Differential expression of glucose metabolism-related genes and AMP-activated protein kinases in crop tissue of male and female pigeons (Columba livia domestica) during the incubation and chick-rearing periods.

The objective of this study was to explore the carbohydrate contents of crop milk, insulin and glucose concentrations in serum and the expression patterns of AMP-activated protein kinases (AMPKs) and genes related to glucose metabolism in pigeon crops during the breeding period. Crop milk was collected from squabs of rearing Day 1 (R1) to R6. Contents of total sugar and reducing sugar increased to the maximum levels at R6 (p < 0.05). Forty-two pairs of adult pigeons were allotted to seven groups by different breeding stages, and their crops and serum were sampled. No significant differences were found in either insulin or glucose levels in serum. The glucose transporter 2 gene level was the greatest at R15 in females, whereas it was at R7 in males. However, sodium-dependent glucose transporters 1 expression in both sexes decreased from incubation Day 17 (I17) to R7. In females, glucokinase expression peaked at R1, and at R1 and R7 in males. Pyruvate kinase mRNA levels peaked at R7 in females and at R15 males. The mRNA abundance of fructose-1,6-bisphosphatase 1 in both sexes and glucose-6-phosphatase in females decreased after I10. While phosphoenolpyruvate carboxykinase 1 expression decreased after I17 (p < 0.05). Protein levels of AMPKα in crops were minimized at R1 (p < 0.05). In females, expression of AMPKα1 and AMPKα2 was inhibited at I17 and R1 (p < 0.05). In males, AMPKα1 expression was decreased at R7 (p < 0.05) and AMPKα2 was reduced at I10 and R1. pAMPK expression was the lowest at I17 in females, and it was at R7 and R25 in males. Conclusively, glycolysis in pigeon crops was enhanced during chick-rearing, while gluconeogenesis was significantly inhibited. The stability of the insulin level suggests that it was probably not involved in the regulation of glucose metabolism in crop tissues.

Comparative Proteomic Analysis Reveals the Effects of Exogenous Calcium against Acid Rain Stress in Liquidambar formosana Hance Leaves.

Acid rain (AR) impacts forest health by leaching calcium (Ca) away from soils and plants. Ca is an essential element and participates in various plant physiological responses. In the present study, the protective role of exogenous Ca in alleviating AR stress in Liquidambar formosana Hance at the physiological and proteomic levels was examined. Our results showed that low Ca condition resulted in the chlorophyll content and photosynthesis decreasing significantly in L. formosana leaves; however, these effects could be reversed by high Ca supplementation. Further proteomic analyses successfully identified 81 differentially expressed proteins in AR-treated L. formosana under different Ca levels. In particular, some of the proteins are involved in primary metabolism, photosynthesis, energy production, antioxidant defense, transcription, and translation. Moreover, quantitative real time polymerase chain reaction (qRT-PCR) results indicated that low Ca significantly increased the expression level of the investigated Ca-related genes, which can be reversed by high Ca supplementation under AR stress. Further, Western blotting analysis revealed that exogenous Ca supply reduced AR damage by elevating the expression of proteins involved in the Calvin cycle, reactive oxygen species (ROS) scavenging system. These findings allowed us to better understand how woody plants respond to AR stress at various Ca levels and the protective role of exogenous Ca against AR stress in forest tree species.

Regulation of the calcium-sensing receptor in both stomatal movement and photosynthetic electron transport is crucial for water use efficiency and drought tolerance in Arabidopsis.

Production per amount of water used (water use efficiency, WUE) is closely correlated with drought tolerance. Although stomatal aperture can regulate WUE, the underlying molecular mechanisms are still unclear. Previous reports revealed that stomatal closure was inhibited in the calcium-sensing receptor (CAS) antisense line of Arabidopsis (CASas). Here it is shown that decreased drought tolerance and WUE of CASas was associated with higher stomatal conductance due to improper regulation of stomatal aperture, rather than any change of stomatal density. CASas plants also had a lower CO2 assimilation rate that was attributed to a lower photosynthetic electron transport rate, leading to higher chlorophyll fluorescence. Gene co-expression combined with analyses of chlorophyll content and transcription levels of photosynthesis-related genes indicate that CAS is involved in the formation of the photosynthetic electron transport system. These data suggest that CAS regulates transpiration and optimizes photosynthesis by playing important roles in stomatal movement and formation of photosynthetic electron transport, thereby regulating WUE and drought tolerance.

Comparative proteomic analysis on wild type and nitric oxide-overproducing mutant (nox1) of Arabidopsis thaliana.

Nitric oxide (NO) as a ubiquitous signal molecule plays an important role in plant development and growth. Here, we compared the proteomic changes between NO-overproducing mutant (nox1) and wild-type (WT) of Arabidopsis thaliana using two-dimensional electrophoresis coupled with MALDI-TOF MS. We successfully identified 59 differentially expressed proteins in nox1 mutant, which are predicted to play potential roles in specific cellular processes, such as post-translational modification, energy production and conversion, metabolism, transcription and signal transduction, cell rescue and defense, development and differentiation. Particularly, expression levels of five anti-oxidative enzymes were altered by the mutation; and assays of their respective enzymatic activities indicated an enhanced level of oxidative stress in nox1 mutant. Finally, some important proteins were further confirmed at transcriptional level using quantitative real-time PCR revealing the systemic changes between WT and nox1. The result suggests that obvious morphological changes in the nox1 mutant may be regulated by different mechanisms and factors, while excess endogenous NO maybe one of the possible reasons.

Comparative proteomic analysis of differential responses of Pinus massoniana and Taxus wallichiana var. mairei to simulated acid rain.

Acid rain (AR), a serious environmental issue, severely affects plant growth and development. As the gymnosperms of conifer woody plants, Pinus massoniana (AR-sensitive) and Taxus wallichiana var. mairei (AR-resistant) are widely distributed in southern China. Under AR stress, significant necrosis and collapsed lesions were found in P. massoniana needles with remarkable yellowing and wilting tips, whereas T. wallichiana var. mairei did not exhibit chlorosis and visible damage. Due to the activation of a large number of stress-related genes and the synthesis of various functional proteins to counteract AR stress, it is important to study the differences in AR-tolerance mechanisms by comparative proteomic analysis of tolerant and sensitive species. This study revealed a total of 65 and 26 differentially expressed proteins that were identified in P. massoniana and T. wallichiana var. mairei, respectively. Among them, proteins involved in metabolism, photosynthesis, signal transduction and transcription were drastically down-regulated in P. massoniana, whereas most of the proteins participating in metabolism, cell structure, photosynthesis and transcription were increased in T. wallichiana var. mairei. These results suggest the distinct patterns of protein expression in the two woody species in response to AR, allowing a deeper understanding of diversity on AR tolerance in forest tree species.

A transcriptomic study reveals differentially expressed genes and pathways respond to simulated acid rain in Arabidopsis thaliana.

Acid rain, as a worldwide environmental issue, can cause serious damage to plants. In this study, we provided the first case study on the systematic responses of arabidopsis (Arabidopsis thaliana (L.) Heynh.) to simulated acid rain (SiAR) by transcriptome approach. Transcriptomic analysis revealed that the expression of a set of genes related to primary metabolisms, including nitrogen, sulfur, amino acid, photosynthesis, and reactive oxygen species metabolism, were altered under SiAR. In addition, transport and signal transduction related pathways, especially calcium-related signaling pathways, were found to play important roles in the response of arabidopsis to SiAR stress. Further, we compared our data set with previously published data sets on arabidopsis transcriptome subjected to various stresses, including wound, salt, light, heavy metal, karrikin, temperature, osmosis, etc. The results showed that many genes were overlapped in several stresses, suggesting that plant response to SiAR is a complex process, which may require the participation of multiple defense-signaling pathways. The results of this study will help us gain further insights into the response mechanisms of plants to acid rain stress.

Calcium-sensing receptor regulates stomatal closure through hydrogen peroxide and nitric oxide in response to extracellular calcium in Arabidopsis.

The Arabidopsis calcium-sensing receptor CAS is a crucial regulator of extracellular calcium-induced stomatal closure. Free cytosolic Ca(2+) (Ca(2+)(i)) increases in response to a high extracellular calcium (Ca(2+)(o)) level through a CAS signalling pathway and finally leads to stomatal closure. Multidisciplinary approaches including histochemical, pharmacological, fluorescent, electrochemical, and molecular biological methods were used to discuss the relationship of hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) signalling in the CAS signalling pathway in guard cells in response to Ca(2+)(o). Here it is shown that Ca(2+)(o) could induce H(2)O(2) and NO production from guard cells but only H(2)O(2) from chloroplasts, leading to stomatal closure. In addition, the CASas mutant, the atrbohD/F double mutant, and the Atnoa1 mutant were all insensitive to Ca(2+)(o)-stimulated stomatal closure, as well as H(2)O(2) and NO elevation in the case of CASas. Furthermore, it was found that the antioxidant system might function as a mediator in Ca(2+)(o) and H(2)O(2) signalling in guard cells. The results suggest a hypothetical model whereby Ca(2+)(o) induces H(2)O(2) and NO accumulation in guard cells through the CAS signalling pathway, which further triggers Ca(2+)(i) transients and finally stomatal closure. The possible cross-talk of Ca(2+)(o) and abscisic acid signalling as well as the antioxidant system are discussed.

Physiological, Proteomic Analysis, and Calcium-Related Gene Expression Reveal Taxus wallichiana var. mairei Adaptability to Acid Rain Stress Under Various Calcium Levels.

As one of the serious environmental problems worldwide, acid rain (AR) has always caused continuous damage to the forestry ecosystem. Studies have shown that AR can leach calcium ions from plants and soil. Calcium (Ca) is also a crucial regulator of the plant stress response, whereas there are few reports on how Ca regulates the response of AR-resistant woody plants to AR stress. In this study, by setting different exogenous Ca levels, we study the physiological and molecular mechanism of Ca in regulating the Taxus wallichiana var. mairei response to AR stress. Our results showed that low Ca level leads to photosynthesis, and antioxidant defense system decreases in T. wallichiana var. mairei leaves; however, these negative effects could be reversed at high Ca level. In addition, proteomic analyses identified 44 differentially expressed proteins in different Ca level treatments of T. wallichiana var. mairei under AR stress. These proteins were classified into seven groups, which include metabolic process, photosynthesis and energy pathway, cell rescue and defense, transcription and translation, protein modification and degradation, signal transduction, etc. Furthermore, the study found that low Ca level leads to an obvious increase of Ca-related gene expression under AR stress in T. wallichiana var. mairei using qRT-PCR analyses and however can be reversed at high Ca level. These findings would enrich and extend the Ca signaling pathways of AR stress in AR-resistant woody plants and are expected to have important theoretical and practical significance in revealing the mechanism of woody plants tolerating AR stress and protecting forestry ecosystem in soil environment under different Ca levels.

Comparative proteomic analysis of proteins in response to simulated acid rain in Arabidopsis.

A proteomic study using 2-D gel electrophoresis and MALDI-TOF MS was performed to characterize the responses of Arabidopsis thaliana plants to simulated acid rain (SiAR) stress, which is a global environmental problem and has become a serious issue in China in recent years. The emphasis of the present study was to investigate the overall protein expression changes when exposed to SiAR. Out of over 1000 protein spots reproducibly resolved, 50 of them changed their abundance by at least 2-fold. Analysis of protein expression patterns revealed that a set of proteins associated with energy production, metabolism, cell rescue, cell defense and protein folding, etc., could play important roles in mediating plant response to SiAR. In addition to this, some proteins involved in stress responses and jasmonic acid pathway are also involved in plant response to SiAR. More interestingly, the expression of several ubiquitination-related proteins changed dramatically after 32-h SiAR treatment, suggesting that they may act as a molecular marker for the injury phenotype caused by SiAR. Based on our results, we proposed a schematic model to explain the mechanisms associated with the systematic response of Arabidopsis plants to SiAR.

Elevated nitrogen metabolism and nitric oxide production are involved in Arabidopsis resistance to acid rain.

Acid rain (AR) can induce great damages to plants and could be classified into different types according to the different SO42-/NO3- ratio. However, the mechanism of plants' responding to different types of AR has not been elucidated clearly. Here, we found that nitric-rich simulated AR (N-SiAR) induced less leaves injury as lower necrosis percentage, better physiological parameters and reduced oxidative damage in the leaves of N-SiAR treated Arabidopsis thaliana compared with sulfate and nitrate mixed (SN-SiAR) or sulfuric-rich (S-SiAR) simulated AR treated ones. Of these three types of SiAR, N-SiAR treated Arabidopsis maintained the highest of nitrogen (N) content, nitrate reductase (NR) and nitrite reductase (NiR) activity as well as N metabolism related genes expression level. Nitric oxide (NO) content showed that N-SiAR treated seedlings had a higher NO level compared to SN-SiAR or S-SiAR treated ones. A series of NO production and elimination related reagents and three NO production-related mutants were used to further confirm the role of NO in regulating acid rain resistance in N-SiAR treated Arabidopsis seedlings. Taken together, we concluded that an elevated N metabolism and enhanced NO production are involved in the tolerance to different types of AR in Arabidopsis.

Proteomic analysis reveals differences in tolerance to acid rain in two broad-leaf tree species, Liquidambar formosana and Schima superba.

Acid rain (AR) is a serious environmental issue inducing harmful impacts on plant growth and development. It has been reported that Liquidambar formosana, considered as an AR-sensitive tree species, was largely injured by AR, compared with Schima superba, an AR-tolerant tree species. To clarify the different responses of these two species to AR, a comparative proteomic analysis was conducted in this study. More than 1000 protein spots were reproducibly detected on two-dimensional electrophoresis gels. Among them, 74 protein spots from L. formosana gels and 34 protein spots from S. superba gels showed significant changes in their abundances under AR stress. In both L. formosana and S. superba, the majority proteins with more than 2 fold changes were involved in photosynthesis and energy production, followed by material metabolism, stress and defense, transcription, post-translational and modification, and signal transduction. In contrast with L. formosana, no hormone response-related protein was found in S. superba. Moreover, the changes of proteins involved in photosynthesis, starch synthesis, and translation were distinctly different between L. formosana and S. superba. Protein expression analysis of three proteins (ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, ascorbate peroxidase and glutathione-S-transferase) by Western blot was well correlated with the results of proteomics. In conclusion, our study provides new insights into AR stress responses in woody plants and clarifies the differences in strategies to cope with AR between L. formosana and S. superba.

Comparative proteomic analysis of differentially expressed proteins induced by hydrogen sulfide in Spinacia oleracea leaves.

Hydrogen sulfide (H2S), as a potential gaseous messenger molecule, has been suggested to play important roles in a wide range of physiological processes in plants. The aim of present study was to investigate which set of proteins is involved in H2S-regulated metabolism or signaling pathways. Spinacia oleracea seedlings were treated with 100 µM NaHS, a donor of H2S. Changes in protein expression profiles were analyzed by 2-D gel electrophoresis coupled with MALDI-TOF MS. Over 1000 protein spots were reproducibly resolved, of which the abundance of 92 spots was changed by at least 2-fold (sixty-five were up-regulated, whereas 27 were down-regulated). These proteins were functionally divided into 9 groups, including energy production and photosynthesis, cell rescue, development and cell defense, substance metabolism, protein synthesis and folding, cellular signal transduction. Further, we found that these proteins were mainly localized in cell wall, plasma membrane, chloroplast, mitochondria, nucleus, peroxisome and cytosol. Our results demonstrate that H2S is involved in various cellular and physiological activities and has a distinct influence on photosynthesis, cell defense and cellular signal transduction in S. oleracea leaves. These findings provide new insights into proteomic responses in plants under physiological levels of H2S.

Photosynthetic and antioxidant responses of Liquidambar formosana and Schima superba seedlings to sulfuric-rich and nitric-rich simulated acid rain.

To study whether differential responses occur in photosynthesis and antioxidant system for seedlings of Liquidambar formosana, an acid rain (AR)-sensitive tree species and Schima superba, an AR-tolerant tree species treated with three types of pH 3.0 simulated AR (SiAR) including sulfuric-rich (S-SiAR), nitric-rich (N-SiAR), sulfate and nitrate mixed (SN-SiAR), we investigated the changes of leaf necrosis, chlorophyll content, soluble protein and proline content, photosynthesis and chlorophyll fluorescence characteristics, reactive oxygen species production, membrane lipid peroxidation, small molecular antioxidant content, antioxidant enzyme activities and related protein expressions. Our results showed that SiAR significantly caused leaf necrosis, inhibited photosynthesis, induced superoxide radical and hydrogen peroxide generation, aggravated membrane lipid peroxidation, changed antioxidant enzyme activities, modified related protein expressions such as Cu/Zn superoxide dismutase (SOD), l-ascorbate peroxidase (APX, EC 1. 11. 1. 11), glutathione S transferase (GST, EC 2. 5. 1. 18) and Rubisco large subunit (RuBISCO LSU), altered non-protein thiols (NPT) and glutathione (GSH) content in leaves of L. formosana and S. superba. Taken together, we concluded that the damages caused by SiAR in L. formosana were more severe and suffered from more negative impacts than in S. superba. S-SiAR induced more serious damages for the plants than did SN-SiAR and N-SiAR.

Nitric oxide mediates root K+/Na+ balance in a mangrove plant, Kandelia obovata, by enhancing the expression of AKT1-type K+ channel and Na+/H+ antiporter under high salinity.

It is well known that nitric oxide (NO) enhances salt tolerance of glycophytes. However, the effect of NO on modulating ionic balance in halophytes is not very clear. This study focuses on the role of NO in mediating K(+)/Na(+) balance in a mangrove species, Kandelia obovata Sheue, Liu and Yong. We first analyzed the effects of sodium nitroprusside (SNP), an NO donor, on ion content and ion flux in the roots of K. obovata under high salinity. The results showed that 100 µM SNP significantly increased K(+) content and Na(+) efflux, but decreased Na(+) content and K(+) efflux. These effects of NO were reversed by specific NO synthesis inhibitor and scavenger, which confirmed the role of NO in retaining K(+) and reducing Na(+) in K. obovata roots. Using western-blot analysis, we found that NO increased the protein expression of plasma membrane (PM) H(+)-ATPase and vacuolar Na(+)/H(+) antiporter, which were crucial proteins for ionic balance. To further clarify the molecular mechanism of NO-modulated K(+)/Na(+) balance, partial cDNA fragments of inward-rectifying K(+) channel, PM Na(+)/H(+) antiporter, PM H(+)-ATPase, vacuolar Na(+)/H(+) antiporter and vacuolar H(+)-ATPase subunit c were isolated. Results of quantitative real-time PCR showed that NO increased the relative expression levels of these genes, while this increase was blocked by NO synthesis inhibitors and scavenger. Above results indicate that NO greatly contribute to K(+)/Na(+) balance in high salinity-treated K. obovata roots, by activating AKT1-type K(+) channel and Na(+)/H(+) antiporter, which are the critical components in K(+)/Na(+) transport system.

Emissions of nitric oxide from 79 plant species in response to simulated nitrogen deposition.

To assess the potential contribution of nitric oxide (NO) emission from the plants grown under the increasing nitrogen (N) deposition to atmospheric NO budget, the effects of simulated N deposition on NO emission and various leaf traits (e.g., specific leaf area, leaf N concentration, net photosynthetic rate, etc.) were investigated in 79 plant species classified by 13 plant functional groups. Simulated N deposition induced the significant increase of NO emission from most functional groups, especially from conifer, gymnosperm and C(3) herb. Moreover, the change rate of NO emission was significantly correlated with the change rate of various leaf traits. We conclude that the plants grown under atmospheric N deposition, especially in conifer, gymnosperm and C(3) herb, should be taken into account as an important biological source of NO and potentially contribute to atmospheric NO budget.

The changes of nitric oxide production during the growth of Microcystis aerugrinosa.

This study characterized the changes of nitric oxide (NO) production during the growth of Microcystis aerugrinosa, a cyanobacterium which usually cause cyanobacterial blooms. Results showed a drastic NO release accompanying with cell density and Chl-a content sharp rises when M. aerugrinosa grew from fifth day to sixth day. Moreover, high N:P ratio accelerated the cyanobacterial growth and NO burst. Sodium nitroprusside, an exogenous NO donor, promoted M. aerugrinosa growth with the optimal concentration of 0.1 mg/L. Experiments by supplementing with sodium nitrite and L-arginine demonstrated NO production in M. aerugrinosa cells was mainly through nitrate reductase (NR) pathway while minorly through NO synthase pathway. All these data suggested M. aerugrinosa produced increasing NO during its growth mainly by NR pathway, during which NO positively regulated the growth of M. aerugrinosa.

Effects of calcium on seed germination, seedling growth and photosynthesis of six forest tree species under simulated acid rain.

We selected six tree species, Pinus massoniana Lamb., Cryptomeria fortunei Hooibr. ex Otto et Dietr., Cunninghamia lanceolata (Lamb.) Hook., Liquidambar formosana Hance, Pinus armandii Franch. and Castanopsis chinensis Hance, which are widely distributed as dominant species in the forest of southern China where acid deposition is becoming more and more serious in recent years. We investigated the effects and potential interactions between simulated acid rain (SiAR) and three calcium (Ca) levels on seed germination, radicle length, seedling growth, chlorophyll content, photosynthesis and Ca content in leaves of these six species. We found that the six species showed different responses to SiAR and different Ca levels. Pinus armandii and C. chinensis were very tolerant to SiAR, whereas the others were more sensitive. The results of significant SiAR × Ca interactions on different physiological parameters of the six species demonstrate that additional Ca had a dramatic rescue effect on the seed germination and seedling growth for the sensitive species under SiAR. Altogether, we conclude that the negative effects of SiAR on seed germination, seedling growth and photosynthesis of the four sensitive species could be ameliorated by Ca addition. In contrast, the physiological processes of the two tolerant species were much less affected by both SiAR and Ca treatments. This conclusion implies that the degree of forest decline caused by long-term acid deposition may be attributed not only to the sensitivity of tree species to acid deposition, but also to the Ca level in the soil.

Soil acidity reconstruction based on tree ring information of a dominant species Abies fabri in the subalpine forest ecosystems in southwest China.

To assess the suitability of dendrochemistry as an indicator of soil acidification, soil chemistry and tree ring information of Abies fabri were measured at two distinct sites (severe acid deposition site-Emei Mountain and clean site-Gongga Mountain) of the subalpine forest ecosystems of western Sichuan, southwest China. The actual soil acidity (pH) was significantly correlated with some of the recent xylem cation (Ca, Mg, Mn, Al, Sr and Ba) concentrations and their molar ratios. Xylem Ca/Mg and Ca/Mn of A. fabri were ultimately selected to reconstruct the historical changes of soil pH in Emei Mountain and Gongga Mountain, respectively. The validity of those rebuild was also verified to a certain extent. We conclude that xylem cation molar ratios of A. fabri were superior to the single cation concentrations in soil acidity rebuild at the study sites due to normalizing for concentration fluctuations.