Molecular Mechanism Of BpYAB1,a Downstream Target Gene Of BpPHD4 In Betula Platyphylla × B.pendula,Involved In Regulating Leaf Curling

Leaf is one of the important vegetative organs of plants,crucial to plant growth and development.As a key component of leaves,leaf veins play a key role in supporting leaves.Previously,we constructed the transcriptional regulatory network of leaf vein development of Betula pendula ’Dalecarlica’,and identified that the BpPHD4(PLANT HOMEODOMAIN 4)of Betula platyphylla × B.pendula plays an important regulatory role in leaf morphogenesis and leaf vein development.Here,I analyzed the expression characteristics of BpPHD4,identified BpYAB1 is the downstream target gene of BpPHD4,and clarified the function of BpYAB1,uncovered the main results are as follows:(1)Expression patterns of BpPHD4.Birch BpPHD4 was mainly located in nucleus.q RTPCR results showed that BpPHD4 gene respond to ABA,GA,Na Cl and PEG treatment.The expression of BpPHD4 gene in the fourth leaf,stem and root was significantly higher than that in terminal bud.GUS-stained confirmed the higher expression level of BpPHD4 in leaf veins.(2)BpPHD4 binds the promoter and promote the expression of BpYAB1.Ch IP-PCR and yeast single hybridization(Y1H)experiments showed that BpPHD4 could bind to the promoter of BpYAB1.q RT-PCR preliminarily proved that BpPHD4 positive regulated the expression of BpYAB1.Furthermore,it was verified that seven elements could be recognized by BpPHD4 by Y1 H experiment and tobacco transient co-expression system,and six of them were found on the promoter of BpYAB1.(3)Expression patterns of BpYAB1.The BpYAB1 in birch belongs to YABBY family.Sequence analysis shows that YABBY family genes of birch is highly conserved with Arabidopsis thaliana and Populus trichocarpa at amino acids 15-64 and 115-174,and their gene structure are also similar.q RT-PCR showed that the BpYAB1 was most expressed in terminal buds,but not in roots.It was also confirmed by the staining of Pro BpYAB1::GUS transgenic birch,in which the staining mainly appeared in terminal buds and young leaves.The expression of BpYAB1 was significantly down-regulated after 3 h,6 h,and 12 h during ABA,Na Cl,and PEG treatment,and was significantly up-regulated after 3 h of GA treatment.(4)Construction of transgenic lines of BpYAB1.Based on CRISPR/Cas9 gene editing technology,a three-target knockout vector aiming BpYAB1 was constructed.6 knockout lines were obtained by Agrobacterium-mediated transformation using zygotic embryo method.Analysis of the obtained knockout lines showed that 5 lines had base deletions,base insertions or missing fragments between two target sites,and their coding sequences were changed.The35S::BpYAB1-FLAG vector was constructed by the gateway method,and the BpYAB1 overexpressed birch was obtained.(5)Overexpression of BpYAB1 promote multiple morphogenesis including leaf curling.The leaf morphology observation of transgenic birch showed that there was no obvious change between the BpYAB1 knockout line and WT.However,the overexpressed birch showed abaxial leaves,and the leaf area,seedling height,ground diameter and internode decreased,the leaf color became darker and chlorophyll content increased.Paraffin section showed that within100 microns,the upper and lower epidermal cells number in the overexpression lines increased,upper epidermal cells increased by a higher multiple.The palisade tissue/sponge tissue increased significantly,and the xylem area decreased.Scanning electron microscope observation of leaf epidermis showed that compared with the WT line,the BpYAB1 knockout line had no obvious change,while the adaxial cells of the overexpressed birch leaves were irregularly arranged,and the epidermis of Y2(overexpression line 2)and Y9(overexpression line 9)lines appeared swirling.In addition,the stomatal aperture of the overexpressed lines increased,and the water loss rate increased.(6)Overexpression of BpYAB1 induce multiple downstream signaling.After RNA-seq analysis,compared with WT and YK8(knockout line 8),genes only differentially expressed in overexpressed strains Y2(overexpression line 2)were found.Among the up-regulated genes,GO pathways enriched in positive regulation of plant epidermal cell differentiation,response to auxin,response to injury and stress,the down-regulated gene GO pathways enriched in mitochondria and chloroplast.These differentially expressed genes are widely involved in various hormone related pathways and plant development progress.In conclusion,BpYAB1 is the downstream target gene of BpPHD4 involved in regulation of leaf curling.BpYAB1 affects the expression of hormone-related genes,growth and development-related genes.Compared with WT,there was no obvious change in knockout lines,but lines overexpressed BpYAB1 produced various phenotypic defects such as leaf downward curling,plant dwarfing,and increased water loss,the changes in its microstructure may be direct cause.Our results provide important basis for the further elucidating the mechanisms of BpYAB1 regulating developments in birch.