our analysis of the human genome identified two novel NR sequences. Both these contained stop codons within the coding regions, indicating that both are pseudogenes. One (HNF4 γ-related) contained no introns and expressed no detectable mRNA, whereas the other (FXR-related) produced mRNA at relatively high levels in testis. If translated, the latter is predicted to encode a short, non-functional protein. Our analysis indicates that there are fewer than 50 functional human NRs, dramatically fewer than in Caenorhabditis elegans and about twice as many as in Drosophila. Using the complete human NR set we made comparisons with the NR sets of C. elegans and Drosophila. Searches for the >200 NRs unique to C. elegans revealed no human homologs. The comparative analysis also revealed a Drosophila member of NR subfamily NR3, confirming an ancient metazoan origin for this subfamily.

Conclusions

This work provides the basis for new insights into the evolution and functional relationships of NR superfamily members.     

Nutrient resupplementation arrests bio-oil accumulation in Phaeodactylum tricornutum

Phaeodactylum tricornutum is a marine diatom in the class Bacillariophyceae and is important ecologically and industrially with regards to ocean primary production and lipid accumulation for biofuel production, respectively. Triacylglyceride (TAG) accumulation has been reported in P. tricornutum under different nutrient stresses, and our results show that lipid accumulation can occur with nitrate or phosphate depletion. However, greater lipid accumulation was observed when both nutrients were depleted as observed using a Nile Red assay and fatty acid methyl ester (FAME) profiles. Nitrate depletion had a greater effect on lipid accumulation than phosphate depletion. Lipid accumulation in P. tricornutum was arrested upon resupplementation with the depleted nutrient. Cells depleted of nitrogen showed a distinct shift from a lipid accumulation mode to cellular growth post-resupplementation with nitrate, as observed through increased cell numbers and consumption of accumulated lipid. Phosphate depletion caused lipid accumulation that was arrested upon phosphate resupplementation. The cessation of lipid accumulation was followed by lipid consumption without an increase in cell numbers. Cells depleted in both nitrate and phosphate displayed cell growth upon the addition of both nitrate and phosphate and had the largest observed lipid consumption upon resupplementation. These results indicate that phosphate resupplementation can shut down lipid accumulation but does not cause cells to shift into cellular growth, unlike nitrate resupplementation. These data suggest that nutrient resupplementation will arrest lipid accumulation and that switching between cellular growth and lipid accumulation can be regulated upon the availability of nitrogen and phosphorus.     

The effect of phosphate and flavin adenine dinucleotide on nitrate reductase activity of some unicellular marine algae

Nitrate reductase (NR) activity was measured in cell-free extracts from seven species of unicellular, marine algae. For most species Tris buffer was not a satisfactory assay buffer unless supplemented with phosphate. With some species (e.g. Brachiomonas submarina Bohlin, Nannochloropsis oculata Droop, Phaeodactylum tricomutum Bohlin) maximum NR activity required the addition of phosphate and flavin adenine dinucleotide (FAD) to assays; with extracts of P. tricornutum, the requirement for FAD increased after partial purification of extracts. Like phosphate, arsenate stimulated NR activity in extracts, but selenate, sulphate, and tungstate were less effective. Addition of manganese to partially purified extracts of P. tricomutum resulted in an inhibition of NR activity which was alleviated by phosphate or EDTA.     

Fine-root dynamics in sugi (Cryptomeria japonica) under manipulated soil nitrogen conditions

Aims

Nitrogen deposition affect fine-root dynamics, a key factor in forest carbon and nutrient dynamics. This study aimed to elucidate the effects of increased soil inorganic nitrogen (N) levels on the fine-root dynamics of Cryptomeria japonica, which is tolerant to excess N load.

Methods

An ammonium nitrate solution (28 kg ha^?1 month^?1) was applied for 3 years to plots (1 m?×?2 m) in a C. japonica plantation. The elongation and disappearance of the fine roots were examined using the minirhizotron technique.

Results

The N fertilization increased soil inorganic N content and lowered the soil pH. Fine-root elongation rates increased with fertilization, whereas patterns of their seasonal changes were not affected. The ratio of cumulative disappearance to cumulative elongation of fine roots was lower in the N-fertilized plots than in the control plots. The mean diameter of the fine roots was not affected by N fertilization.

Conclusions

Our results suggest that C. japonica can respond to increased levels of soil inorganic N by increasing both the production and residence time of the fine roots. However, the effects of the changing soil N content are less evident for the phenology and morphology of the fine roots in C. japonica.     

Can plant sugars mediate the effect of nitrogen fertilization on lettuce susceptibility to two necrotrophic pathogens: Botrytis cinerea and Sclerotinia sclerotiorum?

Aims

Nitrogen (N) fertilization is known to modify a plant’s susceptibility to necrotrophic diseases. However, the effect of N nutrition on defence is not well known. It was hypothesized that not only molecules related to the N metabolism but also main sugars could mediate the effect of plant fertilization on its susceptibility to pathogens.

Methods

Two necrotrophic fungi, Botrytis cinerea and Sclerotinia sclerotiorum were inoculated on leaves of lettuce plants grown at five nitrate (NO₃ ^?) fertilization levels, in three independent experiments. Variations in plant composition at the time of inoculation were linked to the size of lesions observed after 5–6 days.

Results

Both diseases were favoured by high NO₃ ^? fertilization. However, the highest disease levels were not found in the same experiment for B. cinerea and S. sclerotiorum. Among the components measured, NO₃ ^? and sucrose (SUC) were positively and negatively correlated to the two diseases in the three experiments, but the relationship between SUC and lesion size was more significant for S. sclerotiorum. Water content, N and total carbon (C) were also significantly correlated to the diseases, but the relationships were less straightforward. The ratios of SUC over total sugars and fructose (FRU) over total sugars fitted, very closely for S. sclerotiorum, a negative and positive exponential relationship respectively with lesion size. Absolute or relative glucose levels were not linked to the diseases.

Conclusions

Plant metabolic modifications induced by variations of N availability conferred the plant variable defence ability, which seemed, at least for S. sclerotiorum, mainly mediated by variations in host SUC and FRU levels. The generalization of these findings to other species would be of interest.     

Auxin transport in maize roots in response to localized nitrate supply

Background and Aims

Roots typically respond to localized nitrate by enhancing lateral-root growth. Polar auxin transport has important roles in lateral-root formation and growth; however, it is a matter of debate whether or how auxin plays a role in the localized response of lateral roots to nitrate.

Methods

Treating maize (Zea mays) in a split-root system, auxin levels were quantified directly and polar transport was assayed by the movement of [³H]IAA. The effects of exogenous auxin and polar auxin transport inhibitors were also examined.

Key Results

Auxin levels in roots decreased more in the nitrate-fed compartment than in the nitrate-free compartment and nitrate treatment appeared to inhibit shoot-to-root auxin transport. However, exogenous application of IAA only partially reduced the stimulatory effect of localized nitrate, and auxin level in the roots was similarly reduced by local applications of ammonium that did not stimulate lateral-root growth.

Conclusions

It is concluded that local applications of nitrate reduced shoot-to-root auxin transport and decreased auxin concentration in roots to a level more suitable for lateral-root growth. However, alteration of root auxin level alone is not sufficient to stimulate lateral-root growth.     

Using optical full-field measurement based on digital image correlation to measure strain on a tree subjected to mechanical load

Key message

The results indicate the usability of DIC technique in tree biomechanics is advantageous compared to standard procedure since it reveals the induced strain in full-field way, accurately and noninvasively.

Abstract

The goal of this study was to analyze and measure the displacement and strain response behavior of the tree (Juglans regia L.) subjected to a mechanical load. The analysis addresses issues of tree biomechanics that call for a high-quality deformation data measured on trees’ surfaces in a noninvasive way. For these purposes, the 3D digital image correlation (3D-DIC) was proposed and tested against standard extensometers. The measured tree was subjected to a bending by a pulling test to induce strain on the tree stem. The DIC technique successfully provided strain field on a tree bark despite its highly complicated geometry. Vertical averaging of the strain field obtained by the DIC revealed that a tree stem behaves according to beam theory exhibiting compression and tension parts. The absolute values of strain measured by both techniques agree with each other in order of magnitude, DIC returning lower values (approximately 21.1 and 40.8 % for compression and tension part, respectively). The results reveal necessity for future study of stress/strain transfer from xylem to bark. The sensitivity analysis shows that the computed strain highly depends on subset size used in the DIC computation. Based on the projection error and strain, the optimal subset size was found out to be between 21 and 31 pixels. The measurement proved that the DIC method can be successfully used in tree biomechanics. In general, our results and the character of DIC technique that allows testing at different scales depending on imaging methods indicate its big potential in plant biomechanics.     

Kinetic exploration of nitrate-accumulating microalgae for nutrient recovery

Within sustainable resource management, the recovery of nitrogen and phosphorus nutrients from waste streams is becoming increasingly important. Although the use of microalgae has been described extensively in environmental biotechnology, the potential of nitrate-accumulating microalgae for nutrient recovery has not been investigated yet. The ability of these marine microorganisms to concentrate environmental nitrate within their biomass is remarkable. The aim of this study was to investigate the application potential of nitrate-accumulating diatoms for nutrient recovery from marine wastewaters. The intracellular nitrate storage capacity was quantified for six marine diatom strains in synthetic wastewater. Amphora coffeaeformis and Phaeodactylum tricornutum stored the highest amount of nitrate with respectively 3.15 and 2.10 g N L^?1 of cell volume, which accounted for 17.3 and 4.6 %, respectively, of the total nitrogen content. The growth and nitrate and phosphate uptake of both diatoms were further analyzed and based on these features P. tricornutum showed the highest potential for nutrient recovery. A mathematical model was developed which included intracellular nitrate storage and the kinetic parameters were derived for P. tricornutum. Furthermore, a simulation study was performed to compare the performance of a proposed microalgal nutrient recovery unit with a conventional denitrification system for marine wastewater treatment. Overall, this study demonstrates the potential application of P. tricornutum for saline wastewater treatment with concurrent nitrogen and phosphorus recycling.     

Effects of plateau pika (Ochotona curzoniae) on net ecosystem carbon exchange of grassland in the Three Rivers Headwaters region, Qinghai-Tibet, China

Background and aim

Because the indigenous burrowing lagomorph plateau pika (Ochotona curzoniae) is considered to have negative ecological impacts on alpine meadow steppe grasslands of the Headwaters Region of the Yellow, Yangtze and Mekong Rivers we investigated its effects on ecosystem productivity and soil properties, and especially net ecosystem carbon flux.

Methods

We measured net ecosystem CO₂ exchange (NEE) and its components gross ecosystem productivity (GEP) and ecosystem respiration (ER) at peak aboveground biomass by the chamber method with reference to plant and soil characteristics of areas of alpine meadow steppe with different densities of pika burrows.

Results

Higher burrow density decreased NEE, GEP and ER. Above-ground biomass, species number, plant cover and leaf area index decreased with increasing pika density. Higher burrow density was associated with lower soil moisture and higher soil temperature. Responses of NEE were related to changes of abiotic and biotic factors affecting its two components. NEE was positively related to soil moisture, soil ammonium nitrogen, plant cover, leaf area index and above-ground biomass but was negatively correlated with higher soil nitrate nitrogen.

Conclusion

Decrease of NEE by plateau pika may reduce the carbon sink balance of Qinghai-Tibet plateau grassland. Such effects may be influenced by grazing pressure from domestic livestock, population levels of natural predators, and climate change.