Mechanism Of Calcium Receptor Protein(CAS) Involved In Nitrogen Fertilizer Regulation Of Flowering Time In Arabidopsis Thaliana

Calcium receptor protein（CAS）is located on thylakoid membrane of chloroplast.Our previous studies have found that the loss of CAS gene function can lead to the senescence of Arabidopsis leaves and the advance of flowering time,and plant nitrogen deficiency will show the physiological phenomenon of leaf etiolation and early flowering.However,whether CAS is involved in the absorption,transportation and utilization of nitrogen fertilizer in Arabidopsis roots is rarely reported.In order to reveal how CAS participates in plant nitrogen regulation and flowering process,Arabidopsis thaliana was used as material in this paper,and the results were verified by phenotypic observation and molecular biology.The main conclusions are as follows:1.Phylogenetic tree analysis of CAS in more than 40 plants using MEGA-X software showed that CAS was a common calcium receptor protein in plants.Most plant species had only one CAS like protein,but for some shade plants and diurnal neutral plants,there were 2-3 CAS like proteins.Since CAS is a phosphorylated protein controlled by light stress,it is speculated that CAS in plants may be closely related to its photoadaptation.2.In the experiment of hypocotyl elongation and phototropism,there was no significant difference between wild-type Arabidopsis（WT）and CAS mutant（CASas）under red light and dark conditions.But,under blue light,the hypocotyl of CASas was3.3 mm longer than WT,which was similar to cry1,a blue light receptor cryptochrome mutant.CASas had weaker phototropism than WT under strong blue light,which indicated that CASas responded to blue light signal.The results showed that:（1）the expression of HY5 gene in CASas was significantly lower than that in WT;（2）the content of CRY1 gene in CASas mutant was 40%lower than that in WT under blue and red light conditions,and there was no significant difference between them under dark conditions;（3）long day（LD）and short day（SD）The content of CRY2gene in CASas was low in SD,about 40%lower in LD than in WT,and about 70%lower in SD.The protein expression of CRY1 and CRY2 was consistent with that of gene expression.In conclusion,CAS is involved in plant blue light signal pathway.3.Under the treatments of low calcium（0.1 mM）,normal calcium（1 mM）and high calcium（10 mM）,the expression of FLC,FT and LHY in CASas was higher than that in WT.In CASas the lowest expression time of FLC was 22:00,6:00 and 14:00under low calcium,normal calcium and high calcium treatment,respectively,the highest expression was 2:00.The expression of FT in CASas and WT under low and high calcium treatments was lower than that under normal calcium treatment,which was only half of that under normal calcium treatment.Under high calcium condition,LHY expression in CASas was the highest at 10:00,which was four hours later than that of low calcium and normal calcium.These results suggest that CAS and its coupled calcium signaling are involved in the regulation of plant flowering.The results of calcium and nitrogen flowering experiment showed that WT flowered early under low nitrogen（0.2 mM）and normal nitrogen（2 mM）treatments.With the increase of calcium,the flowering time of CASas and WT was closer.Under high nitrogen（20 mM）treatment,CASas flowered earlier than WT and did not respond to the increase of calcium,suggesting that CAS and calcium were involved in nitrogen regulation of plant flowering.4.The results of observe the phenotype showed that CASas had the characteristics of early flowering,early senescence,less rosette leaves and leaf yellowing.Under LD condition,the rosette leaves of CASas were 5 pieces less than WT,and the flowering time was 7 days earlier.Under SD condition,the number of rosette leaves of CASas was 25 less than that of WT,and the flowering time of CASas was 40 days earlier.For the SPAD and photosynthetic rate,those of WT were 25 and5.95μmol·m²·s^-1,while those of CASas were 18 and 3.90μmol·m²·s^-1.The photosynthetic parameters of WT were significantly higher than those of CASas,suggesting that CAS affected plant nitrogen metabolism.5.The experiment proved that the transcription level of NRT1.5 and NRT2.5 in CASas was significantly lower than that in WT.The nitrogen content in CASas（81.3mg/kg）was significantly lower than that in WT（110.9 mg/kg）,which proved that CAS was involved in the absorption and transportation of nitrogen in plants.