Livelihood Resilience Perception: Gender Equalisation of Resettlers from Rural Reservoirs—Empirical Evidence from China

Hydropower engineering has brought unprecedented benefits to the world while causing massive displacement of people. Since the implementation of the Post-Relocation Support (PReS) policy for reservoir resettlers in China in 2006, the distribution of perceived livelihood resilience by gender of resettlers has gradually become more equal. Based on data from a survey of rural reservoir resettlers’ livelihoods in nine regions of Guizhou Province, China, this data examines the distribution of resettlers’ perceived livelihood resilience across genders using logit regression and then explores the contribution to gender equality. The empirical results show that, unlike previous studies, household economic conditions do not bring about more gender differences in perceived livelihood resilience among resettlers (gender contribution ratio = 1.12). Gender differences in perceived livelihood resilience among resettlers were influenced by household workforce levels (e.g., gender contribution ratio = 1.23 at high workforce levels), education level (e.g., contribution ratio = 1.87 in primary education), and resettlement methods (e.g., contribution ratio = 4.53 at external resettlement). The implementation of the PReS policy also contributes to the gender equality of these resettlers’ perceived livelihood resilience. For rural resettlers in different regions with different livelihoods, resettlement patterns, capital, and gender differences of resettlers should be understood through different livelihood resilience perspectives. Improving capacity building of resettlers’ livelihoods resilience through site-specific, participatory development and resource interoperability to promote high quality, sustainable and simultaneous development in resettlement areas and reservoirs

Enzyme Inhibitor Studies Reveal Complex Control of Methyl-D-Erythritol 4-Phosphate (MEP) Pathway Enzyme Expression in <i>Catharanthus roseus</i>

<div><p>In <i>Catharanthus roseus</i>, the monoterpene moiety exerts a strong flux control for monoterpene indole alkaloid (MIA) formation. Monoterpene synthesis depends on the methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we have explored the regulation of this pathway in response to developmental and environmental cues and in response to specific enzyme inhibitors. For the MEP pathway entry enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a new (type I) DXS isoform, CrDXS1, has been cloned, which, in contrast to previous reports on type II CrDXS, was not transcriptionally activated by the transcription factor ORCA3. Regulation of the MEP pathway in response to metabolic perturbations has been explored using the enzyme inhibitors clomazone (precursor of 5-ketochlomazone, inhibitor of DXS) and fosmidomycin (inhibitor of deoxyxylulose 5-phosphate reductoisomerase (DXR)), respectively. Young leaves of non-flowering plants were exposed to both inhibitors, adopting a non-invasive <i>in vivo</i> technique. Transcripts and proteins of DXS (3 isoforms), DXR, and hydroxymethylbutenyl diphosphate synthase (HDS) were monitored, and protein stability was followed in isolated chloroplasts. Transcripts for <i>DXS1</i> were repressed by both inhibitors, whereas transcripts for <i>DXS2A</i>&<i>B</i>, <i>DXR</i> and <i>HDS</i> increased after clomazone treatment but were barely affected by fosmidomycin treatment. DXS protein accumulated in response to both inhibitors, whereas DXR and HDS proteins were less affected. Fosmidomycin-induced accumulation of DXS protein indicated substantial posttranscriptional regulation. Furthermore, fosmidomycin effectively protected DXR against degradation <i>in planta</i> and in isolated chloroplasts. Thus our results suggest that DXR protein stability may be affected by substrate binding. In summary, the present results provide novel insight into the regulation of DXS expression in <i>C. roseus</i> in response to MEP-pathway perturbation.</p></div

Effect of paraquat treatment on the expression of MEP pathway genes at protein and transcript level, respectively, in leaf discs obtained from mature leaves of 6-week-old <i>C. roseus</i> plants.

<p>Leaf discs (5 mm diameter) were collected from fully expanded leaves and floated on distilled water in the presence or absence of 0.5 µM paraquat for the indicated time intervals at 25°C under continuous light (270 µmol m⁻² s⁻¹). <b>(A)</b> DXS, DXR and HDS proteins were detected by immunoblot. Equal sample loading was confirmed by Coomassie staining. The arrow marks the position of mature CrDXS2A protein.<b>(B)</b> transcript amounts for DXS (isoforms 1, 2A & 2B), DXR and HDS were determined by qPCR relative to the geometric mean of multiple reference genes according to Vandesompele et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062467#pone.0062467-Vandesompele1" target="_blank">[60]</a>. The experiment was performed 3 times; values from a representative experiment are presented ± SD.</p

Time course of <i>in vivo</i> clomazone treatment on the expression of MEP pathway genes, and subsequent degradation of DXS and DXR proteins in isolated chloroplasts.

<p>For each plant, two pairs of mature leaves were injected with a 50 µM clomazone solution (or water for control) until the entire leaf blades were fully soaked, using a 1 ml needleless syringe applied to the lower epidermis. For each time point, young leaves were pooled from three independent plants and processed for MEP pathway protein and transcript analysis, respectively. <b>(A)</b> DXS, DXR and HDS proteins were detected by immunoblot. Equal sample loading was confirmed by Coomassie staining. The arrow marks the position of mature CrDXS2A protein. <b>(B)</b> transcript amounts for DXS isoforms (1, 2A & 2B), DXR and HDS were determined by qPCR relative to the geometric mean of multiple reference genes according to Vandesompele et al.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062467#pone.0062467-Vandesompele1" target="_blank">[60]</a>. The experiment was performed 3 times, values from a representative experiment are presented ± SD. <b>(C)</b> chloroplasts were isolated from young leaves of 6-week-old soil-grown <i>C. rosues</i> control and 50 µM clomazone-treated plants (see <b>(A)</b> this Figure) 78 hours after treatment. Chloroplasts were incubated for 1 h in the light (100 µmol m⁻² s⁻¹) at 25°C in the presence of 5 mM ATP. Aliquots were taken at 0, 15, 30 and 60 minutes and used for protein extraction. DXS and DXR proteins were detected by immunoblot. Note that to obtain similar signal intensity at time point 0, the loading amount of protein from control samples (chloroplast isolated from water infiltrated plants) was twice that of clomazone samples.</p

Time course of <i>in vivo</i> fosmidomycin treatment on the expression of MEP pathway genes in young leaves of 6-week-old <i>C. roseus</i> plants at protein and transcript level, respectively.

<p>For each plant, two pairs of mature leaves were injected with a 50 µM fosmidomycin solution. Application procedure and time course of sampling was as described for clomazone treatment (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062467#pone-0062467-g005" target="_blank">Figure 5</a>). <b>(A)</b> DXS, DXR and HDS proteins were detected by immunoblot. Equal sample loading was confirmed by Coomassie staining. The arrow marks the position of mature CrDXS2A protein. <b>(B)</b>, transcript amounts for DXS isoforms (1, 2A & 2B), DXR, and HDS were determined by qPCR, relative to the geometric mean of multiple reference genes according to Vandesompele et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062467#pone.0062467-Vandesompele1" target="_blank">[60]</a>. The experiment was performed 3 times, values from a representative experiment are presented ± SD.</p

Tissue-specific expression of MEP pathway genes in young leaves, mature leaves and roots of 6-week-old non-flowering <i>C. roseus</i> plants, at protein and transcript level, respectively.

<p>Soil-grown periwinkle (<i>Catharanthus roseus</i>) plants were cultivated in a growth chamber at 25°C with 14 hrs light period (170 µmol m⁻² s⁻¹) and 22°C during dark period. <b>(A)</b> DXS, DXR, and HDS proteins were detected by immunoblot with polyclonal antisera raised against recombinant CrDXS2A, DXR and HDS proteins. Equal sample loading was confirmed by Coomassie staining. The arrow marks the position of mature CrDXS2A protein. <b>(B)</b> transcript amounts for DXS isoforms (1, 2A & 2B), DXR, and HDS were determined by qPCR (the experiment was performed 3 times, values from a representative experiment are presented ± SD), relative to the geometric mean of multiple reference genes according to Vandesompele et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062467#pone.0062467-Vandesompele1" target="_blank">[60]</a>.</p

Schematic view of plastidic methylerythritol 4-phosphate (MEP) pathway providing the precursors for secologanin (monoterpene) synthesis.

<p>Enzymes analyzed in the present study are in bold face (DXS, 1-deoxy-D-xylulose 5-phosphate synthase; DXR, deoxyxylulose 5-phosphate reductoisomerase; HDS, hydroxymethylbutenyl diphosphate synthase). GAP, glyceraldehyde 3-phosphate; DXP, deoxyxylulose 5-phosphate; MEP, methylerythritol 4-phosphate; MEcPP, methylerythritol 2,4-cyclodiphosphate; HMBPP, hydroxymethylbutenyl diphosphate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; GGPP, geranylgeranyl diphosphate. ABA, abscisic acid. The step branching to thiamine from DXP is indicated. Furthermore, inhibition of DXS and DXR by 5-ketoclomazone (formed <i>in planta</i> from clomazone) and fosmidomycin, respectively, is highlighted. Dashed arrows indicate multiple steps.</p

Activation of DXS isoforms' promoters (<i>CrDXS1p, CrDXS2Ap and CrDXS2Bp</i>) by the transcription factor ORCA3, analyzed via transient expression of promoter-luciferase fusions in leaves of 6-week old <i>C. roseus</i> plants.

<p>Results are expressed as fold change of promoter activation, based on four independent assays. The promoter activity was determined by the ratio of luciferase (LUC) activity to renilla expression. Dual <i>CaMV</i> 35S promoter was employed as a positive control showing high LUC activity, while promoterless LUC control (pGreenII 0800-LUC vector) reflected the LUC background value. <i>DXS</i> promoter sequences were fused to the firefly luciferase and transiently co-transformed with ORCA3 or without (mock control). <i>Agrobacteria</i>-mediated transient transformation was carried via leaf infiltration of 6-week-old soil-grown <i>C. roseus</i> plants. Two days after transformation, infiltrated leaves were harvested and subjected to dual-luciferase reporter assay. Error bars represent the standard deviation of four independent experiments for promoter activity analysis. The asterisks represent significant difference (** <i>p</i><0.001, * <i>p</i><0.1 by student's t-test).</p