The Mechanisms Of Heterotrimeric G-protein In The Regulation Of Auxin-dependent Lateral Root Development In Arabidopsis

Roots systems, as one of the major organs of plant, perform essential tasks of absorbing water and nutrients from soil, and anchoring plants. The development and morphology of lateral roots play a crucial role in the construction of root systems. A lot of studies have shown that plant hormone auxin plays vital roles in the development of lateral roots. Recent studies showed that the heterotrimeric G-protein (G protein) is involved in auxin-dependent lateral root development. However, at present, the physiological and molecular mechanisms concerning G protein-mediated auxin-dependent lateral root development are largely unknown. In present study, we investigated the mechanisms of Arabidopsis heterotrimeric G-protein in the regulation of auxin-dependent lateral root development by using heterotrimeric G-proteinα/βsubunit null mutants and the transgenic cells and plants of overexpressed GPA1 /AGB1 plants as experimental materials with different approaches. The results showed that1. Mutation of the heterotrimeric G-proteinαsubunit andβsubunit affected significantly lateral root development of Arabidopsis. The lateral root numbers (LRNs) and the primary root length of G-proteinβsubunit mutant (agb1-2) and double mutant (gpa1agb1) were greater significantly than those of Col. No obvious difference was observed in main root length between G-proteinαsubunit mutant (gpa1-3) and wildtype, while the LRNs of gpa1-3 mutant were significantly lower than those of Col.2. The lateral root development was markedly affected by auxin. Under the condition of inhibiting auxin polar transport by using TIBA, an auxin polar transport inhibitor, exogenous IAA significantly increased the number of emerging lateral roots in heterotrimeric G protein mutants and wild type, which means that lateral roots formation in G protein mutants is also auxin-depend. However, significant differences were observed in the responses of lateral roots formation to auxin treatment among different genotypes investigated. As compared with Col, the lateral roots formation of gpa1-3 mutant was relatively insensitive and agb1-2 and gpa1agb1double mutant super-insensitive to auxin treatment3. The initiation of lateral roots occurred about at day 2 after seed germination and no obvious difference was detected in the numbers of lateral root primordia (LRP) between gpa1-3 and agb1-2 mutant, but all were significantly higher than that of Col. After 4 days of germination, when the auxin formed in the shoots began to transport to roots, the total number of lateral roots (LRs) and LRPs of gpa1-3 mutant was similar to that of Col, but significantly lower than that of gpa1agb1. Furthermore, About 70% of LRPs of gpa1-3 after 4 days germination were at 3-5 stages, while most of LRPs of agb1-2 and gpa1agb1 were at 5-7stages in agb1-2 and gpa1agb1.4. In the case of aerial tissue removal at 4 days after germination (DAG), the relative number of emerging and fully developed LRs decreased drastically compared to the intact seedlings, and no differences were observed among genotypes investigated. At the earlier stage of lateral root development (about 4 days after seed germination), excision of root tips (primary roots) significantly increased LRs except agb1-2 mutant. However, when root tips were excised after 7 days of germination, the increased LRs of mutant gpa1-3 was significantly lower than Col and other genotypes.5. Protein localization analysis using GFP fusion protein technique showed that GPA1 was mainly located in root apical and pericycle parts while AGB1 was distributed through the whole root. The density of LRs in GPA1 overexpressed seedlings was significantly higher than that of wild type plants, while no significant difference in LRNs was observed between AGB1 overexpressed seedlings and Col. The development of LRs of AGB1 overexpressed seedlings were less response to exogenous auxin and hypersensitive to TIBA while the response to auxin and TIBA in GPA1 overexpressed seedlings was similar to Col. The LRPs of AGB1 overexpressed seedlings at 4 days after germination were mainly distributed at stages 3-4, while LRPs in GPA1 overexpressed seedlings were mainly distributed at stages 3-5.6. There were significantly differences in cell division and cell elongation responses to 2,4-dichlorophenoxyacetic acid (2,4-D) andα-naphthalene acetic acid (NAA). The division of agb1-2 and GPA1-overexpressed cells was insensitive to 2,4-D (2μM) treatment and cell elongation of those genotypes was super-sensitive to NAA (2μM) treatment. RT-PCR result showed that, cell cyclin gene AtCYCD4;1 was highly expressed in agb1-2 suspension cells without exogenous auxin treatment. Gene ABP1, which was related to cell elongation, was highly expressed in agb1-2 and GPA1-overexpressed suspension cells treated with NAA. According to the different transport patyway of 2,4-D and NAA, it could be concluded that agb1-2 mutate and GPA1-overexpressed promoted auxin export capacity and enhanced the cell mitosis ability.7. Responsive to gravistimulation shown that agb1-2, GPA1-overexpressed seedlings were more responsive to gravistimulation than Col and gpa1-3 while AGB1- overexpressed seedlings were less responsive than the wild type Col.8. Real-time PCR analysis showed that the expression of PIN1 was significantly stimulated in roots of agb1-2 and GPA1 overespressed seedlings.Based on the results above, it was suggested that G protein-mediated lateral root development was largely through affecting auxin basipetal transport in roots. AGB1 negatively regulated this process by inhibiting the expression of PIN1, as a result, more lateral roots were formed in agb1-2 mutant.