Genome-Wide Identification and Expression Analysis of the Cyclic Nucleotide-Gated Channel Gene Family in Zoysia japonica under Salt Stress.

Salt stress severely inhibits plant growth. Understanding the mechanism of plant salt tolerance is highly important to improving plant salt tolerance. Previous studies have shown that nonselective cyclic nucleotide-gated ion channels (CNGCs) play an important role in plant salt tolerance. However, current research on CNGCs mainly focuses on CNGCs in glycophytic plants, and research on CNGCs in halophytes that exhibit special salt tolerance strategies is still scarce. This study used the halophilic plant Zoysia japonica, an excellent warm-season turfgrass, as the experimental material. Through bioinformatics analysis, 18 members of the CNGC family were identified in Zoysia japonica; they were designated ZjCNGC1 through ZjCNGC18 according to their scaffold-level chromosomal positions. ZjCNGCs are divided into four groups (I-IV), with the same groups having differentiated protein-conserved domains and gene structures. ZjCNGCs are unevenly distributed on 16 scaffold-level chromosomes. Compared with other species, the ZjCNGCs in Group III exhibit obvious gene expansion, mainly due to duplication of gene segments. The collinearity between ZjCNGCs, OsCNGCs, and SjCNGCs suggests that CNGCs are evolutionarily conserved among gramineous plants. However, the Group III ZjCNGCs are only partially collinear with OsCNGCs and SjCNGCs, implying that the expansion of Group III ZjCNGC genes may have been an independent event occurring in Zoysia japonica. Protein interaction prediction revealed that ZjCNGCs, calcium-dependent protein kinase, H+-ATPase, outwardly rectifying potassium channel protein, and polyubiquitin 3 interact with ZjCNGCs. Multiple stress response regulatory elements, including those involved in salt stress, are present on the ZjCNGC promoter. The qPCR results revealed differences in the expression patterns of ZjCNGCs in different parts of the plant. Under salt stress conditions, the expression of ZjCNGCs was significantly upregulated in roots and leaves, with ZjCNGC8 and ZjCNGC13 showing the greatest increase in expression in the roots. These results collectively suggest that ZjCNGCs play an important role in salt tolerance and that their expansion into Group III may be a special mechanism underlying the salt tolerance of Zoysia japonica.

Identification of Shaker Potassium Channel Family Members and Functional Characterization of SsKAT1.1 in Stenotaphrum secundatum Suggest That SsKAT1.1 Contributes to Cold Resistance.

Stenotaphrum secundatum is an excellent shade-tolerant warm-season turfgrass. Its poor cold resistance severely limits its promotion and application in temperate regions. Mining cold resistance genes is highly important for the cultivation of cold-resistant Stenotaphrum secundatum. Although there have been many reports on the role of the Shaker potassium channel family under abiotic stress, such as drought and salt stress, there is still a lack of research on their role in cold resistance. In this study, the transcriptome database of Stenotaphrum secundatum was aligned with the whole genome of Setaria italica, and eight members of the Shaker potassium channel family in Stenotaphrum secundatum were identified and named SsKAT1.1, SsKAT1.2, SsKAT2.1, SsKAT2.2, SsAKT1.1, SsAKT2.1, SsAKT2.2, and SsKOR1. The KAT3-like gene, KOR2 homologous gene, and part of the AKT-type weakly inwardly rectifying channel have not been identified in the Stenotaphrum secundatum transcriptome database. A bioinformatics analysis revealed that the potassium channels of Stenotaphrum secundatum are highly conserved in terms of protein structure but have more homologous members in the same group than those of other species. Among the three species of Oryza sativa, Arabidopsis thaliana, and Setaria italica, the potassium channel of Stenotaphrum secundatum is more closely related to the potassium channel of Setaria italica, which is consistent with the taxonomic results of these species belonging to Paniceae. Subcellular location experiments demonstrate that SsKAT1.1 is a plasma membrane protein. The expression of SsKAT1.1 reversed the growth defect of the potassium absorption-deficient yeast strain R5421 under a low potassium supply, indicating that SsKAT1.1 is a functional potassium channel. The transformation of SsKAT1.1 into the cold-sensitive yeast strain INVSC1 increased the cold resistance of the yeast, indicating that SsKAT1.1 confers cold resistance. The transformation of SsKAT1.1 into the salt-sensitive yeast strain G19 increased the resistance of yeast to salt, indicating that SsKAT1.1 is involved in salt tolerance. These results suggest that the manipulation of SsKAT1.1 will improve the cold and salt stress resistance of Stenotaphrum secundatum.

The rectification control and physiological relevance of potassium channel OsAKT2.

AKT2 potassium (K+) channels are members of the plant Shaker family which mediate dual-directional K+ transport with weak voltage-dependency. Here we show that OsAKT2 of rice (Oryza sativa) functions mainly as an inward rectifier with strong voltage-dependency and acutely suppressed outward activity. This is attributed to the presence of a unique K191 residue in the S4 domain. The typical bi-directional leak-like property was restored by a single K191R mutation, indicating that this functional distinction is an intrinsic characteristic of OsAKT2. Furthermore, the opposite R195K mutation of AtAKT2 changed the channel to an inward-rectifier similar to OsAKT2. OsAKT2 was modulated by OsCBL1/OsCIPK23, evoking the outward activity and diminishing the inward current. The physiological relevance in relation to the rectification diversity of OsAKT2 was addressed by functional assembly in the Arabidopsis (Arabidopsis thaliana) akt2 mutant. Overexpression (OE) of OsAKT2 complemented the K+ deficiency in the phloem sap and leaves of the mutant plants but did not significantly contribute to the transport of sugars. However, the expression of OsAKT2-K191R overcame both the shortage of phloem K+ and sucrose of the akt2 mutant, which was comparable to the effects of the OE of AtAKT2, while the expression of the inward mutation AtAKT2-R195K resembled the effects of OsAKT2. Additionally, OE of OsAKT2 ameliorated the salt tolerance of Arabidopsis.

Regulation of Cytosolic pH: The Contributions of Plant Plasma Membrane H+-ATPases and Multiple Transporters.

Cytosolic pH homeostasis is a precondition for the normal growth and stress responses in plants, and H+ flux across the plasma membrane is essential for cytoplasmic pH control. Hence, this review focuses on seven types of proteins that possess direct H+ transport activity, namely, H+-ATPase, NHX, CHX, AMT, NRT, PHT, and KT/HAK/KUP, to summarize their plasma-membrane-located family members, the effect of corresponding gene knockout and/or overexpression on cytosolic pH, the H+ transport pathway, and their functional regulation by the extracellular/cytosolic pH. In general, H+-ATPases mediate H+ extrusion, whereas most members of other six proteins mediate H+ influx, thus contributing to cytosolic pH homeostasis by directly modulating H+ flux across the plasma membrane. The fact that some AMTs/NRTs mediate H+-coupled substrate influx, whereas other intra-family members facilitate H+-uncoupled substrate transport, demonstrates that not all plasma membrane transporters possess H+-coupled substrate transport mechanisms, and using the transport mechanism of a protein to represent the case of the entire family is not suitable. The transport activity of these proteins is regulated by extracellular and/or cytosolic pH, with different structural bases for H+ transfer among these seven types of proteins. Notably, intra-family members possess distinct pH regulatory characterization and underlying residues for H+ transfer. This review is anticipated to facilitate the understanding of the molecular basis for cytosolic pH homeostasis. Despite this progress, the strategy of their cooperation for cytosolic pH homeostasis needs further investigation.

Function and Regulation of Ammonium Transporters in Plants.

Ammonium transporter (AMT)-mediated acquisition of ammonium nitrogen from soils is essential for the nitrogen demand of plants, especially for those plants growing in flooded or acidic soils where ammonium is dominant. Recent advances show that AMTs additionally participate in many other physiological processes such as transporting ammonium from symbiotic fungi to plants, transporting ammonium from roots to shoots, transferring ammonium in leaves and reproductive organs, or facilitating resistance to plant diseases via ammonium transport. Besides being a transporter, several AMTs are required for the root development upon ammonium exposure. To avoid the adverse effects of inadequate or excessive intake of ammonium nitrogen on plant growth and development, activities of AMTs are fine-tuned not only at the transcriptional level by the participation of at least four transcription factors, but also at protein level by phosphorylation, pH, endocytosis, and heterotrimerization. Despite these progresses, it is worth noting that stronger growth inhibition, not facilitation, unfortunately occurs when AMT overexpression lines are exposed to optimal or slightly excessive ammonium. This implies that a long road remains towards overcoming potential limiting factors and achieving AMT-facilitated yield increase to accomplish the goal of persistent yield increase under the present high nitrogen input mode in agriculture.

Screening of environmental stimuli for the positive regulation of stomatal aperture in centipedegrass.

Grasslands, the largest carbon pool in China, possess enormous potential for carbon sequestration. Increasing the stomatal aperture to increase the CO2 absorption capacity is a potential method to improve plant photosynthetic efficiency and ultimately enhance the carbon sequestration capacity of grass plants. Research on stomatal aperture regulation has focused mostly on Arabidopsis or crops, while research on grass plants in these areas is scarce, which seriously restricts the implementation of this grassland carbon sequestration strategy. Here, a widely used ecological grass, centipedegrass, was used as the experimental material. First, a convenient method for observing the stomatal aperture was developed. The leaves were floated in a potassium ion-containing open solution (67 mM KCl, pH 6.0) with the adaxial surface rather than the abaxial surface in contact with the solution and were cultivated under light for 1.5 h. Then, nail polish was applied on the adaxial surface, and a large number of open stomata were imprinted. Second, with the help of this improved method, the concentration‒response characteristics of the stomatal aperture to eleven environmental stimuli were tested. The stomatal aperture is dependent on these environmental stimuli in a concentration-dependent manner. The addition of 100 µM brassinolide led to the maximal stomatal aperture. This study provided a technical basis for manipulating stomatal opening to increase the carbon sequestration capacity of centipedegrass.

An appropriate ammonium: nitrate ratio promotes the growth of centipedegrass: insight from physiological and micromorphological analyses.

Reasonable nitrogen fertilizer application is an important strategy to maintain optimal growth of grasslands, thereby enabling them to better fulfil their ecological functions while reducing environmental pollution caused by high nitrogen fertilizer production and application. Optimizing the ammonium (NH4 +):nitrate (NO3 -) ratio is a common approach for growth promotion in crops and vegetables, but research on this topic in grass plants has not received sufficient attention. Centipedegrass, which is widely used in landscaping and ecological protection, was used as the experimental material. Different NH4 +:NO3 - ratios (0: 100, 25:75, 50:50, 75:25, 100:0) were used as the experimental treatments under hydroponic conditions. By monitoring the physiological and morphological changes under each treatment, the appropriate NH4 +:NO3 - ratio for growth and its underlying mechanism were determined. As the proportion of ammonium increased, the growth showed a "bell-shaped" response, with the maximum biomass and total carbon and nitrogen accumulation achieved with the NH4 +:NO3 - ratio of 50:50 treatment. Compared with the situation where nitrate was supplied alone, increasing the ammonium proportion increased the whole plant biomass by 93.2%, 139.7%, 59.0%, and 30.5%, the whole plant nitrogen accumulation by 44.9%, 94.6%, 32.8%, and 54.8%, and the whole plant carbon accumulation by 90.4%, 139.9%, 58.7%, and 26.6% in order. As a gateway for nitrogen input, the roots treated with an NH4 +:NO3 - ratio of 50:50 exhibited the highest ammonium and nitrate uptake rate, which may be related to the maximum total root length, root surface area, average root diameter, root volume, and largest root xylem vessel. As a gateway for carbon input, leaves treated with an NH4 +:NO3 - ratio of 50:50 exhibited the highest stomatal aperture, stomatal conductance, photosynthetic rate, transpiration rate, and photosynthetic products. The NH4 +:NO3 - ratio of 50:50 treatment had the largest stem xylem vessel area. This structure and force caused by transpiration may synergistically facilitate root-to-shoot nutrient translocation. Notably, the change in stomatal opening occurred in the early stage (4 hours) of the NH4 +:NO3 - ratio treatments, indicating that stomates are structures that are involved in the response to changes in the root NH4 +:NO3 - ratio. In summary, we recommend 50:50 as the appropriate NH4 +:NO3 - ratio for the growth of centipedegrass, which not only improves the nitrogen use efficiency but also enhances the carbon sequestration capacity.

Roles of plastid-located phosphate transporters in carotenoid accumulation.

Enhanced carotenoid accumulation in plants is crucial for the nutritional and health demands of the human body since these beneficial substances are acquired through dietary intake. Plastids are the major organelles to accumulate carotenoids in plants and it is reported that manipulation of a single plastid phosphate transporter gene enhances carotenoid accumulation. Amongst all phosphate transport proteins including phosphate transporters (PHTs), plastidial phosphate translocators (pPTs), PHOSPHATE1 (PHO1), vacuolar phosphate efflux transporter (VPE), and Sulfate transporter [SULTR]-like phosphorus distribution transporter (SPDT) in plants, plastidic PHTs (PHT2 & PHT4) are found as the only clade that is plastid located, and manipulation of which affects carotenoid accumulation. Manipulation of a single chromoplast PHT (PHT4;2) enhances carotenoid accumulation, whereas manipulation of a single chloroplast PHT has no impact on carotenoid accumulation. The underlying mechanism is mainly attributed to their different effects on plastid orthophosphate (Pi) concentration. PHT4;2 is the only chromoplast Pi efflux transporter, and manipulating this single chromoplast PHT significantly regulates chromoplast Pi concentration. This variation subsequently modulates the carotenoid accumulation by affecting the supply of glyceraldehyde 3-phosphate, a substrate for carotenoid biosynthesis, by modulating the transcript abundances of carotenoid biosynthesis limited enzyme genes, and by regulating chromoplast biogenesis (facilitating carotenoid storage). However, at least five orthophosphate influx PHTs are identified in the chloroplast, and manipulating one of the five does not substantially modulate the chloroplast Pi concentration in a long term due to their functional redundancy. This stable chloroplast Pi concentration upon one chloroplast PHT absence, therefore, is unable to modulate Pi-involved carotenoid accumulation processes and finally does affect carotenoid accumulation in photosynthetic tissues. Despite these advances, several cases including the precise location of plastid PHTs, the phosphate transport direction mediated by these plastid PHTs, the plastid PHTs participating in carotenoid accumulation signal pathway, the potential roles of these plastid PHTs in leaf carotenoid accumulation, and the roles of these plastid PHTs in other secondary metabolites are waiting for further research. The clarification of the above-mentioned cases is beneficial for breeding high-carotenoid accumulation plants (either in photosynthetic or non-photosynthetic edible parts of plants) through the gene engineering of these transporters.

Functional and Regulatory Characterization of Three AMTs in Maize Roots.

Maize grows in nitrate-dominated dryland soils, but shortly upon localized dressing of nitrogen fertilizers, ammonium is retained as a noticeable form of nitrogen source available to roots. Thus in addition to nitrate, the absorption of ammonium can be an important strategy that promotes rapid plant growth at strong nitrogen demanding stages. The present study reports the functional characterization of three root-expressed ammonium transporters (AMTs), aiming at finding out functional and regulatory properties that correlate with efficient nitrogen acquisition of maize. Using a stable electrophysiological recording method we previously established in Xenopus laevis oocytes that integrates the capture of currents in response to voltage ramps with onsite stability controls, we demonstrate that all three ZmAMT1s engage NH4 + uniporting as ammonium uptake mechanisms. The K m value for ZmAMT1.1a, 1.1b, or ZmAMT1.3 is, respectively, 9.9, 15.6, or 18.6 µM, indicating a typical high-affinity transport of NH4 + ions. Importantly, the uptake currents of these ZmAMT1s are markedly amplified upon extracellular acidification. A pH drop from 7.4 to 5.4 results in a 140.8%, 64.1% or a 120.7% increase of ammonium uptake activity through ZmAMT1.1a, 1.1b, or ZmAMT1.3. Since ammonium uptake by plant roots accompanies a spontaneous acidification to the root medium, the functional promotion of ZmAMT1.1a, 1.1b, and ZmAMT1.3 by low pH is in line with the facilitated ammonium uptake activity in maize roots. Furthermore, the expression of the three ZmAMT1 genes is induced under ammonium-dominated conditions. Thus we describe a facilitated ammonium uptake strategy in maize roots by functional and expression regulations of ZmAMT1 transporters that may coordinate with efficient acquisition of this form of nitrogen source when available.

Functional Characterization of the Arabidopsis Ammonium Transporter AtAMT1;3 With the Emphasis on Structural Determinants of Substrate Binding and Permeation Properties.

AtAMT1;3 is a major contributor to high-affinity ammonium uptake in Arabidopsis roots. Using a stable electrophysiological recording strategy, we demonstrate in Xenopus laevis oocytes that AtAMT1;3 functions as a typical high-affinity NH4 + uniporter independent of protons and Ca2+. The findings that AtAMT1;3 transports methylammonium (MeA+, a chemical analog of NH4 +) with extremely low affinity (K m in the range of 2.9-6.1 mM) led to investigate the mechanisms underlying substrate binding. Homologous modeling and substrate docking analyses predicted that the deduced substrate binding motif of AtAMT1;3 facilitates the binding of NH4 + ions but loosely accommodates the binding of MeA+ to a more superficial location of the permeation pathway. Amongst point mutations tested based on this analysis, P181A resulted in both significantly increased current amplitudes and substrate binding affinity, whereas F178I led to opposite effects. Thus these 2 residues, which flank W179, a major structural component of the binding site, are also important determinants of AtAMT1;3 transport capacity by being involved in substrate binding. The Q365K mutation neighboring the histidine residue H378, which confines the substrate permeation tunnel, affected only the current amplitudes but not the binding affinities, providing evidence that Q365 mainly controls the substrate diffusion rate within the permeation pathway.

RNA-Seq analysis of differentially expressed genes in rice under varied nitrogen supplies.

Ammonium is the main inorganic nitrogen source in paddy soil. Rice (Oryza sativa), an ammonium-preferring and -tolerant grain crop, is a valuable resource for researching ammonium-uptake mechanism and understanding the molecular networks that the plant copes with ammonium variation. To generate a broad survey of early responses affected by varied ammonium supplies in rice, RNA samples were prepared from the roots and shoots of rice plants subjected to nitrogen-free (0mM ammonium), 1mM ammonium and high ammonium (10mM ammonium) for a short period of 4h (1mM ammonium treatment as the control), respectively, and the transcripts were sequenced using the Illumina/HiSeq™ 2000 RNA sequencing (RNA-Seq) platform. By comparative analysis, 394 differentially expressed genes (DEGs) were identified in roots, among which, 143 and 251 DEGs were up- and down-regulated under nitrogen-free condition, respectively. In shoots, 468 (119 up-regulated/349 down-regulated) DEGs were found under such condition. However, with high ammonium treatment, only 63 genes (6 up-regulated/57 down-regulated) in roots and 115 genes in shoots (93 up-regulated/22 down-regulated) were differentially expressed. According to KEGG analysis, when exposed to nitrogen-free condition, DEGs participating in the carbohydrate and amino acid metabolisms were down-regulated (with 1 exception) in roots as well as in shoots, implying reduced carbohydrate and nitrogen metabolisms. Under high ammonium supply, all DEGs associated with carbohydrate and amino acid metabolisms were down-regulated in roots and to the contrary, up-regulated in shoots. Aldehyde dehydrogenase (ALDH, NAD(+)) [EC: 1.2.1.3] seemed to have played an important role in rice shoots under high ammonium condition, analysis results implicated a coordinative regulation of carbohydrate with amino acid metabolisms under nitrogen deficiency as well as the high ammonium conditions during a short period of several hours in rice. Moreover, transcripts with abundance variation might be precious gene resources in responding to different ammonium supplies in rice.