Studies On Physiological And Molecular Mechanisms Of Highland Barley In Response To Low Nitrogen Stress

Nitrogen（N）is an essential nutrient element for crop growth and also one of the limiting factors affecting crop yield.However,excessive application of N fertilizers has caused serious environmental pollution and ecological imbalance.Agricultural production urgently needs to reduce the heavy dependence on N fertilizers.Breeding crop varieties with high N use efficiency is a useful and effective approach to achieve the goal of reducing N fertilizer dependence.Therefore,it is of great significance to study the physiological and molecular mechanisms of low N tolerant crops for breeding high NUE crop varieties.Highland barley,one of the unique barley varieties,is widely cultivated in the Qinghai-Tibet Plateau of China.It has adapted to the extreme environments for a long time and has high tolerance to barren soil.In this study,by using Kunlun14（highland barley）and Ganpi6（common barley）as experimental material,we explored the molecular mechanisms of low N tolerance in highland barley through physiological,molecular,transcriptomic,genetic and other methodologies.In addition,the mechanism of auxin participation in regulating root morphology and carbon and nitrogen metabolism of highland barley under low N stress was also investigated.Furthermore,we analyzed the physiological and molecular functions of glucose 6-phosphate dehydrogenase（G6PDH）,especially Pla-G6PDH3（Plastic G6PDH3）in highland barley in response to low N stress.The main findings are as follows:1 Physiological and molecular mechanisms of highland barley in response to low N stress（1）Low N significantly reduced the plant height and above-ground dry and fresh weight of Kunlun14 and Ganpi6,but promoted the root length and biomass.Interestingly,the relative growth and the Root/Shoot ratio in Kunlun14 were increased,but not in Ganpi6,suggesting that Kunlun14 has stronger tolerance to low N than Ganpi6.（2）Under low N condition,the contents of free amino acids,soluble proteins and nitrogen in Kunlun14 were decreased,but still higher than those in Ganpi6;the activities of nitrate reductase（NR）and glutamine synthetase（GS）in Kunlun14 and Ganpi6 were also decreased,but the decrease in Ganpi6 was greater than that in Kunlun14;the activities of nitrite reductase（Ni R）and glutamate synthetase（GOGAT）were increased,especially in Kunlun14,which was significantly higher than that in Ganpi6.These results indicate that stronger N assimilation ability in Kunlun14 may be one of the reasons for its high tolerance to low N.（3）Under low N stress,compared with Ganpi6,the expression of nitrate transporter coding genes,NRT2.1,NRT2.2,NRT2.5,NRT2.8 and NAR3.1,in Kunlun14 was significantly up-regulated;moreover,the activity of PM H⁺-ATPase was significantly increased in the roots of Kunlun14;¹⁵N tracing analysis showed that the NO₃^-acquisition and transport activities in Kunlun14 were 21.5%and 33.4%higher than those in Ganpi6,respectively.These results suggest that Kunlun14 has higher NO₃^-uptake and translocation capacity in comparison with Ganpi6 under low N stress.（4）Under low N stress,the chlorophyll contents in Ganpi6 and Kunlun14 were decreased,but the extent in Ganpi6 was greater;the net photosynthetic rates in Ganpi6 and Kunlun14 were also decreased,but the rate in Kunlun14 was higher than that in Ganpi6;the photosynthetic nitrogen utilization rate in Kunlun14 was increased significantly by 16.6%,while the rate in Ganpi6 had no significant changes.These results showed that Kunlun14 has higher photosynthetic rate and photosynthetic N utilization capacity under low N stress.（5）Principal component analysis（PCA）analysis between varieties and low N tolerance indices showed that root length,root dry weight and root/shoot ratio may be the main indices of low N tolerance in Kunlun14.The results of Orthogonal Partial Least Squares Discriminant Analysis（OPLS-DA）showed that the VIP values of root NO₃^-content and root amino acid content were greater than 1,which could be used as physiological substances to distinguish Ganpi6 from Kunlun14.（6）The results of transcriptomic analysis showed that under low N stress,there were 1575 and 1594 DEGs in Kunlun14 and Ganpi6,respectively.KEGG functional enrichment analysis revealed that pathways of DEGs only expressed in Kunlun14were mainly enriched in flavonoid biosynthesis,phenylpropane biosynthesis,nitrogen metabolism,starch and sucrose metabolism,and plant hormone signal transduction;while the pathways of DEGs only exppressed in Ganpi6 were mainly enriched in plant pathogen interaction,phenylpropane biosynthesis,MAPK signal pathway,flavonoids synthesis,and starch and sucrose metabolism.Especially in N metabolism,the nitrate or ammonium transporter DEGs enriched in Kunlun14 were more than those in Ganpi6.Moreover,most of them were up-regulated.In addition,DEGs related to auxin signal transduction,root development and cell wall expansion proteins were also up-regulated in Kunlun14.（7）The weighted gene co-expression network（WGCNA）method was used to construct the gene co-expression networks of the two materials at different nitrogen concentrations,and six modules with positive or negative correlation with low N tolerance were obtained.MEmagenta module was significantly positively correlated with root length(r=0.93,P=8.0×10^–6),root dry weight(r=0.96,P=5×10^-7)and root/shoot ratio(r=0.91,P=5×10^-5).The KEGG enrichment analysis of the first 150 core genes of MEmagenta module showed that the phenylpropanoid metabolism,plant hormone signal transduction,lipid metabolism could respond to low N stress in Kunlun14.In addition,two genes related to plant hormones（IAA14,IAA26）and xyloglucosidase/hydrolase proteins（XTH1,XTH4）were identified in this module,which were also in the key position in the co-expression network.2 Molecular mechanism of auxin in regulating root morphology and carbon/nitrogen metabolism balance in highland barley under low N stress（1）Under low N condition,the root length of Kunlun14 was significantly higher than that of Ganpi6;PI staining showed that the cell length of elongation zone cells in Kunlun14 roots was 12.9%higher than that in Ganpi6,and the number of meristem cells in Kunlun14 was 17.8%higher than that of Ganpi6.（2）The auxin content in Kunlun14 roots was increased and significantly higher than that in Ganpi6 under low N.When the auxin transport inhibitor NPA（100μM）was added,the elongation of primary roots of Ganpi6 and Kunlun14 was significantly inhibited by 30.4%and 23.2%,respectively;the uptake capacity of NO₃^-was decreased by 38.6%and 18.1%,and the transport activity decreased by 20.2%and27.2%in Ganpi6 and Kunlun14,respectively.These results showed that auxin affects root development,which further affects the capacity of NO₃^-uptake and transport under low N condition.（3）Under low N treatment,exogenous NPA also reduced the shoot carbon content by 6.5%and 12.3%and nitrogen content by 5.9%and 6.9%,in Kunlun14 and Ganpi6,respectively.The carbon/nitrogen ratio was also significantly decreased by15.9%and 16.1%in Kunlun14 and Ganpi6,respectively.（4）The analysis of photosynthetic parameters showed that the photosynthetic rate（Photo）,the maximum photochemical efficiency（Fv/Fm）,the photochemical quenching coefficient（q P）,the electron transfer rate（ETR）,and the actual photochemical efficiency（YII）of PSII in Kunlun14 and Ganpi6 were significantly decreased,while the non-photochemical quenching coefficient（q N）was increased after NPA was added under low N treatment.Results also showed that auxin affected the photosynthetic efficiency of plants under low N stress.Furthermore,the analysis of thylakoid membrane protein complex by Blue-Native PAGE showed that low N stress significantly reduced the level of PSII protein complex in leaves of two barley,and the decrease extent in Ganpi6 was greater than that in Kunlun14.When exogenous NPA was added,the content of PSII was further reduced,and the contents of PSII proteins and LHCII were also significantly reduced.3 Overexpression of highland barley Pla-Hv G6PDH3 increases the low N tolerance in Arabidopsis thaliana.（1）Low N markedly induced the Tot-G6PDH activity in both barley roots,especially the Pla-G6PDH activity in Kunlun14.The nitrate uptake and assimilation capacity were significantly reduced when G6PDH function was inhibited by Glc N under low N condition.Dysfunction of G6PDH markedly decreased the expressions of NRT2.5 and NAR3.1.Moreover,the activities of NR,Ni R,GS and GOGAT were decreased obviously in both barley cultivars under low N stress.These results showed that the N acquisition and assimilation efficiency is positively correlated with the G6PDH activity in barley tolerance to low N stress.（2）Phylogenetic analysis of five G6PDH protein-encoding genes in highland barley（Hv G6PDHs）showed that Hv G6PDH1,Hv G6PDH3 and Hv G6PDH4 are localized in plastids,while Hv G6PDH2 and Hv G6PDH5 are localized in the cytoplasm.The results of tissue expression pattern analysis showed that Hv G6PDH1is mainly expressed in primary root,second leaf and inflorescence axis;Hv G6PDH3is mainly expressed in primary root,adventitious root and second leaf;Hv G6PDH4 is mainly expressed in the second leaf;Hv G6PDH2 and Hv G6PDH5 are mainly expressed in adventitious roots,inflorescences and flag leaves.（3）qRT-PCR results showed that the Hv G6PDH3 expression was significantly induced by low N.When Hv G6PDH3 was overexpressed in Arabidopsis thaliana,the biomass and root length of overexpression lines（OE-1,OE-2,OE-5）were superior to those of wild type（WT）,and the number of siliques and yield per plant in OE lines were higher than those of WT.These results showed that overexpression of Hv G6PDH3 improves the low N tolerance of Arabidopsis.（4）Compared with WT,the expression of nitrate transporter-coding genes At NRT1.5 and At NRT2.1 was significantly up-regulated in overexpression lines under low N stress;the activities of NR and Ni R were markedly increased in overexpression lines.¹⁵N tracing experiment showed that the NO₃^-uptake activity was increased by23.2%,29.3%and 30.8%in OE-1,OE-2 and OE-5,respectively;while the transport activity was increased by 32.9%,37.5%and 35.6%in OE-1,OE-2 and OE-5,respectively,compared with that in WT under low N condition.The above results imply that overexpression of Hv G6PDH3 in Arabidopsis increases nitrate uptake and translocation capacity,which results in higher tolerance to low N stress.In conclusion,the root length,R/S ratio,and relative root growth rate in Kunlun14 are significantly higher than those in Ganpi6 under N stress,suggesting that Kunlun14 has higher low N tolerance.Compared to Ganpi6,Kunlun14 has higher tolerance to low N due to increased capacity of N uptake,translocation,assimilation capacity and photosynthetic nitrogen utilization capacity.The nitrate uptake and assimilation capacity are significantly reduced when G6PDH function is inhibited by Glc N under low N condition.qRT-PCR and RNA-Seq analysis showed that Pla-G6PDH3 is markedly induced by low N,especially in Kunlun14.Overexpression of Hv G6PDH3 in Arabidopsis up-regulates At NRT1.5 and At NRT2.1 transcriptions and increases nitrate uptake and translocation capacity,which results in high seed yield under low N stress.The transcriptomic analysis results showed that the DEGs involved in plant hormones,TFs,and cell wall biosynthesis play key roles in the root development and N metabolism in highland barley.Auxin can positively regulate the tolerance to low N by regulating the root morphological changes,photosynthetic capacity and C/N metabolism balance in highland barley.