Functional characterization of Arabidopsis thaliana metallothionein-deficient mutants

Matheus Romanos Benatti, Purdue University

Abstract

In an effort to understand the functions of MT genes in plants, a reverse genetic approach was used in this study. Arabidopsis thaliana lines containing T-DNA insertions in each of the MT genes were identified. Because mutants that were deficient in a single MT showed few dramatic or interesting phenotypes, the insertion lines were crossed and used to develop mutant plants lacking the expression of multiple MT genes. Arabidopsis mutant plants carrying insertions in all four of the MT genes that are expressed in vegetative tissues, here referred to as the quad-MT mutant (mt1a-2/mt2a-1/mt2b-1/mt3-1 ), were then used for this study. The MT-deficient plants were subjected to a wide variety of abiotic stresses including growth under toxic levels of CuSO4, ZnSO4 and paraquat. Additional experiments were conducted to test the sensitivity of MT-deficient mutants to the fungal pathogens Botrytis cinerea and Alternaria brassicicola, and the bacterial pathogen Pseudomonas syringae. These experiments have revealed MT-deficient mutants are no more sensitive than wild-type plants to the abiotic and biotic stresses described above. In addition, I also tested whether MTs play a direct role in the leaf senescence program. MT-deficient plants also have a normal senescence program. These results suggest that MTs are not directly involved in metal ion and oxidative stress tolerance, defense against various pathogens, or in the leaf senescence program. Additional experiments were conducted by using a hydroponic system to grow Arabidopsis plants under conditions where the supply of Cu could be carefully controlled. These experiments allowed investigation of the role that MTs play an important role in Cu homeostasis in Arabidopsis. Interestingly, the MT-deficient plants develop normally under standard conditions. However, the senescent leaves of quad-MT mutants accumulate more Cu while mature seeds contain less Cu than in wild-type plants. Quad-MT mutants also showed decreased accumulation of Cu in roots and shoots when plants were exposed to excess copper, compared to wild-type plants. When quad-MT mutants were grown under conditions that limit the availability of Cu, significant growth inhibition was observed in these plants compared to the wild-type. MT-deficient plants were grown in hydroponic medium containing modified ratios of the stable isotopes of Cu (63Cu and 65Cu). Throughout plant development Cu accumulation in leaves and seeds of both wild-type and quad-MT mutant plants is accomplished by direct transport of Cu from the nutrient solution. No significant remobilization of Cu out of senescent leaves to seeds takes place. In addition, quad-MT mutant plants take up Cu at a higher rate than wild-type plants. The impact of MT-deficiency on tolerance to Cu-deficiency was also examined. MT-deficient mutant plants had an increased sensitivity to two Cu-specific chelators, TM and BCS. Growth of quad-MT mutant plants was also significantly inhibited when plants were grown in hydroponic culture with very limited supply of Cu. RNA expression analysis has revealed that quad-MT deficient mutants have altered expression of at least two genes regulated by Cu supply, FSD1 and ZIP2. These observations indicate that MT-deficient mutant seedlings behave as Cu-deficient under Cu-sufficient conditions. Overall, these experiments provide new evidence that MTs play an important role in Cu homeostasis in Arabidopsis and that MT-deficiency can lead to a significant decrease in both vegetative and reproductive plant growth when Cu availability is limited.

Degree

Ph.D.

Advisors

Goldsbrough, Purdue University.

Subject Area

Molecular biology|Horticulture|Plant sciences

COinS