Nitrogen(N)is a key component in plants and their biological macromolecules,having a profound effect on developmental stages,such as germination,vegetative growth,and flowering.However,the mechanism of nitrogen-regulated flowering time remains unclear.In this study,CmNLP7 was isolated from the chrysanthemum cultivar‘Jinba'and was characterized.CmNLP7 is a transcription factor localized in the nucleus but has no transcriptional activity.Tissue expression pattern analysis showed that CmNLP7 was mainly transcribed in leaves and roots.Knocking down CmNLP7 through the artificial-miRNA method in chrysanthemum resulted in early flowering under optimal nitrogen(ON)and low nitrogen(LN)conditions;whereas overexpression lines showed delayed flowering under LN conditions.Transcriptome sequencing analysis showed that the nitrate transporters NRT2.5,NPF3.1,and NPF4.6;SBP-like genes SPL7 and SPL12,and flowering integration factor FT were significantly up-regulated in the knockdown lines.Based on the KEGG pathway enrichment analysis,the differentially transcribed genes were enriched in phenylpropanoid biosynthesis and starch and sucrose metabolism pathways,which indicated their alleged function in nitrogen-regulated flowering and development in chrysanthemum.Furthermore CmPP6 as a homolog of the Arabidopsis phosphatase PP6,was verified as an interacting protein of CmNLP7 by yeast two-hybrid,BiFC,pull-down and Biacore in vitro and in vivo,and the knockdown line of CmPP6(amiR-CmPP6)flowered earlier compared to that of the wild-type chrysanthemum‘Jinba'.Collectively,these results demonstrated that CmPP6 interacts with CmNLP7 to regulate chrysanthemum flowering,and CmNLP7 could regulate flowering time in response to nitrogen,which lays a foundation for the regulation of flowering and molecular breeding of chrysanthemum through changes in nutrient signaling.
Nitrogen(N)is a limiting factor that determines the yield and quality of chrysanthemum.Genetic variation in N use efficiency(NUE)has been reported among chrysanthemum genotypes.We performed a transcriptome analysis of two chrysanthemum genotypes,'Nannonglihuang'(LH,N-efficient genotype)and'Nannongxuefeng"(XF,N-inefficient genotype),under low N(0.4 mmol L^(-1)N)and normal N(8 mmol L^(-1)N)treatments for 15 d and an N recovery treatment for 12 h(low N treatment for 15 d and then normal N treatment for 12 h)to understand the genetic factors impacting NUE in chrysanthemum.The two genotypes exhibited contrasting responses to the different N treatments.The N-efficient genotype LH had significant superiority in agronomic traits,N accumulation and glutamine synthase activity under both normal N and low N treatments.Low N treatment promoted root growth in LH,but inhibited root growth in XF.Transcriptome analysis revealed that the low N treatment increased the expression of some N metabolism genes,genes related to auxin and abscisic acid signal transduction in the roots of both genotypes,as well as genes related to gibberellin signal transduction in roots of LH.The N recovery treatment just increased the expression of genes related to cytokinin signal transduction in roots of LH.The expression levels of the NRT2.1,AMT1.1,and Gln1 genes related to gibberellin and cytokinin signal transduction were higher in roots of LH than in XF under different N treatments,suggesting that the genes related to N metabolism and hormone(auxin,abscisic acid,gibberellin,and cytokinin)signal transduction in roots of LH are more sensitive to different N treatments than those of XF.Co-expression network analysis(WGCNA)also identified hub genes like bZIP43,bHLH93,NPF6.3,IBR10,MYB62,PP2C,PP2C06 and NLP7,which may be the key regulators of N-mediated responses in chrysanthemum and play crucial roles in enhancing NUE and resistance to low N stress in the N-efficient chrysanthemum genotype.These results revealed the key factors involved in regulating N
