Dr. Joe E Lepo

Sanguinarine has strong inhibitory effects against the cyanobacterium Microcystis aeruginosa. However, previous studies were mainly limited to laboratory tests. The efficacy of sanguinarine for mitigation of cyanobacterial blooms under field conditions, and its effects on aquatic microbial community structure remain unknown. To elucidate these issues, we carried out in situ cyanobacterial bloom mitigation tests. Our results showed that sanguinarine decreased population densities of the harmful cyanobacteria Microcystis and Anabaena. The inhibitory effects of sanguinarine on these cyanobacteria lasted 17 days, after which the harmful cyanobacteria recovered and again became the dominant species. Concentrations of microcystins in the sanguinarine treatments were lower than those of the untreated control except during the early stage of the field test. The results of community DNA pyrosequencing showed that sanguinarine decreased the relative abundance of the prokaryotic microorganisms Cyanobacteria, Actinobacteria, Planctomycetes and eukaryotic microorganisms of Cryptophyta, but increased the abundance of the prokaryotic phylum Proteobacteria and eukaryotic microorganisms within Ciliophora and Choanozoa. The shifting of prokaryotic microbial community in water column was directly related to the toxicity of sanguinarine, whereas eukaryotic microbial community structure was influenced by factors other than direct toxicity. Harmful cyanobacteria mitigation efficacy and microbial ecological effects of sanguinarine presented in this study will inform the broad application of sanguinarine in cyanobacteria mitigation.
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•Sanguinarine effectively decreases the cell density of harmful cyanobacteria.•Sanguinarine treatment decreases microcystins concentration in water column.•Sanguinarine treatment improves water quality.•Prokaryotic community shifting is different from that of eukaryotic microbes.

•Reduced V-ATPase and V-PPase activity improved nitrogen use efficiency.
•Reduced V-ATPase and V-PPase activity decreased Cd2+ tolerance.•Decreased NO3− vacuolar sequestration capacity (VSC) enhanced Atclca-2 Cd2+ VSC.
•Enhanced Cd2+ VSC decreased NO3− VSC in AtCAX4-OE.•Regulating Cd2+ and NO3− vacuolar accumulation enhances NUE and Cd2+ tolerance.

The transmembrane transport of NO3− and Cd2+ into plant cell vacuoles relies on the energy from their tonoplast proton pumps, V-ATPase and V-PPase. If the activity of these pumps is reduced, it results in less NO3− and Cd2+ being transported into the vacuoles, which contributes to better nitrogen use efficiency (NUE) and lower Cd2+ tolerance in plants. The physiological mechanisms that regulate the balance between NUE and Cd2+ tolerance remain unknown. In our study, two Brassica napus genotypes with differential NUEs, xiangyou 15 and 814, and Atclca-2 mutant and AtCAX4 over-expression line (AtCAX4-OE) of Arabidopsis thaliana, were used to investigate Cd2+ stress responses. We found that the Brassica napus genotype, with higher NUE, was more sensitive to Cd2+ stress. The AtCAX4-OE mutant, with higher Cd2+ vacuolar sequestration capacity (VSC), limited NO3− sequestration into root vacuoles and promoted NUE. Atclca-2 mutants, with decreased NO3− VSC, enhanced Cd2+ sequestration into root vacuoles and conferred greater Cd2+ tolerance than the WT. This may be due to the competition between Cd2+ andNO3− in the vacuoles for the energy provided by V-ATPase and V-PPase. Regulating the balance between Cd2+ and NO3− vacuolar accumulation by inhibiting the activity of CLCa transporter and increasing the activity of CAX4 transporter will simultaneously enhance both the NUE and Cd2+ tolerance of Brassica napus, essential for improving its Cd2+ phytoremediation potential.

A high concentration of ammonium (NH4+) as the sole source of nitrogen in the growth medium often is toxic to plants. The nitrate transporter NRT1.1 is involved in mediating the effects of NH4+ toxicity; however, the mechanism remains undefined. In this study, wild-type Arabidopsis (Arabidopsis thaliana Columbia-0 [Col-0]) and NRT1.1 mutants (chl1-1 and chl1-5) were grown hydroponically in NH4NO3 and (NH4)(2)SO4 media to assess the function of NRT1.1 in NH4+ stress responses. All the plants grew normally in medium containing mixed nitrogen sources, but Col-0 displayed more chlorosis and lower biomass and photosynthesis than the NRT1.1 mutants in (NH4)(2)SO4 medium. Grafting experiments between Col-0 and chli-5 further confirmed that NH4+ toxicity is influenced by NRT1.1. In (NHASO, medium, NRT1.1 induced the expression of NH4+ transporters, increasing NH4+ uptake. Additionally, the activities of glutamine synthetase and glutamate synthetase in roots of Col-0 plants decreased and soluble sugar accumulated significantly, whereas pyruvate kinase-mediated glycolysis was not affected, all of which contributed to NH4+ accumulation. By contrast, the NRT1.1 mutants showed reduced NH4+ accumulation and enhanced NH4+ assimilation through glutamine synthetase, glutamate synthetase, and glutamate dehydrogenase. Moreover, the up-regulation of genes involved in ethylene synthesis and senescence in Col-0 plants treated with (NH4)(2)SO4 suggests that ethylene is involved in NH4+ toxicity responses. This study showed that NH4+ toxicity is related to a nitrate-independent signaling function of NRT1.1 in Arabidopsis, characterized by enhanced NH4+ accumulation and altered NH4+ metabolism, which stimulates ethylene synthesis, leading to plant senescence.

Soil in short-term crop rotation systems (STCR) is still in the initial development stage of farmland soil, whereas after long-term crop rotation treatment (LTCR), soil properties are significantly different. This study compares STCR (4 years) and LTCR (30 years) rice-rice-fallow, rice-rice-rape rotation practices under the same soil type background and management system. To reveal ecosystem mechanisms within soils and their effects on rice yield following LTCR, we analyzed the physical, chemical, and microbiological properties of soils with different rotations and rotation times. Relative to STCR, LTCR significantly reduced soil water-stable aggregate (WSA) content in the < 0.053-mm range, while > 2 mm WSA content significantly increased. Soil organic matter increased in fields under LTCR, mainly in > 2 mm, 2–0.25 mm, and < 0.053 mm soil WSA in 0–10 cm soil layer. LTCR was associated with significantly increased total soil organic matter, at the same time being associated with increasing the amount of active organic matter in the 0–20 cm soil layer. The two crop rotation regimes significantly differed in soil aggregate composition as well as in soil N and P, microbial biomass, and community composition. Relative to STCR, LTCR field soils had significantly higher soil organic matter, active organic matter content, soil enzyme activities, and overall microbial biomass, while soil WSA and microbial community composition was significantly different. Our results demonstrate that LTCR could significantly improve soil quality and rice yield and suggest that length of rotation time and rice-rice-rape rotation are critical factors for the development of green agriculture.

The effect of exogenous abscisic acid (ABA) on the nitrogen use efficiency (NUE) of Brassica napus was studied by pot experiments using ABA (1 mg L−1) daubed on the leaves at the early siliquing stage. The results showed that activities of proteases and glutamine synthetase (GS) were significantly promoted by the ABA treatment, which increased the capacity of degradation of foliar proteins and biosynthesis of glutamine (N-transport compounds), both of which promote decreased N in leaves of the ABA treatment. L-glutamine, other free amino acids, and soluble sugar content in the phloem sap of the ABA treatment were significantly higher than those of the control. A 15N label trial showed that more 15N was distributed from leaves to the grain by the ABA treatment, which resulted in a significantly higher NUE in the ABA treatment relative to the control.

We characterized the responses of plant dry biomass, nitrogen (N) distribution and N-utilization efficiency (NUtE) to changes in CO2 concentration through exposure and culture of winter rape under normal-(380 mu mol center dot mol(-1)) and elevated-CO2 (760 mu mol center dot mol(-1)) conditions. Brassica napus (Xiangyou 15) was used as an agriculturally important model plant. Plants were cultivated in a greenhouse with sand culture under normal- (15 mmol center dot L-1) and limited-N (5 mmol center dot L-1) conditions. NUtE increased with elevated CO2 regardless of whether N was limited. NUtE was higher under N limitation than under normal N conditions for both normal- and elevated-CO2 conditions. N-15 labeling was used to assess the distribution of N from vegetative- to reproductive-organs.Ndistribution within the plant and during different developmental stages was affected by CO2 concentration and the level of N application. A higher proportion of N was found in siliques at the harvest stage for N-limited plants compared to normal-N plants. The proportion of N absorbed into siliques after the stem elongation stage under elevated-CO2 conditions was significantly higher than under normal CO2. The proportion of N transport, as well as the total amount of N, absorbed at the stem elongation stage from vegetative organs into siliques under elevated CO2 was significantly lower than under normal-CO2 conditions. However, the proportion of N absorbed at the stem elongation stage and thus lost from the silique under elevated CO2 was significantly higher than under normal CO2. In conclusion, limited N or elevated CO2 generally benefitted plant NUtE. In addition, after the stem elongation stage, elevated CO2 promoted the redistribution of N from plant vegetative tissues to reproductive organs; however, elevated CO2 during or before stem elongation had the opposite effect.

Enhancing nitrogen use efficiency (NUE) in crop plants is an important breeding target to reduce excessive use of chemical fertilizers, with substantial benefits to farmers and the environment. In Arabidopsis (Arabidopsis thaliana), allocation of more NO3 (-) to shoots was associated with higher NUE; however, the commonality of this process across plant species have not been sufficiently studied. Two Brassica napus genotypes were identified with high and low NUE. We found that activities of V-ATPase and V-PPase, the two tonoplast proton-pumps, were significantly lower in roots of the high-NUE genotype (Xiangyou15) than in the low-NUE genotype (814); and consequently, less vacuolar NO3 (-) was retained in roots of Xiangyou15. Moreover, NO3 (-) concentration in xylem sap, [(15)N] shoot:root (S:R) and [NO3 (-)] S:R ratios were significantly higher in Xiangyou15. BnNRT1.5 expression was higher in roots of Xiangyou15 compared with 814, while BnNRT1.8 expression was lower. In both B. napus treated with proton pump inhibitors or Arabidopsis mutants impaired in proton pump activity, vacuolar sequestration capacity (VSC) of NO3 (-) in roots substantially decreased. Expression of NRT1.5 was up-regulated, but NRT1.8 was down-regulated, driving greater NO3 (-) long-distance transport from roots to shoots. NUE in Arabidopsis mutants impaired in proton pumps was also significantly higher than in the wild type col-0. Taken together, these data suggest that decrease in VSC of NO3 (-) in roots will enhance transport to shoot and essentially contribute to higher NUE by promoting NO3 (-) allocation to aerial parts, likely through coordinated regulation of NRT1.5 and NRT1.8.

Nitrate, once taken up by plants, can either be stored in vacuoles or reduced by nitrate reductase in the cytoplasm. High accumulation of NO3 − in the vacuole occurs when assimilation into the cytoplasm is saturated. This study elucidates how proton pumps at the tonoplast (V-ATPase and V-PPase) affect the NO3 − content of Brassica napus by controlling the distribution of NO3 − between the cytoplasm and vacuole. Pot experiments were conducted in a greenhouse under normal N (15.0 mM nitrate) conditions using B. napus genotypes that demonstrated either high (Xiangyou15) or low (814) nitrogen use efficiency (NUE). The NO3 − content of the high NUE genotype was significantly lower than that of the low NUE genotype, whereas the total N per plant of the two genotypes was almost the same, suggesting that the different NUE between the two genotypes is not due to the difference of NO3 − uptake. The relative expression levels of V-ATPase (vha-a3) and V-PPase (avp1) genes in the high NUE genotype were significantly lower than in the low NUE genotype, resulting in lower V-ATPase and V-PPase activities in the high NUE genotype. The transport of NO3 − and protons from the cytoplasm to the vacuole is powered by V-ATPase and V-PPase, so their lower activities increase H+ efflux from and reduce NO3 − influx into the vacuoles of the high NUE genotype. We conclude that the lower activity of proton pumps at the tonoplast is the main reason the high NUE genotype possesses lower NO3 − content and higher N-use efficiency.

Nitrate (NO3-) can accumulate in high concentrations in plant cell vacuoles if it is not reduced, reutilized or transported into the cytoplasm. Such accumulation of NO3- in the vacuole occurs when mechanisms for NO3- assimilation in the cytoplasm are saturated. Moreover, other processes such as efflux across the plasma membrane might affect NO3- accumulation in the vacuole. These are the main reasons limiting nitrogen use efficiency (NUE) in plants. This study elucidates mechanisms for NO3- transport from the cytoplasm to vacuoles by the V-proton pump (V-ATPase and V-PPase) and their relationship with different NUE in four Brassica napus genotypes. Pot experiments were conducted in a greenhouse under normal (15.0 mmol L-1) and limited N (7.5 mmol L-1) concentrations of nitrate using B. napus genotypes that demonstrated either high (742 and Xiangyou 15) or low (814 and H8) NUE (g g(-1)). Specific inhibitors of V-ATPase and V-PPase increased nitrate reductase (NR) activity, resulting in greatly decreased NO3- in plant tissues. Nitrate reductase activity and NO3- content correlated more highly to V-PPase activity than they did to V-ATPase activity, and correlation between V-PPase activity and NO3- content was significantly higher than it was to V-ATPase. Genotypes with high NUE had significantly lower activities of V-ATPase and V-PPase than those with low NUE. In the high-NUE plants, lower activities of V-proton pump underlie mechanisms that result in significantly lower NO3- content in plant tissues of the high-NUE genotypes than those found in plant tissues of the low-NUE genotypes. Our results show that the tonoplast proton pumps V-PPase and V-ATPase strongly negatively affect NR activity and positively affect NO3- content. V-PPase contributed more to this regulatory mechanism than did V-ATPase.

To assess the pollution situation and human health risks, the concentrations of heavy metals Pb, Cd, Cu, and Zn in paddy soils and white rice around seven mining-affected areas in Hunan Province were analyzed. The ranges of concentrations of Pb (23.9-1595.8 mg kg(-1)), Cd (0.3-9.5 mg kg(-1)), Cu (31.2-321.5 mg kg(-1)) and Zn (56.1-3478.9 mg kg(-1)) in all paddy soils were significantly higher than Hunan background values and even exceeded the maximum permissible concentrations for paddy soil quality recommended by the Ministry of Environmental Protection of China. The geoaccumulation index (I- (geo) ) showed that Cd (1.42-6.33) was the predominant pollutant in all paddy soils, while Zn was the least important element. The concentrations of Pb, Cd, Cu, and Zn in white rice ranged from 0.18 to 0.72 mg kg(-1), 0.10 to 1.32 mg kg(-1), 3.83 to 5.95 mg kg(-1), and 8.64 to 18.18 mg kg(-1), respectively. Human health risk, associated with these heavy metals, was assessed based on hazard quotients (HQ) and hazard indexes (HI) for adults through consumption of white rice. HQ values of heavy metals (except for Cd) in most of mining-affected areas were below 1.0, while the HI of all heavy metals in all mine areas was higher than 1.0, the maximum acceptable level, suggesting that consumption of such contaminated white rice was a health risk. Except for the Leng Shui-Jiang mine area, Cd was the major contributor to the risk in the mine areas through white rice consumption, amounting to over 67 % of the HI, while Zn was a minimal contributor compared to the other metals.