Screening Of Magnesium Stress Mutants And The Mechanism Of The Plant Responding To Magnesium Toxicity In Arabidopsis

Magnesium (Mg2+) is an abundant divalent cation in a living cell and plays fundamental role in massive physiological and biochemical processes. However, the regulatory mechanisms responsing to magnesium stresses in plant are known little so far. In this study, we established a reasonable screening system of magnesium stress mutants, which has a great significance for clarifying the physiological and molecular mechanisms of plants responding to magnesium stress. The results obtained here elucidate that the responding characteristics of Arabidopsis seedling morphogenesis to magnesium toxicity (MgT) at least partly dependend on the activity of the properties of ABA-ABI1-mediated growth repressors DELL As.1. Screening of Magnesium Stress Mutants.1.5mM (Low Magnesium, LM) Mg2+is the most appropriate concentration for Arabidopsis growth. Death is overhanging to the wild-type Arabidopsis when the concentration levels of Mg2+was higher than28mM (High Magnesium, HM) or lower than0.05mM (Magnesium Deficiency, DM). Therefore, in the present study,28mM and0.05mM of Mg2+were respectively used as the critical concentrations for Magnesium toxicity and Magnesium deficiency to screening the Magnesium stress responding mutants. Total50lines of EMS mutants (HM mutants:34; DM mutants:16) were obtained.2. Mg toxicity and exogenous ABA regulate root growth and architecture. Young leaves and roots of the Arabidopsis plants (WT) were seriously inhibited by MgT and exogenous ABA application whereas root hairs were stimulated by the treatments. Interestingly, under MgT contiditions, a Q-DELLA mutant exhibited less1/3times of the length of roots and root hairs but higher biomass, starch content, chlorophyll level and some metal contents as compared to WT. The Q-DELLA mutant also showed a lower magnesium level.in the leaves of the plants. However, these phenotypes of Q-DELLA exhibited under MgT could be eliminated by adding exogenous ABA although the ability of bioactive ABA was less increased in Q-DELLA mutant under MgT. The roots of an ABA-insensitive mutant abil-1did not exhibite the growth-inhibitory effects of both ABA and MgT treatments resulted in the WT plant.3. Mg toxicity treatment regulates Mg2+uptake efficiency and starch degradation probably via a DELLA-Dependent mechanism. The seedlings of both WT and the Q-DELLA mutant showed lower accumulation of starch (Ler:5.42; Q:2.24) but higher levels of Magnesium (Ler:2.80; Q:1.93) under HM-growing conditions as compared to those under LM-growing conditions. Apparently, the effects were lower in the Q-DELLA mutant than were in WT. Transcripts of SSI, SS2, GBSS1, APL1and APL2genes in Q-DELLA mutant were significantly down-regulated by MgT but the transcripts of AT AMY1, BAM1and BAM2genes were up-regulated. These genes are responsible for the reduction of starch biosynthesis or the increase of starch degradation in plants. In addition, the expressions of all magnesium transport genes were up-regulated in Q-DELLA mutant versus wild-type under HM conditions.4. Mg Toxicity induces accumulation of bioactive ABA and expression of genes encoding the enzymes-related to ABA Metabolism and Signaling. The ABA concentration of WT (6.33) and Q-DELLA mutant (4.56) was markedly higher under HM conditions than was under LM conditions, and it was notably higher (1.57) in WT than was in Q-DELLA mutant under HM. Except for AB13and AB14, whose expression were slightly up-regulated in O-DELLA plants, all the other genes related to ABA biosynthesis(ABA1, ABA2, ABA3, NCED1and AAO3) and its negative regulation (ABI1, ABI2, ABI3, ABI4and ABI5) were down-regulated in the mutant.5. Mg toxicity and exogenous ABA application promote accumulation of DELLA protein. The accumulation of green fluorescent protein-tagged DELLA (GFP-RGA) proteins in the nucleis of root tip cells was observed in WT plants with both MgT-treated plants for24h and ABA-treated plants for2h. However, the accumulation of GFP-RGA fusion protein could be promptly destroyed by following10μM GA treatment for1.5h in the absence or presence of28mM Mg2+or20uM exogenous ABA.6. Transcription changes between Q-DELLA mutants and wild-type plant associated with Mg toxicity. The network analyses of gene interaction and metabolical pathways of differentially expressed genes showed that DELLA proteins regulate ABA metabolism and signaling, starch metabolism and Mg+uptake efficiency. Furthermore, great changes in transcript of a set of genes encoding WRKY transcription factor family proteins, protein kinase superfamily proteins, and peroxidase superfamily proteins were also observed, indicating their crucial roles in transmission and transduction of Mg toxicity stress signaling.As a whole, all the results obtained here suggest that ABI1-dependent ABA-mediated enhancement of DELLA restraint contributes to the response of Arabidospsis plants to MgT and a systematic remodeling of stress signaling, transporters, and starch metabolism might be involved in the responding process. Our research provides a solid basis for further understanding the regulatory mechanism of plants responding to magnesium toxicity.