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.

The ratio of Zn to Cd supply as a determinant of metal-homeostasis gene expression in tobacco and its modulation by overexpressing the metal exporter AtHMA4.

This study links changes in the tobacco endogenous metal-homeostasis network caused by transgene expression with engineering of novel features. It also provides insight into the concentration-dependent mutual interactions between Zn and Cd, leading to differences in the metal partitioning between wild-type and transgenic plants. In tobacco, expression of the export protein AtHMA4 modified Zn/Cd root/shoot distribution, but the pattern depended on their concentrations in the medium. To address this phenomenon, the expression of genes identified by suppression subtractive hybridization and the Zn/Cd accumulation pattern were examined upon exposure to six variants of low/high Zn and Cd concentrations. Five tobacco metal-homeostasis genes were identified: NtZIP2, NtZIP4, NtIRT1-like, NtNAS, and NtVTL. In the wild type, their expression depended on combinations of low/high Zn and Cd concentrations; co-ordinated responses of NtZIP1, NtZIP2, and NtVTL were shown in medium containing 4 µM Cd, and at 0.5 µM versus 10 µM Zn. In transgenics, qualitative changes detected for NtZIP1, NtZIP4, NtIRT1-like, and NtVTL are considered crucial for modification of Zn/Cd supply-dependent Zn/Cd root/shoot distribution. Notwithstanding, NtVTL was the most responsive gene in wild-type and transgenic plants under all concentrations of Zn and Cd tested; thus it is a candidate gene for the regulation of metal cross-homeostasis processes involved in engineering new metal-related traits.

HMA4 expression in tobacco reduces Cd accumulation due to the induction of the apoplastic barrier.

Ectopic expression in tobacco (Nicotiana tabacum v. Xanthi) of the export protein AtHMA4 (responsible in Arabidopsis for the control of Zn/Cd root to shoot translocation) resulted in decreased Cd uptake/accumulation in roots and shoots. This study contributes to understanding the mechanisms underlying this Cd-dependent phenotype to help predict the consequences of transgene expression for potential phytoremediation/biofortification-based strategies. Microarray analysis was performed to identify metal homeostasis genes that were differentially expressed in roots of Cd-exposed AtHMA4-expressing tobacco relative to the wild type. It was established that down-regulation of genes known to mediate Cd uptake was not responsible for reduced Cd uptake/accumulation in AtHMA4 transformants. The transcript levels of NtIRT1 and NtZIP1 were higher in transgenic plants, indicating an induction of the Fe and Zn deficiency status due to AtHMA4 expression. Interestingly, upon exposure to Cd, genes involved in cell wall lignification (NtHCT, NtOMET, and NtPrx11a) were up-regulated in transformants. Microscopic analysis of roots demonstrated that expression of AtHMA4 caused an induction of cell wall lignification in the external cell layers that was accompanied by enhanced H2O2 accumulation. Further study showed that the concentration of other elements (B, Co, Cu, Ni, Mo, and Zn) was reduced in AtHMA4 transformants in the presence of Cd. In conclusion, due to ectopic expression of 35S::AtHMA4, the physical apoplastic barrier within the external cell layer developed, which is likely to be responsible for the reduction of Cd uptake/accumulation.

Differential regulation of alanine aminotransferase homologues by abiotic stresses in wheat (Triticum aestivum L.) seedlings.

Wheat (Triticum aestivum L.) seedlings contain four alanine aminotransferase (AlaAT) homologues. Two of them encode AlaAT enzymes, whereas two homologues act as glumate:glyoxylate aminotransferase (GGAT). To address the function of the distinct AlaAT homologues a comparative examination of the changes in transcript level together with the enzyme activity and alanine and glutamate content in wheat seedlings subjected to low oxygen availability, nitrogen and light deficiency has been studied. Shoots of wheat seedlings were more tolerant to hypoxia than the roots as judging on the basis of enzyme activity and transcript level. Hypoxia induced AlaAT1 earlier in roots than in shoots, while AlaAT2 and GGAT were unaffected. The increase in AlaAT activity lagged behind the increase in alanine content. Nitrogen deficiency has little effect on the activity of GGAT. In contrast, lower activity of AlaAT and the level of mRNA for AlaAT1 and AlaAT2 in wheat seedlings growing on a nitrogen-free medium seems to indicate that AlaAT is regulated by the availability of nitrogen. Both AlaAT and GGAT activities were present in etiolated wheat seedlings but their activity was half of that observed in light-grown seedlings. Exposure of etiolated seedlings to light caused an increase in enzyme activities and up-regulated GGAT1. It is proposed that hypoxia-induced AlaAT1 and light-induced peroxisomal GGAT1 appears to be crucial for the regulation of energy availability in plants grown under unfavourable environmental conditions. Key message In young wheat seedlings, both AlaAT and GGAT are down-regulated by nitrogen deficiency, whereas AlaAT1 is upregulated by hypoxia and GGAT1 by light.

Properties of serine:glyoxylate aminotransferase purified from Arabidopsis thaliana leaves.

The photorespiratory enzyme L-serine:glyoxylate aminotransferase (SGAT; EC 2.6.1.45) was purified from Arabidopsis thaliana leaves. The final enzyme was approximately 80% pure as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with silver staining. The identity of the enzyme was confirmed by LC/MS/MS analysis. The molecular mass estimated by gel filtration chromatography on Sephadex G-150 under non-denaturing conditions, mass spectrometry (matrix-assisted laser desorption/ionization/time of flight technique) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 82.4 kDa, 42.0 kDa, and 39.8 kDa, respectively, indicating dimer as the active form. The optimum pH value was 9.2. The enzyme activity was inhibited by aminooxyacetate and beta-chloro-L-alanine both compounds reacting with the carbonyl group of pyridoxal phosphate. The enzyme's transaminating activity with L-alanine and glyoxylate as substrates was approximately 55% of that observed with L-serine and glyoxylate. The lower Km value (1.25 mM) for L-alanine, compared with that of other plant SGATs, and the kcat/Km(Ala) ratio being approximately 2-fold higher than kcat/Km(Ser) suggested that, during photorespiration, Ala and Ser are used by Arabidopsis SGAT with equal efficiency as amino group donors for glyoxylate. The equilibrium constant (Keq), derived from the Haldane relation, for the transamination reaction between L-serine and glyoxylate with the formation of hydroxypyruvate and glycine was 79.1, strongly favoring glycine synthesis. However, it was accompanied by a low Km value of 2.83 mM for glycine. A comparison of some kinetic properties of the studied enzymes with the recombinant Arabidopsis SGATs previously obtained revealed substantial differences. The ratio of the velocity of the transamination reaction with L-alanine and glyoxylate as substrates versus that with L-serine and glyoxylate was 1:1.8 for the native enzyme, whereas it was 1:7 for the recombinant SGAT. Native SGAT showed a much lower Km value for L-alanine compared to the recombinant enzyme.

Functional Analysis of NtZIP4B and Zn Status-Dependent Expression Pattern of Tobacco ZIP Genes.

Tobacco is frequently considered as a plant useful for phytoremediation of metal-contaminated soil, despite the mechanisms for regulation of uptake and accumulation being largely unknown. Here we cloned and characterized a new tobacco Zn and Cd transporter NtZIP4B from the ZIP family (ZRT-IRT-Like proteins). It complemented the Zn-uptake defective yeast mutant zrt1zrt2, and rendered the wild type DY1457 yeast more sensitive to Cd. Bioinformatic analysis and transient expression of the NtZIP4B-GFP fusion protein in tobacco leaves indicated its localization to the plasma membrane. Real-time q-PCR based analysis showed that it is expressed in all vegetative organs with the highest level in leaves. The Zn status determined transcript abundance; NtZIP4B was upregulated by Zn-deficiency and downregulated by Zn excess. At the tissue level, in roots NtZIP4B is expressed in the vasculature of the middle part of the roots and in surrounding tissues including the root epidermis; in leaves primarily in the vasculature. Bioinformatic analysis identified two copies of ZIP4 in tobacco, NtZIP4A and NtZIP4B with 97.57% homology at the amino acid level, with the same expression pattern for both, indicating a high degree of functional redundancy. Moreover, the present study provides new insights into the coordinated function of NtZIP1, NtZIP2, NtZIP4, NtZIP5, NtZIP8, NtIRT1, and NtIRT1-like in response to low-to-high Zn status. Leaves were the major site of NtZIP4, NtZIP5, and NtZIP8 expression, and roots for NtZIP1, NtZIP2, NtIRT1, and NtIRT1-like. Contrasting expression level in the apical and basal root parts indicates distinct roles in root-specific processes likely contributing to the regulation of Zn root-to-shoot translocation. In summary, new insight into the role of ZIP genes in Zn homeostasis pointing to their overlapping and complementary functions, offers opportunities for strategies to modify Zn and Cd root/shoot partition in tobacco.

Contribution of NtZIP1-Like to the Regulation of Zn Homeostasis.

Tobacco has frequently been suggested as a candidate plant species for use in phytoremediation of metal contaminated soil but knowledge on the regulation of its metal-homeostasis is still in the infancy. To identify new tobacco metal transport genes that are involved in Zn homeostasis a bioinformatics study using the tobacco genome information together with expression analysis was performed. Ten new tobacco metal transport genes from the ZIP, NRAMP, MTP, and MRP/ABCC families were identified with expression levels in leaves that were modified by exposure to Zn excess. Following exposure to high Zn there was upregulation of NtZIP11-like, NtNRAMP3, three isoforms of NtMTP2, three MRP/ABCC genes (NtMRP5-like, NtMRP10-like, and NtMRP14 like) and downregulation of NtZIP1-like and NtZIP4. This suggests their involvement in several processes governing the response to Zn-related stress and in the efficiency of Zn accumulation (uptake, sequestration, and redistribution). Further detailed analysis of NtZIP1-like provided evidence that it is localized at the plasma membrane and is involved in Zn but not Fe and Cd transport. NtZIP1-like is expressed in the roots and shoots, and is regulated developmentally and in a tissue-specific manner. It is highly upregulated by Zn deficiency in the leaves and the root basal region but not in the root apical zone (region of maturation and absorption containing root hairs). Thus NtZIP1-like is unlikely to be responsible for Zn uptake by the root apical region but rather in the uptake by root cells within the already mature basal zone. It is downregulated by Zn excess suggesting it is involved in a mechanism to protect the root and leaf cells from accumulating excess Zn.

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 10µM 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 0.25µM 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.

Biochemical characterization and kinetic properties of alanine aminotransferase homologues partially purified from wheat (Triticum aestivum L.).

Four homologues of alanine aminotransferase have been isolated from shoots of wheat seedlings and purified by saline precipitation, gel filtration, preparative electrophoresis and anion exchange chromatography on Protein-Pak Q 8HR column attached to HPLC. Alanine aminotransferase 1 (AlaAT1) and 2 (AlaAT2) were purified 303- and 452-fold, respectively, whereas l-glutamate: glyoxylate aminotransferase 1 (GGAT1) and 2 (GGAT2) were purified 485- and 440-fold, respectively. Consistent inhibition of AlaAT (EC 2.6.1.2) and GGAT (EC 2.6.1.4) activities by p-hydroxymercuribenzoate points on participation of cysteine residues in the enzyme activity. The molecular weight of AlaAT1 and AlaAT2 was estimated to be 65kDa and both of them are monomers in native state. Nonsignificant differences between K(m) using alanine as substrate and catalytic efficiency (k(cat)/K(m)) for l-alanine in reaction with 2-oxoglutarate indicate comparable kinetic constants for AlaAT1 and AlaAT2. Similar kinetic constants for l-alanine in reaction with 2-oxoglutarate and for l-glutamate in reaction with pyruvate for all four homologues suggest equally efficient reaction in both forward and reverse directions. GGAT1 and GGAT2 were able to catalyze transamination between l-glutamate and glyoxylate, l-alanine and glyoxylate and reverse reactions between glycine and 2-oxoglutarate or pyruvate. Both GGATs also consisted of a single subunit with molecular weight of about 50kDa. The estimated K(m) for GGAT1 (3.22M) and GGAT2 (1.27M) using l-glutamate as substrate was lower in transamination with glyoxylate than with pyruvate (9.52 and 9.09mM, respectively). Moreover, distinctively higher values of catalytic efficiency for l-glutamate in reaction with glyoxylate than for l-glutamate in reaction with pyruvate confirm involvement of these homologues into photorespiratory metabolism.