Gene And Protein Expression Profile Analysis Of ARL1 Regulation During Rice Adventitious Root Development

Rice adventitious root development is a post-embryonic organogenesis, which is regulated by auxin signaling. Although considerable progress has been made in understanding of auxin signaling regulation of cell cycle progression, the molecular mechanisms by which auxin regulates the rice adventitious root primordia formation remains poorly understood. To investigate the effects of auxin on the cell cycle regulation during adventitious root primordia development, the adventitious rootless rice mutant arl1 were used by LCM sampling strategy to compare on a genome and proteome-wide level the transcriptional and translational changes in the pericycle of shoot-borne parts of the wild type and arl1 mutant. The results are summarized as follows:1. Transverse sections of the shoot-base of the wild-type ZH11 and arl1 mutant seedlings revealed that the mutant arl1 does not initiate adventitious root primordia, and cannot be restored by exogenous auxin. As ARL1 is directly regulated by auxin, suggesting that ARL1 is a key factor to link auxin signaling to adventitious root primordia initiation.2. Cell-type-specific transcriptome profiling analyses of the shoot-base pericycle of arl1 mutant and wild-type, using laser capture microdissection(LCM) sampling and rice affymetrix microarray analysis, revealed that G1/S-specific genes, such as E2Fa,D type cyclin,cdc2 and CycA1;1, were significantly down-regulated in the ar/imutant pericycle together with the DNA replication genes H3,H4,ORC,CDC6b,CDT2,MCMs,PCNA and NAF/CAF, suggesting that the machinery of activation and progression through the G1/S phase transition of the cell cycle are strongly impaired in arl1 mutants.3. The expression of early auxin signal response genes AUX/IAA,TIR1,ARF36,AUX1 and PIN1 were down-regulated in the shoot-base pericycle of arl1 mutants. Also, NAC7 and IAA-amido synthetase GH3 are down-regulated. These results suggest that a negative feedback mechanism interfering with the early auxin signaling cascades and homeostasis may be changed during the adventitious root primordia initiation in shoot-base pericycle cells of arl1 mutants.4. Affymetrix microarray and semi-quantity of RT-PCR analysis showed that cell fate respecification genes, such as WUSCHEL-related homeobox(WOX) and SCR were greatly down-regulated in the shoot-base pericycle of arl1 mutant. Because,the lateral root primordia were normally initiated in arl1 mutant, the results suggest that WUS and SCR genes, which acquired different cell fates and are indispensable for induction of asymmetric division, may be also needed to turn on adventitious root organogenesis.5. Via a combination of laser capture microdissection and fluorescence two-dimensional difference gel electrophoresis (2D DIGE) and mass Spectrometry as well as mono-antibody immunobloting analysis, at least eleven proteins controlling cell cycle G1/S phase transition, such as G1 to S phase transition protein 1 homolog/EF-alpha-related GTP-binding protein, high mobility group protein,cell cycle switch protein, D type cyclin, CycA1;1, Histone 3,replication factor C110 kDa subunit, DNA-directed RNA polymerase and DNA repair protein, were found to be repressed in the shoot-base pericycle of arl1 mutant, In addition, expression of early auxin signal protein F-box containing TIR1 were also repressed in arl1 mutant, this is in accordance with the results from affymetrix microarray analysis. These results indicate that ARL1 is a key factor linking auxin signaling with G1/S phase cell cycle reactivation of shoot-base pericycle .6. One gene for heterotrimeric GTP-binding protein alpha subunit(GPA1) were specific expressed in the shoot-base pericycle of the wild-type, and is strongly induced in transcript and translation level by exogenous auxin (NAA). The results suggests that GPA1 could be involved in auxin signaling cascades and the cell cycle reactivation of shoot-base pericycle.GPA1 and ARL1 may co-regulate the G1/S phase transition, this needs to be further investigated.