Functional Characterization And Molecular Mechanism Analysis Of ABP7-Like,a B HLH Transcription Factor,in Maize Leaf And Kernel Development

Maize(Zea mays L.),is the third most important cereal crop species in the world(after wheat and rice),which has multiple uses such that can be used as human foods,animal feeds and for the manufacture of pharmaceutical and industrial products.Food shortage is becoming the main problem in this century because the world population is increasing day by day but the arable lands are limited.So,to meet the future demand,we have to concentrate on increasing the yield of food crops.Through photosynthesis process maize leaf can affect on kernel yield.The final yield of maize depends on the kernel traits.The identification of factors responsible for leaf and kernel development is very important for enhancing the maize yield.In our lab,ABP7(ABRE Binding Protein 7),a G-Box binding bHLH transcription factor was identified using Yeast-one hybrid assay after screening a cDNA library of maize 17 DAP embryos.Over expression of ABP7 produced larger siliques with increased seed size and seed weight in Arabidopsis.Through Bioinformatics analysis,we have searched for the homologs of ABP7 and found one homolog with 89% DNA sequence and 97% amino acid sequence similarity.We named the newly identified gene as ABP7-Like.In this study,the research focused on the functional and molecular mechanism analysis of ABP7-Like in leaf and kernel development.Through sequence comaparison with other known bHLH transcription factors it was identified that ABP7-Like is a bHLH transcription factor containing the conserved bHLH region.The biochemical studies suggested that ABP7-Like is a transcriptional activator and binds to G-box cis-element ABRE can form heterodimer with ABP7.ABP7-Like has ABRE binding activity.From the expression analysis of ABP7-Like it was observed that ABP7-Like express throughout both vegetative and reproductive developmental stages of maize.Higher expression was observed in young leaves,cobs,pericarps,the PC region,BETL cell layers and embryos.For the functional analysis of ABP7-Like,four different constructs i.e.ABP7-Like-OE,ABP7-LikeRNAi,ABP7-Like-SRDX and ABP7-Like-VP64 were transformed and phenotyped.In ABP7-Like-OE transgenic events,defective seedling development,pale green leaves and leaf senescence were observed whereas in ABP7-Like-RNAi events no such phenotypes were observed.The function of ABP7-Like was further verified using ABP7-Like-VP64 and ABP7-Like-SRDX plants.It showed that ABP7-Like-VP64 transgenic events produced shorter plants with pale green and early senescence leaves compared to the non-transgenic plants which were similar with ABP7-Like-OE transgenic events.On the other hand,Leaves of ABP7-Like-SRDX plants were dark green without showing any senescence.ABP7-Like-OE transgenic plants produced wider,longer and heavier kernels compared to the non-transgenic plants while ABP7-Like-RNAi transgenic plants produced smaller and lighter kernels.These phenotypes indicated that ABP7-Like regulates both leaf and kernel development.Results of histochemical staining and quantitative measurement of ROS species and chlorophyll quantification showed that ABP7-Like regulates ROS accumulation and chlorophyll in leaves.From the grain filling rate it was observed that ABP7-Like may not directly affect endosperm development during the grain filling stage.The difference in kennel dry weight accumulation between the transgenic and non-transgenic events may reflect an effect of ABP7-Like on sporaphytic development.The transcriptomic analysis of leaves at seedling stage identified genes encoding proteins such as Expansin-B4,O-Glycosyl hydrolases family 17 protein,Beta-glucosidase,Alcohol dehydrogenase-like 2,Peroxidase 53,Thioredoxin H-type,GDSL esterase/lipase,Serine carboxypeptidase-like 33 as potential target of ABP7-Like in regulating leaf development,which are involved in several biological processes including hydrogen peroxide catabolic process,response to oxidative stress,carbohydrate metabolic process,cell redox homeostasis etc.The molecular mechanism underlying ABP7-Like-regulated kernel development was revealed by RNA-Seq and ChIP-Seq analysis and identified genes encoding proteins such as High affinity nitrate transporter,Expansin-B4,Mitogen-activated protein kinase YODA,SWEET 3a,Protein NRT1/ PTR FAMILY 4.4,Thioredoxin F2 as potential direct targets of ABP7-Like,which are involved in the biological processes such as death,transport,cell wall organization,organ development,cellular metabolic process etc.In summary,ABP7-Like is a bHLH-type G-box-binding transcriptional activator that plays important roles in leaf and kernel development.