| Maize is an important cereal crop for feed,food and industry raw material.Despite its highest yield and largest planting acreage among major crops,there is still a big gap between maize production and the increasing demands.In this case,improving the per unit area yield will be crucial.Traits of kernels are key components of grain yield and impact the final yield a lot.Maize kernel is composed of multiple tissues and their development is controlled by a complex regulatory network involving multiple genes.Although some of these genes have been identified,the whole structure of the network remains largely unknown.Therefore,further identification and functional studies on genes involved in maize kernel development will be of immense value in both theoretic research and breeding practice.ABA plays a key role in maize kernel development through regulating genes containing cis-element G_box.A bHLH transcription factor ABP7(ABRE Binding Protein 7)which binds to G-box was identified by Yeast One Hybrid screening a cDNA library of 17 DAP maize embryos previously.Over expression of ABP7 inArabidopsis promoted the development of transgenic plants and led to larger siliques,as well as increased seed size and weight.Based on these results,in this study,the research focused on the function and molecular mechanism of ABP7 in the regulation of maize kernel development and reached conclusions as described below.Previous work showed that ABP7 binds to G-box specifically.In this study,ABP7 was further identified as a transcriptional activator by protoplast transient expression as well as yeast transcriptional activation assays.Furthermore,serial deletion form N and C terminal of ABP7 uncovered that ABP7 has at least three transactivation domains,which two of them locate at C terminal(255-287 aa,321-353 aa),and one in N terminal(1-33 aa).In addition,17 proteins interacting with ABP7 were detected by Yeast Two Hybrid,and this will shed light on the regulators of ABP7.Expression profiles of ABP7 in different tissues during maize development suggest that ABP7 is expressed ubiquitously,but has two peaks,with one at early germination,and the other at early kernel development indicating that ABP7 may function during kernel development,in combination with the seed phenotype of transgenic Arabidopsis.Transgenic maize plants respectively expressing ABP70E,ABP7RNAi,ABP7VP64,ABP7SRDX and ABP7FLAG were obtained for functional analysis.ABP70E plants showed enlarged ears in diameter as well as larger and heavier seeds than non-transgenic control,while ABP7RNAi plants decreased ear diameter and seeds size.Moreover,ABP7VP64 and ABP7SRDX plants exhibited similar phenotype with that of ABP70E and ABP7RNAi,respectively.All this result demonstrates that ABP7 functions as a transcriptional activator,and regulates the seed size positively.Importantly,transgenic and non-transgenic kernels segregated from the same transgenic ear showed no difference in kernel size and weight,suggesting that effect of ABP7 on kernel development may be exerted through maternal but not filial tissues,which is further supported by the similar linear grain filling rate and the changed size of seed coat cells of transgenic lines in comparison with non-transgenic plants.To reveal the targets of ABP7,ABP70E,ABP7RNAi,ABP7VP64,ABP7SRDX transgenic plants were used for RNA-Seq while ABP7FLAG was subjected to ChIP-Seq.RNA-Seq and ChIP-Seq results showed that various biological processes were affected,and ABP7 mainly regulated genes like expAl,RD22,LRX3,BXL4 involved in cell wall formation and genes like ZmTCRR-1,ZmMRP-1,Mn1,ZmSWEET4c and BAP2/BETL2 involved in BETL development and sugar transport,directly or indirectly.These results provide valuable information on the specific role of ABP7 during kernel development.In summary,ABP7 is a G-box binding bHLH transcription activator and promotes kernel development,possibly though regulating the expression in maternal tissues of genes involved in cell wall metabolism,glucose metabolism,nutrients transport. |
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