PHO1;H4 Is Involved In Pi Long-distance Transport In Arabidopsis Thaliana

Phosphorus(P)is a macronutrient element essential for plant growth and development,and is a constituent of important compounds such as nucleic acids,phospholipids,coenzymes and ATP.In order to adapt to the changing Pi environment,the plant has evolved a set of Pi steady-state regulation mechanisms.Proteins containing the SPX domain play an important role in the steady state regulation of plant Pi.There are four families of SPX domain-containing proteins in Arabidopsis:SPX-MFS,SPX-EXS,SPX-RING and SPX.Previous studies in our laboratory revealed the function of a Pi transporter SPX-MFS1(VPT1)localized to the tonoplast,which regulates the transport of Pi from the cytoplasm to the vacuole,it is crucial for the Pi steady-state of Arabidopsis.The SPX-EXS family is predicted to be a Pi transporter family,and its representative member PHO1 has been shown to regulate the transport of Pi from root to shoot.We speculate that other members may also have Pi transport function,according to the evolutionary relationship of this family,we choose PHO1;H4,PHO1;H5 and PHO1;H9 as the objects of our study,we performed phenotypic screening of mutants pho1;h4?pho1;h5 and pho1;h9,genetic techniques,molecular biology methods and histochemical staining techniques were used to focus on the Pi transport function of PHO1;H4.The main conclusions are as follows:(1)PHO1;H4 is not sensitive to Pi and other stress screening conditionsFirst,we identified the mutants pho1;h4,pho1;h5 and pho1;h9 by PCR and RT-PCR,and constructed the double mutants pho1;h4/h5,pho1;h4/h9 and pho1;h5/h9 and triple mutants pho1;h4/h5/h9 by hybridization.The mutants were all homozygous mutants in which the genes were completely knocked out.Subsequently,we screened the mutants for phenotypic results.The results showed that the mutants were not sensitive to different concentrations of Pi,Pi and divalent cations combination screening conditions,salt stress and osmotic stress.There were no significant differences in root length,fresh weight and growth status between the mutants and wild type(Col-0).(2)PHO1;H4 is mainly expressed in leaves and vascular tissues of rootsIn order to obtain more information about the research object and help the next step,we performed histochemical staining(GUS)on the transgenic plants and obtained the tissue localization of PHO1;H4,PHO1;H5,PHO1;9.The results showed that PHO1;H4 was expressed in the fibril of seedlings(3?4d),in the leaves of seedlings(6?2 1d),and in the vascular column of roots;PHO1;H5 and PHO1:H4 had similar tissue localization patterns,also in fibril of seedlings(3?4d)?leaves of seedlings(5?18d)and vascular tissues of roots;PHO1;H9 was not expressed in seedlings,and was expressed in the hydathodes of seedlings(5?7d),and small amount of expression was observed in the leaves,petioles and roots of the seedlings.(3)PHO1;H4 limits long-distance transport of PiWe determined the Pi content of the mutants and found that the shoot Pi content of the pho1;h4 mutant was about 20%higher than that of the wild type under the treatment of 40?M,130?M and 500?M Pi,while there was no significant difference between the mutants and the wild type in root,indicating that PHO1;H4 played a role in limiting the transport of Pi from root to shoot.Pi content of the shoot of pho1;h4/h5,pho1;h4/h9 were slightly higher than that of wild type,but lower than that of pho1;h4 mutant,It was indicated that after the mutation of PHO1;H4,PHO1;H5 and PHO1;H9 mutation caused a decrease in the content of Pi in the shoot.(4)PHO1;H4 mutation affects distribution of Pi in different tissues of Arabidopsis thalianaTo explore the function of PHO1;H4 in Pi distribution,we determined the Pi content of various tissues and organs in pho1;h4 mutant and wild-type seedlings.The results showed that the content of Pi in the flower of pho1;h4 mutant was higher than that in wild type after treatment with 1.3?M Pi.The difference of other tissues and organs was not significant;after treatment with 130?M Pi,the content of Pi in the fruit pod,flower,rosette leaf and stem leaves of pho1;h4 mutant was higher than those of wild type;after treatment with 1.3mM Pi,the content of Pi in the pods,flowers,rosette leaves,stem leaves and stems of pho1;h4 mutants were higher than those of wild type.In addition,PHO1;H4 is mainly expressed in the vigorous growth of tissues and organs such as flowers and pods,and the expression of PHO1;H4 is slightly changed after treatment with different concentrations of Pi.The above results indicate that PHO1;H4 mutation affects Pi distribution in different tissues of Arabidopsis.(5)The function of PHO1;H4 needs PHO1To explore the functional association between PHO1 and PHO1;H4,we first analyzed the expression levels of PHO1 and PHO1;H4 by qPCR and found that under low Pi conditions,the expression of PHO1 and PHO1;H4 in the shoots were up-regulated.Subsequently,we constructed pho1/pho1;h4 double mutants,and compared the Pi content with phol and pho1;h4,the results showed that the Pi content in the shoot of phol and pho1/pho1;h4 mutant was significantly lower than that of wild type and pho1;h4,but there was no significant difference in phol and pho1/pho1;h4,indicating that PHO1;H4 plays a role in restricting the transport of Pi from the root to shoot,and PHO1;H4 requires the participation of PHO1,or the high Pi environment provided by PHO1.(6)PHO1;H4 mutation affects Pi steady-state regulatory networkPHR1-miR399-PHO2 is a signaling pathway important for Arabidopsis in response to Pi stress.To investigate whether PHO1;H4 mutation affects this pathway and whether it affects the expression of other Pi starvation-responsive genes,we treated pho1;h4 mutant and wild-type seedlings with different concentrations of Pi,the expression levels of PHR1,miR399,PHO2 and genes that play a role in Arabidopsis Pi homeostasis(AT4,VPT1,ZIP5,NLA,PHT1;4,PHT1;1,IPS,miR399A,miR399B,RNS1)were tested by q-PCR.The results showed that after the mutant of PHO1;H4,the expression level of PHT1;1 gene was up-regulated to about 4 times under 10?M Pi treatment,and RNS1 was down-regulated to 0.38 times;under the treatment of 1.3mM Pi treatment,the AT4,PHO2,PHT1;1,1PS,and miR399B genes were up-regulated to 5.1-fold,2-fold,4.8-fold,3.3-fold,and 2.3-fold,respectively,RNS1 was down-regulated to 0.13 times.These results indicated that the mutant of PHO1;H4 affected the PHR1-miR399-PHO2 signaling pathway and up-regulated the expression levels of PHO2 and miR399 under the treatment of 1.3mM Pi,and the mutation of PHO1;H4 disturbed the Pi regulatory network of Arabidopsis.In summary,this research identified a protein PHO1;H4 that restricted the long-distance transport of Pi.After the mutation of PHO1;H4,the Pi content in the shoots of Arabidopsis thaliana was about 20%higher than that of the wild type,the distribution of Pi between different tissues was also affected,and the function of PHO1;H4 requires PHO1.In this research,the preliminary study on the Pi transport function of PHO1;H4 provides a theoretical basis and practical basis for the cultivation of crops that use phosphorus efficiently.