| Brassinosteroids?BRs?are a class of newly found phytohormones with a broader spectrum of physiological and cellular functions,higher activity and special structure.Both DWF4 and CPD were believed to be the rate-limited enzyme genes in BRs biosynthesis.Over the past twenty years,with the development of research technology and discovery of many BRs-biosynthesis defective and-insensitive mutants,many important progresses have been made in the research on BRs.To date,the pathways of BRs biosynthesis have been basically clear and the majority of BRs biosynthesis genes have been successfully cloned.However,these studies were mostly focused on annual herbaceous plants,such as Arabidopsis thaliana,Oryza sativa,Zea mays,and few studies have been performed in other plants,especially in woody plants with vast ecological and economical benefit.For the study of differences in BRs biosytheisis and genetic mechanism between herbs and woody plants,the full length cDNA which homologous with A.thaliana CPD?AtCPD?was cloned previously from Populus euphratica in our lab?named PeCPD?,and the function of PeCPD was studied simply by establishing Arabidopsis-PeCPD transgenic line?PeCPD-TL??Wu et al.;2014?.In this study,the full length cDNA which homologous with A.thaliana DWF4?AtDWF4?was also cloned from P.euphratica?named PeDWF4?and Arabidopsis-PeDWF4 transgenic line?PeDWF4-TL?was established.Then,Arabidopsis-PeDWF4-PeCPD co-transgenic line?PeCP/DW-TL?was established by crossing of PeDWF4-TL and PeCPD-TL.On this basis,under the completely same conditions a comparative study was conducted among PeDWF4-TL,PeCPD-TL and PeCP/DW-TL in the light of morphology,physiology,genetics,anatomy,biochemistry,molecular biology and proteomics.And all those are intended to understand the function of PeDWF4,and the functional differences as well as interplays between PeDWF4 and PeCPD in the regulation of plant growth and development.The main experiment results are as follows:1 Bioinformatics analysis showed that the cloned PeDWF4 cDNA was 1667 bp long,which included a whole encoding frame with 1470 bp.The PeDWF4 encoded a protein with 490 aa?amino acids?.The PeDWF4 belonged to CYP90 B family and share 72.53% and 38.95% aa homology with Arabidosis DWF4?AtDWF4,CYP90B1?and PeCPD?CYP90A?,respectively.In addition,the homology between PeDWF4 and some herbs?Gossypium raimondii,Gossypium hirsutum?CYP90B were higher than the homology between PeDWF4 and some trees?Prunus persica,Eucalyptus grandis?CYP90B.2 Morphological studies found that compared with wild type?WT?,PeDWF4-TL rosette leaf was elongated and curved,and leaf area was decreased.PeCPD-TL leaf shape was similar to WT,while the leaf area was increased.PeCP/DW-TL leaf was similar to PeDWF4-TL,but more slender and curlier than PeDWF4-TL's.PeDWF4-TL,PeCPD-TL and PeCP/DW-TL were all significantly increased in plant height,but PeCPD-TL was higher than PeDWF4-TL,and PeCP/DW-TL was higher than PeCPD-TL.Moreover,the inflorescence stem of PeDWF4-TL became thinner,that of PeCPD-TL was thicker than WT,and of PeCP/DW-TL was between PeDWF4-TL and PeCPD-TL?similar to WT?.3 Genetic analyses displayed that the blotting time of PeDWF4-TL was advanced,that of PeCPD-TL was delayed,and PeCP/DW-TL was between PeDWF4-TL and PeCPD-TL?similar to WT?.The silique length of PeDWF4-TL,PeCPD-TL and PeCP/DW-TL were all significantly increased,but that of PeCPD-TL was longer than that of PeDWF4-TL,and that of PeCP/DW-TL was longer than that of PeCPD-TL.In silique number and seed yield,PeDWF4-TL,PeCPD-TL and PeCP/DW-TL all showed a significant decrease,but the decreasing degree was different for each line: PeDWF4-TL was bigger than PeCPD-TL,and PeCP/DW-TL was bigger than PeDWF4-TL.Besides,there were a lot of abnormal and abortive siliques in PeDWF4-TL,but no one in PeCPD-TL.In PeCP/DW-TL,a greater percentage of the abnormal and abortive siliques than in PeDWF4-TL were observed.In addition,PeDWF4-TL also was completely different from the Arabidopsis AtDWF4-overexpression line previously reported?Choe et al.,2001?in the aspects of bolting time,stem diameter,silique development and seed yield.4 Physiology and biochemistry studies found that compared with WT,the maximal photochemical efficiency of PSII??PSII?,quantum yield?PQY?and electron transfer efficiency?ETR?were all decreased in PeDWF4-TL;in PeCPD-TL,?PSII and PQY were increased,and ETR showed no significant difference;in PeCP/DW-TL,?PSII and PQY were unchanged,and ETR was increased.The chlorophyll a and b?Chl a and Chl b?contents were all decreased in PeDWF4-TL.In PeCPD-TL,Chl b content was increased,and Chl a content was close to WT.In PeCP/DW-TL,Chl a and Chl b contents were also decreased,and its extent was similar to that in PeDWF4-TL.The lignin and cellulose contents were significantly decreased in PeDWF4-TL,but increased in PeCPD-TL.PeCP/DW-TL was between PeDWF4-TL and PeCPD-TL?similar to WT?.5 Microstructure observation showed that compared to WT,the stem cross section and pith area were reduced in PeDWF4-TL;in PeCPD-TL,the cross section,xylem,phloem and pith areas were all increased;in PeCP/DW-TL,the cross section areas was unchanged,however,xylem and phloem area were increased,and pith area was decreased.6 The expression level of PeDWF4,PeCPD,AtDWF4,AtCPD,AtBR6OX2,AtFLC,AtTCP1 and AtGA5 were detected using RT-PCR and RT-qPCR approach.The results showed that only PeDWF4 or PeCPD expressed in PeDWF4-TL and PeCPD-TL.Both PeDWF4 and PeCPD were expressed in PeCP/DW-TL and completely did not express in WT.AtDWF4,AtCPD,AtBR6OX2,AtFLC,AtTCP1 and AtGA5 expressed in all the transgenic lines and WT.In PeDWF4-TL,the expression of PeDWF4 made the expression level of AtDWF4,AtCPD,AtBR6OX2,AtFLC,AtTCP1 and AtGA5 down-regulate.In PeCPD-TL,the expression level of PeCPD made the expression of AtDWF4,AtBR6OX2,AtFLC and AtTCP1down-regulate and that of AtCPD,AtGA5 up-regulate.In PeCP/DW-TL,the expression levels of PeDWF4 and PeCPD were significantly lower than them in PeDWF4-TL and PeCPD-TL.The expression levels of AtDWF4,AtBR6OX2,AtFLC and AtTCP1 were all similar to them in PeDWF4-TL,and down-regulated relative to them in PeCPD-TL.The expression levels of AtCPD and AtGA5 were between them in PeDWF4-TL and PeCPD-TL.7 ELISA analyses revealed that the content of endogenous BL was significantly increased in PeDWF4-TL?2.567 ?g g-1FW?and significantly decreased in PeCPD-TL?1.323 ?g g-1FW?as compared with that in WT?1.937 ?g g-1FW?.The content of endogenous BL in PeCP/DW-TL?2.1731 ?g g-1FW?was lower than that in PeDWF4-TL,but higher than that in PeCPD-TL.8.The protein differences among PeDWF4-TL,PeCPD-TL,PeCP/DW-TL were analyzed using 2D electrophoresis plus MALDI-TOF-TOF tandem mass spectrometry method.The results showed that there were 704,633,732 and 723 protein spots in WT,PeDWF4-TL,PeCPD-TL and PeCP/DW-TL,separately.The proportions of matched proteins were 65.56%?62.6%?between WT and PeDWF4-TL?PeCPD-TL?,and were 39.56%?41.63%?between PeDWF4-TL?PeCPDTL?and PeCP/DW-TL,respectively.The successfully identified 10 protein spots included myrosinase 1?N16?,hypothetical protein ArthMp025?N18?,glycine hydroxymethyltransferase like protein?N19?,putative protein?R1?,thioglucosidase?R2?,endomembrane-associated protein?R3?,AT4g38970/F19H2270?R4?,putative ribose 5-phosphate isomerase?R5?,ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit?R8?,ribulose bisphosphate carboxylase/oxygenase activase?R12?.N16 and R2 were stress resistance related proteins,R8 and R12 were photosynthesis related proteins,and the others belonged to unknown proteins.The above results imply that: 1)PeDWF4 and PeCPD were all involve in BRs biosynthesis,but their roles were not exactly same in regulating plant growth and development.2)PeDWF4 is not exactly the same as AtDWF4 in the regulation of plant growth and development.3)There are complex synergistic or antagonistic effects between PeDWF4 and PeCPD in different developmental stages or organs and tissues.The detailed mechanism of PeDWF4 and PeCPD action and interplay between them should be further studied. |
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