Screening Of Salt-tolerance Germplasms And Identification Of Lignin Biosynthesis Related Genes Of Sorghum Bicolor

We are facing more and more serious energy crisis due to exhaustion of fossil fuel reserves, such as coal, crude oil and natural gas。This situation is worse in China owing to the larger population, fewer resources per capita and more energy demand. Thus, it becomes increasingly necessary to the development of renewable, sustainable, clean and efficient energy sources. As the only renewable energy source of liquid fuel, ethanol is the most promising type of renewable energy sources in future and has received lots of attention in China. Increased exploitation of biomass energy from starch or oil crops will threaten food supplies through competition for land, which make lignocellulosic biomass a kind of ideal bioenergy resource. The presence of abundant of saline-alkali and marginal soils in China makes it very meaningful to exploit biomass resources not only produce sufficient biomass for converting to biofuels but also with stress-resistance which suitable for growing on the soils.As a C4plant, sorghum accumulates a significantly greater amount of carbon during photosynthesis than do C3plants. Most importantly, sorghum shows high tolerance to drought and low nutrition, which makes it possible for grown on lean soils and still achieves high yields. The availability of genome sequence informations and abundant number of germplasms together with a great deal of bmr mutants make sorghum not only a bioenergy crop which can be grown in salty and dry land, but also a model of lignin synthesis and regulation research.Plant cell walls comprise most of the dry body mass synthesized through photosynthesis, which mainly comprised of cellulose, semicellulose and lignin. The lignin in lignocellulosic biomass can lower the efficiency of converting cellulose to ethanol, thus, understanding the molecular mechanisms of lignin biosynthesis and regulation and identifying key genes related to lignin content is necessary for genetically improving the feedstock quality of bioenergy crops with lower lignin content. The brown-midrib (bmr) is a kind of genetic mutations found in a few species which often associated with reduced lignin biosynthesis. This genetic mutation may serve as a model of developing strategies for manipulating lignin content. Although the reduced lignin content in the bmr mutant plant is evidenced, at present very little is known concerning where the bmr mutations occur in the lignin biosynthesis pathway. In order to identify saline and alkali resistant sources in sorghum, we have completed a project on evaluation of the entire U.S. sorghum germplasm collections. Meanwhile, we have constructed forward and reverse subtracted cDNA library with bmr mutants and wild-type sorghum. Thousands of cDNA sequences have been selected from the SSH cDNA libraries and then were used to print cDNA microarrays for transcript analysis. According to the transcript profiling, we identified some differently expressed genes involved in lignin synthesis and regulation. Subsequent characterization and functional analysis of their role involved in cell wall biosynthesis will benefit for genetically improvement of cell wall components of sorghum and other bioenergy crops.The main results of this work are summarized as follows.1. Screening for salt-resistant germplasm line of sorghumWe have evaluated the salt resistance of717out of1200sweet sorghums and4222out of more than40,000grain sorghums in U.S. sorghum germplasm collections (treatment for25-40d under150-200mmol/L NaC1). For sweet sorghums, we obtained83salt-resistant lines,65of which survived after transplant in field or pots and46of them got seeds. For grain sorghums, we found312salt-resistant lines, and most of them can finish the life cycle.A total of31salt-resistant sweet sorghum lines were planted in the alkali field and saline-alkali land. The rate of germination in alkali field is higher than that in saline-alkali field. The overall biomass of sweet sorghums in the two kinds of field is almost the same, but the individual one of seedling in saline-alkali field is higher. The brix degree of most sweet sorghums in saline-alkali field is higher than that in alkali field. The seed yields of each sweet sorghum line in these two kinds of field are very different, with the mature time of sorghums in saline-alkali field is5-to-7-day earlier.2. Identification of differentially expressed genes in sorghum (Sorghum bicolor L.) brown midrib mutants using cDNA subtraction and microarray analysisFor dissecting genes involved in the cell wall metabolism that leading to the brown midrib phenotypes and their regulatory mechanisms in sorghum, suppression subtractive hybridization (SSH) combined with cDNA microarray profiling was performed to identify the differentially expressed genes in13sorghum bmr mutants. A total of153differentially expressed genes were identified, of which43genes showed up-regulated expression while the other110down-regulated in the bmr mutants. The expression pattern of12candidate genes through semi-quantitive RT-PCR confirmed the accuracy of the data from microarray analysis. All the differentially expressed cDNAs with significant protein homology could be classified into11functional groups: metabolism, photosynthesis, genetic information processing, stress responsive, protein fate, signal transduction, transport, lignin synthesis, cell process and mobility, development and regulation, and others. The most abundant group in the differentially expressed genes was metabolism. In the photosynthesis category,16of the17differentially expressed genes were down-regulated which coincident with the yield reduction phenotype of some bmr mutants. A few amount of differently expressed lignin synthesis related genes were identified, of which the expression of CYP was repressed in the bmr mutants which might responsible for the reduction of lignin content of the bmr mutants. However, the expression of another lignin biosynthetic gene cinnamic acid4-hydroxylase (C4H) was enhanced in the bmr mutants, which might indicated that monolignol biosynthesis from L-phenylalanine might occur by more than one route. No differentially expressed MYB, NAC and Lim transcription factors were found in the bmr mutants, however, the expression of a bHLH transcription factor was up-regulated in several bmr mutants. We then dissected the function of these three genes in lignin biosynthesis and regulation.3. Functional analysis of lignin biosynthesis related genes in sorghumFunctional analysis of SbHLHSbHLH was one of the most up-regulated transcription factors identified from SSH and microarray analysis and it showed obviously up-regulated expression in7of the13bmr mutants at7leaf stage. SbHLH appears six times in cDNA microarray analysis, which might indicated that SbHLH may be involved in lignin-biosynthesis regulation of several bmr mutants.The SbHLH showed different expression levels in roots, stems and leaves, with the highest level in leaves and lowest in roots, which correlated with the lignified degree of different organs. The full length SbHLH showed transcriptional activation activity in yeast which proved it a transcription factor. The deletion of the low complex N terminal region will lead to the lost of transcriptional activation activity even though the HLH domain is intact, which indicated the importance of this region to the activity of this transcription factor. Contrary, the deletion of the C terminal has no effect on the transcriptional activation activity of SbHLH. Transient expression of SbHLH-GFP fusion gene in onion epidermal cells indicated that SbHLH was located in the nucleus. Transgenic Arabidopsis overexpressing SbHLH showed no phenotypic alterations and could finish normal life cycle. However, the lignin content was reduced in the stem of transgenic plants. The overexpression of SbHLH down-regulated the expression of several genes of the lignin pathway and flavonoid/anthocyanin biosynthesis, such as PAL1,4CL, CCR1, HCT, COMT, CHI, UGT. Together, these results suggest that the sorghum SbHLH is a transcription factor repressing the phenylpropanoid biosynthetic pathway in Arabidopsis.Functional analysis of SbCYPThe expression of SbCYP was down-regulated in several bmr mutants at7-leaf-stage; however, it was up regulated in wild sorghum BTx623from5-leaf-stage to7-leaf-stage. Transgenic Arabidopsis plants overexpressing SbCYP could finish normal life cycle, with obviously increased lignin content in8-week stem although no obvious lignin content changes was observed in6-week stem. The overexpression of SbCYP induced the expression of several lignin biosynthesis related genes such as PAL1,4CL1, CCoAOMT and CCR1, which suggest that the sorghum SbCYP may contribute to the lignin biosynthesis and/or regulation. The differential expression of this gene between wild sorghum and bmr mutants would partly explain the lower lignin content of the bmr mutant, although this might due to the regulation of upper genes in the regulatory network (for example SbHLH).Functional analysis of SbC4H The expression of SbC4H was up-regulated in several bmr mutants. The involvement of thesorghum SbC4H in the lignin biosynthesis was investigated by overexpressing in Arabidopsis thaliana where the lignin content was reduced in6-week stem of transgenic plants when compared with wild type plants. The overexpression of SbC4H in Arabidopsis down-regulated the expression of several lignin biosynthesis genes such as4CL1,4CL and F5H1.In conclusion, the differently expressed genes in bmr mutants, SbHLH, SbCYP and SbC4H, may be part of the lignin biosynthesis and regulatory network according to the functional analysis. Furthermore, all the results have not only enriched our knowledge about the bmr mutant, lignin biosynthesis and regulation, but also provide new resources and theoretical basis for genetic improvement of lignocellulosic biomass with improved bioethanol production.