Abstract
Main conclusion
Differences in cell wall components between two BNF-contrasting sugarcane genotypes might result from genetic variations particular to the genotype and from the efficiency in diazotrophic bacteria association.
Abstract
Sugarcane is a plant of the grass family (Poaceae) that is highly cultivated in Brazil, as an important energy resource. Commercial sugarcane genotypes may be successfully associated with beneficial endophytic nitrogen-fixing bacteria, which can influence several plant metabolic pathways, such as cell division and growth, synthesis of hormones, and defense compounds. In this study, we investigated how diazotrophic bacteria associated with sugarcane plants could be involved in the regulation of cell wall formation pathways. A molecular and structural characterization of the cell wall was compared between two genotypes of sugarcane with contrasting rates of Biological Nitrogen Fixation (BNF): SP70-1143 (high BNF) and Chunee (low BNF). Differentially expressed transcripts were identified in transcriptomes generated from SP70-1143 and Chunee. Expression profiles of cellulose and lignin genes, which were more expressed in SP70-1134, and callose genes, which were more expressed in Chunee, were validated by RT-qPCR and microscopic analysis of cell wall components in tissue sections. A similar expression profile in both BNF-contrasting genotypes was observed in naturally colonized plants and in plants inoculated with G. diazotrophicus. Cell walls of the high BNF genotype have a greater cellulose content, which might contribute to increase biomass. In parallel, callose was concentrated in the vascular tissues of the low BNF genotype and could possibly represent a barrier for an efficient bacterial colonization and dissemination in sugarcane tissues. Our data show a correlation between the gene profiles identified in the BNF-contrasting genotypes and a successful association with endophytic diazotrophic bacteria.
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- BNF:
-
Biological nitrogen fixation
- DET:
-
Differentially expressed transcripts
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Acknowledgements
Brazilian Institute for Science and Technology on Biological Nitrogen Fixation (INCT-FBN), Brazilian National Council for Scientific and Technological Development (CNPq), Foundation for Research Support of the State of Rio de Janeiro (FAPERJ), Funding Authority for Studies and Projects (FINEP), and Coordination for the Improvement of Higher Education Personnel (CAPES) funded this work. INCT/CNPq, FAPERJ, and CAPES supported HGFB and TLGC for postgraduate fellowships. ACR is supported by CNPq for Ph.D. fellowships. ASH receives support from CNPq and FINEP research grants.
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The raw RNA-seq data are available in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra) under the BioProject accession number PRJNA226750 and BioSamples accession numbers SRX375173–SRX375188.
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Supplementary file1 (TIF 11022 kb) Fig. S1 Hierarchical clustering of differentially expressed transcripts from gene families involved in cell wall functions in sugarcane stalks. Expression pattern of a Glycosyltransferase (GT) family, b Laccases, c Nucleotide-sugar, d Peroxidases, e Glycoside-hydrolase-family-17, and f Proteases represented in naturally colonized roots (NC-root) and naturally colonized shoots (NC-shoot) from 15-day-old germinated stalks. Colors represent differential gene regulation as log2 fold change, calculated as SP root/CH root and SP shoot/CH shoot. Blue represents higher expressions in Chunee (CH) genotype and red represents higher expressions in SP70-1143 (SP) genotype. The two columns for each comparison represent the two biological replicates. For specific analysis of each gene after hierarchical clustering, see Table S3
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Supplementary file2 (TIF 5792 kb) Fig. S2 Root morphology of naturally colonized plants germinated from stalks, in two contrasting genotypes. a–c SP70-1143 roots, d–f Chunee roots. a–f Representative sections of three analyzed regions of the roots: a, d upper zone; b, e middle zone; c, f basal zone. Roots of SP70-1143 and Chunee were stained with Safranin O, 0.5 mg/mL. g Measurement of length of naturally colonized plant roots. The graph represents the average length of 30 roots ± SD. h Schematic representation of tissue areas of representative root sections of SP70-1143 and Chunee contrasting genotypes. The asterisk over the bar indicates that the differences between the means are statistically significant with p value ≤ 0.05
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Supplementary file3 (TIF 4684 kb) Fig. S3 Cellulose, callose, and lignin staining in shoots of SP70-1143 and Chunee genotypes. Cellulose and callose in leaf cell walls were stained with Calcofluor white and Aniline blue, respectively, and visualized through confocal microscopy. Relative fluorescence was quantified (see Materials and methods for details). Lignified structures in leaf cell walls were stained with phloroglucinol and visualized through light microscopy. a, b Confocal micrographs of cellulose staining. c, d Confocal micrographs of callose staining. e, f Light microscopy images of lignin staining. g Comparative analyses of cellulose relative fluorescence quantified in vascular tissues of stalk shoots of SP70-1143 and Chunee genotypes. h Comparative analyses of relative callose fluorescence quantified in vascular tissues of stalk shoots of SP70-1143 and Chunee genotypes. Bars indicate mean ± standard deviation (n= 30). t test (p value ≤ 0.05) was performed. Asterisks (*) indicate significant changes between SP70-1143 and Chunee samples. Bar = 150 µm; arrow, epidermis; arrowhead: vascular tissue; e, sclerenchyma
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Supplementary file4 (TIF 8294 kb) Fig. S4 Comparison of expression profiles of cell wall genes between roots naturally colonized and roots free of diazotrophic bacteria. The graph shows the expression profiles from RNA-seq of naturally colonized stalk roots (STALK) and plantlet roots cultivated for 15 days in hydroponic condition, from SP70-1143 and Chunee genotypes. Values were presented as SP70-1143 compared with Chunee and transformed in log2 fold change. Bars represented mean ± SD of two biological replicates of Stalk and Hydroponic transcriptomes
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Supplementary file8 (XLSX 111 kb) Table S1 Annotated DETs between SP70-1143 (SP) and Chunee (CH) germinated stalk tissues belonging to the cell wall functional category
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Supplementary file6 (XLS 11 kb) Table S2 Primers used for validation of compounds synthesis of assembly architecture and growth genes in stalk and inoculated plants
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Supplementary file7 (XLS 499 kb) Table S3 Cell wall pathway annotation. Gene families involved in cell wall biosynthesis pathway were identified in the reference transcriptome RT1 through local alignment annotation using protein sequence database of two monocotyledons (rice and maize) and one eudicotyledon (A. thaliana). The homologs in S. spontaneum, S. hybrid, and S. bicolor are also presented. The transcriptome database was aligned at a minimum of 70% identity and e value ≤ 10-5
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Supplementary file5 (TIF 143 kb) Fig. S8 Colonization by G. diazotrophicus of SP70-1143 (SP) and Chunee (CH) plantlets, after diazotrophic bacterial inoculation. Relative levels of G. diazotrophicus colonizing a roots and b shoots of inoculated and control mock plants were evaluated by RT-qPCR, 15 days after bacterial inoculation. Bacterial 23S rRNA levels were normalized with rice 28S rRNA and sugarcane GAPDH levels. Values of all three biological replicates are presented in the graph. Bars indicate mean ± SD
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Ballesteros, H.G.F., Rosman, A.C., Carvalho, T.L.G. et al. Cell wall formation pathways are differentially regulated in sugarcane contrasting genotypes associated with endophytic diazotrophic bacteria. Planta 254, 109 (2021). https://doi.org/10.1007/s00425-021-03768-0
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DOI: https://doi.org/10.1007/s00425-021-03768-0