Analysis Of Root Growth Mechanism Of Arabidopsis Thaliana RLD1 Transcription Factors Regulating Etiolated Seedlings

Light is not only the energy source of photosynthesis of plants,but also an important environmental signal involved in regulating the growth and development of plants.Under natural condition,above ground parts of plant seedlings and root of growing in the dark can feel the changes of light quality in the external environment.Through regulating the directional movement of hypocotyl,promoting root growth and development to complete the seedling morphology,plant can improve their adaptability to environment.The primary root of plants can be able to participate in the fixation of plants growth,absorption and storage of nutrition and moisture,and interactions between plants and soil microenvironment and so on.It plays a crucial important role in growth and development of plants.At present,it is not clear that the mechanism of light promoting root elongation and how light interacts with other signals to regulate the growth and development of plant roots.In view of the effect of light on the growth and development of plant roots,we screened the root growth mutant under dark culture condition.Flavonoids can be stored in the seed.It pays a crucial role in resisting external stress and injury,ensuring normal growth and development of plants.So we filtered out the growth and development of mutant seedlings of some important genes in the flavonoid synthesis pathway,rld1 mutant was successfully obtained under the condition of dark culture.In addition to the dominant growth of the dark root,the rld1 mutant showed that the cotyledons of the etiolated seedling were expanded.It is suggested that RLD1 plays a key role in inhibiting the elongation of the primary root and expansion of cotyledons.The cytological observation showed that RLD1 mutation could increase the apical meristem and increase the number of cells in the apical meristem region,resulting in the elongation of the primary root.RLD1(root length-related in the dark)is a transcription factor of WD40-repeat.It is not only the key factor to control the synthesis of anthocyanin,but also involved in the biological processes of controlling the skin color,surface fur and the fate of the root hair cells.We found that high blue light induced anthocyanin accumulation of wild-type depended on exogenous sugars,while the mutant rld1 lacked high blue light induced anthocyanin accumulation.It is suggested that RLD1 regulating root elongation may be directly related to the uptake and utilization of sugar,but not to anthocyanin accumulation.Consistently,We found seedlings grown on sugar-free medium have no significant difference in root length between WT and the mutant rld1,and the mutant has no advantages in cotyledon expansion.Through the treatment of exogenous sugar,it is proved that sucrose,not glucose or fructose,is the real promoter of rld1 mutant root growth.It was found that only the seeds were germinated in the sugar medium to support the root growth.And if the cotyledon is guaranteed to contact with sugar,it can promote the expansion of rld1 mutant cotyledons.The results showed that the sufficient supply of sucrose during seed germination is essential for the root growth in rld1 dark culture.We speculate that it may be due to the enhancement of sucrose uptake and utilization in rld1 mutants during seed germination stage,resulting in cotyledons expanding and at the same time promoting root elongation.In order to validate the theories,we analyzed some sugar transporter(SUS,SUC,SWEET series)quantity expressed in different period of rld1 mutant under the dark condition.We found the expressionof SWEET1,SWEET3,SWEET7,SWEET8,SWEET9,SWEET10,SUS6,and SUC9 increased significantly.During this period,the ability to absorb and transport is enhanced by sugar transporter,promoting root elongation.In order to explore the possible mechanism of sugar promoting the root growth of rld1 mutant,we add all kinds of sugar inhibitor to the medium,such as TOR inhibitors Rapamycin(RAPA),electron transport chain inhibitors Antimycin A(AMA),Mitochondrial uncoupler 2,4-dinitrophenol(DNP),and Glycolysis inhibitor 2-deoxidation-D glucose(2-DG).We can find that under the condition of dark the root elongation advantage also can not exist in the mutant rld1.After the addition of these sugar inhibitors,the plant to the absorption and utilization of the sugar is hindered,thus resulting in the undifferentiated root length between WT and rld1.The dynamic balance of active oxygen content plays an important role in maintaining the cell division and cell differentiation in the primary root.In order to analyze whether the active oxygen content in the primary root of mutant rld1 was changed,the content of H2O2 and O2-· in rld1 was detected by DAB and NBT staining.Results showed that in the primary root of mutant rld1 under the dark condition,the content H2O2 and O2-· is more than wild type.and the content of O2-· increased significantly.RLD1 could control active oxygen content and homeostasis of active oxygen in root involved in regulating the main root length.But the specific role needs further analysis.When we add a suitable concentration of reducing agent to the medium,such as glutathione GSH,dithiothreitol DTT,procyanidins PA to the medium,or on the sugar-free medium,these treatments can reduce the active oxygen content,so that the root elongation of rld1 disappeared.When exogenous hormone ABA is added,the root elongation of rld1 can be inhibited.In summary,according to the experimental results,the transcription factor RLD1 can regulate the plant root elongation through the mediated light signal and the sugar signal.RLD1 mutation can lead to the increase of root-tip division and the elongation of the primary root.The rld1 mutant showed the primary root elongation by absorption and utilization of sugar in the medium with sugar under dark conditions.RLD1 may mediate root elongation by regulating homeostasis of active oxygen in plants.The study of the mechanism of RLD1 is helpful to analyze the molecular mechanism of the light signal and the sugar signal to the plant root elongation.It is of great significance to explain the regulation mechanism of plant formation in transition from heterotrophic to light..