| Environmental pollution caused by excessive use of nitrogen fertilizer has become a key problem to be solved in production.An effective approach to solve nitrogen deficiency is to develop low-N tolerant crop cultivars.Thus,it is of great practical significance for the sustainable development of ecology and agriculture to explore the physiological and molecular mechanisms of low-nitrogen tolerant crops and improve the nitrogen absorption and utilization efficiency of crops.Panicum miliaceum(L.)is a tetraploid cultivar with wide adaptability and strong resistance to drought,salt and alkali,and temperature stress.It may have evolved a unique mechanism of barren tolerance,especially low nitrogen tolerance.Different low-nitrogen tolerant proso millet varieties as experimental materials were selected to study the physiological-molecular mechanism of resistance to low-nitrogen conditions and excavate key genes for low-nitrogen tolerance,so as to provide some enlightenment for improving the nitrogen use efficiency of crops.The main findings were summarized as follows:1.Determining the index system of low nitrogen screening concentration and low nitrogen tolerance identification of proso millet resources.By setting 8 nitrogen concentration gradients of 0,0.1,0.25,0.5,0.75,1,2.5 and 5 m M,0.25 m M N could be used as the suitable nitrogen concentration for the identification of low nitrogen tolerance of proso millet at seedling stage.84 proso millet germplasm resources were identified for low nitrogen tolerance by hydroponic experiments.The plant nitrogen accumulation,dry matter mass,root-shoot ratio and shoot nitrogen use efficiency of the whole plant could be used as the main indexes for low nitrogen tolerance identification.86,184,235 and 298 showed good tolerance to low nitrogen,while 111 and 230 were sensitive to low nitrogen treatment.They can be used to study the mechanism of response to low nitrogen.2.Physiological analysis of response to low nitrogen stress of low-nitrogen tolerant cultivars.Two low nitrogen tolerant(184 and 298)and two low nitrogen sensitive(111 and 230)proso millet cultivars were used as experimental materials.Physiological differences between low nitrogen tolerant and nitrogen sensitive proso millet cultivars in response to low nitrogen stress were analyzed after treatment with 5 m M(control)and 0.25 m M(low nitrogen stress)for 21 d and after re-nitrogen for 7 d.Under the low N condition,the leaf chlorophyll SPAD value,net photosynthetic rate,transpiration rate,stomatal conductance and chlorophyll fluorescence parameters of the low-nitrogen tolerant cultivar decreased slightly,maintained a higher photosynthetic capacity,and promoted the photosynthetic matter production capacity of the leaves.The low-nitrogen tolerant cultivars had higher NPQ induction,which reduced the excess light energy in the photosynthetic system and maintained a higher electron transport rate.Meanwhile,a higher leaf photosynthetic enzymes and nitrogen transport enzyme activity was to ensure the more nitrogen compounds synthesized under low nitrogen stress,and the leaf physiological metabolism was stronger for delaying the senescence of plant.The resistance to low nitrogen and low nitrogen tolerance in low-nitrogen tolerant varieties were obviously higher than those in sensitive varieties,and showed stronger adaptive low nitrogen and the recovery after nitrogen.3.Expression profile analysis of low-nitrogen tolerant cultivars under low nitrogen stress based on RNA-seq technique.The transcriptome changes of one cultivar with low nitrogen tolerance(184)and one cultivar with nitrogen sensitivity(111)were analyzed by RNA-seq sequencing technology after 21 days of low nitrogen treatment and 7 days after re-nitrogen.A total of 5254 differentially expressed genes(DEGs)by transcription spectrum analysis,including cells components,optical system II(PSII),photosynthetic membrane structure,carbohydrate metabolism related genes,mainly involved in nitrogen metabolism,photosynthesis,starch and sucrose metabolism,amino acid,alanine,glutamic acid and aspartic acid,glycine,serine and threonine metabolism and other metabolic process.The high expression levels of nitrogen transport-related enzymes and photosynthetic enzymes in low-nitrogen tolerant cultivars enabled them to absorb and transport nitrogen more efficiently under low nitrogen stress,which may be an important reason for the improvement of low nitrogen tolerance in low nitrogen tolerant cultivars.4.Identification and expression pattern analysis of NRT1 and NRT2 gene family in proso millet.A total of 9 NRT1 and 6 NRT2 family genes were identified from the whole genome of proso millet.NRT1 and NRT2 family members contained PTR2 and MFS-1 protein conserved domains,respectively.Phylogenetic analysis showed that the nine NRT1 genes in proso millet could be divided into five NPF subfamilies,and the same subfamily gene had similar gene structure and protein conserved motif.The promoter regions of members of the NRT1 and NRT2 gene families contain a variety of hormone and stress response cis-acting elements.NRT1.1,NRT1.4,NRT1.9,NRT2.1,NRT2.2,NRT2.6,NRT3.1 and NRT3.2 were all expressed in roots and leaves,and the expression levels of NRT1.4 and NRT2.1 in leaves and roots were the highest and significantly higher than those in other groups,while the expression levels of NRT2.2,NRT2.6,NRT3.1 and NRT3.2 in leaves and roots were extremely low,suggesting that the expression levels of NRT1 and NRT2 genes were tissue specific.Nitrogen induction experiments showed that the maximum time of these genes in roots was earlier than that in leaves,indicating that the root response to nitrogen occurred earlier than that in leaves.Through the analysis of gene expression patterns in different growth stages and different organs in field experiments with different nitrogen levels,it was found that the gene expression patterns were different under different nitrogen levels.In different reproductive periods,it is manifested as:(1)The expression level of each gene in the leaves was higher at the seedling stage,which might be related to the vegetative growth of proso millet mainly relying on leaves at the seedling stage,(2)The expression levels of each gene in root and stem were higher at flowering and grain filling stages,and the accumulation of nitrogen in root and stem reached the maximum during this period,indicating that these genes may be involved in the accumulation and transport of nitrogen,(3)The expression levels of each gene in the panicle reached the maximum at grain filling stage,indicating that these genes may be involved in nitrogen transport during grain formation.5.Exploring on the mechanism of reducing nitrogen and increasing effect of low-nitrogen tolerant proso millet.Four cultivars with low-nitrogen tolerance(86,184,235,298)and two cultivars with low-nitrogen sensitivity(111,230)were treated with different nitrogen levels in the field for two years.Low-nitrogen tolerant cultivars maintained higher dry matter accumulation,SPAD value and photosynthetic characteristics of leaves under low nitrogen condition,which improved the plant's ability to capture and absorb light energy,and promoted the photosynthetic matter production capacity of leaves.The main reason for this advantage in low-nitrogen tolerant varieties is that the photosynthetic active radiation interception and radiation utilization efficiency of the proso millet population were significantly improved in the low-nitrogen tolerant varieties under the low nitrogen condition,which enhanced the efficient utilization of soil water,and promoted the nitrogen absorption and utilization of the low nitrogen tolerant proso millet.Finally,the productivity level of the proso millet was significantly increased. |
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