A tobacco ringspot virus-based vector system for gene and microRNA function studies in cucurbits.

Cucurbits are economically important crops worldwide. The genomic data of many cucurbits are now available. However, functional analyses of cucurbit genes and noncoding RNAs have been impeded because genetic transformation is difficult for many cucurbitaceous plants. Here, we developed a set of tobacco ringspot virus (TRSV)-based vectors for gene and microRNA (miRNA) function studies in cucurbits. A TRSV-based expression vector could simultaneously express GREEN FLUORESCENT PROTEIN (GFP) and heterologous viral suppressors of RNA silencing in TRSV-infected plants, while a TRSV-based gene silencing vector could knock down endogenous genes exemplified by PHYTOENE DESATURASE (PDS) in Cucumis melo, Citrullus lanatus, Cucumis sativus, and Nicotiana benthamiana plants. We also developed a TRSV-based miRNA silencing vector to dissect the functions of endogenous miRNAs. Four representative miRNAs, namely, miR159, miR166, miR172, and miR319, from different cucurbits were inserted into the TRSV vector using a short tandem target mimic strategy and induced characteristic phenotypes in TRSV-miRNA-infected plants. This TRSV-based vector system will facilitate functional genomic studies in cucurbits.

A natural substitution of a conserved amino acid in eIF4E confers resistance against multiple potyviruses.

Eukaryotic translation initiation factor 4E (eIF4E), which plays a pivotal role in initiating translation in eukaryotic organisms, is often hijacked by the viral genome-linked protein to facilitate the infection of potyviruses. In this study, we found that the naturally occurring amino acid substitution D71G in eIF4E is widely present in potyvirus-resistant watermelon accessions and disrupts the interaction between watermelon eIF4E and viral genome-linked protein of papaya ringspot virus-watermelon strain, zucchini yellow mosaic virus or watermelon mosaic virus. Multiple sequence alignment and protein modelling showed that the amino acid residue D71 located in the cap-binding pocket of eIF4E is strictly conserved in many plant species. The mutation D71G in watermelon eIF4E conferred resistance against papaya ringspot virus-watermelon strain and zucchini yellow mosaic virus, and the equivalent mutation D55G in tobacco eIF4E conferred resistance to potato virus Y. Therefore, our finding provides a potential precise target for breeding plants resistant to multiple potyviruses.

[Monitoring Atmospheric CO2 and delta(13)C (CO2) Background Levels at Shangdianzi Station in Beijing, China].

The study presented time series of atmospheric CO2 concentrations from flask sampling at SDZ regional station in Beijing during 2007 and 2013, together with delta(13)CO2) values during 2009 and 2013. The "representative data" of CO2 and delta(13)C (CO2) were selected from the complete data for further analysis. Annual CO2 concentrations increased from 385.6 x 10(-6) in 2007 to 398.1 x 10(-6) in 2013, with an average growth rate of 2.0 x 10(-6) a(-1), while the delta(13)C values decreased from -8.38% per hundred in 2009 to -8.52% per hundred in 2013, with a mean growth rate of -0.03% per hundred x a(-1). The absolute increase of CO2 from 2007 to 2008 reached the lowest level during 2007 and 2013, possibly due to relatively less carbon emissions during the 2008 Olympic Games period. The peak-to-peak amplitudes of atmospheric CO2 and delta(13)C seasonal variations were 23. 9 x 10 -6 and 1. 03%o, respectively. The isotopic signatures of CO2 sources/sinks were also discussed in this study. The delta8 value for heating season I (Jan. 01-Mar. 14) was -21.30% per hundred, while -25.39% per hundred for heating season 11 (Nov. 15-Dec.31) , and for vegetative season (Mar. 15-Nov. 14) the delta(bio) value was estimated to be -21.28% per hundred, likely suggesting the significant impact of fossil fuel and corn straw combustions during winter heating season and biological activities during vegetative season.

[Evaluation on the Impacts of Different Background Determination Methods on CO2 Sources and Sinks Estimation and Seasonal Variations].

To accurately determine background conditions or extract sources and sinks information from the observed atmospheric carbon dioxide (CO2) concentration is crucial for quantitative estimation of regional and global carbon budget and future trends of atmospheric CO2. In this study, the synchronized observed surface winds and carbon monoxide (CO) concentration have been examined to test their effectiveness as filter factors to determine CO2 background conditions at Waliguan site. The results show that the surface winds and CO concentrations can be used as filter factor in winter, but they are not very effective in summer. Three statistical methods, robust estimation of background signal (REBS), Fourier transform algorithm (FTA) and a new developed moving average filtering (MAF), are applied to atmospheric CO2 background selection. The result suggested that our new developed MAF method, which can well estimate the elevated and sequestered CO2 concentrations due to using changing and adjusting filter criteria at every two-week fitting window, is thus better than the other two statistical methods. A good consistency is indicated by the three methods for estimating the elevated CO2 caused by local or regional emissions, but it showed large discrepancies when determining the sequestered CO2. The result suggested that the three methods can reasonable extract those anthropogenic influenced episodes, but only MAF method would well identify those episodes due to terrestrial CO2 fluxes. Mean seasonal amplitude of atmospheric CO2 at Waliguan during 1995-2008 is 10. 3 x 10(-6) estimated by MAF method, which is in good agreement with previous studies. Whereas, the seasonal amplitudes derived by REBS method are much lower, only with a value of ~9. 1 x 10(-6) during 1995-2008, which will result in an underestimation of regional or global CO2 fluxes.

[Gas chromatography with a Pulsed discharge helium ionization detector for measurement of molecular hydrogen(H2) in the atmosphere].

A high precision GC system with a pulsed discharge helium ionization detector was set up based on the commercial Agilent 7890A gas chromatography. The gas is identified by retention time and the concentration is calculated through the peak height. Detection limit of the system is about 1 x 10(-9) (mole fraction, the same as below). The standard deviation of 140 continuous injections with a standard cylinder( concentration is roughly 600 x 10(-9)) is better than 0.3 x 10(-9). Between 409.30 x 10(-9) and 867.74 x 10(-9) molecular hydrogen mole fractions and peak height have good linear response. By using two standards to quantify the air sample, the precision meets the background molecular hydrogen compatibility goal within the World Meteorological Organization/Global Atmosphere Watch (WMO/GAW) program. Atmospheric molecular hydrogen concentration at Guangzhou urban area was preliminarily measured by this method from January to November 2013. The results show that the atmospheric molecular hydrogen mole fraction varies from 450 x 10(-9) to 700 x 10(-9) during the observation period, with the lowest value at 14:00 (Beijing time, the same as below) and the peak value at 20:00. The seasonal variation of atmospheric hydrogen at Guangzhou area was similar with that of the same latitude stations in northern hemisphere.

[Atmospheric CO2 data filtering method and characteristics of the molar fractions at the Longfengshan WMO/GAW regional station in China].

Based on the in-situ observation results of atmospheric CO2 molar fractions at two levels (10 m and 80 m above the ground) at Longfengshan (LFS) regional background station in Heilongjiang Province during January 2009 to December 2011, this study mainly focused on the results from 10 m above the ground level (a. g. l.). The results indicated that the observed data from 10 m were strongly affected by the local sources/sinks. The differences between the 10 m and 80 m results were relatively small during the daytime (08:00-17:00) with values smaller than (0.5 +/- 0.5) x 10(-6). In spring, summer and winter, higher CO2 molar fractions were observed when surface winds came from the E-ESE-SE-SSE sectors, while, in winter, surface winds from the N-NNW-NW-WNW sectors obviously enhanced the observed values. Generally, lower CO2 values were accompanied with higher wind speed in the four seasons. This phenomenon was most obvious in winter. Based on the analysis of the observed diurnal cycles and the local meteorological conditions, the observed data from 10 m were filtered into background/non-background events. About 30.7% valid hourly data were filtered as regional background representative. The background CO2 variation displayed a peak in winter and a valley in summer with a seasonal peak to peak amplitude of (36.3 +/- 1.4) x 10(-6), which was higher than the values at similar latitude from Marine Boundary Layer (MBL) References and WMO/GAW stations. The yearly CO2 increasing rate at LFS was roughly estimated to be 2.4 x 10(-6) a(-1).

[In-situ measurement of atmospheric methyl chloroform at the Shangdianzi GAW regional background station].

An in-situ GC-ECD monitoring system was established at the Shangdianzi GAW regional background station (SDZ) for a 2-year atmospheric methyl chloroform (CH3CCl3) measurement experiment. Robust extraction of baseline signal filter was applied to the CH3CCl3 time series to separate the background and pollution data. The yearly averaged background mixing ratios of atmospheric CH3CCl3 were (9.03 +/- 0.53) x 10(-12) mol x mol(-1) in 2009 and (7.73 +/- 0.47) x 10(-12) in 2010, and the percentages of the background data in the whole data were 61.1% in 2009 and 60.4% in 2010, respectively. The yearly background CH3CCl3 mixing ratios at SDZ were consistent with the northern hemisphere background levels observed at Mace Head and Trinidad Head stations, but lower than the results observed at sites in southern China and some Chinese cities from 2001 to 2005. During the study period, background mixing ratios trends exhibited a decreasing rate of 1.39 x 10 12(-12) a(-1). The wind direction with the maximum CH3CCl3 mixing ratio was from the southwest sector and that with the minimum ratio was from the northeast sector. The differences between the maximum and the minimum average mixing ratios in the 16 wind directions were 0.77 x 10(-12) (2009) and 0.52 x 10(-12) (2010). In the 16 different wind directions, the averaged mixing ratio of CH3CCl3 in 2010 was lower than that in 2009 by 1.03 x 10(-12) -1.68 x 10(-12).

[Distribution of CO at Lin'an station in Zhejiang Province].

Background CO mole fractions were continuously measured at Lian'an background station in Zhejiang province from September, 2010 to February, 2012 using Cavity Ring Down Spectroscopy (CRDS) system. The diurnal variation of CO was strongly influenced by anthropogenic activities with two peaks occurring at 07:00-10:00 and 19:00-20:00 (local time). The average daily mole fraction and amplitude in summer were the lowest among four seasons with values of 314.3 x 10(-9) +/- 7.6 x 10(-9) (mole fraction, the same below) and 50.1 x 10(-9) +/- 47.9 x 10(-9), respectively. The seasonal variations displayed peak values during winter-spring period and valley in summer, which roughly consisted with those observed variations at other sites located at northern hemisphere such as Jungfraujoch in Switzerland and Waliguan in China. However, the average mole fractions were much higher than those from other stations. The amplitude of monthly CO mole fractions was 286.8 x 10(-9) +/- 19.2 x 10(-9). The cluster analysis of backward trajectories and surface wind influence might suggest that the non-background CO mole fractions at Lin'an station were mainly affected by the emissions from the megacities and industrial area on the N-NNE-ENE sectors. The maximum enhancements in spring, summer and winter all occurred on ENE sector, with a maximum value of 106.3 x 10(-9) +/- 58.0 x 10(-9) in winter.

[Establishment and assessment of QA/QC method for sampling and analysis of atmosphere background CO2].

To strengthen scientific management and sharing of greenhouse gas data obtained from atmospheric background stations in China, it is important to ensure the standardization of quality assurance and quality control method for background CO2 sampling and analysis. Based on the greenhouse gas sampling and observation experience of CMA, using portable sampling observation and WS-CRDS analysis technique as an example, the quality assurance measures for atmospheric CO,sampling and observation in the Waliguan station (Qinghai), the glass bottle quality assurance measures and the systematic quality control method during sample analysis, the correction method during data processing, as well as the data grading quality markers and data fitting interpolation method were systematically introduced. Finally, using this research method, the CO2 sampling and observation data at the atmospheric background stations in 3 typical regions were processed and the concentration variation characteristics were analyzed, indicating that this research method could well catch the influences of the regional and local environmental factors on the observation results, and reflect the characteristics of natural and human activities in an objective and accurate way.

[In-situ measurement of background atmospheric HCFC-142b using GC-MS and GC-ECD method].

Custom-made GC-MS and GC-ECD in-situ measurement systems were established at the Shangdianzi GAW Regional station. From May 2010 to May 2011, the precisions for GC-MS and GC-ECD systems were 0.23% and 0.88%, respectively, and the HCFC-142b mole fraction during the observation period ranged from 21 x 10(-12) to 355 x 10(-12). The result of the independent-sample T test was P > 0.05, and there was no significant difference in HCFC-142b mole fraction measured by the two systems. The small difference of HCFC-142b mole fraction measured by GC-MS and GC-ECD might be associated with the different sampling time and precision of the two systems. A statistical filter of "robust local regression" was applied to separate HCFC-142b background and pollution data. The mean difference, median difference, 25 and 75 percent difference of background data measured by GC-MS and GC-ECD were all within the precisions. The pollution events captured by the two systems showed similar characters. Results from both systems showed a higher HCFC-142b level in summer and autumn than in winter. The pollution mole fraction of the two systems showed similar seasonal changes.

[Study on the in-situ measurement of greenhouse gas by an improved FTIR].

The real-time, automatic, highly accurate and efficient system for measuring the mixing ratios of CO2, CH4, CO and N2O has been developed by combining the commercial FTIR system (Wollongong University) with an auto-sampling system and a working standard module. Based on the tests conducted, the FTIR showed the high precision and a relatively low accuracy associated with its poor determination of correction factors. The absolute error of the mixing ratio of CO was above 38.8 x 10(-9), suggesting that FTIR alone could not meet the requirement for the real time measurement. Using the working standard gases to adjust results from the FTIR significantly improved the accuracy of measurements. For both static and dynamic conditions, the discrepancies between the measured results and the real values were below 0.11 x 10(-6), 1.8 x 10(-9), 0.15 x 10(-9) and 0.5 x 10(-9) for CO2, CH4, N2O and CO respectively, meeting the requirements for the atmospheric real-time measurements. During 6 days in-situ measurements of greenhouse gas outside the lab, the precision of target gas can reach 0.05 x 10(-6), 0.2 x 10(-9), 0.07 x 10(-9), 0.5 x 10(-9) for CO2, CH4, N2O, CO, and inaccuracy can be 0.09 x 10(-6), 0.4 x 10(-9), 0.14 x 10(-9), 0.5 x 10(-9), respectively.

[Impacts of meteorological factors on atmospheric methane mole fractions in the background area of Yangtze River delta].

Impacts of surface wind direction, surface wind speed, surface air temperature and sunshine hours on the CH4 concentration at Lin'an regional atmospheric background station were studied based on the results from Jan. 2009 to Dec. 2011. The results revealed that the diurnal variation of atmospheric CH4 concentration presented a single-peak curve at Lin'an regional background station. The diurnal amplitude varied from 19.0 x 10(-9) to 74.7 x 10(-9), with the lowest value observed in the afternoon and the highest at dawn. The monthly mean CH4 concentrations varied from 1955.7 x 10(-9) to 2036.2 x 10(-9), with the highest concentration observed in autumn and the lowest in spring. The wind directions NE-SSE could induce higher CH4 concentrations while SW-NNW wind directions had negative effects on the observed results. The CH4 concentration turned out to be lower with higher surface wind speed. With the increase of surface air temperature or sunshine hours, the CH4 concentration went up first till reaching a peak, and then decreased.

[CH4 concentrations and the variation characteristics at the four WMO/GAW background stations in China].

Background CH4 concentrations were continuously measured at the 4 WMO/GAW stations [Waliguan in Qinghai (WLG), Lin'an in Zhejiang (LAN), Shangdianzi in Beijing (SDZ), and Longfengshan in Heilongjiang (LFS)] by Cavity Ring Down Spectroscopy system. From 2009 to 2010, the diurnal cycle of hourly average CH4 concentration at LAN was found to be similar in all four seasons, with the highest level detected at 05:00 (Beijing Time) and the lowest at about 14:00. Similar CH4 diurnal cycles were observed at LFS in the summer time. However, the daily amplitude was much higher than that at LAN and reached 216. 8 x 10(-9) (molar ratio). For SDZ station, there were similar trends in spring, autumn and winter. The daily average concentration in the summer was much higher than those of the other seasons and reached the highest at about 20:00. No apparent CH4 diurnal cycle was observed at the WLG station during the whole year. The seasonal variations were obvious at the three regional stations (LAN, SDZ, LFS). The background concentration was the lowest in July at LAN while reached the highest level in August at LFS. The yearly background concentration variation at LFS displayed a "W" pattern. At LFS and SDZ, the wintertime CH4 concentrations were higher than those in spring and autumn. WLG represented a clean area and its CH4 value was the lowest among the four stations with the monthly average amplitude to be about 11.5 x 10(-9). At all three regional stations, non-background data accounted for more than 70% of the whole data. Cluster analysis of 3 day backward trajectories corresponding to the high CH4 concentration (WLG: CH4 > 1 870 x 10(-9), LFS: CH4 > 2100 x 10(-9), LAN: CH4 > 2 150 x10(-9), SDZ: CH4 > 2050 x 10(-9)) data points suggested that the high CH4 level measured in summer might be associated with the air mass transportation.

[Study on the in-situ measurement of atmospheric CH4 and CO by GC-FID method at the Shangdianzi GAW regional station].

In-situ GC-FID system for atmospheric CH4 and CO mixing ratio measurements at the Shangdianzi (SDZ) GAW regional station in Beijing was designed and optimized in 2009 based on a comparable system at the Waliguan GAW global station in Qinhai. Results from this study indicate that the system's precisions for CH4 and CO are higher than 0.03% and 0.45% respectively, which can meet the quality target on background greenhouse gas observations by the World Meteorology Organization's Global Atmosphere Watch (WMO/GAW) program. The selection method of working standards for this system was established: two working standards (WH for the high concentration and WL for the low concentration) were selected, the concentrations of CH4 and CO in these two standards can cover the ambient mixing ratios of CH4 (2 007.1 x 10(-9) and 1 809.5 x 10(-9)) and CO (405.6 x 10(-9) and 123.8 x 10(-9)), an injection sequence was programmed so that the two standards were analyzed alternatively for every three runs. The measurement accuracies are high, as shown by the standard deviations less than 1.7 x 10(-9) and 1 x 10(-9), for CH4 and CO, respectively. This method has been applied to in-situ measurement of atmospheric CH4 and CO in North China.

[In situ measurement of atmospheric HCFC-22 at the Shangdianzi GAW regional station].

An in situ GC-ECD monitoring system was established at the Shangdianzi GAW Regional station from April 2007 to March 2008, and the mixing ratio of atmospheric HCFC-22 was (278.1 +/- 113.6) x 10(-12) (mol/mol). A "R" statistical software was applied to the HCFC-22 time series to separate background and pollution data. The background HCFC-22 mixing ratio was (199.5 +/- 5.1) x 10(-12), close to northern hemisphere background level observed at Mace Head and Trinidad Head stations. The pollution HCFC-22 mixing ratio was (312.1 +/- 121.0) x 10(-12) due to anthropogenic emission from various regions and air mass transport. There was no significant seasonal variability of background data. However, pollution data was much higher in summer than that in winter, and elevated up to 100.9 x 10(-12) in July than in January attribute to seasonal emission characteristics. The mean HCFC-22 mixing ratio from southwest wind sector (327.3 x 10(-12)) was much higher than that of northeast sector (236.2 x 10(-12)). The W-WSW-SW wind sector has a positive contribution to the HCFC-22 level, while NNE-N-NE has a large negative contribution.

[Data processing and QA/QC of atmosphere CO2 and CH4 concentrations by a method of GC-FID in-situ measurement at Waliguan station].

To strengthen scientific management and sharing of greenhouse gas data obtained from atmospheric background stations in China, it is important to ensure the standardization of observations and establish the data treatment and quality control procedure so as to maintain consistency in atmospheric carbon dioxide (CO2) and methane (CH4) measurements from different background stations. An automated gas chromatographic system (Hewlett Packard 5890GC employing flame ionization detection) for in situ measurements of atmospheric CO2 and CH4 has been developed since 1994 at the China Global Atmosphere Watch Baseline Observatory at Mt. Waliguan, in Qinhai. In this study, processing and quality control flow of CO2 and CH4 data acquired by HP ChemStation are discussed in detail, including raw data acquisition, data merge, time series inspection, operator flag, principal investigator flag, and the comparison of the GC measurement with the flask method. Atmosphere CO2 and CH4 mixing ratios were separated as background and non-background data using a robust local regression method, approximately 72% and 44% observed values had been filtered as background data for CO2 and CH4, respectively. Comparison of the CO1 and CH, in situ data to the flask sampling data were in good agreement, the relative deviations are within +/- 0.5% for CO2 and for CH4. The data has been assimilated into global database (Globalview-CO2, Globalview-CH4), submitted to the World Data Centre for Greenhouse Gases (WDCGG), and applied to World Meteorological Organization (WMO) Greenhouse Gas Bulletin and assessment reports of the United Nations Intergovernmental Panel on Climate Change (IPCC).

[Study of distinguish root respiration from total soil respiration by root exclusion method in the temperate semi-arid grassland in Inner Mongolia, China].

In order to determine the possibility of measuring the root respiration by using root exclusion method, we conducted field experiments to measured total soil respiration and net soil respiration in three temperate grassland communities including Leymus chinensis free-grazed steppe, stipa grandis free-grazed steppe and Leymus chinensis degenerated free-grazed steppe in the growing season, 2005. At the same time, the proportion of the root respiration to the total soil respiration was estimated in different steppes by this method separately. The results indicated that the root exclusion method was very operable in estimating the proportion of the root respiration to the total soil respiration in temperate grassland communities, and the proportion varied from 25% - 45% with the mean value of 35.66%. The precision of this proportion was markedly improved compared with the correlation studies of both here and abroad, and it was provided with superior applied cost in the field of carbon cycle of grassland ecosystem.