Differential salt stress resistance in male and female Salix linearistipularis plants: insights from transcriptome profiling and the identification of the 4-hydroxy-tetrahydrodipicolinate synthase gene.

MAIN CONCLUSION: Lysine plays an essential role in the growth differences between male and female S. linearistipularis plants under salt stress. Furthermore, SlDHDPS is identified as a vital gene contributing to the differences in saline-alkali tolerance between male and female plants of S. linearistipularis. Soil salinization is a significant problem that severely restricts agricultural production worldwide. High salinity and low nutrient concentrations consequently prevent the growth of most plant species. Salix linearistipularis is the only woody plant (shrub) naturally distributed in the saline-alkali lands of the Songnen Plain in Northeast China, and it is one of the few plants capable of thriving in soils with extremely high salt and alkaline pH (>9.0) levels. However, insufficient attention has been given to the interplay between salt and nitrogen in the growth and development of S. linearistipularis. Here, the male and female plants of S. linearistipularis were subjected to salt stress with nitrogen-starvation or nitrogen-supplement treatments, and it was found that nitrogen significantly affects the difference in salt tolerance between male and female plants, with nitrogen-starvation significantly enhancing the salt stress tolerance of female plants compared to male plants. Transcriptional analyses showed 66 differentially expressed nitrogen-responsive genes in female and male roots, with most of them showing sexual differences in expression patterns under salinity stress. RNA-seq and RT-qPCR analysis demonstrated that six genes had an opposite salt-induced expression pattern in female and male roots. The expression of the 4-hydroxy-tetrahydrodipicolinate synthase encoding gene (SlDHDPS) in female roots was higher than that in male roots. Further treatment with exogenous lysine could significantly alleviate the inhibitory effect of salt stress on the growth of female and male plants. These results indicate that the SlDHDPS in the nitrogen metabolism pathway is involved in the resistance of S. linearistipularis to salt stress, which lays a foundation for further exploring the mechanism of nitrogen on salt tolerance of S. linearistipularis, and has a significant reference value for saline-alkali land management and sustainable agricultural development.

Unraveling the role of urea hydrolysis in salt stress response during seed germination and seedling growth in Arabidopsis thaliana.

Urea is intensively utilized as a nitrogen fertilizer in agriculture, originating either from root uptake or from catabolism of arginine by arginase. Despite its extensive use, the underlying physiological mechanisms of urea, particularly its adverse effects on seed germination and seedling growth under salt stress, remain unclear. In this study, we demonstrate that salt stress induces excessive hydrolysis of arginine-derived urea, leading to an increase in cytoplasmic pH within seed radical cells, which, in turn, triggers salt-induced inhibition of seed germination (SISG) and hampers seedling growth. Our findings challenge the long-held belief that ammonium accumulation and toxicity are the primary causes of SISG, offering a novel perspective on the mechanism underlying these processes. This study provides significant insights into the physiological impact of urea hydrolysis under salt stress, contributing to a better understanding of SISG.

Genome-wide identification and expression analysis of Ubiquitin-specific protease gene family in maize (Zea mays L.).

BACKGROUND: Ubiquitin-specific proteases (UBPs) are a large family of deubiquitinating enzymes (DUBs). They are widespread in plants and are critical for plant growth, development, and response to external stresses. However, there are few studies on the functional characteristics of the UBP gene family in the important staple crop, maize (Zea mays L.). RESULTS: In this study, we performed a bioinformatic analysis of the entire maize genome and identified 45 UBP genes. Phylogenetic analysis indicated that 45 ZmUBP genes can be divided into 15 subfamilies. Analysis of evolutionary patterns and divergence levels indicated that ZmUBP genes were present before the isolation of dicotyledons, were highly conserved and subjected to purifying selection during evolution. Most ZmUBP genes exhibited different expression levels in different tissues and developmental stages. Based on transcriptome data and promoter element analysis, we selected eight ZmUBP genes whose promoters contained a large number of plant hormones and stress response elements and were up-regulated under different abiotic stresses for RT-qPCR analysis, results showed that these genes responded to abiotic stresses and phytohormones to varying degrees, indicating that they play important roles in plant growth and stress response. CONCLUSIONS: In this study, the structure, location and evolutionary relationship of maize UBP gene family members were analyzed for the first time, and the ZmUBP genes that may be involved in stress response and plant growth were identified by combining promoter element analysis, transcriptome data and RT-qPCR analysis. This study informs research on the involvement of maize deubiquitination in stress response.

Metabolomics analysis reveals the differences between Abrus cantoniensis Hance and Abrus mollis Hance.

BACKGROUND: Abrus cantoniensis Hance. (Ac) and Abrus mollis (Am), two edible and medicinal plants with economic value in southern China, belong to the Abrus genus. Due to its growth characteristics, Am often replaces Ac in folk medicine. However, the latest National Pharmacopeia of China only recommends Ac. The differences in the metabolite composition of the plants are directly related to the differences in their clinical efficacy. RESULTS: The difference in metabolites were analyzed using an untargeted metabolomic approach based on ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC‒ESI‒MS/MS). The roots (R), stems (S) and leaves (L) of the two varieties were examined, and 635 metabolites belonging to 8 classes were detected. A comparative study revealed clear variations in the metabolic profiles of the two plants, and the AmR group had more active ingredients (flavonoids and terpenoids) than the AcR group. The metabolites classified as flavonoids and triterpene saponins showed considerable variations among the various samples. Both Ac and Am had unique metabolites. Two metabolites (isovitexin-2''-xyloside and soyasaponin V) specifically belong to Ac, and nine metabolites (vitexin-2"-O-galactoside, ethyl salicylate, 6-acetamidohexanoic acid, rhein-8-O-glucoside, hederagenin-3-O-glucuronide-28-O-glucosyl(1,2)-glucoside, methyl dioxindole-3-acetate, veratric acid, isorhamnetin-3-O-sophoroside-7-O-rhamnoside, and isorhamnetin-3-O-sophoroside) specifically belong to Am. CONCLUSIONS: The metabolite differences between Ac and Am cause the differences in their clinical efficacy. Our findings serve as a foundation for further investigation of biosynthesis pathways and associated bioactivities and provide guidance for the clinical application of traditional Chinese medicine.

Measurement of pressure discomfort threshold in auricular concha for in-ear wearables design.

In an effort to mitigate the homogenization of in-ear wearables, designers have been focusing on finding new solutions to enhance user comfort. While the concept of pressure discomfort thresholds (PDT) in humans has been applied to product design, research on the auricular concha remains scarce. In this study, we conducted an experiment to measure the PDT at six points in the auricular concha of 80 participants. Our results showed that the tragus was the most sensitive area and that gender, symmetry, and Body Mass Index (BMI)had no significant effect on PDT. Based on these findings, we generated pressure sensitivity maps of the auricular concha to aid in the optimization of in-ear wearable design.

Variation in flavonoid and antioxidant activities of Pyrrosia petiolosa (Christ) Ching from different geographic origins.

Pyrrosia petiolosa (Christ) Ching has both medicinal and health benefits in China. The potential antioxidant activities of P. petiolosa, which are mainly attributed to its flavonoids, have attracted much attention in recent years. The present study aimed to determine the concentration of flavonoid components and evaluate the relative antioxidant activities of P. petiolosa from different geographic origins using a UPLC-MRM-MS-based metabolomics approach. In total, 97 flavonoid components were identified, and their concentrations in the samples from different geographic locations showed significant variation. Thirteen flavonoid components were identified as potential biomarkers for distinguishing between the two major regions, Guizhou (GZ) and Guangxi (GX). The GZ group showed higher total flavonoid content, free radical scavenging activities, and ferric reducing antioxidant power. The well positive correlations were found between the antioxidant capacities and some flavonoid markers. The ecogeographic factors, namely altitude and longitude, play a crucial role in the difference of antioxidant activities and flavonoids concentration. These results indicate that P. petiolosa is rich in flavonoid compounds and is a promising source of natural antioxidants, providing a basis for the quality control of P. petiolosa.

An efficient deep learning based predictor for identifying miRNA-triggered phasiRNA loci in plant.

Phasic small interfering RNAs are plant secondary small interference RNAs that typically generated by the convergence of miRNAs and polyadenylated mRNAs. A growing number of studies have shown that miRNA-initiated phasiRNA plays crucial roles in regulating plant growth and stress responses. Experimental verification of miRNA-initiated phasiRNA loci may take considerable time, energy and labor. Therefore, computational methods capable of processing high throughput data have been proposed one by one. In this work, we proposed a predictor (DIGITAL) for identifying miRNA-initiated phasiRNAs in plant, which combined a multi-scale residual network with a bi-directional long-short term memory network. The negative dataset was constructed based on positive data, through replacing 60% of nucleotides randomly in each positive sample. Our predictor achieved the accuracy of 98.48% and 94.02% respectively on two independent test datasets with different sequence length. These independent testing results indicate the effectiveness of our model. Furthermore, DIGITAL is of robustness and generalization ability, and thus can be easily extended and applied for miRNA target recognition of other species. We provide the source code of DIGITAL, which is freely available at https://github.com/yuanyuanbu/DIGITAL.

Integrated Transcriptome and Metabolome Analysis of Rice Leaves Response to High Saline-Alkali Stress.

Rice (Oryza sativa) is one of the most important crops grown worldwide, and saline-alkali stress seriously affects the yield and quality of rice. It is imperative to elucidate the molecular mechanisms underlying rice response to saline-alkali stress. In this study, we conducted an integrated analysis of the transcriptome and metabolome to elucidate the effects of long-term saline-alkali stress on rice. High saline-alkali stress (pH > 9.5) induced significant changes in gene expression and metabolites, including 9347 differentially expressed genes (DEGs) and 693 differentially accumulated metabolites (DAMs). Among the DAMs, lipids and amino acids accumulation were greatly enhanced. The pathways of the ABC transporter, amino acid biosynthesis and metabolism, glyoxylate and dicarboxylate metabolism, glutathione metabolism, TCA cycle, and linoleic acid metabolism, etc., were significantly enriched with DEGs and DAMs. These results suggest that the metabolites and pathways play important roles in rice's response to high saline-alkali stress. Our study deepens the understanding of mechanisms response to saline-alkali stress and provides references for molecular design breeding of saline-alkali resistant rice.

COPPER: an ensemble deep-learning approach for identifying exclusive virus-derived small interfering RNAs in plants.

Antiviral defenses are one of the significant roles of RNA interference (RNAi) in plants. It has been reported that the host RNAi mechanism machinery can target viral RNAs for destruction because virus-derived small interfering RNAs (vsiRNAs) are found in infected host cells. Therefore, the recognition of plant vsiRNAs is the key to understanding the functional mechanisms of vsiRNAs and developing antiviral plants. In this work, we introduce a deep learning-based stacking ensemble approach, named computational prediction of plant exclusive virus-derived small interfering RNAs (COPPER), for plant vsiRNA prediction. COPPER used word2vec and fastText to generate sequence features and a hybrid deep learning framework, including a convolutional neural network, multiscale residual network and bidirectional long short-term memory network with a self-attention mechanism to enable precise predictions of plant vsiRNAs. Extensive benchmarking experiments with different sequence homology thresholds and ablation studies illustrated the comparative predictive performance of COPPER. In addition, the performance comparison with PVsiRNAPred conducted on an independent test dataset showed that COPPER significantly improved the predictive performance for plant vsiRNAs compared with other state-of-the-art methods. The datasets and source codes are publicly available at https://github.com/yuanyuanbu/COPPER.

Biotinylated subunit of 3-methylcrotonyl-CoA carboxylase encoding gene (AtMCCA) participating in Arabidopsis resistance to carbonate Stress by transcriptome analysis.

Soil salinization is a major factor impacting modern agricultural production, and alkaline soils contain large amounts of NaHCO3. Therefore, understanding plant tolerance to high levels of NaHCO3 is essential. In this study, a transcriptome analysis of shoot and root tissues of wild-type Arabidopsis thaliana was conducted at 0, 4, 12, 24 and 48 h after exposure to a 3 mM NaHCO3 stress. We focused on differentially expressed genes (DEGs) in roots identified in the early stages (4 h and 12 h) of the NaHCO3 stress response that were enriched in GO term, carboxylic acid metabolic process, and utilize HCO3-. Six genes were identified that exhibited similar expression patterns in both the RNA-seq and qRT-PCR data. We also characterized the phenotypic response of AtMCCA-overexpressing plants to carbonate stress, and found that the ability of AtMCCA-overexpressing plants to tolerate carbonate stress was enhanced by the addition of biotin to the growth medium.

Description of AtCAX4 in Response to Abiotic Stress in Arabidopsis.

High-capacity tonoplast cation/H+ antiport in plants is partially mediated by a family of CAX transporters. Previous studies have reported that CAX activity is affected by an N-terminal autoinhibitory region. CAXs may be present as heterodimers in plant cells, and this phenomenon necessitates further study. In this study, we demonstrate that there is an interaction between CAX4 and CAX1 as determined by the use of a yeast two-hybrid system and a bimolecular fluorescence complementation assay. More specifically, the N-terminal of CAX4 interacts with CAX1. We further observed the over-expression and either a single or double mutant of CAX1 and CAX4 in response to abiotic stress in Arabidopsis. These results suggest that CAX1 and CAX4 can interact to form a heterodimer, and the N-terminal regions of CAX4 play important roles in vivo; this may provide a foundation for a deep study of CAX4 function in the future.

Sexual Differences in Physiological and Transcriptional Responses to Salinity Stress of Salix linearistipularis.

Willow (Salix), a dioecious plant, is an important ornamental tree species in the world. Salix linearistipularis, a perennial woody plant species naturally distributed on the Songnen Plain saline-alkali land in northeast China, has a high saline condition. To study the sexual differences of S. linearistipularis in salinity tolerance, the physiological and transcriptional responses to salinity were compared between female and male cuttings. Under salinity stress, the female leaves exhibited higher superoxide dismutase and peroxidase activities and photosynthetic capacity, and lower H2O2 contents than those of male leaves. Under salinity stress, sodium (Na+) accumulation in female leaves was lower than that in the male leaves. The non-invasive micro-test showed that the net Na+ efflux in the salt-treated female roots was higher than that in male roots. Physiological responses revealed that female cuttings were more tolerant than males, which may be mainly due to females having lower leaf Na+ accumulation and higher root Na+ efflux capacity than males. Transcriptional analyses showed that 108 differentially expressed salt-responsive genes were identified in both female and male roots; most of these showed sexual differences in expression patterns under salinity stress. RNA-seq combined with qPCR analysis showed that the salt-induced expression of four Na+/H+ antiporter (NHX) genes (SlNHX3, 5, 6, 7) in female roots was higher than that in male roots. Transcriptional analyses revealed that the higher Na+ efflux capacity in female roots than in male roots may be closely related to the differential expression of salt-responsive genes, especially NHX genes.

Biotin plays an important role in Arabidopsis thaliana seedlings under carbonate stress.

Globally, many saline-alkali soils are rich in NaHCO3 and Na2CO3, which are characterized by a high pH Carbonate stress caused by this kind of soil severely damages plant cells and inhibits plant growth. Biotin and HCO3- participate in the first and rate-limiting reaction of the fatty acid biosynthesis pathway, but whether biotin contributes to plant responses to carbonate stress is unclear. In this study, we revealed that high carbonate and biotin concentrations inhibited Arabidopsis (Arabidopsis thaliana) seedling growth. However, specific concentrations of carbonate and biotin decreased the inhibitory effects of the other compound at the germination and seedling stages. Additionally, a carbonate treatment increased the endogenous biotin content and expression of AtBIO2, which encodes a biotin synthase. Moreover, phenotypic analyses indicated that the overexpression of AtBIO2 in Arabidopsis enhanced the tolerance to carbonate stress, whereas mutations to AtBIO2 had the opposite effect. Furthermore, the carbonate stress-induced accumulation of reactive oxygen species was lower in plants overexpressing AtBIO2 than in the wild-type and bio2 mutants. Accordingly, biotin, which is an essential vitamin for plants, can enhance the resistance to carbonate stress.

Arabidopsis V-ATPase d2 Subunit Plays a Role in Plant Responses to Oxidative Stress.

Vacuolar-type H+-ATPase (V-ATPase), a multisubunit proton pump located on the endomembrane, plays an important role in plant growth. The Arabidopsis thaliana V-ATPase d subunit (VHA-d) consists of two isoforms; AtVHA-d1 and AtVHA-d2. In this study, the function of AtVHA-d2 was investigated. Histochemical analysis revealed that the expression of AtVHA-d1 and AtVHA-d2 was generally highly overlapping in multiple tissues at different developmental stages of Arabidopsis. Subcellular localization revealed that AtVHA-d2 was mainly localized to the vacuole. AtVHA-d2 expression was significantly induced by oxidative stress. Analysis of phenotypic and H2O2 content showed that the atvha-d2 mutant was sensitive to oxidative stress. The noninvasive microtest monitoring demonstrated that the net H+ influx in the atvha-d2 roots was weaker than that in the wild-type under normal conditions. However, oxidative stress resulted in the H+ efflux in atvha-d2 roots, which was significantly different from that in the wild-type. RNA-seq combined with qPCR analysis showed that the expression of several members of the plasma membrane H+-ATPase gene (AtAHA) family in atvha-d2 was significantly different from that in the wild-type. Overall, our results indicate that AtVHA-d2 plays a role in Arabidopsis in response to oxidative stress by affecting H+ flux and AtAHA gene expression.

Transcriptome profiling of Puccinellia tenuiflora during seed germination under a long-term saline-alkali stress.

BACKGROUND: Puccinellia tenuiflora is the most saline-alkali tolerant plant in the Songnen Plain, one of the three largest soda saline-alkali lands worldwide. Here, we investigated the physicochemical properties of saline-alkali soils from the Songnen Plain and sequenced the transcriptomes of germinated P. tenuiflora seedlings under long-term treatment (from seed soaking) with saline-alkali soil extracts. RESULTS: We found that the soils from Songnen Plain were reasonably rich in salts and alkali; moreover, the soils were severely deficient in nitrogen [N], phosphorus [P], potassium [K] and several other mineral elements. This finding demonstrated that P. tenuiflora can survive from not only high saline-alkali stress but also a lack of essential mineral elements. To explore the saline-alkali tolerance mechanism, transcriptional analyses of P. tenuiflora plants treated with water extracts from the saline-alkali soils was performed. Interestingly, unigenes involved in the uptake of N, P, K and the micronutrients were found to be significantly upregulated, which indicated the existence of an efficient nutrition-uptake system in P. tenuiflora. Compared with P. tenuiflora, the rice Oryza sativa was hypersensitive to saline-alkali stress. The results obtained using a noninvasive microtest techniques confirmed that the uptake of NO3- and NH4+ and the regulatory flux of Na+ and H+ were significantly higher in the roots of P. tenuiflora than in those of O. sativa. In the corresponding physiological experiments, the application of additional nutrition elements significantly eliminated the sensitive symptoms of rice to saline-alkali soil extracts. CONCLUSIONS: Our results imply that the survival of P. tenuiflora in saline-alkali soils is due to a combination of at least two regulatory mechanisms and the high nutrient uptake capacity of P. tenuiflora plays a pivotal role in its adaptation to those stress. Taken together, our results highlight the role of nutrition uptake in saline-alkali stress tolerance in plants.

The role of ammonium transporter (AMT) against salt stress in plants.

Since NH4+ is one of the most important limiting nitrogen sources for plant growth, ammonium uptake and transport system has particular attention. In plant cells, ammonium transporters (AMTs) are responsible for ammonium uptake and transport. In previous studies, we identified a PutAMT1;1 gene from Puccinellia tenuiflora, which is a monocotyledonous halophyte species that thrives in alkaline soil. The overexpression of PutAMT1;1 in Arabidopsis thaliana enhanced plant growth and increased plant susceptibility to toxic methylammonium (MeA). This transporter might be useful for improving the root to shoot mobilization of MeA (or NH4+). Interestingly, in our other studies, it can be assumed that urease acts on urea to produce NH4+, which may exacerbate salt stress. Overexpression of PutAMT1;1 promoted early root growth after seed germination in transgenic Arabidopsis under salt stress condition. These findings suggest that ammonium transport alleviates ammonia toxicity caused by salt stress. Subcellular localization revealed that PutAMT1;1 is mainly localized in the plasma membrane and the nuclear periphery and endomembrane system of yeast and plant cells. Here, we discuss these recent findings and speculate on the regular dynamic localization of PutAMT1;1 throughout the cell cycle, which may be related to intracellular activity.

Overexpression of AtOxR gene improves abiotic stresses tolerance and vitamin C content in Arabidopsis thaliana.

BACKGROUND: Abiotic stresses are serious threats to plant growth, productivity and result in crop loss worldwide, reducing average yields of most major crops. Although abiotic stresses might elicit different plant responses, most induce the accumulation of reactive oxygen species (ROS) in plant cells leads to oxidative damage. L-ascorbic acid (AsA, vitamin C) is known as an antioxidant and H2O2-scavenger that defends plants against abiotic stresses. In addition, vitamin C is also an important component of human nutrition that has to be obtained from different foods. Therefore, increasing the vitamin C content is important for improving abiotic stresses tolerance and nutrition quality in crops production. RESULTS: Here, we show that the expression of AtOxR gene is response to multiple abiotic stresses (salt, osmotic, metal ion, and H2O2 treatment) in both the leaves and roots of Arabidopsis. AtOxR protein was localized to the Endoplasmic Reticulum (ER) in yeast and Arabidopsis cells by co-localization analysis with ER specific dye. AtOxR-overexpressing transgenic Arabidopsis plants enhance the tolerance to abiotic stresses. Overexpression of AtOxR gene resulted in AsA accumulation and decreased H2O2 content in transgenic plants. CONCLUSIONS: In this study, our results show that AtOxR responds to multiple abiotic stresses. Overexpressing AtOxR improves tolerance to abiotic stresses and increase vitamin C content in Arabidopsis thaliana. AtOxR will be useful for the improvement of important crop plants through moleculer breeding.

Conserved V-ATPase c subunit plays a role in plant growth by influencing V-ATPase-dependent endosomal trafficking.

In plant cells, the vacuolar-type H(+)-ATPases (V-ATPase) are localized in the tonoplast, Golgi, trans-Golgi network and endosome. However, little is known about how V-ATPase influences plant growth, particularly with regard to the V-ATPase c subunit (VHA-c). Here, we characterized the function of a VHA-c gene from Puccinellia tenuiflora (PutVHA-c) in plant growth. Compared to the wild-type, transgenic plants overexpressing PutVHA-c in Arabidopsis thaliana exhibit better growth phenotypes in root length, fresh weight, plant height and silique number under the normal and salt stress conditions due to noticeably higher V-ATPase activity. Consistently, the Arabidopsis atvha-c5 mutant shows reduced V-ATPase activity and retarded plant growth. Furthermore, confocal and immunogold electron microscopy assays demonstrate that PutVHA-c is mainly localized to endosomal compartments. The treatment of concanamycin A (ConcA), a specific inhibitor of V-ATPases, leads to obvious aggregation of the endosomal compartments labelled with PutVHA-c-GFP. Moreover, ConcA treatment results in the abnormal localization of two plasma membrane (PM) marker proteins Pinformed 1 (AtPIN1) and regulator of G protein signalling-1 (AtRGS1). These findings suggest that the decrease in V-ATPase activity blocks endosomal trafficking. Taken together, our results strongly suggest that the PutVHA-c plays an important role in plant growth by influencing V-ATPase-dependent endosomal trafficking.

Adverse effect of urease on salt stress during seed germination in Arabidopsis thaliana.

Seed germination is a critical stage in the development of crops that grow in saline soils. We noticed that seeds of an Arabidopsis urease mutant have significantly increased salt stress tolerance. To understand why, we treated the wild type (WT) with a urease inhibitor and found that its salt stress tolerance was also improved. We hypothesized that urease acting on urea generates NH4⁺, which probably exacerbates salt stress. As expected, the urease inhibitor significantly decreased the NH4⁺ level in WT seeds. These findings suggest that blocking urease activity improves salt tolerance during seed germination by lowering the concentration of NH4⁺.

An efficient method for stable protein targeting in grasses (Poaceae): a case study in Puccinellia tenuiflora.

BACKGROUND: An efficient transformation method is lacking for most non-model plant species to test gene function. Therefore, subcellular localization of proteins of interest from non-model plants is mainly carried out through transient transformation in homologous cells or in heterologous cells from model species such as Arabidopsis. Although analysis of expression patterns in model organisms like yeast and Arabidopsis can provide important clues about protein localization, these heterologous systems may not always faithfully reflect the native subcellular distribution in other species. On the other hand, transient expression in protoplasts from species of interest has limited ability for detailed sub-cellular localization analysis (e.g., those involving subcellular fractionation or sectioning and immunodetection), as it results in heterogeneous populations comprised of both transformed and untransformed cells. RESULTS: We have developed a simple and reliable method for stable transformation of plant cell suspensions that are suitable for protein subcellular localization analyses in the non-model monocotyledonous plant Puccinellia tenuiflora. Optimization of protocols for obtaining suspension-cultured cells followed by Agrobacterium-mediated genetic transformation allowed us to establish stably transformed cell lines, which could be maintained indefinitely in axenic culture supplied with the proper antibiotic. As a case study, protoplasts of transgenic cell lines stably transformed with an ammonium transporter-green fluorescent protein (PutAMT1;1-GFP) fusion were successfully used for subcellular localization analyses in P. tenuiflora. CONCLUSIONS: We present a reliable method for the generation of stably transformed P. tenuiflora cell lines, which, being available in virtually unlimited amounts, can be conveniently used for any type of protein subcellular localization analysis required. Given its simplicity, the method can be used as reference for other non-model plant species lacking efficient regeneration protocols.