Cloning And Functional Characterization Of Arabidopsis CPSF30 Gene In Regulating Nitrate Signaling

Nitrogen is an essential macronutrient for plant growth and development. Improving nitrogen use efficiency to reduce the loss of nitrogen into the environment is a world-wide project for decreasing the use of nitrogen fertilizer and lowering its pollution on the environment. Plants have evolved sophisticated mechanisms to adapt the fluctuating environmental nitrogen availability. However, the underlying genes regulating the response to nitrate are remaining further discovered and characterized.Here, a mutant defective in responding to nitrate was isolated by a forward genetics screen and the mutation was mapped to Cleavage and Polyadenylaton Specificity Factor 30(CPSF30), which encodes a polyadenylation factor subunit. CPSF30 has been characterized to be a 30-kD subunit of cleavage and polyadenylation specificity factor, which has two splicing forms, a larger one (CPSF30-L) and a smaller one (CPSF30-S). Results showed that CPSF30-L could rescue the weak fluorescence phenotype; while CPSF30-S could not. It indicates that CPSF30-L is responsible for the weak fluorescence phenotype in cpsf30-2.Three known nitrate-responsive genes including NIA1, NiR, and NRT2.1 in cpsf30-2 were examined. The result showed that the fold of induction of these three genes by nitrate treatment in cpsf30-2 was significantly decreased than that in WT. The expression of these genes was restored to the WT levels in CPSF30-L/cpsf3 0-2 line, but not in CPSF30-S/cpsf30-2 line, indicating that CPSF30-L plays an important role in nitrate signaling.cpsf30-2 exhibited reduced induction levels for these genes compared to WT after nitrogen starvation, similar to the result obtained under the condition without nitrogen deprivation treatment. These findings indicate that CPSF30 functions in nitrate signaling regardless of nitrogen starvation. In addition, the expression of CPSF30 is slightly induced after 1 h of nitrate treatment and reduced after nitrogen starvation, but not regulated by ammonium treatment.qPCR and GUS analysis suggested that CPSF30 is mainly expressed in the vascular tissues of leaves, stems, and flowers and its protein is localized in the nucleus.Nitrate accumulation in cpsf30-2 was found to be decreased in both roots and shoots. The ability of nitrate uptake was decreased which may have resulted from that CPSF30 may modulate the uptake and translocation of nitrate, at least partially, through regulating the expression of NRT1.1 and NRT1.5 in roots and NRT1.1 and NRT1.8 in shoots. While the nitrate assimilation was enhanced as the expression of nitrate reduction-related genes was significantly upregulated. Accordingly the NR activity and amino acid content in cpsf30-2 roots were observably higher than in WT. All these findings demonstrate that CPSF30 functions as an important regulator in nitrate absorption, translocation, and assimilation.To better understand the relationship of CPSF30 and NRT1.1, a molecular and genetic method was used. The expression of NRT1.1 in cpsf30-2 was significantly decreased and a double mutant cpsf30-2 chl1-13 was obtained and characterized. The YFP fluorescence levels in the double mutant roots were similar to chl1-13 mutant. In addition, the expression of nitrate responsive genes in cpsf30-2 chl1-13 was similar to those in single mutant chl1-13 and much lower than those in WT. Moreover, when NRT1.1 was overexpressed in cpsf30-2, the phenotypes of weak YFP fluorescence, reduced nitrate content, and inhibited induction of nitrate responsive genes were restored to the levels similar to those in WT. These findings clearly indicate that CPSF30 and NRT1.1 work in the same pathway of nitrate signaling and CPSF30 functions upstream of NRT1.1.To comprehend the relationship of CPSF30 and NLP7, qPCR results showed that these two genes do not regulate each other at transcriptional level. Genetic analysis by using single and double mutants of both genes revealed that the YFP fluorescence of cpsf30-2 nlp7-4 was weaker than that of both single mutants, indicating that CPSF30 and NLP7 may function in different pathways in nitrate signaling. In addition, the induction fold of the three nitrate responsive genes tested was significantly lower in the double mutant than in both single mutants, providing further evidence that both genes work independently in nitrate regulation.Transcriptome analysis revealed that four nitrogen-related clusters found to be related to nitrogen including response to nitrogen compound, nitrogen compound transport, response to nitrate, and nitrate transport were enriched in the differentially expressed genes in cpsf30 mutant. Moreover, some known nitrate-inducible and regulatory genes were found in these four clusters. The above data provide more evidences that CPSF30 can regulate many nitrogen-related genes and is an important nitrate regulator.These findings demonstrate a direct involvement of CPSF30 in central nitrate signaling and offer insights into the mechanism of nitrate regulation in plants. CPSF30 mediates nitrate signaling through regulating NRT1.1 expression, adding an important component into the nitrate regulation network.