| Aspartic proteases（APs）are very important subfamily members of the proteolytic enzyme family.It plays an important role in physiological processes such as protein storage and degradation,sexual reproduction,senescence,PCD（programmed cell death）and stress response.In this study,Populus tomentosa as a material was used to study how aspartic proteases（PtoAED3）are involved in regulating xylem development and whether PtoAED3 has a function similar to DUBs（deubiquitinating enzymes）to specifically dissociate covalently bound Ubs.Transgenic technology,real-time fluorescence quantitative PCR,histochemistry,GFP fluorescent labeling,immunochemistry and other experimental methods were used to analyze the formation of cell walls in xylem cells in PtoAED3 material,and a non-stained paraffin section Raman imaging method was established for observing the content of lignin in the xylem area.Using ubiquitinated antibodies to determine the total protein ubiquitination content in Populus tomentosa materials.The above results indicated that PtoAED3 was involved in the hydrolysis of related proteins synthesized in the secondary xylem cell wall,which affected the lignin content in the secondary xylem and regulated the ubiquitination of the whole protein in the plant.The main results of this study are as follows:（1）Analyzing the localized expression of PtoAED3.By detecting the expression of PtoAED3 gene,it showed that PtoAED3 was specific-expressed in roots,stems and leaves using q PCR analysis.Furthermore,the tissue localization of the PtoAED3 gene was detected in petioles,stems,and roots,especially in lateral meristem tissues of axillary buds by Pro PtoAED3::GUS.p Green-Pro PtoAED3-CDS-GFP fluorescent labeling combined with PGIP2-CFP co-localization is located in the cell wall of tobacco epidermal cells.PtoAED3 mainly localized in the secondary wall of xylem wood fiber cells,wood ray cells,and vessel cells by immunocolloidal gold.These results showed that PtoAED3 is involved in the growth and development of various tissues of plants,especially the formation of secondary walls of xylem cells.（2）In order to study the biological function of PtoAED3 in vivo,PtoAED3 transgenic material was obtained by Agrobacterium-mediated leaf disc method,and q RT-PCR and western blotting was used to detect whether the content of PtoAED3 m RNA and proteins were different in different transgenic lines.Finally,three sense lines with higher expression levels and two anti-sense lines with significantly suppressed expression levels were obtained.These materials with significant differences in expression levels were selected for statistical analysis of growth height in different periods.The plant height of anti-sense（AS-2）materials in each period was lower than that of wild-type materials.The height of the plants at the age of one month was lower than that of the wild-type material in the same period.As the plants grew to three-month-old and six-year-old seedlings after transplanting,the height of the OE-3plants began to be higher than the wild-type in the same period.Paraffin sections showed that the thickness of the xylem region of the antisense（AS-2）material was significantly lower than that of the wild type of the same period;the thickness of the xylem region of the overexpressing（OE-3）material was lower than that of the wild type at the same period of one month,and at the age of three months.These results indicated that the cell wall localization of PtoAED3 affects the lignin content in the xylem region.（3）Using transcriptome to reveal the mechanism of PtoAED3.Transcriptome analysis showed that peroxidase expression was up-regulated in over-expressed materials,especially up-regulation of classⅢperoxidases related to cell walls,which was mainly reflected in changes in the total lignin content and inhibited peroxidation in the material.The expression of the substance was down-regulated,and the laccase gene was also down-regulated in the detected differential genes,which were all manifested in the down-regulation of the lignin content.Differential genes are mainly involved in phenylpropane biosynthesis,plant hormone signal transduction,and protein processing.Among them,phenylpropane biosynthesis is mainly related to lignin synthesis such as peroxidase,berberine bridge enzyme,and caffeic acid 3-O-methyltransferase.Enzymes affect the cell wall,and plant hormone signals are mainly reflected in auxin response proteins,which affect auxin synthesis and transport,leading to plant growth and development.Finally,protein processing mainly exists in the molecular chaperone heat shock protein family.Aspartic proteases are involved in the processing of protein substrates.（4）Non-stained paraffin section method was established for the lignin content in the xylem region by Raman imaging in P.tomentosa.The characteristic peaks around 1600cm-1caused by the expansion and contraction of the aromatic ring were used to detect the lignin content of the plant.Compared with wild type P.tomentosa plants,the lignin content in the overexpressing lines was up-regulated,while the lignin content in the expressing lines was suppressed down.The most significant over-expression（OE-3）and anti-sense（AS-2）PtoAED3 transgenic lines with up-and down-regulation of total lignin were selected,and the lignin content of the aspartic protease transgenic aspen material was compared by acetyl bromide.The total lignin overexpression（OE-3）and antisense（AS-2）were increased by 34.82%and down by 29.46%,respectively,compared with the wild type of the same period.（5）The total protein ubiquitination levels of P.tomentosa plants or suspension cells were measured,respectively.The over-expressing OE-3 lines were significantly decreased,while the antisense AS-2lines were not changed.The ubiquitination level of total protein in suspension cells tended to stabilize after 6 days of suspension cell culture.At the same time,when the concentration of inhibitor MG132exceeded 0.08m M,the ubiquitination level of total protein became stable.Treatment of over-expressing OE-3 suspension cells with three inhibitors of MG132,Pepstatin A,and Leupeptin at established levels of full-protein saturated ubiquitination can effectively restore total protein ubiquitin levels in suspension cells.In this study,the PtoAED3 gene of Populus tomentosa was cloned.Pto AED was specific-expressed in the cell wall of different tissues,especially in cell wall of xylem.The xylem area in the antisense-suppressing strains is significantly decreased and the xylem area in the sense-overexpressing strains is increased than that of in wild type.The lignin content in different transgenic materials was detected by Raman spectroscopy and acetyl bromid.The lignin content is significantly increased in overexpression lines and decreased in the antisense lines,respectively.The branch in the over-expressing plants was significantly increased.The total protein ubiquitination level was significantly also changed in transgenic Populus tomentosa plants.It is speculated that the aspartic protease may have a substrate similar to the formation of ubiquitin-protease complex（UPS）involved in the degradation of branching. |
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