title: Engineering high Zn in tomato shoots through expression of AtHMA4 involves tissue-specific modification of endogenous genes.
abstract: To increase the Zn level in shoots, AtHMA4 was ectopically expressed in tomato under the constitutive CaMV 35S promoter. However, the Zn concentration in the shoots of transgenic plants failed to increase at all tested Zn levels in the medium. Modification of Zn root/shoot distribution in tomato expressing 35S::AtHMA4 depended on the concentration of Zn in the medium, thus indicating involvement of unknown endogenous metal-homeostasis mechanisms. To determine these mechanisms, those metal-homeostasis genes that were expressed differently in transgenic and wild-type plants were identified by microarray and RT-qPCR analysis using laser-assisted microdissected RNA isolated from two root sectors: (epidermis + cortex and stele), and leaf sectors (upper epidermis + palisade parenchyma and lower epidermis + spongy parenchyma).
  
  
  
  Zn-supply-dependent modification of Zn root/shoot distribution in AtHMA4-tomato (increase at 5 μM Zn, no change at 0.5 μM Zn) involved tissue-specific, distinct from that in the wild type, expression of tomato endogenous genes. First, it is suggested that an ethylene-dependent pathway underlies the detected changes in Zn root/shoot partitioning, as it was induced in transgenic plants in a distinct way depending on Zn exposure. Upon exposure to 5 or 0.5 μM Zn, in the epidermis + cortex of the transgenics' roots the expression of the Strategy I Fe-uptake system (ethylene-dependent LeIRT1 and LeFER) was respectively lower or higher than in the wild type and was accompanied by respectively lower or higher expression of the identified ethylene genes (LeNR, LeACO4, LeACO5) and of LeChln. Second, the contribution of LeNRAMP2 expression in the stele is shown to be distinct for wild-type and transgenic plants at both Zn exposures. Ethylene was also suggested as an important factor in a pathway induced in the leaves of transgenic plants by high Zn in the apoplast, which results in the initiation of loading of the excess Zn into the mesophyll of "Zn accumulating cells".
  
  
  
  In transgenic tomato plants, the export activity of ectopically expressed AtHMA4 changes the cellular Zn status, which induces coordinated tissue-specific responses of endogenous ethylene-related genes and metal transporters. These changes constitute an important mechanism involved in the generation of the metal-related phenotype of transgenic tomato expressing AtHMA4.
authors: Faculty of Biology, Institute of Experimental Plant Biology and Biotechnology, Department of Plant Anatomy and Cytology, University of Warsaw, Miecznikowa str 1, 02-096, Warszawa, Poland.
authors: Maria
authors: M
authors: Kendziorek
authors: Faculty of Biology, Institute of Experimental Plant Biology and Biotechnology, Department of Plant Anatomy and Cytology, University of Warsaw, Miecznikowa str 1, 02-096, Warszawa, Poland.|Present address: Plant Biochemistry Department, Institute of Biochemistry and Biophysics PAS, Pawińskiego 5a, 02-106, Warszawa, Poland.
authors: Maria
authors: M
authors: Klimecka
authors: Faculty of Biology, Institute of Experimental Plant Biology and Biotechnology, Department of Plant Anatomy and Cytology, University of Warsaw, Miecznikowa str 1, 02-096, Warszawa, Poland.
authors: Anna
authors: A
authors: Barabasz
authors: Department. of Molecular Biology and Genetics, Aarhus University, Forsøgsvej 1, 4200, Slagelse, Denmark.
authors: Sören
authors: S
authors: Borg
authors: Faculty of Biology, Institute of Experimental Plant Biology and Biotechnology, Department of Plant Anatomy and Cytology, University of Warsaw, Miecznikowa str 1, 02-096, Warszawa, Poland.
authors: Justyna
authors: J
authors: Rudzka
authors: Institute of Biochemistry and Biophysics PAS, Pawińskiego 5a, 02-106, Warszawa, Poland.
authors: Paweł
authors: P
authors: Szczęsny
authors: Faculty of Biology, Institute of Experimental Plant Biology and Biotechnology, Department of Plant Anatomy and Cytology, University of Warsaw, Miecznikowa str 1, 02-096, Warszawa, Poland. dma@biol.uw.edu.pl.
authors: Danuta Maria
authors: 0000-0003-4921-0378
authors: DM
authors: Antosiewicz
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D002104:Cadmium / Q000378:metabolism; D016616:Cryoultramicrotomy; D005452:Fluoresceins / Q000737:chemistry; D007501:Iron / Q000378:metabolism; D018551:Solanum lycopersicum / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D020411:Oligonucleotide Array Sequence Analysis; D018517:Plant Roots / Q000737:chemistry / Q000378:metabolism; D018520:Plant Shoots / Q000737:chemistry / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D018749:RNA, Plant / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D059467:Transcriptome; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005452:Fluoresceins; D018749:RNA, Plant; D002104:Cadmium; D007501:Iron; D000251:Adenosine Triphosphatases; D015032:Zinc; C501987:zinpyr-1
keywords: AtHMA4; Cadmium; Laser microdissection; Microarray; Tomato; Zinc