A P-loop NTPase APP1 Maintains Root Stem Cell Niche Identity Through The Regulation Of ROS Homeostasis In Arabidopsis

Plant root stem cell niche (SCN) plays a crucial role in root growth and development. The quiescent center (QC) comprises a small group of cells which divide infrequently and maintain the identity of the surrounding stem cells which are the source of different tissue cells in the Arabidopsis root. In Arabidopsis, the root SCN is regulated by the GRAS transcription factors such as SCARECROW (SCR) and SHORT ROOT (SHR), PLETHORA transcription factors such as PLT1 and PLT2, WUSCHEL-RELATED HOMEOBOX 5 (WOX5) homeodomain transcription factor and various hormones including auxin, cytokinin, ethylene, ABA, GA and so on. However, the molecular mechanism underlying how root SCN is regulated is not understood very well. Hence, our study is devoted to identifying new regulators which are involved in root SCN maintenance, and it will be important to address the regulatory network in this process.In this study, a T-DNA mutant screening was done using Lugol's staining, which normally stains only differentiated starch-containing columella cells, and leads to the identification of the appl-1 mutant that displayed aberrant QC cell divisions along with an inability to maintain its DSCs. In the present study, the molecular mechanism by which APP1 controls root SCN identity was investigated using molecular biology, cell biology, genetics, physiology and other techniques.In the appl mutant, a higher frequency of QC cell division was observed as exhibited by the lugol's staning. In addition, the 5-day-old app1 mutant roots displayed higher rates of DSC differentiation which was shown by the starch granule accumulation, suggested that the stem cell identity was disrupted. Moreover, comparing with the wide type, the QC-specific marker such as QC184, was clearly down-regulated in the app1 mutant, indicating that the QC identity was disrupted in the app1 mutant.To assess the possible roles of APP1 in root growth, we generated pAPP1::GUS and pAPP1::APP1-GFP lines to study the APP1 expression pattern and the APP1 protein sub-cellular localization. Analysis of several pAPP1::GUS and pAPPl::APP1-GFP transgenic lines revealed that APP1 was highly expressed in the root meristem. Further studies showed that APP1-GFP green fluorescence mainly merged with the mitochondrial probe, Mito Tracker Red, indicating a main mitochondria localization of APP1. This result was also confirmed by western blotting analysis which showed that the APP1 protein was detected in the isolated mitochondria proteins from pAPP1::APP1-GFP seedlings.The in vitro ATP hydrolysis assay of APP1 protein confirmed the ATP hydrolysis activity of APP1. Moreover, compared to WT root tips, the app1 mutants accumulated less hydrogen peroxide (indicated by DAB and H2-DCFDA staining) and superoxide (using NBT staining). In addition, the signal of mitochondrial superoxide marker Mito-cpYFP was also greatly reduced in the app1 mutants compared to the WT. In addition, the level of hydrogen peroxide (H2O2) was lower in the app1 mutant compared to the WT control. These results suggest that dysfunction of APP1 reduces ROS production in root tips.To further explore the mechanisms involved in the altered ROS level in app1 mutant, we evaluated the expression of ROS-related genes. The qRT-PCR results showed that the expression of PER11 and PER55, both encoding ROS-scavenging enzymes and belonging to the Class ? peroxidase family, was up-regulated in app1 mutant. Moreover, an inspection of the mitochondrial complex ? protein showed that its specific activity in the app1-1 mutant was only 50% of that in the WT.In order to address if the increased rate of QC cell division and the enhanced root DSC differentiation were attributed to the reduced ROS levels in app1 mutants, the effect on the root SCN of both providing hydrogen peroxide (H2O2) exogenously or exposing the plant to methyl viologen (MV) was examined. Both H2O2 and MV treatments strongly rescued the app1 mutant phenotype in root SCN. When WT seedlings were grown on a medium supplemented with either diphenyleneiodonium (DPI) or catalase, both treatments significantly induced QC cell division and boosted root DSC differentiation.To deeply understand the role of ROS gradient in the maintenance of root stem cell niche, the effect on the root SCN of providing H2O2 exogenously was examined. When WT seedlings were exposed to H2O2, the rate of cell division in the QC and the extent of root DSC differentiation were enhanced. Consistently, in the APP1 over-expression lines, which displayed higher ROS levels, the QC cell division rate and the rate of root DSC differentiation was also increased. The conclusion was that an appropriate ROS level is important for the maintenance of stem cell identity.To determine whether APP1 regulated root SCN is dependent on the well characterized root SCN-defining transcription factors, we examined the expression of GRAS transcription factor genes such as SCR and SHR and AP2 transcription factor genes such as PLT1 and PLT2 in app1. The expression of SHR and SCR were transcriptionally and translationally down-regulated in app1. Other transcription factors such as PLT1 and PLT2 did not show significant expression difference between appl mutants and the WT control. These results indicate that SHR and SCR are involved in APP1 regulated SCN maintenance.Auxin was reported to be involved in the maintenance of root stem cell niche. Here, we found that auxin signaling in the app1 mutant root tip was unaffected, which implied that APP1 might regulate QC cell division and root DSC identity independent of auxin signaling.Since both increasing and decreasing ROS levels induced QC cell division and root DSC differentiation, the expression of SCR, SHR, PLT1 and PLT2 in the presence of either heightened or lowered levels of ROS was of interest. Under high ROS levels, the expression of both PLT1 and PLT2 was attenuated, while that of both SCR and SHR was unaffected. A low level of ROS, suppressed the SCR and SHR transgenes but not the PLT1 or PLT2 ones. The suggestion is that the response to altered levels of ROS might be mediated by distinct signaling pathways.In conclusion, our study indicated that SHR and SCR are important downstream targets of APP1-regulated ROS signaling to control the identity of root QC and DSCs. Moreover, we found that an appropriate ROS level in Arabidopsis root is important for the maintenance of stem cell identity. This finding provides a direct link between ROS distribution and the QC identity and root DSC differentiation in the A. thaliana root tip.