SHINE clade of ERF transcription factors: A significant player in abiotic and biotic stress tolerance in plants.

SHINE (SHN) clade transcription factors (TFs) represents a subfamily of APETALA2/ethylene-responsive factor (AP2/ERF) proteins. The latter, is characterized by its responsiveness to the phytohormone ethylene and the presence of AP2 DNA-binding domain. They are involved in many biological processes and in responses to different environmental constraints. SHN TFs were among the first identified regulators of cuticle formation. Cuticle plays crucial role in plant tolerance to drought, salinity and high temperature as well as in defense against pathogens. In addition, SHN were shown to be involved in the regulation of stomatal development which influences resistance to drought and diseases. Interestingly, recent studies have also shown that SHN TFs are involved in mediating the beneficial effects of arbuscular mycorrhizal fungi (AMF) as well as disease resistance conferred by nanoparticles. To fulfill their roles, SHN TFs are controlled upstream by other TFs and they control, in their turn, different downstream genes. In this review, we highlight the role of SHN TFs in different abiotic and biotic stresses through their involvement in cuticle biosynthesis, stomatal development and molecular regulation of biochemical and physiological traits. In addition, we discuss the regulation of SHN TFs by plant hormones and their influence on hormone biosynthesis and signaling pathways. Knowledge of this complex regulation can be put into contribution to increase multiple abiotic stress tolerances through transgenesis, gene editing and classical breeding.

The ethylene-responsive transcription factor of durum wheat, TdSHN1, confers cadmium, copper, and zinc tolerance to yeast and transgenic tobacco plants.

Cadmium (Cd), copper (Cu), and zinc (Zn) are among the most common heavy metals (HMs) present in polluted soils. While some HMs are required for key biological processes, they are toxic when present in excess. This toxicity damages plant health, decreases crop yields, and can impact human health via the food chain. For example, durum wheat is a staple food that is known to accumulate Cd when grown on polluted soils. Plant response to HM stress is complex and involves several transcription factors (TFs) among which members of the ERF family. Although roles of SHINE-type ERF transcription factors in abiotic stress tolerance have been thoroughly investigated, there is little information concerning their role in HM stress tolerance. In the present study, we investigated the role of durum wheat TdSHN1 TF in HM response and tolerance. Results showed that TdSHN1 expression was strongly induced by Cd, Cu, and Zn in durum wheat seedlings. In addition, TdSHN1 gene promoter directed HM-inducible GUS gene expression in transgenic tobacco. Overexpression of TdSHN1 encoding cDNA in transgenic yeast and tobacco conferred Cd, Cu, and Zn tolerances. Interestingly, transgenic tobacco lines exhibited longer roots and greater biomass accumulation, retained more chlorophyll, and produced less ROS than WT plants, when subjected to excess HMs. In addition, transgenic tobacco lines had higher activities of ROS-scavenging enzymes (SOD and CAT) which might have contributed to their HM tolerance. This study suggested that TdSHN1 is a potential candidate for improving HM tolerance in plants and phytoremediation of HM-contaminated soils.

Significance of vacuolar proton pumps and metal/H+ antiporters in plant heavy metal tolerance.

Soil and water are among the most valuable resources on earth. Unfortunately, their contamination with heavy metals has become a global problem. Heavy metals are not biodegradable and cannot be chemically degraded; therefore, they tend to accumulate in soils or to be transported by streaming water and contaminate both surface and groundwater. Cadmium (Cd) has no known biological function but is one of the most toxic metals. It represents a serious environmental concern since its accumulation in soils is associated with health risks to plants, animals and humans. On the other hand, copper (Cu) and zinc (Zn) are heavy metals that are indispensable to plants but become toxic when their concentration in soils exceeds a certain optimal level. Plants have evolved many mechanisms to cope with heavy metal toxicity; vacuolar sequestration is one of them. Vacuolar sequestration can be achieved through either phytochelatin-dependent or phytochelatin-independent pathways. Most of the transgenic plants meant for phytoremediation described in the literature result from the manipulation of genes involved in the phytochelatin-dependent pathway. However, recent evidence has emerged to support the importance of the phytochelatin-independent pathway in heavy metal sequestration into the vacuole, with metal/H+ antiporters and proton pumps playing an important role. In this review, the importance of vacuolar proton pumps and metal/H+ antiporters transporting Cd, Cu, and Zn is discussed. In addition, the recent advances in the production of transgenic plants with potential application in phytoremediation and food safety through the manipulation of genes encoding V-PPase proton pumps is described.

The barley SHN1-type transcription factor HvSHN1 imparts heat, drought and salt tolerances in transgenic tobacco.

The APETAL2/Ethylene Responsive Factor (AP2/ERF) family was the subject of intensive research which led to the identification of several members involved in different stress responses such as salinity, drought and high temperature. The SHN/WIN clade of AP2/ERF participates in many important processes such as cutin and wax biosynthesis, ethylene signaling and gene expression. Here, we report the functional analysis of SHN1-type transcription factor, HvSHN1, from barely. The overexpression of HvSHN1 under the control of the duplicated 35S promoter in transgenic tobacco plants improved tolerance to salt, water stress and heat stress. Transgenic lines exhibited altered permeability of the cuticle and decreased stomatal density. Under heat stress, HvSHN1 transgenic lines exhibited higher superoxide dismutase (SOD) and catalase (CAT) activity and lower MDA and H2O2 contents than did WT. The overexpression of HvSHN1 upregulated different genes involved in osmotic stress, oxidative stress, sugar metabolism, and wax biosynthesis. To understand the involvement of HvSHN1 in heat stress tolerance, promoter regions of two tobacco genes homologous to Arabidopsis genes HSP90.1 and RAP2.6 were analyzed and DRE cis-elements; binding sites of HvSHN1, were found. Interaction network of HvSHN1, predicted using STRING software, contained proteins with predicted functions related to lipids metabolism and a gene encoding Cyclin-Dependent Kinase. These results suggest that HvSHN1 is an interesting candidate for the improvement of abiotic stress tolerance especially in the context of climate change.

Expression of V-PPase proton pump, singly or in combination with a NHX1 transporter, in transgenic tobacco improves copper tolerance and accumulation.

One of the most important strategies evolved by plants to tolerate heavy metals (HMs) is their sequestration into the vacuole. Recent studies have demonstrated that Cu sequestration into vacuole is dependent on the electrochemical gradient generated by vacuolar proton pumps: the V-H+-PPase and the V-H+-ATPase. In a previous study, we demonstrated that co-expression of V-H+-PPase and a sodium/proton antiporter genes, isolated from wheat, in transgenic tobacco plants significantly increases both H+ pumping activity of the endogenous V-H+-ATPase and V-H+-PPase compared to wild-type (WT) plants, all grown in the absence of stress. In the present study, we evaluated the effect of expression, in tobacco, of vacuolar proton pump, TaVP1, singly or in combination with sodium/proton antiporter, TaNHXS1, on copper (Cu) tolerance and accumulation. Results showed that, when subjected to Cu stress, TaVP1 single transgenic tobacco lines exhibited a more robust root system, greater biomass production, less chlorophyll loss, lower MDA and H2O2 production, and higher catalase activity and accumulated more Cu than did WT. Interestingly, double transgenic tobacco lines exhibited the best Cu tolerance and accumulation than either of the single TaVP1 transgenic lines or WT plants, when subjected to excess Cu. In fact, double transgenic lines accumulated 2.5-fold and 1.9-fold more Cu than did WT and single TaVP1 lines, respectively. Thus, these results clearly demonstrate the usefulness of expression of vacuolar proton pump alone or in combination with sodium/proton antiporter as novel strategy for Cu phytoremediation.

Combination of the endogenous promoter-intron significantly improves salt and drought tolerance conferred by TdSHN1 transcription factor in transgenic tobacco.

Recent years have witnessed a renewed interest in introns as a tool to increase gene expression. We previously isolated TdSHN1 gene encoding a transcription factor in durum wheat. Here we show that TdSHN1 intron contains many CT-stretches and the motif CGATT known to be important for IME. When subjected to bioinformatics analysis using IMEter software, TdSHN1 intron obtained a score of 17.04 which indicates that it can moderately enhance gene expression. TdSHN1 gene including its intron was placed under the control of TdSHN1 endogenous salt and drought-inducible promoter or the constitutive 35S promoter and transferred into tobacco. Transgenic lines were obtained and designated gD (with 35S promoter) and PI (with native promoter). A third construct was also used in which intron-less cDNA was driven by the 35S promoter (cD lines). Results showed that, gD lines exhibited lower stomatal density than cD lines. When subjected to drought and salt stresses, gD lines outperformed intron-less cD lines and WT. Indeed, gD lines exhibited longer roots, higher biomass production, retained more chlorophyll, produced less ROS and MDA and had higher antioxidant activity. qRT-PCR analysis revealed that gD lines had higher TdSHN1 expression levels than cD lines. In addition, expression of ROS-scavengering, stress-related and wax biosynthesis tobacco genes was higher in gD lines compared to cD lines and WT. Interestingly, under stress conditions, PI transgenic lines showed higher TdSHN1 expression levels and outperformed gD lines. These results suggest that TdSHN1 intron enhances gene expression when used alone or in combination with TdSHN1 endogenous promoter.

Ethylene Response Factors (ERF) are differentially regulated by different abiotic stress types in tomato plants.

Plants are sessile organisms, hence to face environmental constrains they developed strategies that rely on the activation of stress-response genes under the control of specific transcription factors. The plant hormone ethylene mediates physiological, developmental and stress responses through the activation of Ethylene Response Factors (ERFs) which belong to a large multigene family of transcription factors. While an increasing number of studies supports the involvement of ERFs in abiotic stress responses, so far the specific role of ERF family members in different abiotic stress conditions remains unexplored. The present work investigates the expression profile of a set of ERFs, representative of different ERF types, in tomato plants subjected to cold, heat, salt, drought and flooding conditions. The study revealed that a group of ERFs is preferentially associated with cold and heat stress responses while another set is expressed in response to salt, water and flooding stresses. Transactivation assays indicated that ERFs can regulate the expression of abiotic stress genes regardless of whether or not they harbor conserved GCC or DRE cis-elements in their promoter region. The outcome of the study provides clue on which ERFs should be targeted when aiming to improve adaptation to a particular stress type.

Molecular cloning and characterization of novel WIN1/SHN1 ethylene responsive transcription factor HvSHN1 in barley (Hordeum vulgare L.).

Barley (Hordeum vulgare L.) is the fourth major cereal crop and shows high adaptive capabilities to diverse environments. Thus, it might represent a potential reservoir of novel genes to improve abiotic stress tolerance. In this study, a novel AP2/ERF transcription factor gene designated as HvSHN1 was isolated from barley. Protein sequence analysis showed that the HvSHN1 protein contained a nuclear localization signal and the conserved AP2/ERF domain. Phylogenetic analysis showed that HvSHN1 belongs to the group Va protein in the ERF subfamily which contains the Arabidopsis genes (SHN1, 2 and 3) and the wheat gene TdSHN1 with which it has 94.7% protein sequence identity. Expression profile analysis revealed that HvSHN1 is strongly induced by heat, cold, salt and drought. Transient expression using tobacco BY-2 protoplast coupled to confocal microscopy analysis revealed that HvSHN1 is exclusively targeted to the nucleus. Interestingly, when constitutively expressed in transgenic tobacco, HvSHN1 up-regulated stress responsive genes known to harbor GCC or DRE motif in their promoter regions. Therefore, HvSHN1 might represent a potential candidate for improvement of abiotic stress tolerance in economically important crops.

Co-expression of vacuolar Na(+)/H(+) antiporter and H(+)-pyrophosphatase with an IRES-mediated dicistronic vector improves salinity tolerance and enhances potassium biofortification of tomato.

Potassium (K) deficiency is a worldwide problem. Thus, the K biofortification of crops is needed to enhance human nutrition. Tomato represents an ideal candidate for such biofortification programs thanks to its widespread distribution and its easy growth on a commercial scale. However, although tomato is moderately tolerant to abiotic stresses, the crop losses due to salinity can be severe. In this study, we generated transgenic tomato plants over-expressing a Na(+)-K(+)/H(+) exchanger gene (TNHXS1), singly or with H(+)-pyrophosphatase (H(+)-PPiase) gene using a bicistronic construct. Transgenic tomato lines co-expressing both genes (LNV) significantly showed higher salinity tolerance than the wild-type (WT) plans or those expressing the TNHXS1 gene alone (LN). Indeed, under salt stress conditions, double transgenic plants produced higher biomass and retained more chlorophyll and catalase (CAT) activity. In addition, they showed earlier flowering and produced more fruits. To address K deficiencies in humans, an increase of 50% in K content of vegetable products was proposed. In this study, ion content analysis revealed that, under salt stress, fruits from double transgenic plants accumulated 5 times more potassium and 9 times less sodium than WT counterparts. Interestingly, the ionomic analysis of tomato fruits also revealed that LNV had a distinct profile compared to WT and to LN plants. Indeed, LNV fruits accumulated less Fe(2+), Ca(2+), Mg(2+) and Zn(2+), but more Mn(2+). This study demonstrates the effectiveness of bicistronic constructs as an important tool for the enhancement of biofortification and salt stress tolerance in crops.

Isolation and molecular characterization of a novel WIN1/SHN1 ethylene-responsive transcription factor TdSHN1 from durum wheat (Triticum turgidum. L. subsp. durum).

Over the last decade, APETALA2/Ethylene Responsive Factor (AP2/ERF) proteins have become the subject of intensive research activity due to their involvement in a variety of biological processes. This research led to the identification of AP2/ERF genes in many species; however, little is known about these genes in durum wheat, one of the most important cereal crops in the world. In this study, a new member of the AP2/ERF transcription factor family, designated TdSHN1, was isolated from durum wheat using thermal asymetric interlaced PCR (TAIL-PCR) method. Protein sequence analysis showed that TdSHN1 contained an AP2/ERF domain of 63 amino acids and a putative nuclear localization signal (NLS). Phylogenetic analysis showed that TdSHN1 belongs to a group Va protein in the ERF subfamily which contains the Arabidopsis ERF proteins (SHN1, SHN2, and SHN3). Expression of TdSHN1 was strongly induced by salt, drought, abscisic acid (ABA), and cold. In planta, TdSHN1 protein was able to activate the transcription of GUS reporter gene driven by the GCC box and DRE element sequences. In addition, TdSHN1 was targeted to the nucleus when transiently expressed in tobacco epidermal cells. In transgenic yeast, overexpression of TdSHN1 increased tolerance to multiple abiotic stresses. Taken together, the results showed that TdSHN1 encodes an abiotic stress-inducible, transcription factor which confers abiotic stress tolerance in yeast. TdSHN1 is therefore a promising candidate for improvement of biotic and abiotic stress tolerance in wheat as well as other crops.

A constitutively active form of a durum wheat Na⁺/H⁺ antiporter SOS1 confers high salt tolerance to transgenic Arabidopsis.

The SOS signaling pathway has emerged as a key mechanism in preserving the homeostasis of Na⁺ and K⁺ under saline conditions. We have recently identified and functionally characterized, by complementation studies in yeast, the gene encoding the durum wheat plasma membrane Na⁺/H⁺ antiporter (TdSOS1). To extend these functional studies to the whole plant level, we complemented Arabidopsis sos1-1 mutant with wild-type TdSOS1 or with the hyperactive form TdSOS1∆972 and compared them to the Arabidopsis AtSOS1 protein. The Arabidopsis sos1-1 mutant is hypersensitive to both Na⁺ and Li⁺ ions. Compared with sos1-1 mutant transformed with the empty binary vector, seeds from TdSOS1 or TdSOS1∆972 transgenic plants had better germination under salt stress and more robust seedling growth in agar plates as well as in nutritive solution containing Na⁺ or Li⁺ salts. The root elongation of TdSOS1∆972 transgenic lines was higher than that of Arabidopsis sos1-1 mutant transformed with TdSOS1 or with the endogenous AtSOS1 gene. Under salt stress, TdSOS1∆972 transgenic lines showed greater water retention capacity and retained low Na⁺ and high K⁺ in their shoots and roots. Our data showed that the hyperactive form TdSOS1∆972 conferred a significant ionic stress tolerance to Arabidopsis plants and suggest that selection of hyperactive alleles of the SOS1 transport protein may pave the way for obtaining salt-tolerant crops.

Phytoremediation potential of Arabidopsis thaliana, expressing ectopically a vacuolar proton pump, for the industrial waste phosphogypsum.

Phosphogypsum (PG) is a by-product of the phosphorus-fertiliser industry and represents an environmental concern since it contains pollutants such as cadmium (Cd). We have recently shown that the overexpression of a proton pump gene (TaVP1) in transgenic tobacco (Nicotiana tabacum) led to an enhanced Cd tolerance and accumulation. The aim of this study was to evaluate the potential of transgenic Arabidopsis thaliana plants harbouring the TaVP1 gene to phytoremediate phosphogypsum. A pot experiment was carried out under greenhouse conditions. Transgenic A. thaliana plants harbouring the TaVP1 gene were grown on various substrates containing phosphogypsum (0, 25, 50 and 100 %) for 40 days. At the end of the growth period, we examined the growth (germination, root length, fresh weight) and physiological parameters (chlorophyll and protein contents, catalase activity and proteolysis) as well as the cadmium, Mg, Ca, and P contents of the A. thaliana plants. In order to evaluate Cd tolerance of the A. thaliana lines harbouring the TaVP1 gene, an in vitro experiment was also carried out. One week-old seedlings were transferred to Murashige and Skoog agar plates containing various concentrations of cadmium; the germination, total leaf area and root length were determined. The growth and physiological parameters of all A. thaliana plants were significantly altered by PG. The germination capacity, root growth and biomass production of wild-type (WT) plants were more severely inhibited by PG compared with the TaVP1 transgenic A. thaliana lines. In addition, TaVP1 transgenic A. thaliana plants maintained a higher antioxidant capacity than the WT. Interestingly, elemental analysis of leaf material derived from plants grown on PG revealed that the transgenic A. thaliana line accumulated up to ten times more Cd than WT. Despite its higher Cd content, the transgenic A. thaliana line performed better than the WT counterpart. In vitro evaluation of Cd tolerance showed that TaVP1 transgenic A. thaliana lines were more Cd-tolerant than the WT plants. These results suggested that ectopic expression of a vacuolar proton pump in A. thaliana plants can lead to various biotechnological applications including the phytoremediation of industrial wastes.

Expression of wheat Na(+)/H(+) antiporter TNHXS1 and H(+)- pyrophosphatase TVP1 genes in tobacco from a bicistronic transcriptional unit improves salt tolerance.

Abiotic stress tolerance of plants is a very complex trait and involves multiple physiological and biochemical processes. Thus, the improvement of plant stress tolerance should involve pyramiding of multiple genes. In the present study, we report the construction and application of a bicistronic system, involving the internal ribosome entry site (IRES) sequence from the 5'UTR of the heat-shock protein of tobacco gene NtHSF-1, to the improvement of salt tolerance in transgenic tobacco plants. Two genes from wheat encoding two important vacuolar ion transporters, Na(+)/H(+) antiporter (TNHXS1) and H(+)-pyrophosphatase (TVP1), were linked via IRES to generate the bicistronic construct TNHXS1-IRES-TVP1. Molecular analysis of transgenic tobacco plants revealed the correct integration of the TNHXS1-IRES-TVP1construct into tobacco genome and the production of the full-length bicistronic mRNA from the 35S promoter. Ion transport analyses with tonoplast vesicles isolated from transgenic lines confirmed that single-transgenic lines TVP1cl19 and TNHXS1cl7 had greater H(+)-PPiase and Na(+)/H(+) antiport activity, respectively, than the WT. Interestingly, the co-expression of TVP1 and TNHXS1 increased both Na(+)/H(+) antiport and H(+)-PPiase activities and induced the H(+) pumping activity of the endogenous V-ATPase. Transgenic tobacco plants expressing TNHXS1-IRES-TVP1 showed a better performance than either of the single gene-transformed lines and the wild type plants when subjected to salt treatment. In addition, the TNHXS1-IRES-TVP1 transgenic plants accumulated less Na(+) and more K(+) in their leaf tissue than did the wild type and the single gene-transformed lines. These results demonstrate that IRES system, described herein, can co-ordinate the expression of two important abiotic stress-tolerance genes and that this expression system is a valuable tool for obtaining transgenic plants with improved salt tolerance.

Transgenic tobacco plants expressing ectopically wheat H⁺-pyrophosphatase (H⁺-PPase) gene TaVP1 show enhanced accumulation and tolerance to cadmium.

Cadmium (Cd) is considered an extremely significant pollutant due to its high toxicity to many organisms. Plants have evolved several mechanisms to cope with Cd, the most important of which is vacuolar sequestration. Cadmium can be directly transported into vacuoles by cations/H(+) exchangers, such as CAXs, which are energized by the pH gradient established by proton pumps. A cDNA (TaVP1) encoding wheat vacuolar H(+)-pyrophosphatase (V-H-PPase) was ectopically expressed in transgenic tobacco to evaluate whether this proton pump expression would enhance Cd tolerance and accumulation in planta. When TaVP1-expressing plants were exposed to various concentrations of Cd, they were found to be more tolerant to Cd compared to wild type plants. Cadmium accumulation in the plant biomass in transgenic plants was higher than that in wild type plants. To the best of our knowledge, this is the first report on the potential for enhancing proton pump expression as a strategy to improve Cd tolerance and accumulation in plants.

Overexpression of wheat dehydrin DHN-5 enhances tolerance to salt and osmotic stress in Arabidopsis thaliana.

Late Embryogenesis Abundant (LEA) proteins are associated with tolerance to water-related stress. A wheat (Triticum durum) group 2 LEA proteins, known also as dehydrin (DHN-5), has been previously shown to be induced by salt and abscisic acid (ABA). In this report, we analyze the effect of ectopic expression of Dhn-5 cDNA in Arabidopsis thaliana plants and their response to salt and osmotic stress. When compared to wild type plants, the Dhn-5 transgenic plants exhibited stronger growth under high concentrations of NaCl or under water deprivation, and showed a faster recovery from mannitol treatment. Leaf area and seed germination rate decreased much more in wild type than in transgenic plants subjected to salt stress. Moreover, the water potential was more negative in transgenic than in wild type plants. In addition, the transgenic plants have higher proline contents and lower water loss rate under water stress. Also, Na(+) and K(+) accumulate to higher contents in the leaves of the transgenic plants. Our data strongly support the hypothesis that Dhn-5, by its protective role, contributes to an improved tolerance to salt and drought stress through osmotic adjustment.

The de novo designed nutritive protein MB-1Trp does not resist proteolytic degradation in alfalfa leaves.

We previously reported on a de novo designed protein "milk bundle-1Trp" (MB-1Trp) as a source of selected essential amino acids (EAA) for ruminant feeding. Here, we attempt to express this de novo designed protein in alfalfa. The microbial version of the gene encoding the protein was modified in order to achieve two expression strategies in transgenic alfalfa plants. Chimeric MB-1Trp genes alone or fused to a signal peptide and an endoplasmic reticulum retention sequence were introduced into alfalfa via Agrobacterium-mediated transformation. Polymerase chain reaction and reverse transcriptase polymerase chain reaction analysis performed on individual transgenic lines demonstrated that the MB-1Trp gene was correctly integrated and transcribed into mRNA. However, under our conditions, it was impossible to detect MB-1Trp protein expression in any of the transgenic plants analyzed. In order to assess MB-1Trp stability in alfalfa, Escherichia coli-derived MB-1Trp was incubated with proteins extracted from leaves of a non-transgenic plant. This study revealed a high susceptibility of mature MB-1Trp to alfalfa proteases, which may have contributed to its lack of accumulation.

Controlling proteolytic degradation of the methionine enriched MB-1Trp protein

Protein design is currently used for the creation of new proteins with desirable traits, which include a superior nutritional value. One of the challenges of protein design in this area is to achieve the production of stable native-like proteins that resist the proteolytic pressure of the organism used for its production (the bioreactor). We report here the identification of a specific peptide bond sensitive to E. coli proteolysis in the designer protein MB-1Trp. In an attempt to reduce proteolysis, we have created a MB-1TrpHis gene library in which the two amino acids surrounding the peptide bond, N44 and L45, were randomized using degenerated oligonucleotides. The initial characterization of MB-1TrpHis N44E/L45V and MB-1TrpHis N44E/L45M, 2 variants of the library that were more resistant than the parent protein, was performed in order to investigate the nature of the mutants' resistance. Our results suggest that the mutants behaved like MB-1Trp regarding folding and thermal stability, and that proteolytic resistance is due to the elimination of the protease recognition site.