Leaf Protoplasts Research Articles

Impact of protein domains on the MEL2 granule, a cytoplasmic ribonucleoprotein complex maintaining faithful meiosis progression in rice.

Cytoplasmic ribonucleoprotein (RNP) granules are membraneless structures composed of various RNAs and proteins that play important roles in post-transcriptional regulation. While RNP granules are known to regulate the meiotic entry in some organisms, little is known about their roles in plants. In this study, we observed the cytoplasmic granular structures of rice RNA-binding protein MEIOSIS ARRESTED AT LEPTOTENE2 (MEL2), which contributes to the control of meiotic entry timing, in leaf protoplasts and spore mother cells. We performed colocalization analysis with known cytoplasmic RNP factors, and domain deletion analysis to assess their impact on granule formation and meiosis progression. Conservation of MEL2 domains across plant species was also explored. Our results indicated that MEL2 granules colocalized with processing body and stress granule factors. The maintenance of granule properties modulated by LOTUS domain and the intrinsically disordered region (IDR) is essential for proper MEL2 function in meiosis progression. MEL2-like proteins widely found in plant kingdom conserved LOTUS domain followed by the IDR despite their diverse domain structures, suggesting the functional conservation of these domains among plant species. This study highlights the role of MEL2 granule dynamics and its impact on meiotic transition and progression.

Arabidopsis and maize terminator strength is determined by GC content, polyadenylation motifs and cleavage probability

The 3’ end of a gene, often called a terminator, modulates mRNA stability, localization, translation, and polyadenylation. Here, we adapted Plant STARR-seq, a massively parallel reporter assay, to measure the activity of over 50,000 terminators from the plants Arabidopsis thaliana and Zea mays. We characterize thousands of plant terminators, including many that outperform bacterial terminators commonly used in plants. Terminator activity is species-specific, differing in tobacco leaf and maize protoplast assays. While recapitulating known biology, our results reveal the relative contributions of polyadenylation motifs to terminator strength. We built a computational model to predict terminator strength and used it to conduct in silico evolution that generated optimized synthetic terminators. Additionally, we discover alternative polyadenylation sites across tens of thousands of terminators; however, the strongest terminators tend to have a dominant cleavage site. Our results establish features of plant terminator function and identify strong naturally occurring and synthetic terminators.

Effects of Salts on Helical Protoplast-Callose-Fiber Formation and Cell Division in Leaf Protoplast Culture of Arabidopsis thaliana: Ultrastructure of PCF Using Transmission Electron Microscopy

Effects of four salts addition (10-200 mM), NaCl, KCl, MgCl2, and CaCl2 were examined on the growth of protoplasts and formation of helical 1.5 mm long, protoplast-callose-fibers (PCF) in Arabidopsis thaliana liquid leaf protoplast cultures. The basal medium was Murashige and Skoog’s medium containing 1 μM 2,4-dichlorophenoxyacetic acid and 0.1 μM benzyladenine, 3% sucrose, and 0.4 M mannitol. The protoplast division was highly stimulated by the addition of 50 mM Ca2+ ion but was totally inhibited by 50 mM Mg2+ ion. Inhibition by K+ and Na+ ions was in-between. By contrast, PCF formation, whose numbers were counted under a fluorescence inverted microscope after Aniline Blue staining for β-1,3-glucan (callose), was stimulated by both K+ and Ca2+ ions but inhibited by Mg2+ ion. After selecting Arabidopsis PCF using a micromanipulator, the sub-fibril ultrastructure was examined by transmission electron microscopy (TEM). The PCFs of Betula platyphylla and Larix leptolepis, cultured with 200 mM Ca2+ and 50 mM Mg2+ ions, respectively, after long-term storage and rehydration, were examined by TEM. The effects of different factors were discussed on PCF formation and sub-structures in different herbaceous and tree plant species.

Microfluidics identify moso bamboo and henon bamboo by leaf protoplast subpopulations with single-cell analysis.

Current techniques identifying herbal medicine species require marker labeling or lack systematical accuracy (expert authentication). There is an emerging interest in developing an accurate and label-free tool for herbal medicine authentication. Here, a high-resolution microfluidic-based method is developed for identifying herbal species by protoplast subpopulations. Moso bamboo and henon bamboo are used as a model to be differentiated based on protoplast. Their biophysical properties factors are characterized to be 7.09(±0.39) × 108V/m2 and 6.54(±0.26) × 108V/m2, respectively. Their biophysical distributions could be distinguished by the Cramér-von Mises criterion with a 94.60% confidence level. The subpopulations of each were compared with conventional flow cytometry indicating the existence of subpopulations and the differences between the two species. The subsets divided by a biophysical factor of 8.05(±0.51) × 108V/m2 suggest good consistency with flow cytometry. The work demonstrated the possibility of microfluidics manipulation on protoplast for medication safety use taking advantage of dielectrophoresis. The device is promising in developing a reliable and accurate way of identifying herbal species with difficulties in authentication.

Enabling High-throughput Transgene Expression Studies Using Automated Liquid Handling for Etiolated Maize Leaf Protoplasts.

The field of plant biotechnology has witnessed remarkable advancements in recent years, revolutionizing the ability to manipulate and engineer plants for various purposes. However, as research in this field increases in diversity and becomes increasingly sophisticated, the need for early, efficient, dependable, and high-throughput transient screening solutions to narrow down strategies proceeding to stable transformation is more apparent. One method that has re-emerged in recent years is the utilization of plant protoplast, for which methods of isolation and transfection are available in numerous species, tissues, and developmental stages. This work describes a simple automated protocol for the randomized preparation of plasmid within a 96-well plate, a method for the isolation of etiolated maize leaf protoplast, and an automated transfection procedure. The adoption of automated solutions in plant biotechnology, exemplified by these novel liquid handling protocols for plant protoplast transfection, represents a significant advancement over manual methods. By leveraging automation, researchers can easily overcome the limitations of traditional methods, enhance efficiency, and accelerate scientific progress.

Optimization of preparation and transformation of protoplasts from Populus simonii × P. nigra leaves and subcellular localization of the major latex protein 328 (MLP328)

BackgroundPopulus simonii × P. nigra is an ideal material for studying the molecular mechanisms of woody plants. In recent years, research on Populus simonii × P. nigra has increasingly focused on the application of transgenic technology to improve salt tolerance. However, the rapid characterization of gene functions has been hampered by the long growth cycle and exceedingly poor transformation efficiency. Protoplasts are an important tool for plant gene engineering, which can assist with challenging genetic transformation and the protracted growth cycle of Populus simonii × P. nigra. This study established an optimized system for the preparation and transformation of protoplasts from Populus simonii × P. nigra leaves, making genetic research on Populus simonii × P. nigra faster and more convenient. Major Latex Protein (MLP) family genes play a crucial role in plant salt stress response. In the previous study, we discovered that PsnMLP328 can be induced by salt treatment, which suggested that this gene may be involved in response to salt stress. Protein localization is a suggestion for its function. Therefore, we conducted subcellular localization analysis using protoplasts of Populus simonii × P. nigra to study the function of the PsnMLP328 gene preliminarily.ResultsThis study established an optimized system for the preparation and transformation of Populus simonii × P. nigra protoplasts. The research results indicate that the optimal separation scheme for the protoplasts of Populus simonii × P. nigra leaves included 2.5% cellulase R-10, 0.6% macerozyme R-10, 0.3% pectolyase Y-23, and 0.8 M mannitol. After enzymatic digestion for 5 h, the yield of obtained protoplasts could reach up to 2 × 107 protoplasts/gFW, with a high viability of 98%. We carried out the subcellular localization analysis based on the optimized transient transformation system, and the results indicated that the MLP328 protein is localized in the nucleus and cytoplasm; thereby proving the effectiveness of the transformation system.ConclusionIn summary, this study successfully established an efficient system for preparing and transforming leaf protoplasts of Populus simonii × P. nigra, laying the foundation for future research on gene function and expression of Populus simonii × P. nigra.

Preparation of leaf protoplasts from Populus (Populus × xiaohei T. S. Hwang et Liang) and establishment of transient expression system

Poplar, as a typical woody plant, is an ideal raw material for the production of lignocellulose biofuel. However, the longer life cycle is not conducive to the rapid identification of poplar genes. At present, protoplasts have been used for gene function identification and high-throughput analysis in many model plants. In this paper, a simplified and efficient protoplast isolation and transient expression system of Populus (Populus × xiaohei T. S. Hwang et Liang) is described. Firstly, we proposed an efficient enzyme hydrolysis method for isolating protoplasts from leaves of Populus × xiaohei. Secondly, we optimized the conditions of protoplast transformation mediated by PEG, and established an efficient transient expression system of protoplasts of Populus × xiaohei. Finally, the subcellular localization of three identified Dof transcription factors (PnDof19, PnDof20 and PnDof30) was also observed in the nucleus by using this scheme, which proved that the method was feasible. In general, this efficient method of protoplast isolation and transformation can be used for the study of protein subcellular localization and can be applied to other fields of molecular biology, such as protein interaction, gene activation and so on.

Comet assay as an early predictor tool to detect ozone enhanced sensitivity of vegetation in a free-air controlled long-term exposure

Although ozone (O3) is a strong oxidant with serious impacts on plants, few studies have investigated the effects on DNA damage. Here we tested the Comet assay combined with the enzymatic isolation of leaf protoplasts. Two species differing in O3 sensitivity were used, i.e. the moderately tolerant Arbutus unedo and the very sensitive Populus maximowiczii Henry × berolinensis (or Oxford clone), exposed to three O3 levels (ambient [40.3 ppb], 1.5 times ambient O3 concentration, twice ambient O3 concentration) for one (A. unedo) or two (Oxford clone) growing seasons in an O3 Free Air Controlled Exposure (FACE) facility. We found that O3 has strong plant genotoxicity capacity and the negative effects increased with increasing exposure or stomatal uptake in both species. Interestingly, both ecophysiological and DNA damage were detected in the sensitive species, while no ecophysiological injury was observed in A. unedo although DNA was damaged before other symptoms occurred. Overall, DNA damage increased when the ecophysiological parameters decreased, confirming that DNA damage is a good proxy of the functional status of a plant. We conclude that the enzymatic isolation and Comet assay could be used as a predictor tool to reveal plant O3 DNA damage even in moderately O3 tolerant species, able to recover the abiotic stress.

ScRNA-seq Reveals the Mechanism of Fatty Acid Desaturase 2 Mutation to Repress Leaf Growth in Peanut (Arachis hypogaea L.).

Fatty Acid Desaturase 2 (FAD2) controls the conversion of oleic acids into linoleic acids. Mutations in FAD2 not only increase the high-oleic content, but also repress the leaf growth. However, the mechanism by which FAD2 regulates the growth pathway has not been elucidated in peanut leaves with single-cell resolution. In this study, we isolated fad2 mutant leaf protoplast cells to perform single-cell RNA sequencing. Approximately 24,988 individual cells with 10,249 expressed genes were classified into five major cell types. A comparative analysis of 3495 differentially expressed genes (DEGs) in distinct cell types demonstrated that fad2 inhibited the expression of the cytokinin synthesis gene LOG in vascular cells, thereby repressing leaf growth. Further, pseudo-time trajectory analysis indicated that fad2 repressed leaf cell differentiation, and cell-cycle evidence displayed that fad2 perturbed the normal cell cycle to induce the majority of cells to drop into the S phase. Additionally, important transcription factors were filtered from the DEG profiles that connected the network involved in high-oleic acid accumulation (WRKY6), activated the hormone pathway (WRKY23, ERF109), and potentially regulated leaf growth (ERF6, MYB102, WRKY30). Collectively, our study describes different gene atlases in high-oleic and normal peanut seedling leaves, providing novel biological insights to elucidate the molecular mechanism of the high-oleic peanut-associated agronomic trait at the single-cell level.

Optimization of Protoplast Preparation System from Leaves and Establishment of a Transient Transformation System in Apium graveolens

Protoplast culture and transformation technology offer a novel method for developing new plant varieties. Nonetheless, the effective preparation of protoplasts and transformation technology specific to celery has yet to be achieved. This study utilized celery seedling leaves as the primary materials to examine the key factors influencing protoplast isolation. The aim was to prepare leaf protoplasts with a high yield and of high quality and subsequently conduct transient gene transformation and expression. The findings indicated that the most effective procedure for isolating and purifying protoplasts was enzymatic digestion using an enzyme solution consisting of 2.0% cellulase, 0.1% pectolase, and 0.6 M mannitol for a duration of 8 h. Subsequently, the protoplasts were filtered through a 400-mesh sieve and purified through centrifugation at 200× g. Within this system, the overall protoplast yield was exceptionally high, reaching a viability rate of up to 95%. The transient transformation system yielded a maximum transformation efficiency of approximately 53%, as evaluated using the green fluorescent protein (GFP) as a reporter gene. The parameters of the transient transformation system were as follows: a protoplast concentration of 5 × 105 cells·mL−1, exogenous DNA concentration of 500 μg·mL−1, final concentration of PEG4000 at 40%, and transformation duration of 15 min. The transient transformation system was also utilized to further analyze the protein localization characteristics of the celery transcription factor AgMYB80. The findings indicated that AgMYB80 predominantly localizes in the nucleus, thereby confirming the reliability and effectiveness of the transient transformation system. This study successfully established an efficient system for isolating, purifying, and transforming celery protoplasts, and will serve as a basis for future studies on molecular biology and gene function.

Genome-wide identification of accessible chromatin regions by ATAC-seq upon induction of the transcription factor bZIP11 in Arabidopsis

Basic leucine zipper 11 (bZIP11) is a transcription factor that is activated under low energy conditions in plants and plays a crucial role in enabling plants to adapt to starvation situations. Although previous results indicate that bZIP11 regulates chromatin accessibility based on evidence obtained from single genomic loci, to what extent this transcription factor regulates the chromatin landscape at the whole genome level remains unknown. Here we addressed this by performing an ATAC-seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing) on Arabidopsis thaliana (Arabidopsis) leaf protoplasts to obtain a profile of chromatin patterning in response upon bZIP11 induction. We identified, on average, 10,000 differentially accessible regions upon bZIP11 induction, corresponding to over 8,420 different genes out of the 25,000 genes present in the Arabidopsis genome. Our study provides a resource for understanding how bZIP11 regulates the genome at the chromatin level and provides an example of the impact of a single transcription factor on a whole plant genome.

Using A Protoplast Transformation System to Enable Functional Studies in Mangifera indica L.

Mangoes (Mangifera indica L.) are an important kind of perennial fruit tree, but their biochemical testing method and transformation technology were insufficient and had not been rigorously explored. The protoplast technology is an excellent method for creating a rapid and effective tool for transient expression and transformation assays, particularly in plants that lack an Agrobacterium-mediated plant transformation system. This study optimized the conditions of the protoplast isolation and transformation system, which can provide a lot of help in the gene expression regulation study of mango. The most beneficial protoplast isolation conditions were 150 mg/mL of cellulase R-10 and 180 mg/mL of macerozyme R-10 in the digestion solution at pH 5.6 and 12 h of digestion time. The 0.16 M and 0.08 M mannitol in wash solution (WI) and suspension for counting (MMG), respectively, were optimal for the protoplast isolation yield. The isolated leaf protoplasts (~5.4 × 105 cells/10 mL) were transfected for 30 min mediated by 40% calcium-chloride-based polyethylene glycol (PEG)-4000-CaCl2, from which 84.38% of the protoplasts were transformed. About 0.08 M and 0.12 M of mannitol concentration in MMG and transfection solutions, respectively, were optimal for protoplast viability. Under the florescence signal, GFP was seen in the transformed protoplasts. This showed that the target gene was successfully induced into the protoplast and that it can be transcribed and translated. Experimental results in this paper show that our high-efficiency protoplast isolation and PEG-mediated transformation protocols can provide excellent new methods for creating a rapid and effective tool for the molecular mechanism study of mangoes.

Molecular characterization of SrSTP14, a sugar transporter from thermogenic skunk cabbage, and its possible role in developing pollen.

In floral thermogenesis, sugars play an important role not only as energy providers but also as growth and development facilitators. Yet, the mechanisms underlying sugar translocation and transport in thermogenic plants remain to be studied. Asian skunk cabbage (Symplocarpus renifolius) is a species that can produce durable and intense heat in its reproductive organ, the spadix. Significant morphological and developmental changes in the stamen are well-characterized in this plant. In this study, we focused on the sugar transporters (STPs), SrSTP1 and SrSTP14, whose genes were identified by RNA-seq as the upregulated STPs during thermogenesis. Real-time PCR confirmed that mRNA expression of both STP genes was increased from the pre-thermogenic to the thermogenic stage in the spadix, where it is predominantly expressed in the stamen. SrSTP1 and SrSTP14 complemented the growth defects of a hexose transporter-deficient yeast strain, EBY4000, on media containing 0.02, 0.2, and 2% (w/v) glucose and galactose. Using a recently developed transient expression system in skunk cabbage leaf protoplasts, we revealed that SrSTP1 and SrSTP14-GFP fusion proteins were mainly localized to the plasma membrane. To dig further into the functional analysis of SrSTPs, tissue-specific localization of SrSTPs was investigated by in situ hybridization. Using probes for SrSTP14, mRNA expression was observed in the microspores within the developing anther at the thermogenic female stage. These results indicate that SrSTP1 and SrSTP14 transport hexoses (e.g., glucose and galactose) at the plasma membrane and suggest that SrSTP14 may play a role in pollen development through the uptake of hexoses into pollen precursor cells.

Development of an efficient leaf protoplast isolation and transient expression system for Artemisia japonica

Development of an efficient leaf protoplast isolation and transient expression system for Artemisia japonica

Optimization of ATAC-seq in wheat seedling roots using INTACT-isolated nuclei

BackgroundThe genetic information contained in the genome of an organism is organized in genes and regulatory elements that control gene expression. The genomes of multiple plants species have already been sequenced and the gene repertory have been annotated, however, cis-regulatory elements remain less characterized, limiting our understanding of genome functionality. These elements act as open platforms for recruiting both positive- and negative-acting transcription factors, and as such, chromatin accessibility is an important signature for their identification.ResultsIn this work we developed a transgenic INTACT [isolation of nuclei tagged in specific cell types] system in tetraploid wheat for nuclei purifications. Then, we combined the INTACT system together with the assay for transposase-accessible chromatin with sequencing [ATAC-seq] to identify open chromatin regions in wheat root tip samples. Our ATAC-seq results showed a large enrichment of open chromatin regions in intergenic and promoter regions, which is expected for regulatory elements and that is similar to ATAC-seq results obtained in other plant species. In addition, root ATAC-seq peaks showed a significant overlap with a previously published ATAC-seq data from wheat leaf protoplast, indicating a high reproducibility between the two experiments and a large overlap between open chromatin regions in root and leaf tissues. Importantly, we observed overlap between ATAC-seq peaks and cis-regulatory elements that have been functionally validated in wheat, and a good correlation between normalized accessibility and gene expression levels.ConclusionsWe have developed and validated an INTACT system in tetraploid wheat that allows rapid and high-quality nuclei purification from root tips. Those nuclei were successfully used to performed ATAC-seq experiments that revealed open chromatin regions in the wheat genome that will be useful to identify cis-regulatory elements. The INTACT system presented here will facilitate the development of ATAC-seq datasets in other tissues, growth stages, and under different growing conditions to generate a more complete landscape of the accessible DNA regions in the wheat genome.

Functional analysis of MdSUT2.1, a plasma membrane sucrose transporter from apple

Sugar content is a determinant of apple (Malus×domestica Borkh.) sweetness. However, the molecular mechanism underlying sucrose accumulation in apple fruit remains elusive. Herein, the role of the sucrose transporter MdSUT2.1 in the regulation of sucrose accumulation in apple was reported. The MdSUT2.1 gene encodes a protein with 612 amino acid residues that could be localized at the plasma membrane when expressed in tobacco leaf protoplasts. MdSUT2.1 was highly expressed in fruit and was positively correlated with sucrose accumulation during apple fruit development. Moreover, the sucrose transport activity of MdSUT2.1 was validated by complementary growth assays in a yeast mutant. MdSUT2.1 overexpression in apple and tomato resulted in significant increases in sucrose, fructose, and glucose contents compared to WT. Further analysis revealed that the expression levels of sugar metabolism- and transport-related genes SUSYs, NINVs, FRKs, HXKs, and TSTs increased in apple and tomato with MdSUT2.1 overexpression compared to WT. Finally, unlike the tonoplast sugar transporters MdTST1 and MdTST2, the promoter of MdSUT2.1 was not inducible by exogenous sugars. These findings provide valuable insights into the molecular mechanism underlying sugar accumulation in apple.

ASR1 and ASR2, Two Closely Related ABA-Induced Serine-Rich Transcription Repressors, Function Redundantly to Regulate ABA Responses in Arabidopsis.

The plant hormone abscisic acid (ABA) is able to regulate the expression of ABA-responsive genes via signaling transduction, and thus plays an important role in regulating plant responses to abiotic stresses. Hence, characterization of unknown ABA response genes may enable us to identify novel regulators of ABA and abiotic stress responses. By using RT-PCR analysis, we found that the expression levels of ABA-induced Serine-rich Repressor 1 (ASR1)and ASR2, two closely related unknown function genes, were increased in response to ABA treatment. Amino acid sequence analyses show that ASR1 contains an L×L×L motif and both ASR1 and ASR2 are enriched in serine. Transfection assays in Arabidopsis leaf protoplasts show that ASR1 and ASR2 were predominantly localized in the nucleus and were able to repress the expression of the reporter gene. The roles of ASRs in regulating ABA responses were examined by generating transgenic Arabidopsis plants over-expressing ASR1 and ASR2, respectively, and CRISPR/Cas9 gene-edited single and double mutants for ASR1 and ASR2. In both the seed germination and cotyledon greening assays, ABA sensitivity remained largely unchanged in the over-expression transgenic plants and the single mutants of ASR1 and ASR2, but greatly increased ABA sensitivity was observed in the asr1 asr2 double mutants. In root elongation assays, however, decreased ABA sensitivity was observed in the 35S:ASR1 and 35S:ASR2 transgenic plants, whereas increased ABA sensitivity was observed in the asr1 and asr2 single mutants, and ABA sensitivity was further increased in the asr1 asr2 double mutants. Transcriptome analysis show that the differentially expressed genes (DEGs) down-regulated in the 35S:ASR1 transgenic plant seedlings, but up-regulated in the asr1 asr2 double mutant seedlings were highly enriched in processes including responses to plant hormones and stress stimuli. Taken together, our results show that ASR1 and ASR2 are closely related ABA response genes, ASR1 and ASR2 are serine-rich novel transcription repressors, and they negatively regulate ABA responses in Arabidopsis in a redundant manner.

Constructing the maize inflorescence regulatory network by using efficient tsCUT&Tag assay

Maize ear development determines the crop yield, and many important transcription factors (TFs) have been identified to function in this process. However, their transcriptional regulation mechanisms are still elusive. In this study, we generated the genome-wide DNA binding sites for 8 TFs which are known to function or highly expressed in inflorescence by applying the tsCUT&Tag method in maize leaf protoplast. We exposed a regulatory grid of 4 WUSCHEL-related homeobox (WOX) genes and revealed their potential regulatory mechanisms. In addition, a hierarchical regulation network for the determinacy and specification of maize inflorescence meristems were also constructed using the binding profiles of a floral development gene INDETERMINATE FLORAL APEX1 (IFA1) and 3 MADS-box genes. Our study provides an in-depth understanding and new insights of the regulatory mechanisms during maize inflorescence development.

Evaluation of genome and base editing tools in maize protoplasts.

Despite its rapid worldwide adoption as an efficient mutagenesis tool, plant genome editing remains a labor-intensive process requiring often several months of in vitro culture to obtain mutant plantlets. To avoid a waste in time and money and to test, in only a few days, the efficiency of molecular constructs or novel Cas9 variants (clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9) prior to stable transformation, rapid analysis tools are helpful. To this end, a streamlined maize protoplast system for transient expression of CRISPR/Cas9 tools coupled to NGS (next generation sequencing) analysis and a novel bioinformatics pipeline was established. Mutation types found with high frequency in maize leaf protoplasts had a trend to be the ones observed after stable transformation of immature maize embryos. The protoplast system also allowed to conclude that modifications of the sgRNA (single guide RNA) scaffold leave little room for improvement, that relaxed PAM (protospacer adjacent motif) sites increase the choice of target sites for genome editing, albeit with decreased frequency, and that efficient base editing in maize could be achieved for certain but not all target sites. Phenotypic analysis of base edited mutant maize plants demonstrated that the introduction of a stop codon but not the mutation of a serine predicted to be phosphorylated in the bHLH (basic helix loop helix) transcription factor ZmICEa (INDUCER OF CBF EXPRESSIONa) caused abnormal stomata, pale leaves and eventual plant death two months after sowing.

Glycine betaine increases salt tolerance in maize (Zea mays L.) by regulating Na+ homeostasis.

Improving crop salt tolerance is an adaptive measure to climate change for meeting future food demands. Previous studies have reported that glycine betaine (GB) plays critical roles as an osmolyte in enhancing plant salt resistance. However, the mechanism underlying the GB regulating plant Na+ homeostasis during response to salinity is poorly understood. In this study, hydroponically cultured maize with 125 mM NaCl for inducing salinity stress was treated with 100 μM GB. We found that treatment with GB improved the growth of maize plants under non-stressed (NS) and salinity-stressed (SS) conditions. Treatment with GB significantly maintained the properties of chlorophyll fluorescence, including Fv/Fm, ΦPSII, and ΦNPQ, and increased the activity of the antioxidant enzymes for mitigating salt-induced growth inhibition. Moreover, GB decreased the Na+/K+ ratio primarily by reducing the accumulation of Na+ in plants. The results of NMT tests further confirmed that GB increased Na+ efflux from roots under SS condition, and fluorescence imaging of cellular Na+ suggested that GB reduced the cellular allocation of Na+. GB additionally increased Na+ efflux in leaf protoplasts under SS condition, and treatment with sodium orthovanadate, a plasma membrane (PM) H+-ATPase inhibitor, significantly alleviated the positive effects of GB on Na+ efflux under salt stress. GB significantly improved the vacuolar activity of NHX but had no significant effects on the activity of V type H+-ATPases. In addition, GB significantly upregulated the expression of the PM H+-ATPase genes, ZmMHA2 and ZmMHA4, and the Na+/H+ antiporter gene, ZmNHX1. While, the V type H+-ATPases gene, ZmVP1, was not significantly regulated by GB. Altogether these results indicate that GB regulates cellular Na+ homeostasis by enhancing PM H+-ATPases gene transcription and protein activities to improve maize salt tolerance. This study provided an extended understanding of the functions of GB in plant responses to salinity, which can help the development of supportive measures using GB for obtaining high maize yield in saline conditions.