Zinc (Zn) shortage is a common micronutrient deficiency affecting plants worldwide, while Zn toxicity may occur when this metal is in excess. Ectomycorrhizal (ECM) fungi are known to be able to modulate the transfer of macro- and microelements, among them Zn, to the plant. However, the underlying mechanisms are not well understood. We identified the HcZnT2 gene from the ECM fungus Hebeloma cylindrosporum, encoding a member of the Cation Diffusion Facilitator (CDF) family including Zn transporters, and analyzed its transcriptional regulation, the transport function by yeast complementation experiments, and its subcellular localization using a GFP fusion protein in yeast. HcZnT2 is highly induced during mycorrhization of Pinus pinaster, and upregulated in presence of the host plant root even without any direct contact. However, HcZnT2 is repressed by Zn excess conditions. By functional expression in yeast, our results strongly support the ability of HcZnT2 to transport Zn and, to a lesser extent, manganese. HcZnT2 localization was associated with the endoplasmic reticulum of yeast. Mycorrhizal gene activation at low external Zn suggests that the Zn transporter HcZnT2 might be important for the early establishment of the ECM symbiosis during Zn deficiency, rather than under Zn excess. HcZnT2 arises as an extremely remarkable candidate playing a key role in Zn homeostasis and regulation in ectomycorrhiza.

Ionic and metal toxicity in plants is still a global problem for the environment, agricultural productivity and ultimately poses human health threats when these metal ions accumulate in edible organs of plants. Metal and ion transport from cytosol to the vacuole is considered an important component of metal and ion tolerance and a plant\'s potential utility in phytoremediation. Finger millet (Eleusine coracana) is an orphan crop but has prominent nutritional value in comparison to other cereals. Previous transcriptomic studies suggested that one of the calcium/proton exchanger (EcCAX3) is strongly upregulated during different developmental stages of spikes development in plant. This finding led us to speculate that high calcium accumulation in the grain might be because of CAX3 function. Moreover, phylogenetic analysis shows that EcCAX3 is more closely related to foxtail millet, sorghum and rice CAX3 protein. To decipher the functional role of EcCAX3, we have adopted complementation of yeast triple mutant K677 (Δpmc1Δvcx1Δcnb1), which has defective calcium transport machinery. Furthermore, metal tolerance assay shows that EcCAX3 expression conferred tolerance to different metal stresses in yeast. The gain-of-function study suggests that EcCAX3 overexpressing Arabidopsis plants shows better tolerance to higher concentration of different metal ions as compared to wild type Col-0 plants. EcCAX3-overexpression transgenic lines exhibits abundance of metal transporters and cation exchanger transporter transcripts under metal stress conditions. Furthermore, EcCAX3-overexpression lines have higher accumulation of macro- and micro-elements under different metal stress. Overall, this finding highlights the functional role of EcCAX3 in the regulation of metal and ion homeostasis and this could be potentially utilized to engineer metal fortification and generation of stress tolerant crops in near future.

Loss of function of the STRADA gene, an upstream mTOR inhibitor, causes a rare neurodevelopmental disorder characterized by polyhydramnios, megalencephaly, and symptomatic epilepsy (PMSE syndrome). Patients display a homogeneous phenotype including early-onset drug-resistant epilepsy, severe psychomotor delay, multisystemic comorbidities, and increased risk of premature death. The administration of sirolimus, an mTOR inhibitor, is helpful in controlling seizures in this syndrome. We report the electroclinical phenotype of two novel patients and the development of a yeast model to validate the pathogenicity of missense variants. Patient 1 harbored a missense STRADA variant and had a peculiar electroclinical phenotype with a relatively mild epilepsy course. Patient 2 harbored a truncating STRADA variant and showed a typical PMSE phenotype and a favorable response to early treatment with sirolimus. When we modeled the p.(Ser264Arg) STRADA change in its yeast homolog SPS1, it impaired SPS1 function. The results underlie the importance of a timely molecular diagnosis in these patients and show that yeast is a simple yet effective model to validate the pathogenicity of missense variants.

Prohibitins (PHBs) are highly conserved pleiotropic proteins as they have been shown to mediate key cellular functions. Here, we characterize PHBs encoding putative genes ofPlasmodium falciparum by exploiting different orthologous models. We demonstrated that PfPHB1 (PF3D7_0829200) and PfPHB2 (PF3D7_1014700) are expressed in asexual and sexual blood stages of the parasite. Immunostaining indicated hese proteins as mitochondrial residents as they were found to be localized as branched structures. We further validated PfPHBs as organellar proteins residing in Plasmodium mitochondria, where they interact with each other. Functional characterization was done in Saccharomyces cerevisiae orthologous model by expressing PfPHB1 and PfPHB2 in cells harboring respective mutants. The PfPHBs functionally complemented the yeast PHB1 and PHB2 mutants, where the proteins were found to be involved in stabilizing the mitochondrial DNA, retaining mitochondrial integrity and rescuing yeast cell growth. Further, Rocaglamide (Roc-A), a known inhibitor of PHBs and anti-cancerous agent, was tested against PfPHBs and as an antimalarial. Roc-A treatment retarded the growth of PHB1, PHB2, and ethidium bromide petite yeast mutants. Moreover, Roc-A inhibited growth of yeast PHBs mutants that were functionally complemented with PfPHBs, validating P. falciparum PHBs as one of the molecular targets for Roc-A. Roc-A treatment led to growth inhibition of artemisinin-sensitive (3D7), artemisinin-resistant (R539T) and chloroquine-resistant (RKL-9) parasites in nanomolar ranges. The compound was able to retard gametocyte and oocyst growth with significant morphological aberrations. Based on our findings, we propose the presence of functional mitochondrial PfPHB1 and PfPHB2 in P. falciparum and their druggability to block parasite growth.

In the 1980s, it was found that addition of N-terminal Arg to proteins induces their ubiquitination and degradation by the N-end rule pathway. While this mechanism applies only to the proteins which also have other features of the N-degron (including a closely adjacent Lys that is accessible for ubiquitination), several test substrates have been found to follow this mechanism very efficiently after ATE1-dependent arginylation. Such property enabled researchers to test ATE1 activity in cells indirectly by assaying for the degradation of such arginylation-dependent substrates. The most commonly used substrate for this assay is E. coli beta-galactosidase (beta-Gal) because its level can be easily measured using standardized colorimetric assays. Here, we describe this method, which has served as a quick and easy way to characterize ATE1 activity during identification of arginyltransferases in different species.

Although recent physiological studies demonstrate that flue-cured tobacco preferentially utilizes nitrate ( NO 3 - ) or ammonium nitrate (NH4NO3), and possesses both high- and low-affinity uptake systems for NO 3 - , little is known about the molecular component(s) responsible for acquisition and translocation in this crop. Here we provide experimental data showing that NtNRT1.1B with a 1,785-bp coding sequence exhibited a function in mediating NO 3 - transport associated with tobacco growth on NO 3 - nutrition. Heterologous expression of NtNRT1.1B in the NO 3 - uptake-defective yeast Hp△ynt1 enabled a growth recovery of the mutant on 0.5 mM NO 3 - , suggesting a possible molecular function of NtNRT1.1B in the import of NO 3 - into cells. Transient expression of NtNRT1.1B::green fluorescent protein (GFP) in tobacco leaf cells revealed that NtNRT1.1B targeted mainly the plasma membrane, indicating the possibility of NO 3 - permeation across cell membranes via NtNRT1.1B. Furthermore, promoter activity assays using a GFP marker clearly indicated that NtNRT1.1B transcription in roots may be down-regulated by N starvation and induced by N resupply, including NO 3 - , after 3 days\' N depletion. Significantly, constitutive overexpression of NtNRT1.1B could remarkably enhance tobacco growth by showing a higher accumulation of biomass and total N, NO 3 - , and even NH 4 + in plants supplied with NO 3 - ; this NtNRT1.1B-facilitated N acquisition/accumulation could be strengthened by short-term 15N- NO 3 - root influx assays, which showed 15%-20% higher NO 3 - deposition in NtNRT1.1B-overexpressors as well as a high affinity of NtNRT1.1B for NO 3 - at a K m of around 30-45 µM. Together with the detection of NtNRT1.1B promoter activity in the root stele and shoot-stem vascular tissues, and higher NO 3 - in both xylem exudate and the apoplastic washing fluid of NtNRT1.1B-transgenic lines, NtNRT1.1B could be considered as a valuable molecular breeding target aiming at improving crop N-use efficiency by manipulating the absorption and long-distance distribution/transport of nitrate, thus adding a new functional homolog as a nitrate permease to the plant NRT1 family.

Chrysanthemum (Chrysanthemum morifolium Ramat.) is one of the largest cut flowers in the world. Phosphate transporter Pht1 family member CmPht1;2 protein (CmPT2) plays an important role in response to low-phosphate (LP) stress in chrysanthemum. Post-translational modification (PTM) can modulate the function of proteins in multiple ways. Here, we used yeast and rice systems to study the role of putative PTM in CmPT2 by determining the effect of mutation of key amino acid residues of putative glycosylation, phosphorylation, and myristoylation sites. We chose nine amino acid residues in the putative PTM sites and mutated them to alanine (A) (Cmphts). CmPT2 recovered the growth of yeast strain MB192 under LP conditions. However, G84A, G222A, T239A, Y242A, and N422A mutants could not grow normally under LP conditions. Analysis of phosphorus absorption kinetics showed that the Km of CmPT2 was 65.7 μM. Among the nine Cmphts, the expression of five with larger Km (124.4-397.5 μM) than CmPT2 was further evaluated in rice. Overexpression of CmPT2-OE increased plant height, effective panicle numbers, branch numbers, and yield compared with that of wild type \'Wuyunjing No. 7\' (W7). Overexpression of Cmphts-OE led to decreased plant height and effective panicle numbers compared with that of the CmPT2-OE strain. The Pi content in roots of CmPT2-OE was higher than that of the W7 under both high (normal) phosphate (HP) and LP conditions. However, the Pi content in the leaves and roots was significantly lower in the N422A-OE strain than in the CmPT2-OE strain under both HP and LP conditions. Under LP conditions, the phosphorus starvation response (PSR) genes in CmPT2-OE were inhibited at the transcription level. The expression patterns of phosphorus-related genes in T239A, Y242A, and N422A-OE under LP conditions were different from those of CmPT2-OE. In conclusion, these five post-translational modification residues of CmPT2 play key roles in modulating the function of CmPT2. This work boosters our understanding of the function of phosphate transporters and provides genetic resources for improving the efficiency of phosphorus utilization in crop plants.

In plants, the trehalose biosynthetic pathway plays key roles in the regulation of carbon allocation and stress adaptation. Engineering of the pathway holds great promise to increase the stress resilience of crop plants. The synthesis of trehalose proceeds by a two-step pathway in which a trehalose-phosphate synthase (TPS) uses UDP-glucose and glucose-6-phosphate to produce trehalose-6 phosphate (T6P) that is subsequently dephosphorylated by trehalose-6 phosphate phosphatase (TPP). While plants usually do not accumulate high amounts of trehalose, their genome encodes large families of putative trehalose biosynthesis genes, with many members lacking obvious enzymatic activity. Thus, the function of putative trehalose biosynthetic proteins in plants is only vaguely understood. To gain a deeper insight into the role of trehalose biosynthetic proteins in crops, we assessed the enzymatic activity of the TPS/TPP family from tomato (Solanum lycopersicum L.) and investigated their expression pattern in different tissues as well as in response to temperature shifts. From the 10 TPS isoforms tested, only the 2 proteins belonging to class I showed enzymatic activity, while all 5 TPP isoforms investigated were catalytically active. Most of the TPS/TPP family members showed the highest expression in mature leaves, and promoter-reporter gene studies suggest that the two class I TPS genes have largely overlapping expression patterns within the vasculature, with only subtle differences in expression in fruits and flowers. The majority of tomato TPS/TPP genes were induced by heat stress, and individual family members also responded to cold. This suggests that trehalose biosynthetic pathway genes could play an important role during temperature stress adaptation. In summary, our study represents a further step toward the exploitation of the TPS and TPP gene families for the improvement of tomato stress resistance.

CONCLUSIONS: Plasma membrane-localized AtAVT6D importing aspartic acid can be targeted to develop plants with enhanced osmotic and nitrogen-starvation tolerance. AtAVT6D promoter can be exploited as a stress-inducible promoter for genetic improvements to raise stress-resilient crops. The AtAVT6 family of amino acid transporters in Arabidopsis thaliana has been predicted to export amino acids like aspartate and glutamate. However, the functional characterization of these amino acid transporters in plants remains unexplored. The present study investigates the expression patterns of AtAVT6 genes in different tissues and under various abiotic stress conditions using quantitative Real-time PCR. The expression analysis demonstrated that the member AtAVT6D was significantly induced in response to phytohormone ABA and stresses like osmotic and drought. The tissue-specific expression analysis showed that AtAVT6D was strongly expressed in the siliques. Taking together these results, we can speculate that AtAVT6D might play a vital role in silique development and abiotic stress tolerance. Further, subcellular localization study showed AtAVT6D was localized to the plasma membrane. The heterologous expression of AtAVT6D in yeast cells conferred significant tolerance to nitrogen-deficient and osmotic stress conditions. The Xenopus oocyte studies revealed that AtAVT6D is involved in the uptake of Aspartic acid. While overexpression of AtAVT6D resulted in smaller siliques in Arabidopsis thaliana. Additionally, transient expression studies were performed with the full-length AtAVT6D promoter and its deletion constructs to study the effect of ACGT-N24-ACGT motifs on the reporter gene expression in response to abiotic stresses and ABA treatment. The fluorometric GUS analyses revealed that the promoter deletion construct-2 (Pro.C2) possessing a single copy of ACGT-N24-ACGT motif directed the strongest GUS expression under all the abiotic conditions tested. These results suggest that Pro.C2 can be used as a stress-inducible promoter to drive a significant transgene expression.

The zinc/iron-regulated transporter-like protein (ZIP) family has a crucial role in Zn homeostasis of plants. Although the ZIP genes have been systematically studied in many plant species, the significance of this family in wild emmer wheat (Triticum turgidum ssp. dicoccoides) is not yet well understood. In this study, a genome-wide investigation of ZIPs genes based on the wild emmer reference genome was conducted, and 33 TdZIP genes were identified. Protein structure analysis revealed that TdZIP proteins had 1 to 13 transmembrane (TM) domains and most of them were predicted to be located on the plasma membrane. These TdZIPs can be classified into three clades in a phylogenetic tree. They were annotated as being involved in inorganic ion transport and metabolism. Cis-acting analysis showed that several elements were involved in hormone, stresses, grain-filling, and plant development. Expression pattern analysis indicated that TdZIP genes were highly expressed in different tissues. TdZIP genes showed different expression patterns in response to Zn deficiency and that 11 genes were significantly induced in either roots or both roots and shoots of Zn-deficient plants. Yeast complementation analysis showed that TdZIP1A-3, TdZIP6B-1, TdZIP6B-2, TdZIP7A-3, and TdZIP7B-2 have the capacity to transport Zn. Overexpression of TdZIP6B-1 in rice showed increased Zn concentration in roots compared with wild-type plants. The expression levels of TdZIP6B-1 in transgenic rice were upregulated in normal Zn concentration compared to that of no Zn. This work provides a comprehensive understanding of the ZIP gene family in wild emmer wheat and paves the way for future functional analysis and genetic improvement of Zn deficiency tolerance in wheat.