Evolution of metal hyperaccumulation required cis-regulatory changes and triplication of HMA4

Little is known about the types of mutations underlying the evolution of species-specific traits. The metal hyperaccumulator Arabidopsis halleri has the rare ability to colonize heavy-metal-polluted soils, and, as an extremophile sister species of Arabidopsis thaliana, it is a powerful model for research on adaptation. A. halleri naturally accumulates and tolerates leaf concentrations as high as 2.2% zinc and 0.28% cadmium in dry biomass. On the basis of transcriptomics studies, metal hyperaccumulation in A. halleri has been associated with more than 30 candidate genes that are expressed at higher levels in A. halleri than in A. thaliana. Some of these genes have been genetically mapped to broad chromosomal segments of between 4 and 24 cM co-segregating with Zn and Cd hypertolerance. However, the in planta loss-of-function approaches required to demonstrate the contribution of a given candidate gene to metal hyperaccumulation or hypertolerance have not been pursued to date. Using RNA interference to downregulate HMA4 (HEAVY METAL ATPASE 4) expression, we show here that Zn hyperaccumulation and full hypertolerance to Cd and Zn in A. halleri depend on the metal pump HMA4. Contrary to a postulated global trans regulatory factor governing high expression of numerous metal hyperaccumulation genes, we demonstrate that enhanced expression of HMA4 in A. halleri is attributable to a combination of modified cis-regulatory sequences and copy number expansion, in comparison to A. thaliana. Transfer of an A. halleri HMA4 gene to A. thaliana recapitulates Zn partitioning into xylem vessels and the constitutive transcriptional upregulation of Zn deficiency response genes characteristic of Zn hyperaccumulators. Our results demonstrate the importance of cis-regulatory mutations and gene copy number expansion in the evolution of a complex naturally selected extreme trait. The elucidation of a natural strategy for metal hyperaccumulation enables the rational design of technologies for the clean-up of metal-contaminated soils and for bio-fortification

Alergeni iz plijesni Fusarium solani u iranskih bolesnika s astmom utvrđeni s pomoću imunoblot-testa

We extracted Fusarium solani antigens to evaluate specifi c anti-F. solani IgE in fifty-one patients with asthma (33 men and 18 women) and in 22 non-atopic healthy subjects (15 men and 7 women). F. solani strains were cultured in Sabouraud glucose agar and subjected to cell disruption using the freeze-and-thaw method. The obtained cytoplasmic extracts were analysed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Sensitisation to F. solani antigens has been evaluated in asthmatic patients using the immunoblotting assay. The SDS-PAGE identifi ed 29 protein bands in the cytoplasmic extracts of F. solani isolates, with molecular weights ranging from 24 kDa to 112 kDa. Immunoblotting detected specific anti-F. solani IgE antibody in all asthma patients, but not in the control group. The predominant reactive allergens in patients corresponded to the bands with molecular weights of 24 kDa, 58.5 kDa, 64.5 kDa, 69 kDa, 72 kDa, and 97 kDa. Our results suggest that various allergenic components of F. solani may produce symptoms of asthma in susceptible individuals and they call for further research.Ekstrahirali smo antigene plijesni Fusarium solani kako bismo izmjerili imunosni odgovor, tj. razine anti-F. solani IgE u 52 bolesnika s astmom (33 muškarca i 18 žena) te u 22 zdrava ispitanika bez atopija (15 muškaraca i sedam žena). Sojevi F. solani uzgojeni su na podlozi glukoznoga Sabouraudova agara i podvrgnuti razbijanju stanica s pomoću metode smrzavanja i odmrzavanja. Dobiveni citoplazmatski ekstrakti analizirani su s pomoću elektroforeze u poliakrilamidnom gelu u prisutnosti natrijeva dodecilsulfata (engl. sodium dodecyl sulphate-polyacrylamide gel electrophoresis, krat. SDS-PAGE). Senzibilizacija na antigene F. solani u bolesnika s astmom utvrđena je s pomoću imunoblot-testa. S pomoću SDS-PAGE-a u citoplazmatskim ekstraktima izolata F. solani dokazano je 29 proteinskih vrpca. Molekulske mase razdvojenih proteina kretale su se u rasponu od 24 kDa do 112 kDa. Imunoblot-test otkrio je specifi čno anti-F. solani IgE- protutijelo u svih bolesnika s astmom, ali niti u jednog ispitanika iz kontrolne skupine. Najčešći su alergeni u bolesnika s astmom odgovarali proteinima ovih molekulskih masa: 24 kDa, 58,5 kDa, 64,5 kDa, 69 kDa, 72 kDa i 97 kDa. Naši nalazi upućuju na to da simptome astme u osjetljivih pojedinaca može izazvati više alergena soja F. solani te da ih treba podrobnije istražiti