Chloroplastic Glutamine Synthetase Research Articles

A phytoplasma effector destabilizes chloroplastic glutamine synthetase inducing chlorotic leaves that attract leafhopper vectors

Leaf yellowing is a well-known phenotype that attracts phloem-feeding insects. However, it remains unclear how insect-vectored plant pathogens induce host leaf yellowing to facilitate their own transmission by insect vectors. Here, we report that an effector protein secreted by rice orange leaf phytoplasma (ROLP) inhibits chlorophyll biosynthesis and induces leaf yellowing to attract leafhopper vectors, thereby presumably promoting pathogen transmission. This effector, designated secreted ROLP protein 1 (SRP1), first secreted into rice phloem by ROLP, was subsequently translocated to chloroplasts by interacting with the chloroplastic glutamine synthetase (GS2). The direct interaction between SRP1 and GS2 disrupts the decamer formation of the GS2 holoenzyme, attenuating its enzymatic activity, thereby suppressing the synthesis of chlorophyll precursors glutamate and glutamine. Transgenic expression of SRP1 in rice plants decreased GS2 activity and chlorophyll precursor accumulation, finally inducing leaf yellowing. This process is correlated with the previous evidence that the knockout of GS2 expression in rice plants causes a similar yellow chlorosis phenotype. Consistently, these yellowing leaves attracted higher numbers of leafhopper vectors, caused the vectors to probe more frequently, and presumably facilitate more efficient phytoplasma transmission. Together, these results uncover the mechanism used by phytoplasmas to manipulate the leaf color of infected plants for the purpose of enhancing attractiveness to insect vectors.

Ilex danxiaensis (Aquifoliaceae), a Distinct New Tree Species Endemic to Danxia Mountain in Guangdong Province, China, Based on Molecular and Morphological Evidence

Many holly species are dominant species in evergreen broad-leaved forests, and they play an important role in the formation and maintenance of forest ecosystems in tropical and subtropical regions. Here, a new holly species (Ilex danxiaensis K. W. Xu & Q. Fan) of Aquifoliaceae was discovered from Guangdong, China, when conducting biodiversity investigations in the Danxia landform. We inferred the phylogenetic position of the new species based on sequences from three nuclear markers, namely internal transcribed spacer (ITS), external transcribed spacer (ETS), and chloroplast glutamine synthetase gene (nepGS). The results demonstrated a close relationship among the new species and members of I. sect. Lioprinos. The inferred phylogenetic position of the new species is also supported by morphological evidence that all of these species are characterized by having axillary cymes inflorescences and abaxially 1-sulcate pyrenes. However, the new species can be distinguished from others by having puberulent branchlets, petioles, peduncles, and pedicel; (1–)2–3-flowered cymes of male inflorescences; 1-flowered cymes of female inflorescences; ciliate sepals, 4-merous flowers, pink or white female flowers; and 4 pyrenes.

Unraveling the mechanism of resistance in a glufosinate-resistant Palmer amaranth (Amaranthus palmeri) accession

AbstractGlufosinate resistance in Palmer amaranth (Amaranthus palmeri S. Watson) was recently detected in three accessions from Arkansas, USA. Amaranthus palmeri is the first and only broadleaf weed species resistant to this herbicide, and the resistance mechanism is still unclear. A previous study characterized the glufosinate resistance level in the accessions from Arkansas. A highly glufosinate-resistant accession was further used to investigate the mechanism conferring glufosinate resistance in A. palmeri. Experiments were designed to sequence the herbicide target enzyme cytosolic and chloroplastic glutamine synthetase isoforms (GS1 and GS2, respectively) and quantify copy number and expression. Absorption, translocation, and metabolism of glufosinate using the 14C-labeled herbicide were also evaluated in the resistant and susceptible accessions. The glufosinate-resistant accession had an increase in copy number and expression of GS2 compared with susceptible plants. All accessions showed only one GS1 copy and no differences in expression. No mutations were identified in GS1 or GS2. Absorption (54% to 60%) and metabolism (13% to 21%) were not different between the glufosinate-resistant and glufosinate-susceptible accessions. Most residues of glufosinate (94% to 98%) were present in the treated leaf. Glufosinate translocation to tissues above the treated leaf and in the roots was not different among accessions. However, glufosinate translocation to tissues below the treated leaf (not including roots) was greater in the resistant A. palmeri (2%) compared with the susceptible (less than 1%) accessions. The findings of this paper strongly indicate that gene amplification and increased expression of the chloroplastic glutamine synthetase enzyme are the mechanisms conferring glufosinate resistance in the A. palmeri accession investigated. Thus far, no additional resistance mechanism was observed, but further investigations are ongoing.

Isolation and characterization of chloroplastic glutamine synthetase gene (CsGS2) in tea plant Camellia sinensis

Tea plant (Camellia sinensis) is an ammonium preferring plant species. However, little is known about the mechanism underlying this preference. Herein, a chloroplastic glutamine synthetase gene (CsGS2), which is vital for nitrogen assimilation in mesophyll tissue, was isolated from tea cultivar C. sinensis cv. ‘Longjing43’. The full length cDNA of CsGS2 was 1622 bp, having a 1299 bp open reading frame encoding a 432-amino acid protein. Homology search and sequence analysis demonstrated that CsGS2 protein carried the basic characteristics of a canonical GS2 domain and shared high identity with GS2s from other plant species. Subcellular localization and immunolocalization of CsGS2 revealed that it is localized in chloroplast. qRT-PCR and Western blot analyses showed that CsGS2 was expressed in a leaf-specific pattern, such that both CsGS2 and its protein were most abundant in mature leaves. Temporal expression patterns of CsGS2 showed minor differences in response to ammonium and nitrate nutrition. The transcript level of CsGS2 was significantly induced in mature leaves during the development of new shoots, whereas darkness inhibited this induction significantly. These results suggested that CsGS2 does not play a role in the differential utilization mechanisms of differing nitrogen forms in tea, and imply a light dependent transcription regulation in mature leaves during the development of new shoots.

Exogenous DCPTA Increases the Tolerance of Maize Seedlings to PEG-Simulated Drought by Regulating Nitrogen Metabolism-Related Enzymes

2-(3,4-Dichlorophenoxy) triethylamine (DCPTA) regulates plant development; however, the molecular basis of this regulation is poorly understood. In this study, RNA sequencing (RNA-seq) analysis and physiological indexes of maize seedlings (three-leaf stage) treated with 15% polyethylene glycol (PEG) with/without DCPTA were investigated to explore the possible mechanism of exogenous DCPTA-improved drought tolerance. In the library pair comparisons of DCPTA vs. the control, PEG vs. the control, and PEG + DCPTA vs. PEG, totals of 19, 38 and 20 differentially expressed genes (DEGs) were classified as being involved in metabolic processes, respectively; totals of 5, 11, and 6 DEGs were enriched in the nitrogen (N) metabolic pathway, respectively. The genes encoding nicotinamide adenine dinucleotide-nitrate reductase (NADH-NR), ferredoxin-nitrite reductase (Fd-NiR), reduced ferredoxin- glutamate synthase (Fd-GOGAT), and chloroplastic glutamine synthetase (GS 2) were common in response to PEG-simulated drought stress with/without DCPTA treatment. Moreover, DCPTA maintained stable gene relative expression levels and protein abundances of NADH-NR, Fd-NiR, GS2, and Fd-GOGAT. Moreover, exogenous DCPTA partially mitigated PEG-simulated drought-induced reductions in the enzymatic activities of NR, nitrite reductase (NiR), glutamine synthase (GS), glutamine oxoglutarate aminotransferase (GOGAT), and transaminase, as well as in the contents of nitrate (NO3−), nitrite (NO2−) and soluble proteins and increases in the contents of ammonium (NH4+) and free amino acids. Together, our results indicate that exogenous DCPTA improved plant growth and drought tolerance by regulating N-mechanism enzymatic activities involved in transcription and enzymatic protein synthesis.

Arabidopsis thaliana mutants devoid of chloroplast glutamine synthetase (GS2) have non-lethal phenotype under photorespiratory conditions

Chloroplast located Glutamine Synthetase (GS2) is believed to play a major role in the reassimilation of ammonium generated by photorespiration, being GS2 knockout mutants unable to grow under photorespiratory conditions (low-CO2 atmosphere) in the species characterized so far (Barley, Lotus). To investigate the importance of GS2 in A. thaliana nitrogen metabolism mutant plants devoid of this GS isoenzyme were characterized. It was shown that GS2 mutants although smaller, slightly chlorotic and with the nitrogen metabolism impaired, were able to grow and complete their life cycle under ordinary air conditions. Surprisingly, GS2 mutants were more tolerant to salt stress than wild-type plants. The lack of GS2 seems to be compensated by higher expression of some GS cytosolic isogenes, namely GLN1;2 and GLN1;3 and by glutamate dehydrogenase, whose activity and expression is enhanced in the GS2 mutant plants and might account for the increased tolerance to salt stress. Under conditions that minimize photorespiration (CO2-enriched atmosphere) plant growth and ammonium assimilation impairment is less evident in the GS2 mutant plants and is accompanied by an adjustment of levels of expression of the cytosolic isogenes, with an increase in the expression of GLN1;3 and a decrease in the expression of the GLN1;1 and GLN1;2. Altogether the results confirm a major role of GS2 in the assimilation of ammonium released during photorespiration, but suggest a redundancy of activity with cytosolic GSs and GDH and further support the involvement of the chloroplastic isoenzyme in primary nitrogen assimilation and plant growth and development in A. thaliana.

Coordinated Expression of Cytosolic and Chloroplastic Glutamine Synthetase During Reproductive Stage and Its Impact in GS1 RNAi Transgenic Rice

To understand the reallocation of organic nitrogen from leaf to the flower head of rice, the role of glutamine synthetase (GS) was investigated by characterizing GS1 RNAi transgenic rice, which revealed a significant reduction in panicle number and number of seeds per panicle. We observed the expression of GS isotypes at transcriptional and protein levels in flag leaves, leaf sheaths and panicles at three different flower development stages. The mRNA expression of GS1;1 was clearly suppressed in flag leaves, especially at the flowering stage. GS1 protein was barely detectable in flag leaves until the flowering stage, while GS1 protein was compromised in the leaf sheath and panicle, with transient expression of GS2 protein at the flowering stage. The glutamine level in transgenic plants was significantly reduced in both flag leaves and panicles, but ammonium was highly accumulated. The level of other amino acids, including aspartate and asparagine, tended to be higher in RNAi transgenic plants than the wild type plants during the reproductive stage. In addition, accumulation of toxic ammonium in panicles with low glutamine level might have caused low seed-setting in the transgenic rice. These results indicated that nitrogen reallocation was critical for panicle development, and that multiple GS isotypes functioned cooperatively to complete the rice life cycle when leaf nitrogen was remobilized to the developing reproductive organs.

Expression Analysis of Nitrogen Metabolism-Related Genes Reveals Differences in Adaptation to Low-Nitrogen Stress between Two Different Barley Cultivars at Seedling Stage.

The excess use of nitrogen fertilizers causes many problems, including higher costs of crop production, lower nitrogen use efficiency, and environmental damage. Crop breeding for low-nitrogen tolerance, especially molecular breeding, has become the major route to solving these issues. Therefore, in crops such as barley (Hordeum vulgare L.), it is crucial to understand the mechanisms of low-nitrogen tolerance at the molecule level. In the present study, two barley cultivars, BI-04 (tolerant to low nitrogen) and BI-45 (sensitive to low nitrogen), were used for gene expression analysis under low-nitrogen stress, including 10 genes related to primary nitrogen metabolism. The results showed that the expressions of HvNIA2 (nitrite reductase), HvGS2 (chloroplastic glutamine synthetase), and HvGLU2 (ferredoxin-dependent glutamate synthase) were only induced in shoots of BI-04 under low-nitrogen stress, HvGLU2 was also only induced in roots of BI-04, and HvGS2 showed a rapid response to low-nitrogen stress in the roots of BI-04. The expression of HvASN1 (asparagine synthetase) was reduced in both cultivars, but it showed a lower reduction in the shoots of BI-04. In addition, gene expression and regulation differences in the shoots and roots were also compared between the barley cultivars. Taken together, the results indicated that the four above-mentioned genes might play important roles in low-nitrogen tolerance in barley.

Corrigendum: Chloroplast Glutamine Synthetase, the Key Regulator of Nitrogen Metabolism in Wheat, Performs Its Role by Fine Regulation of Enzyme Activity via Negative Cooperativity of Its Subunits

[This corrects the article on p. 191 in vol. 9, PMID: 29520285.].

Chloroplast Glutamine Synthetase, the Key Regulator of Nitrogen Metabolism in Wheat, Performs Its Role by Fine Regulation of Enzyme Activity via Negative Cooperativity of Its Subunits.

Glutamine synthetase (GS) is of central interest as the main route of ammonia assimilation in plants, and as a connection point between the organic and inorganic worlds. Even though GS activity is critical for producing high yields of crop plants, the autoregulation of substrate consumption of wheat GS remained unknown until now. Here we show kinetic evidence, that the chloroplast localized GS isoform (GS2) of wheat (Triticum aestivum L. cv. Jubilejnaja-50) takes place at the carbon-nitrogen metabolic branch point, where it is a mediator, and its enzymatic activity is regulated in a negatively cooperative allosteric manner. We have discovered that GS2 activity is described by a tetraphasic kinetic curve in response to increasing levels of glutamate supply. We constructed a model that explains the kinetic properties of glutamate consumption and this unique allosteric behavior. We also studied the subunit composition of both wheat leaf GS isoenzymes by a combination of two dimensional gel electrophoresis and protein blotting. Both leaf isozymes have homogeneous subunit composition. Glutamate is both a substrate, and an allosteric regulator of the biosynthetic reaction. We have concluded on the basis of our results and previous reports, that wheat GS2 is probably a homooctamer, and that it processes its substrate in a well-regulated, concentration dependent way, as a result of its negatively cooperative, allosteric activity. Thus, GS2 has a central role as a regulator between the nitrogen and the carbon cycles via maintaining glutamine-glutamate pool in the chloroplast on the level of substrates, in addition to its function in ammonia assimilation.

Delaying chloroplast turnover increases water-deficit stress tolerance through the enhancement of nitrogen assimilation in rice

Abiotic stress-induced senescence in crops is a process particularly affecting the photosynthetic apparatus, decreasing photosynthetic activity and inducing chloroplast degradation. A pathway for stress-induced chloroplast degradation that involves the CHLOROPLAST VESICULATION (CV) gene was characterized in rice (Oryza sativa) plants. OsCV expression was up-regulated with the age of the plants and when plants were exposed to water-deficit conditions. The down-regulation of OsCV expression contributed to the maintenance of the chloroplast integrity under stress. OsCV-silenced plants displayed enhanced source fitness (i.e. carbon and nitrogen assimilation) and photorespiration, leading to water-deficit stress tolerance. Co-immunoprecipitation, intracellular co-localization, and bimolecular fluorescence demonstrated the in vivo interaction between OsCV and chloroplastic glutamine synthetase (OsGS2), affecting source-sink relationships of the plants under stress. Our results would indicate that the OsCV-mediated chloroplast degradation pathway is involved in the regulation of nitrogen assimilation during stress-induced plant senescence.

スギには光呼吸に必須な葉緑体型グルタミン合成酵素（GS2）が存在しない

スギには光呼吸に必須な葉緑体型グルタミン合成酵素（GS2）が存在しない

The identification of new cytosolic glutamine synthetase and asparagine synthetase genes in barley (Hordeum vulgare L.), and their expression during leaf senescence.

Glutamine synthetase and asparagine synthetase are two master enzymes involved in ammonium assimilation in plants. Their roles in nitrogen remobilization and nitrogen use efficiency have been proposed. In this report, the genes coding for the cytosolic glutamine synthetases (HvGS1) and asparagine synthetases (HvASN) in barley were identified. In addition to the three HvGS1 and two HvASN sequences previously reported, two prokaryotic-like HvGS1 and three HvASN cDNA sequences were identified. Gene structures were then characterized, obtaining full genomic sequences. The response of the five HvGS1 and five HvASN genes to leaf senescence was then studied. Developmental senescence was studied using primary and flag leaves. Dark-exposure or low-nitrate conditions were also used to trigger stress-induced senescence. Well-known senescence markers such as the chlorophyll and Rubisco contents were monitored in order to characterize senescence levels in the different leaves. The three eukaryotic-like HvGS1_1, HvGS1_2, and HvGS1_3 sequences showed the typical senescence-induced reduction in gene expression described in many plant species. By contrast, the two prokaryotic-like HvGS1_4 and HvGS1_5 sequences were repressed by leaf senescence, similar to the HvGS2 gene, which encodes the chloroplast glutamine synthetase isoenzyme. There was a greater contrast in the responses of the five HvASN and this suggested that these genes are needed for N remobilization in senescing leaves only when plants are well fertilized with nitrate. Responses of the HvASN sequences to dark-induced senescence showed that there are two categories of asparagine synthetases, one induced in the dark and the other repressed by the same conditions.

Reduced glutamine synthetase activity plays a role in control of photosynthetic responses to high light in barley leaves

The chloroplastic glutamine synthetase (GS, EC 6.3.1.2) activity was previously shown to be the limiting step of photorespiratory pathway. In our experiment, we examined the photosynthetic high-light responses of the GS2-mutant of barley (Hordeum vulgare L.) with reduced GS activity, in comparison to wild type (WT). The biophysical methods based on slow and fast chlorophyll fluorescence induction, P700 absorbance, and gas exchange measurements were employed. Despite the GS2 plants had high basal fluorescence (F0) and low maximum quantum yield (Fv/Fm), the CO2 assimilation rate, the PSII and PSI actual quantum yields were normal. On the other hand, in high light conditions the GS2 had much higher non-photochemical quenching (NPQ), caused both by enhanced capacity of energy-dependent quenching and disconnection of PSII antennae from reaction centers (RC). GS2 leaves also maintained the PSII redox poise (QA−/QA total) at very low level; probably this was reason why the observed photoinhibitory damage was not significantly above WT. The analysis of fast chlorophyll fluorescence induction uncovered in GS2 leaves substantially lower RC to antenna ratio (RC/ABS), low PSII/PSI ratio (confirmed by P700 records) as well as low PSII excitonic connectivity.

Characterization of a glutamine synthetase gene DvGS2 from Dunaliella viridis and biochemical identification of DvGS2-transgenic Arabidopsis thaliana

The salt-tolerant green alga Dunaliella has remarkable capability to survive in some extreme environments such as nitrogen starvation, which makes Dunaliella be a proper model for mining novel genes on nitrogen uptake or assimilation. In this study, a glutamine synthetase (GS) gene DvGS2 with amino acid identity of 72% to other homologous GS proteins, was isolated and characterized from Dunaliella viridis. Phylogenetic comparison with other GSs revealed that DvGS2 occupied an independent phylogenetic position. Expressional analysis in D. viridis cells under nitrogen starvation confirmed that DvGS2 increased its mRNA level in 12h. Subcellular localization study and functional analysis in a GS-deficient Escherichia coli mutant proved that DvGS2 was a chloroplastic and functional GS enzyme. In order to investigate the potential application of DvGS2 in higher plants, the transgenic studies of DvGS2 in Arabidopsis thaliana were carried out. Results showed that the transgenic lines expressed the DvGS2 gene and demonstrated obviously enhanced root length (29%), fresh weight (40%–48% at two concentrations of nitrate supplies), stem length (21%), leaf size (39%) and silique number (44%) in contrast with the wild-type Arabidopsis. Furthermore, the transgenic lines had higher total nitrogen content (35%–43%), total GS activity (39%–45%) and soluble protein concentration (23%–24%) than the wild type. These results indicated that the overexpression of DvGS2 in A. thaliana resulted in higher biomass and the improvement of the host's nitrogen use efficiency.

Stromal protein degradation is incomplete in Arabidopsis thaliana autophagy mutants undergoing natural senescence

BackgroundDegradation of highly abundant stromal proteins plays an important role in the nitrogen economy of the plant during senescence. Lines of evidence supporting proteolysis within the chloroplast and outside the chloroplast have been reported. Two extra-plastidic degradation pathways, chlorophagy and Rubisco Containing Bodies, rely on cytoplasmic autophagy.ResultsIn this work, levels of three stromal proteins (Rubisco large subunit, chloroplast glutamine synthetase and Rubisco activase) and one thylakoid protein (the major light harvesting complex protein of photosystem II) were measured during natural senescence in WT and in two autophagy T-DNA insertion mutants (atg5 and atg7). Thylakoid-localized protein decreased similarly in all genotypes, but stromal protein degradation was incomplete in the two atg mutants. In addition, degradation of two stromal proteins was observed in chloroplasts isolated from mid-senescence leaves.ConclusionsThese data suggest that autophagy does contribute to the complete proteolysis of stromal proteins, but does not play a major degenerative role. In addition, support for in organello degradation is provided.

The toxic effects of diethyl phthalate on the activity of glutamine synthetase in greater duckweed (Spirodela polyrhiza L.)

The toxic effects of diethyl phthalate (DEP), a potent allelochemical, on the enzyme activity and polypeptide accumulation of glutamine synthetase (GS) in greater duckweed were investigated. In our previous studies, DEP induced oxidative responses at concentrations from 0.5 to 2mM in greater duckweed and the antioxidant enzymes played important roles in the defense strategy against DEP stress. In this study, DAB-H2O2 and NBT stain for superoxide radicals (O2.−), lipid peroxidation, HSP70, and ammonia accumulation in DEP-treated duckweed tissues revealed adverse effect of DEP in plant growth. Biochemical analysis and physiological methods were combined to investigate GS activity and polypeptide accumulation under DEP-induced stress. The results showed that GS activity was reduced with the increasing concentration of DEP, indicative of enhanced toxic effect. Immunoblot analysis with chloroplast soluble fractions indicated that the chloroplastic GS (GS2) polypeptide from greater duckweed was degraded under DEP stress conditions. The response of GS2 to the DEP stress may be modulated by means of redox change in plant tissues, chloroplasts, and chloroplast lysates. The results suggest that DEP is toxic to the greater duckweed by inhibition of the GS isoenzymes in nitrogen assimilation and the GS2 plays important roles in the adaptation strategy against DEP toxicity.

Phylogeography of Cyananthus delavayi (Campanulaceae) in Hengduan Mountains inferred from variation in nuclear and chloroplast DNA sequences

Abstract Phylogeographic studies on alpine plants endemic to the Hengduan Mountains of the southeastern Qinghai–Tibet Plateau are still limited in number. In this study, we used sequence variation of one nuclear gene (ncpGS, which encodes the chloroplastic glutamine synthetase) and in two chloroplast DNA segments to investigate the phylogeographic structure and population demographic history of Cyananthus delavayi, a narrow‐range species endemic to this region. We identified eight chlorotypes and 16 nuclear genotypes in a survey of 10 populations sampled throughout the range of the species. The results of both phylogenetic and network analyses suggested that the genealogical relationships of both chlorotypes and nuclear genotypes showed a clear geographical correlation. High total genetic diversity, low levels of within‐population diversity, and strong population differentiation (chloroplast DNA: hT= 0.827, hS= 0.087, NST= 0.899, GST= 0.895; nuclear DNA: hT= 0.910, hS= 0.348, NST= 0719, GST= 0.618) were identified. Based on the mismatch distribution analyses, no evidence of recent demographic population expansion was found for this species. Nested clade analyses of both chlorotypes and nuclear genotypes indicated that restricted gene flow resulting from isolation by distance and allopatric fragmentation were likely to have been the major processes that shaped their present‐day spatial distribution. Our dating of the genetic divergences between three major geographic lineages suggested that the largest glaciation of the early Quaternary developed in the Qinghai–Tibet Plateau and mountainous isolation may have together led to deep intraspecific vicariance within this species.

The amino-terminal domain of chloroplast Hsp93 is important for its membrane association and functions in vivo.

Chloroplast 93-kD heat shock protein (Hsp93/ClpC), an Hsp100 family member, is suggested to have various functions in chloroplasts, including serving as the regulatory chaperone for the ClpP protease in the stroma and acting as a motor component of the protein translocon at the envelope. Indeed, although Hsp93 is a soluble stromal protein, a portion of it is associated with the inner envelope membrane. The mechanism and functional significance of this Hsp93 membrane association have not been determined. Here, we mapped the region important for Hsp93 membrane association by creating various deletion constructs and found that only the construct with the amino-terminal domain deleted, Hsp93-ΔN, had reduced membrane association. When transformed into Arabidopsis (Arabidopsis thaliana), most atHsp93V-ΔN proteins did not associate with membranes and atHsp93V-ΔΝ failed to complement the pale-green and protein import-defective phenotypes of an hsp93V knockout mutant. The residual atHsp93V-ΔN at the membranes had further reduced association with the central protein translocon component Tic110. However, the degradation of chloroplast glutamine synthetase, a potential substrate for the ClpP protease, was not affected in the hsp93V mutant or in the atHSP93V-ΔN transgenic plants. Hsp93-ΔN also had the same ATPase activity as that of full-length Hsp93. These data suggest that the association of Hsp93 with the inner envelope membrane through its amino-terminal domain is important for the functions of Hsp93 in vivo.

Circadian Patterns of the Major Nitrogen Metabolism–Related Enzymes and Metabolites in Creeping Bentgrass and the Influence of Cytokinin and Nitrate

Creeping bentgrass (Agrostis stolonifera L.) is a major low‐cut turfgrass species worldwide. Information is limited on the diurnal variation in its N metabolism, particularly the impacts of cytokinin (CK) and nitrate (NO3−). To gain insight into the diurnal fluctuation of N metabolism and the effects of CK and nitrate, the transcript abundance of the major N metabolism enzymes in shoots, including nitrate reductase (NR), nitrite reductase, chloroplastic glutamine synthetase (GS2), ferredoxin‐dependent glutamate synthase (Fd‐GOGAT), and glutamate dehydrogenase, were studied during the day/night cycle (14/10 h). All measured parameters exhibited clear diurnal changes with some differences among treatments (control, CK at 50 μM, and nitrate at 7.5 kg N ha−1), except GS2 expression and total protein. Both NR expression and nitrate content showed a peak at 0430 h (8.5 h in dark). Nitrate nutrition increased diurnal change of nitrate content, showing an over two‐fold diurnal difference compared to 63 and 81% differences for the control and CK treatment, respectively. Simultaneously, CK shifted the diurnal in vivo NR activity pattern. In addition, creeping bentgrass appears to be able to maintain N assimilation at night comparable to that during the day, demonstrated by either similar or even higher levels of measured parameters between certain phases of light and dark periods. This study provides further information for understanding how CK and nitrate regulate N metabolism in creeping bentgrass.