Soil and Herbage Heavy Metal/ Trace Element Variability and Relationships at Farm and Regional Level.

End of Project ReportVariability of heavy metal trace/element amounts in soil and herbage from 3 farms encompassing 26.5, 33.6 and 21.6 ha and in a 230 km2 surrounding area has been estimated. Variability was least in the farm with the greatest soil physical uniformity. It increased in line with increasing sampling area. In soil, variability was least (CV 10%) for sesquioxide metals, iron and aluminium, and elements chromium and nickel. It was highest where there was evidence of geochemical contamination with cadmium and selenium. In general, variability was higher (>20% CV) for extractable components including copper, zinc and manganese. Regression analysis of elements in soil showed up a number of useful associations particularly for the volatiles selenium, sulphate sulphur, mercury, cobalt, vanadium and fluorine with soil organic matter. Associations were also found between fluorine, vanadium and chromium. • Elements in herbage exhibited two types of associations (i) those reckoned to be within the plant and confined mostly to major elements and (ii) those involving metals associated primarily with soil and indicative of the extent to which herbage was contaminated by soil. • Elements that showed lower variability in soil exhibited large variability in herbage. These included iron, aluminium, chromium, vanadium and fluorine, where the cause was attributed to contamination of herbage by soil. It was suggested that iron could be used almost equally as well as titanium as an indicator of the degree to which herbage has been contaminated by soil. • Long term monitoring of herbage for selenium content highlighted the unreliability of relying on a single determination to characterise the selenium status of pasture

Effects of Deficiency and Excess of Zinc on Morphophysiological Traits and Spatiotemporal Regulation of Zinc-Responsive Genes Reveal Incidence of Cross Talk between Micro- and Macronutrients

Zinc (Zn) is an essential micronutrient which affects plant growth and development in deficiency and can be toxic when present in excess. In Arabidopsis thaliana, different families of cation transporters play pivotal roles in Zn homeostasis. In the present study, we evaluated the effects of Zn in its deficiency (0 μM; Zn−) and excess (75 μM; Zn++) on various morphophysiological and molecular traits. Primary root length was reduced in Zn– seedlings, whereas there were significant increases in the number and length of lateral roots under Zn– and Zn++ conditions, respectively. Concentration of various macro- and microelements showed variations under different Zn regimes and notable among them was the reduced level of iron (Fe) in Zn++ seedlings compared to Zn+. Certain members of the ZIP family (<i>ZIP4</i>,<i> ZIP9</i>, and <i>ZIP12</i>) showed significant induction in roots and shoots of the Zn– seedlings. Their suppression under Zn++ condition indicated their transcriptional regulation by Zn and their roles in the maintenance of its homeostasis. Zn-deficiency-mediated induction of <i>HMA2</i> in roots and shoots suggested its role in effluxing Zn into xylem for long-distance transport. Attenuation in the expression of Fe-responsive <i>FRO2</i> and <i>IRT1</i> in Zn– roots and their induction in Zn++ roots provided empirical evidence toward the prevalence of a cross talk between Zn and Fe homeostasis. Variable effects of Zn– and Zn++ on the expression of subset of genes involved in the homeostasis of phosphate (Pi), potassium (K), and sulfur (S) further highlighted the prevalence of cross talk between the sensing and signaling cascades of Zn and macronutrients. Further, the inducibility of <i>ZIP4</i> and <i>ZIP12</i> in response to cadmium (cd) treatment could be harnessed by tailoring them in homologous or heterologous plant system for removing pollutant toxic heavy metals from the environment

Silencing of <i>HaAce1</i> gene by host-delivered artificial microRNA disrupts growth and development of <i>Helicoverpa armigera</i>

<div><p>The polyphagous insect-pest, <i>Helicoverpa armigera</i>, is a serious threat to a number of economically important crops. Chemical application and/or cultivation of <i>Bt</i> transgenic crops are the two strategies available now for insect-pest management. However, environmental pollution and long-term sustainability are major concerns against these two options. RNAi is now considered as a promising technology to complement <i>Bt</i> to tackle insect-pests menace. In this study, we report host-delivered silencing of <i>HaAce1</i> gene, encoding the predominant isoform of <i>H</i>. <i>armigera</i> acetylcholinesterase, by an artificial microRNA, <i>HaAce1</i>-amiR1. Arabidopsis pre-miRNA164b was modified by replacing miR164b/miR164b* sequences with <i>HaAce1</i>-amiR1/<i>HaAce1</i>-amiR1* sequences. The recombinant <i>HaAce1</i>-preamiRNA1 was put under the control of CaMV 35S promoter and NOS terminator of plant binary vector pBI121, and the resultant vector cassette was used for tobacco transformation. Two transgenic tobacco lines expressing <i>HaAce1</i>-amiR1 was used for detached leaf insect feeding bioassays. Larval mortality of 25% and adult deformity of 20% were observed in transgenic treated insect group over that control tobacco treated insect group. The reduction in the steady-state level of <i>HaAce1</i> mRNA was 70–80% in the defective adults compared to control. Our results demonstrate promise for host-delivered amiRNA-mediated silencing of <i>HaAce1</i> gene for <i>H</i>. <i>armigera</i> management.</p></div

Designing and construction of pBI::HAR1.

<p>Primer extension PCR was done with a pair of overlapping primers, <i>HAR-</i>F & <i>HAR</i>-R (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194150#pone.0194150.s006" target="_blank">S1 Table</a>) containing <i>Xba</i>I and <i>Sac</i>I recognition sequences at their 5′ ends. <i>Xba</i>I and <i>Sac</i>I digested PCR product was ligated onto pUC19. pBI121 backbone was generated by digestion with <i>Xba</i>I and <i>Sac</i>I that released <i>GUS</i> coding sequence. After sequence confirmation, <i>HaAce1</i>-preamiRNA1 DNA fragment was released from recombinant pUC19 and ligated onto pBI121 backbone to get pBI::HAR1.</p

Additional file 3: Table S3. of Transcript profiling of genes expressed during fibre development in diploid cotton (Gossypium arboreum L.)

Differentially expressed transcripts encoding transcription factors (TFs) in Gossypium arboreum fuzzy-lintless line (Fl) as compared to fuzzy-linted (FL) at fibre itiation stage (0 dpa) and fibre elongation stage (10 dpa). (DOC 75 kb

Expression of <i>HaAce1</i>-amiR1 in transgenic tobacco lines and down regulation of <i>HaAce1</i> in <i>H</i>. <i>armigera</i> adults.

<p>(A) Northern blot showing <i>HaAce1</i>-amiR1 expression in the transgenic tobacco lines, 17R1 and 29R1. Small RNAs were hybridized with DIG end labelled Anti-amiR<i>Ace</i> probe of 21 nt. C, pBI121 transformed tobacco (vector control); 29R1 & 17R1, <i>HaAce1</i>-amiR1 expression in the selected two transgenic tobacco line, 29R1 and 17R1, respectively; P, labelled Anti-amiR<i>Ace</i> probe. (B) Relative expression of <i>HaAce1</i> in the <i>H</i>. <i>armigera</i> adults. Vector control, <i>HaAce1</i> transcript abundance in <i>H</i>. <i>armigera</i> adults emerged from vector control tobacco treated group; 17R1 and 29R1, <i>HaAce1</i> transcript abundance in deformed <i>H</i>. <i>armigera</i> adults emerged from 17R1 and 29R1 transgenic tobacco treatment groups, respectively. <i>β</i>-<i>Actin</i> gene of <i>H</i>. <i>armigera</i> was used as an internal control. Real time PCR data was analysed using delta-delta Ct method. One way ANOVA test was used to perform the statistical analysis of the data. *** Extremely significant at P<0.001. The test was performed three times.</p

Mortality and developmental deformity in <i>H</i>. <i>armigera</i>.

<p>Thirty second instar larvae of <i>H</i>. <i>armigera</i> were fed continuously on each transgenic line (Line 17R1 and 29R1) and vector control separately (one larva per plate) till their active feeding stage<b>.</b> (A) Mortality percentage of <i>H</i>. <i>armigera</i> larvae. Vector control, mortality percentage in larvae fed on pBI121 transformed tobacco leaves; 17R1, mortality percentage in larvae fed on 17R1 transgenic tobacco line; 29R1, mortality percentage in larvae fed on 29R1 transgenic tobacco line. (B) Deformity percentage of <i>H</i>. <i>armigera</i> adults. Vector control, percentage of emergence of deformed adults from larvae fed on pBI121 transformed tobacco leaves; 17R1, percentage of emergence of deformed adults from larvae fed on 17R1 transgenic tobacco line; 29R1, percentage of emergence of deformed adults from larvae fed on 29R1 transgenic tobacco line. (C) Phenotype of <i>H</i>. <i>armigera</i> adults. Upper panel: Normal adults developed from vector control fed larvae; Lower panel: Deformed adults developed from transgenic fed larvae. One way ANOVA test was used to perform statistical analysis of the data. *** denotes extremely significant differences at P<0.001. The test was repeated three times.</p

Additional file 8: Table S8. of Transcript profiling of genes expressed during fibre development in diploid cotton (Gossypium arboreum L.)

Short listed view of differentially expressed transcripts present in various metabolic processes based on MapMan (version 3.5) visualization software in Gossypium arboreum fuzzy-lintless line (Fl) at 10 dpa. (DOC 93 kb