Involvement of the ammonium assimilation mediated by glutamate dehydrogenase in response to heat stress in the scleractinian coral Pocillopora damicornis

Glutamate dehydrogenase(GDH)plays an important role in the ammonium assimilation and nitrogen metabolism by catalyzing the reversible oxidative deamination of L-glutamate toα-ketoglutarate.In the present study,the potential functions of GDH in response to heat stre ss were explored in the scleractinian coral Pocillopora damicornis(designated as PdGDH).The cDNA of PdGDH contained an open reading frame of 1611 bp encoding a polypeptide of 536 amino acids,which exhibited the highest sequence identity to GDH of Stylophora pistillata(96%identity),and the deduced PdGDH protein was predicted to contain one GdhA domain(from Val95 to Tyr525).The recombinant protein of PdGDH(rPdGDH)was expressed in Escherichia coli BL21(DE3)-Transetta,and its catalytic activity was measured under different temperatures,pH conditions and epigallocatechin-3-gallate(EGCG,a GDH inhibitor)concentrations.The purified rPdGDH only used reduced coenzyme nicotinamide adenine dinucleotide(NADH)as coenzyme,and its highe st activity was observed at 35℃and pH 7.5,re spectively.The rPdGDH activity was negatively correlated with the concentration of EGCG,and was inhibited by more than half(65%,P<0.05)at 10mol/L EGCG.No significant alteration of PdGDH mRNA expression was detected at 12 h after exposure to heat and ammonium(P>0.05).Furthermore,the activities of NADH-GDH in the scleractinian coral P.damicornis increased significantly at 12 h after the heat and ammonium stress,and the NADH-GDH activity in the heat stress group(32.66 U/mg,P<0.05)was significantly higher than that in the heat and ammonium stress group(11.26 U/mg).These results collectively suggested that PdGDH,as a homologue of glutamate dehydrogenase in the scleractinian coral P.damicornis,could respond to heat stress at the protein level,which would have ability to further promote ammonium assimilation to increase the heat acclimatization of the coral-Symbiodiniaceae symbiotic association.

Nitrogen assimilation in plants:current status and future prospects

Nitrogen(N)is the driving force for crop yields;however,excessive N application in agriculture not only increases production cost,but also causes severe environmental problems.Therefore,comprehensively understanding the molecular mechanisms of N use efficiency(NUE)and breeding crops with higher NUE is essential to tackle these problems.NUE of crops is determined by N uptake,transport,assimilation,and remobilization.In the process of N assimilation,nitrate reductase(NR),nitrite reductase(Ni R),glutamine synthetase(GS),and glutamine-2-oxoglutarate aminotransferase(GOGAT,also known as glutamate synthase)are the major enzymes.NR and Ni R mediate the initiation of inorganic N utilization,and GS/GOGAT cycle converts inorganic N to organic N,playing a vital role in N assimilation and the final NUE of crops.Besides,asparagine synthetase(ASN),glutamate dehydrogenase(GDH),and carbamoyl phosphate synthetase(CPSase)are also involved.In this review,we summarize the function and regulation of these enzymes reported in three major crops—rice,maize,and wheat,also in the model plant Arabidopsis,and we highlight their application in improving NUE of crops via manipulating N assimilation.Anticipated challenges and prospects toward fully understanding the function of N assimilation and further exploring the potential for NUE improvement are discussed.

A Critical Role of AMT2;1 in Root-To-Shoot Translocation of Ammonium in Arabidopsis

Ammonium uptake in plant roots is mediated by AMT/MEP/Rh-type ammonium transporters. Out of five AMTs being expressed in Arabidopsis roots, four AMT1-type transporters contribute to ammonium uptake, whereas no physiological function has so far been assigned to the only homolog belonging to the MEP subfamily, AMT2;1. Based on the observation that under ammonium supply, the transcript levels of AMT2;1 increased and its promoter activity shifted preferentially to the pericycle, we assessed the contribution of AMT2;1 to xylem loading. When exposed to ^15N-labeled ammonium, amt2;1 mutant lines translocated less tracer to the shoots and contained less ammonium in the xylem sap. Moreover, in an amtl;1 amtl;2 amtl ;3 amt2;1 quadruple mutant （qko）, co-expression of AMT2;1 with either AMT1;2 or AMT1;3 significantly enhanced ^15N translocation to shoots, indicating a cooperative action between AMT2;1 and AMT1 transporters. Under N deficiency, proAMT2;1-GFP lines showed enhanced promoter activity predominantly in cortical root cells, which coincided with elevated ammonium influx conferred by AMT2;1 at millimolar sub- strate concentrations. Our results indicate that in addition to contributing moderately to root uptake in the low-affinity range, AMT2;1 functions mainly in root-to-shoot translocation of ammonium, depending on its Cell-type-specific expression in response to the plant nutritional status and to local ammonium gradients.

The Plastid-Localized NAD-Dependent Malate Dehydrogenase Is Crucial for Energy Homeostasis in Developing Arabidopsis thaliana Seeds

In the absence of photosynthesis, ATP is imported into chloroplasts and non-green plastids by ATP/ADP transporters or formed during glycolysis, the latter requiring continuous regeneration of NAD＋, supplied by the plastidial isoform of NAD-MDH. During screening for T-DNA insertion mutants in the plNAD-MDH gene of Arabidopsis, only heterozygous plants could be isolated and homozygous knockout mutants grew only after complementation. These heterozygous plants show higher transcript levels of an alternative NAD＋-regenerating enzyme, NADH-GOGAT, and, remarkably, improved growth when ammonium is the sole N-source. In situ hybridization and GUS-histochemical stain- ing revealed that plNAD-MDH was particularly abundant in male and female gametophytes. Knockout plNAD-MDH pollen exhibit impaired tube growth in vitro, which can be overcome by adding the substrates of NADH-GOGAT. In vivo, knockout pollen is able to fertilize the egg cell. Young siliques of selfed heterozygous plants contain both green and white seeds corresponding to wild-type/heterozygous （green） and homozygous knockout mutants （white） in a （1：2）：1 ratio. Embryos of the homozygous knockout seeds only reached the globular stage, did not green, and developed to tiny wrinkled seeds. Complementation with the gene under the native promoter rescued this defect, and all seeds developed as wild-type. This suggests that a blocked major physiological process in plNAD-MDH mutants stops both embryo and endosperm development, thus avoiding assimilate investment in compromised offspring.