| Cell wall lignification is a process of lignin deposition on cell wall. This process occured in plant secondary xylem of vascular bundle, leads to enhancement of cell wall strength and mechanical supporting. Lignin also reinforces the ability of transport of water and nutrients, defense of pathogens'attack and resistance to mechanical damage. Physiological processes of cell wall lignificaiton are not only a metabolism issue, but also a development issue. Because of complexed mechanism and numerous genes involved, understanding of cell wall lignification is still limited so far. Therefore, further research of the regulation mechanism of cell wall lignificaiton is of great benefit not only to the understanding of plant growth and development, but also to the crop and tree improvement in the terms of cell wall material formation.The phenomenon of monolignol glycosylation in planta is very ubiquitous. Although monolignol glucosides were reported in planta in the 1960s-1970s,whether they were important for cell wall lignification was still obscure.To further explore the function of monolignol glycosylation in lignin biosynthesis and cell wall lignification, glycosyltransferase mutants involved in the lignification abnormality were screened and the function of glycosyltransferase UGT72B1 was analyzed in detail. Some important progresses gained in this study are as follows.1. Screening of ugt72bl mutant and its phenotype analysisTwo ugt72b1 T-DNA insertion mutant lines ?SALK049597 and SAIL611E04? were identified by using three primers method and RT-PCR.They showed same phenotypes. Their shoot tips exhibited anthocyanin accumulation and growth repression after bolting. After introducing the cDNA of UGT72B1 into the ugt72b1 mutant, the phenotype of rescued lines was restored to wild-type, which confirmed that the phenotype of ugt72bl was cause by the loss of UGT72B1 function. The cross-sections of mutant floral stems were stained by Wiesner staining and Maule staining. It was found that mutant stems exhibited aggravated and ectopic lignification. Cell wall thickness in ugt72bl mutants was also increased, which was observed by transmission electron microscopy.These results indicated that UGT72B1 was involved in the process of cell wall lignification. Its loss of function could cause the ectopic and enhanced lignification and affect plant growth and development.2. Identification of biochemical activity of glycosyltransferase UGT72B1The prokaryotic expression vector of UGT72B1 was constructed, and then the recombinant protein was expressed and purified from E.Coli. The in vitro enzyme activity of UGT72B1 was analyzed by using dozens of natural compounds as potential substrates including plant hormones, flavonoids, phenylpropanoids and lignin precursors. HPLC-MS analysis of reaction products indicated that UGT72B1 has the glucosylating activity toward coniferyl alcohol, dihydroconiferyl alcohol, coniferyl aldehyde, p-coumaryl alcohol and p-coumaryl aldehyde. At the same time, the UGT72B1 overexpression lines were constructed and used to detect the in vivo enzyme activity. We found that overexpression lines contained much higher level of coniferin than wild type and rescued lines, suggesting the same catalytic activity of UGT72B1 toward monolignols in vivo as in vitro. Enzyme kinetics of,,UGT72Bl showed that the UGT72B1 had a higher enzymatic activity toward coniferyl alcohol than toward coniferyl aldehyde. The specific activity and Kcat/Km toward coniferyl alcohol were 6.6 nkat/mg and 0.74 mM-1s-1 respectively, while toward coniferyl aldehyde were 3.1 nkat/mg and 0.30 mM-1s-1 respectively.In addition, the recombinant proteins of UGT72B2 and UGT72B3, the closest paralogs of UGT72B1, were also obtained and used to detect their biochemical activity. UGT72B3 could glucosylated the monolignols:sinapyl aldehyde and coniferyl aldehyde, whereasUGT72B2 activity was not found. In consistence with this biochemical activity, UGT72B3 but not UGT72B2 was found to be able to restore the phenotype of ugt72b1 when UGT72B2 and UGT72B3 were overexpressed in ugt72b1 mutants. Thus, our results indicated that UGT72B1 was able to glycosylate monolignols in plant cells, and UGT72B1 and UGT72B3 have the similar biochemical activity toward monolignols.3. Gene expression pattern analysisThe UGT promoter::GUS system was constructed. GUS histochemical staining showed that, in seedling stage, UGT72B1 was mainly expressed in shoot bud and new leaf vein. After bolting, UGT72B1 expression was mainly in vascular tissue of stem tip, main vein of young cauline leaf, pedicle and petals. Cross-sections of mutant stems was made and used for GUS staining. In the young stem, UGT72B1 was expressed in almost every tissue such as cortex, xylem and pith, but only the xylem showed GUS reaction in older stem. Our qRT-PCR analysis showed the similar results with GUS reaction for UGT72B1 expression pattern in different tissues. The expression patterns of UGT72B2, UGT72B3 and UGT72E2 were also analyzed using GUS staining and qRT-PCR.The result showed that UGT72B2 was mainly expressed in the leaf vein, but no obvious expression was observed in xylem vessel. UGT72B3 mainly expressed in the base of petiole and the water outlet hole of young leaves, while a weak expression in xylem vessel was also observed. UGT72E2 was mainly expressed in the seedling stage, but a small amount of expression in the root and stem. In addition, we detected the expression level of UGT72B3 and UGT72E2 in ugt72bl mutant, and found that their expression levels were both enhanced.These results indicated that different glycosyltransferase genes had different expression patterns. The main expression site of UGT72B1 in stems was consistent with its mutant phenotype resulted from its loss of function.4. qRT-PCR and transcriptomic profiling of ugt72bl mutantsBy using qRT-PCR technology, lignin biosynthesis related genes ?including monolignol biosynthesis and transport, lignin polymerization and related transcription factor? were examined in the ugt72bl mutant background. It was found that the monolignol synthesis related genes including CCR, COMT, HCT, CAD,4CL, C4H, CCOATOT and PAL were significantly up-regulated. Up-regulated genes included also those genes related to monolignol transport and lignin polymerization such as ABCQ RBOH, PRX and LAC, those genes of transcription factors regulating lignin biosynthesis such as MYB58, MYB61, MYB63, MYB643 and NAC family members NST1 and NST3. In addition, the DFR of anthocyanin biosynthesis pathway and CHS of flavonoid biosynthesis pathway were also significantly up regulated in ugt72b1 mutants. These results indicated that the whole pathway of lignin biosynthesis was strengthened. In order to know global transcription changes of ugt72bl mutants, transcriptomic profiling was carried out. A total of 931 genes changed expression level dramatically, among them 705 were up regulated and 226 were down regulated. Pathway enrichment enalysis indicated that differentially expressed genes were mainly enriched in those pathways like phenylpropanoid biosynthesis, flavonoid biosynthesis, plant-pathogen interaction, starch and sucrose metabolism, and phenylalanine metabolism. For Phenylpropanoid and lignin biosynthesis pathway, almost every gene corresponding to each step was up regulated in mutants, which wasin accordance with the results of qRT-PCR analysis.These results suggest that loss function of UGT72B1 could activated whole lignin biosynthesis pathway and even linked pathways such as flavonoid and anthocyanin biosynthesis, which may be a reason leading to the ectopic and enhanced lignification accompanied with increased anthocyanin accumulation and stunted growth in mutant stems.In conclusion, the results of this research not only increased our understanding of the function of glycosyltransferase family in plant kingdom, but also provided the first evidence that monolignol glycosylation is essential for the normal cell wall lignification, thus giving unambiguous answer for an unresolved scientific issue that plagued us nearly half a century. This research will open the door to further study the mechanism controlling lignin metabolism and cell wall lignification, and will also provide an opportunity for getting new insight into the function of monolignol glycosylation in modulating lignin pathway and cell wall lignification. |
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