| Root is one of the important organs of plants.In addition to fixing plants,the root system is also responsible for absorbing mineral nutrients and water,and it plays important roles in plant growth and development.Plant root stem cell niche(SCN)not only participates in plant growth and development,but also serves as the master modulator for plants to adapt to the environment.In Arabidopsis,the root SCN is regulated by the GRAS transcription factors such as SCARECROW and SHORT ROOT,PLETHORA transcription factors such as PLT1 and PLT2,WUSCHEL RELATED HOMEOBOX 5 homeodomain transcription factor,environmental factors and various hormones.However,the underlying mechanism of how root stem cell niche is maintained is still not understood very well.Hence,our research is committed to looking for a new regulatory factor involved in root stem cell niche.In this study,the model plant Arabidopsis thaliana was used as the experimental material.A T-DNA mutant with obviously short primary root was found in the Cystatin/monellin superfamily protein,named prod1-1,and its expression analysis,functional identification and regulating mechanism were studied.The main results are as follows:(1)A T-DNA insertion mutant of At5G7150,belonging to the Cystatin/monellin superfamily,exhibited apparently short primary root.We rename it prod1-1.(2)We analyzed the primary root length of prod1-1 and WT during seedling stage.We found that the prod1-1 mutant had the phenotype of root short.We also generated the overexpression lines of PROD1,and verified that the expression level of RT-PCR was higher than that of WT.However,no significant difference in root length between overexpressing lines and WT was observed.(3)In order to study whether prod1-1 still has short root phenotype at adult stage,the seedlings of WT and prod1-1 mutants grown in incubator for 7 days were transferred to soil.The phenotype was observed under long daylight at 22? for 15 days,and the root length was measured.The results showed that the prod1-1 mutant also had a short root phenotype at the adult stage.However,there was no obvious developmental defect phenotype in the shoots.(4)The tissue expression patterns of PROD1 was analyzed by semi-quantitative RT-PCR.The results showed that PROD1 was expressed in rosette leaf,cauline leaf,stem,flower,root,developing silique and seed of Arabidopsis.Relatively abundant in cauline leaf,developing slique and flower.As prod1-1 has a salt-sensitive phenotype at the seedling stage,we also found that PROD1 was up-regulated by salt stress using semi-quantitative RT-PCR analysis.Subcellular distribution data showed that PROD1 was mainly located in the cytoplasm.(5)Through propidium iodide(PI)staining,we found that the length of root apical meristem in prod1-1was shorter than that of WT,and the number of meristem cells were smaller than WT,suggesting that PROD1 can regulate the growth and development of root apical meristem.(6)To investigate the functional mechanism of PROD1,we introduced DR5::GUS,auxin exporters PIN1(PIN-FORMED1),PIN2 and PIN7 into the homozygous prod1-1 mutant by hybridization,which can quickly and sensitively reflect the auxin level of root tip.It was found that the prod1-1 mutant accumulated more auxin and less PIN protein than that in WT seedlings.(7)We measured the content of hydrogen peroxide and superoxide in mutant and wild type by DAB staining and NBT staining,respectively.The content of hydrogen peroxide and superoxide in mutant was much higher than that in wild type.These results suggest that PROD1 may regulate the growth and development of primary root by affecting the accumulation of reactive oxygen species.(8)Given that prod1-1 belongs to the Cystatin/monellin superfamily protein,we isolatedand purified PROD1 by prokaryotic expression system and then carried out casein hydrolysis experiment.The results revealled that PROD1 did not have the activity of cysteine protease inhibitor.(9)By yeast two-hybrid screening,DFP(DUF1677 family protein,AT1G72510)displayed strong interaction with PROD1. |
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