Knockout Of SUMO E3 Ligase OsSIZ1 Gene Affects The Modification Of Root Architecture Structure In Rice

Phosphorus (P) is one of the essential macro-nutrients for plant growth and development, and it plays an important role in almost all metabolic activities of plants. However, since P is readily chelated by cations and precipitated in the soil and the only form of P available for plant uptake is inorganic ortho-phosphate (Pi), the mobility and availability of P is poor. Plants have evolved a suite of responses to P-deprived environment, modification of root architecture system(RAS) is one of the most notable adaptations of plants. In the past decades, a series of regulator mechanisms underlying the Pi starvation -induced modification of root architecture have been intensively studied.SUMOylation (Small Ubiquitin-related Modifier) is identified as an post-translational modification in recent years. Similar to ubiquitination, SUMOylation occurs in a series of biochemical steps referred as maturation, activation, conjugation, ligation and deconjugation. SIZ (SAP and Miz finger domain) is the SUMO E3 ligase which determines its specific targets. SUMOylation mediated by SIZ has been proved implicated in many important processes in plants, including growth and development, biotic stresses, and many abiotic stresses (drought stress, cold stress, heat shock and salt stress).The effects of AtSIZl on RAS have been associated under low Pi stress with the regulator of AtSIZl on the auxin accumulation by Miura. The results suggested that SIZ1 negatively regulates Pi starvation-induced RSA remodeling through the inhibition of auxin transfer from root tips to lateral root primordia. But, this research basically not yet for OsSIZl, homologous gene of AtSIZl in rice. Here, we studied the effects of knockout SUMO E3 ligase gene OsSIZ1 on the root development and architecture, and the relationship between low Pi stress and the role of auxin using ossizl mutants. The main results were summarized as follows:1. We observed the growth of the roots of wild-type(WT, dongjin) seedlings and ossizl mutant seedlings. Statistical analysis indicated that the growth rate of seminal root of ossizl mutant seedlings is smaller than that of WT, and the seminal root length of ossizl mutant seedlings is notably shorter than that of WT. Demonstrating that knockout of OsSIZ1 leads to the shorten of seminal root length and the decrease of the growth rate of of seminal root in ossizl mutant seedlings. We speculate that OsSIZl regulates the seminal root growth via regulating the growth rate of seminal root of ossizl mutant seedlings.2. Hydroponic cultivation with different Pi concentrations treatments showed that compared with WT, the ossizl mutants cause the shortened of adventitious roots, increased lateral root density and root hair proliferation, and the response is more evident under Pi-deficiency treatment. It means that OsSIZ1 regulates the modification of RAS. In addition, and the regulation may be related with the Pi concentration.3. Hydroponic cultivation with different Pi concentrations treatments also showed that compared with WT, more adventitious roots with large lateral root(llr) formation in clumps near the root tips of the ossizl mutant. The OsSIZl expression is strong in tips of seminal roots and lateral roots. We speculate that knockout of OsSIZl affects llr formation and its growth area, and it may be one of the mechanisms that OsSIZl negatively regulates lateral root formation.4. The modification of RAS of ossizl mutant seedlings influenced its Pi absorption and transport. By comparing the P concentrations in roots and shoots of WT and ossizl mutant plants at seedling stage, our results show that the total Pi concentrations in shoots and roots of ossizl mutant seedlings are notably higher than that of WT. And, P translocation from roots to shoots in ossizl mutant seedlings increases as compared with that of WT. Probably, OsSIZl may regulate Pi absorption and transport via changing the RAS.5. Our preliminary experiment demonstrated that 0.5μM is the most suitable concentration to promote roots growth of WT seedlings. Based on this result, we set hydroponic experiments with Pi-sufficiency(+P), Pi-deficiency(-P), Pi-sufficiency and application of exogenous IAA(++P), Pi-deficiency and application of exogenous IAA(-+P). The data demonstrated that exogenous auxin promoted adventitious roots elongation, and increase of lateral root density and root hair proliferation. Phosphate starvation-induced RAS remodeling of ossizl mutant can be obtained through applying exogenous IAA, but the effect of exogenous IAA is related with the phosphorus concentration in medium, and affected by knockout of OsSIZ1. It suggests that the OsSIZ1 regulation of Pi deficiency induced root architecture remodeling involves auxin accumulation. In addition, there may be some interaction between auxin and phosphorus nutrition.6. Monitoring of the auxin responsive reporter DR5::GUS indicated that auxin accumulation level in ossizl root tips is lower as compared with WT. The GUS expression is similar in WT root tips during Pi starvation, but it is weakened at early stages of the Pi starvation response in ossizl root tips. Expression of auxin biosynthesis gene and auxin efflux transporter genes was detected by qRT-PCR in WT and ossizl mutant seedlings under Pi treatment and exogenous IAA. The results demonstrated that relative expression levels of OsYUCCA1、OsPIN1a、OsPIN1b changed. However, the range of increase or decrease of relative expression level is different under different Pi or exogenous IAA concentrations treatments. These results link OsSIZ1 with the auxin response of the RSA in low-Pi conditions, which likely involves auxin patterning.Taken together, SUMO E3 ligase OsSIZl gene regulates the modification of RAS, and the effect is more. apparent under low Pi stress. Moreover, it likely involves auxin patterning. Although these findings need to be further search and the concrete mechanism also need deeper research, these results provide theoretical and material foundation for charactering the function of SUMO E3 ligase OsSIZ1 gene in the growth and development of rice roots and efficient phosphorus utilization.