Engineering high Zn in tomato shoots through expression of AtHMA4 involves tissue-specific modification of endogenous genes.

BACKGROUND: 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). RESULTS: 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". CONCLUSIONS: 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

Mutations in the Non-Catalytic Subunit Dpb2 of DNA Polymerase Epsilon Affect the Nrm1 Branch of the DNA Replication Checkpoint

To preserve genome integrity, the S-phase checkpoint senses damaged DNA or nucleotide depletion and when necessary, arrests replication progression and delays cell division. Previous studies, based on two pol2 mutants have suggested the involvement of DNA polymerase epsilon (Pol ε) in sensing DNA replication accuracy in Saccharomyces cerevisiae. Here we have studied the involvement of Pol ε in sensing proper progression of DNA replication, using a mutant in DPB2, the gene coding for a non-catalytic subunit of Pol ε. Under genotoxic conditions, the dpb2-103 cells progress through S phase faster than wild-type cells. Moreover, the Nrm1-dependent branch of the checkpoint, which regulates the expression of many replication checkpoint genes, is impaired in dpb2-103 cells. Finally, deletion of DDC1 in the dpb2-103 mutant is lethal supporting a model of strand-specific activation of the replication checkpoint. This lethality is suppressed by NRM1 deletion. We postulate that improper activation of the Nrm1-branch may explain inefficient replication checkpoint activation in Pol ε mutants

Approach to engineer tomato by expression of AtHMA4 to enhance Zn in the aerial parts

The aim of this work was to assess the potential for using AtHMA4 to engineer enhanced efficiency of Zn translocation to shoots, and to increase the Zn concentration in aerial tissues of tomato. AtHMA4, a P1B-ATPase, encodes a Zn export protein known to be involved in the control of Zn root-to-shoot translocation. In this work, 35S::AtHMA4 was expressed in tomato (Lycopersicon esculentum var. Beta). Wild-type and transgenic plants were tested for Zn and Cd tolerance; Zn, Fe and Cd accumulation patterns, and for the expression of endogenous Zn/Fe-homeostasis genes. At 10 ?M Zn exposure, a higher Zn concentration was observed in leaves of AtHMA4-expressing lines compared to wild-type, which is promising in terms of Zn biofortification. AtHMA4 also transports Cd and at 0.25 ?M Cd the transgenic plants showed similar levels of this element in leaves to wild-type but lower levels in roots, therefore indicating a reduction of Cd uptake due to AtHMA4 expression. Expression of this transgene AtHMA4 also resulted in distinct changes in Fe accumulation in Zn-exposed plants, and Fe/Zn-accumulation in Cd-exposed plants, even though Fe is not a substrate for AtHMA4. Analysis of the transcript abundance of key Zn/Fe-homeostasis genes showed that the pattern was distinct for transgenic and wild-type plants. The reduction of Fe accumulation observed in AtHMA4-transformants was accompanied by up-regulation of Fe-deficiency marker genes (LeFER, LeFRO1, LeIRT1), whereas down-regulation was detected in plants with the status of Fe-sufficiency. Furthermore, results strongly suggest the importance of the up-regulation of LeCHLN in the roots of AtHMA4-expressing plants for efficient translocation of Zn to the shoots. Thus, the modifications of Zn/Fe/Cd translocation to aerial plant parts due to AtHMA4 expression are closely related to the alteration of the endogenous Zn–Fe–Cd cross-homeostasis network of tomato

Model for replication checkpint activation and response in <i>dpb2-103</i> cells.

<p><b>(A)</b> In <i>ddc1Δ</i> and <i>dpb2-103</i> cells replication checkpoint activation is partially impared whereas in <i>ddc1Δ dpb2-103</i> cells is abolished. (<b>B)</b> The two branches i. e. DDR (Dun1/Crt1) and CC (Nrm1/MBF) are activated in wild type cells and inactive in <i>rad53</i> or <i>mec1</i> checkpoint mutants. In <i>dpb2-103</i> cells the Nrm1/MBF branch is inactive.</p

The expression of G1/S transition genes (repressed by Nrm1) from the MBF pathway is not activated in <i>dpb2-103</i> cells under replication stress.

<p>(<b>A)</b> quantitative RT-PCR analysis of <i>TOS2</i>, <i>TOS4</i>, <i>MCD1</i>, <i>CDC21</i> transcripts in wild-type (solid line) and <i>dpb2-103</i> (dashed line) cells under unperturbed growth conditions (green) or under replication stress generated by 200 mM HU (red). Transcript levels were normalized to G1-synchronized wild-type cells. Yeast cultures were synchronized in G1 and released from α-factor in YNBD or YNBD 200 mM HU. Samples were taken at time 0 (G1 arrest), 30, 60, 90, 120 and 180 minutes after release from G1 block. Standard deviations were omitted for clarity and are presented in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006572#pgen.1006572.s006" target="_blank">S1 Table</a></b>. (<b>B)</b> Flow cytometry analysis of DNA content (cell cycle progression) at 0, 30, 60, 90, 120 and 180 minutes time points in wild-type (solid line) and <i>dpb2-103</i> (dashed line) cells under unperturbed growth conditions (green) or under replication stress generated by 200 mM HU (red). <b>(C and D)</b> Deletion of <i>NRM1</i> gives viable <i>dpb2-103 ddc1Δ nrm1Δ</i> cells. <b>(C)</b> Deletion of <i>DDC1</i> in <i>dpb2-103</i> cells carrying plasmid pMJDPB2 (source of <i>DPB2</i>). Transformants were were grown exponentially and dilutions were spotted on YNBD or YNBD with 5-FOA. <b>(D)</b> Tetrad analysis of a <i>dpb2-103</i>/<i>DPB2</i>, <i>ddc1Δ</i>/<i>DDC1 nrm1Δ/ nrm1Δ</i> strain.</p

The Crt1/Dun1 pathway of the replication stress checkpoint is activated in <i>dpb2-103</i> cells under MMS or HU treatment.

<p><b>(A)</b> Western-Blot detection of Sml1 degradation. Extracts from WT or <i>dpb2-103</i> yeast cells treated with 0,05% MMS were analyzed. <b>(B)</b> Quantitative RT-PCR analysis of <i>RNR3</i> and <i>HUG1</i> transcripts in yeast cells released from G1-arrest in YNBD (green) or YNBD with 200 mM HU (red). Transcript levels were analyzed after 120 and 240 minutes of growth and normnalized to wild-type G1-synchronized cells.</p

Synthetic lethality <i>dpb2-103 ddc1</i>Δ cells.

<p><b>(A)</b> Tetrad analysis of a heterozygous <i>dpb2-103</i>/<i>DPB2</i>, <i>ddc1Δ</i>/<i>DDC1</i> strain. <b>(B)</b> Indicated strains with plasmid pMJDPB2 (source of <i>DPB2</i> allele) were grown exponentially, serially diluted and spotted on YNBD or YNBD supplemented with 5-FOA.</p

Primers used in this work.

<p>Primers used in this work.</p

<i>dpb2-103</i> mutation affects yeast viability under genotoxic or replication stress.

<p>(<b>A)</b> Cultures of indicated strains were grown exponentially, serially diluted and spotted on YNBD supplemented with MMS or HU. Plates were incubated at 23°C for 5 days. (<b>B)</b> Log-phase cultures of yeast strains were appropriately diluted and plated on YNBD medium without MMS or supplemented with 0,03% or 0,05% MMS. Plates were incubated at 23°C for 5 days. (<b>C)</b> Log-phase cultures of yeast strains were supplemented with HU to final concentration 200 mM. Samples were collected at 2 hours intervals, plated on YNBD medium, and incubated at 23°C for 5 days.</p