The Arabidopsis domain of unknown function 1218 (DUF1218) containing proteins, MODIFYING WALL LIGNIN-1 and 2 (At1g31720/MWL-1 and At4g19370/MWL-2) function redundantly to alter secondary cell wall lignin content

Mewalal, Ritesh; Mizrachi, Eshchar; Coetzee, Berdine; Mansfield, Shawn D.; Myburg, Alexander Andrew

The Arabidopsis domain of unknown function 1218 (DUF1218) containing proteins, MODIFYING WALL LIGNIN-1 and 2 (At1g31720/MWL-1 and At4g19370/MWL-2) function redundantly to alter secondary cell wall lignin content

Mewalal, Ritesh; Mizrachi, Eshchar; Coetzee, Berdine; Mansfield, Shawn D.; Myburg, Alexander Andrew

Date: 2016-03-01

Abstract:

DUF1218 is a land plant-specific innovation and has previously been shown to be associated with cell wall biology, vasculature patterning and abiotic/biotic stress response. The Arabidopsis genome encodes 15 members, two of which (At1g31720 and At4g27435) are preferentially expressed in the secondary cell wall depositing inflorescence stems. To further our understanding of the roles of DUF1218-containing proteins in secondary cell wall biology, we functionally characterized At1g31720 (herein referred to as MODIFYING WALL LIGNIN-1 or MWL-1). Since related gene family members may contribute to functional redundancy, we also characterized At4g19370 (MWL-2), the most closely related gene to MWL-1 in the protein family. Subcellular localization revealed that both Arabidopsis proteins are targeted to the cell periphery. The single T-DNA knockout lines, mwl-1 and mwl-2, and independent overexpression lines showed no significant differences in plant growth or changes in total lignin content relative to wild-type (WT) control plants. However, the double homozygous mutant, mwl-1/mwl-2, had smaller rosettes with a significant decrease in rosette fresh weight and stem height relative to the WT control at four weeks and six weeks, respectively. Moreover, mwl-1/mwl-2 showed a significant reduction in total lignin content (by ca. 11% relative to WT) and an increase in syringyl/guaiacyl (S/G) monomer ratio relative to the control plants. Our study has identified two additional members of the DUF1218 family in Arabidopsis as novel contributors to secondary cell wall biology, specifically lignin biosynthesis, and these proteins appear to function redundantly.

Description:

S1 Fig. Phylogenetic and bioinformatics analysis of all members of the Arabidopsis domain of unknown function 1218 (DUF1218) family, and expression profiling of the candidate members, MODIFYING WALL LIGNIN-1 (MWL-1, At1g31720) and MWL-2 (At4g19370). (A) Neighbor-joining phylogenetic tree of Arabdiopsis DUF1218-containing proteins. ClustalW was used to align protein sequences from TAIR and the alignment thereafter used to construct the tree using p-distance and pairwise deletion with 1000 bootstrap replicates in MEGA5 [16]. Prediction of subcellular localization, signal peptide and number of transmembrane domains was done using SUBA3 [31], Signal-3L [18] and TMHMM[19] respectively, with default settings. Highlighted in pink are the related MWL-1 and 2 sequences. (B) Arabidopsis expression profiles for MWL-1 and MWL-2 across different tissues during development, exctracted from The Bio-Analytic Resource for Plant Biology (http://bar. utoronto.ca/welcome.htm) [20]. Preferential expression is seen at distinct developmental stages, however, there is overlap in the secondary cell wall depositing, 2nd internode region. (DOCX)

S2 Fig. Gene ontology enrichment of MWL-1 top 300 co-expressed genes in Arabidopsis. Co-expressed genes were extracted from ATTED-II [10]. GO-full was conducted in Cytoscape 2.8.2 [22] using BiNGO 2.44 [21], while overrepresentation summary enrichment was performed with the REVIGO server [23]. (DOCX)

S3 Fig. Phenotypic analysis of At1g31720 (MWL-1) single T-DNA knockout line mutants and MWL-1 overexpression lines. (A) RT-PCR detection of endogenous MWL-1 transcript in the wildtype (WT) plants and absence in the single knockout mutant. (B) Semi-quantitative RT-PCR analysis of MWL-1 overexpression lines 1 to 3 showing detection of MWL-1 transgene in the transgenic lines. Actin2 was used as a control gene and RT-PCR was performed on cDNA from stem tissue. Actin2 and MWL-1 gene-specific oligonucleotide sequences can be found in S1 Table. Rosette size (C) and mass (D) of MWL-1 single T-DNA knockout line and overexpression lines 1–3 relative to (WT) control line at four weeks. Qualitative (E) and quantitative (F) stem length of MWL-1 single T-DNA knockout line and overexpression lines relative to WT control at six weeks. For rosette mass n = 3 and for quantitative stem length n = 66. Error bars indicate the standard error. Scale bar, 3 cm. Based on a two-tailed Student’s t-test (P-value 0.05) no significant differences were seen in the growth and development of the single mutant and transgenic OE lines in comparison to the WT controls.

S4 Fig. Phenotypic analysis of At4g19370 (MWL-2) single T-DNA knockout line mutants and MWL-2 overexpression lines. (A) RT-PCR detection of endogenous MWL-2 transcript in the wildtype (WT) plants and absence in the single knockout mutant. (B) Semi-quantitative RT-PCR analysis of MWL-2 overexpression lines 1 to 3 showing detection of MWL-2 transgene in the transgenic lines except for OE1 which could be indicative of positional effect (position in the genome), or co-suppression dominant repression. Actin2 was used as a control gene and RT-PCR was performed on stem tissue. Actin2 and MWL-2 gene-specific oligonucleotide sequences can be found in S1 Table. Rosette size (C) and mass (D) of MWL-2 single T-DNA knockout line and overexpression lines 1–3 relative to (WT) control line at four weeks. Qualitative (E) and quantitative (F) stem length of MWL-2 single T-DNA knockout line and overexpression lines relative to WT control at six weeks. For rosette mass n = 3 and for quantitative stem length n = 66. Error bars indicate the standard error while significant difference from the WT based on a two-tailed Student’s t-test (P-value 0.05) is indicated by . Scale bar, 3 cm. No significant differences were seen in the growth and development of the single mutant and transgenic OE lines in comparison to the WT controls except for OE-Line 2. (DOCX)

S5 Fig. Transverse sections of six-week-old stem tissue stained with phloroglucinol from At1g31720 (MWL-1) and At4g19370 (MWL-2) T-DNA knockout mutant and overexpression (OE) lines. Transverse sections from wildtype (WT) (A), At1g31720 mutant (B), At4g19370 mutant (C), At1g31720 x At4g19370 double knockdown mutant (D), OEAt1g31720 line 1 (E), line 2 (F), line 3 (G), OEAt4g19370 line 1(H), line 2 (I), line 3 (J). Scale bar, 100μm (indicated in red). No discernible differences were seen in the transverse sections of the single and double mutant as well as the transgenic OE lines in comparison to the WT controls. (DOCX)

S1 Table. List of oligonucleotides used in the study. (DOCX)

S2 Table. Top 300 Arabidopsis co-expressed genes for MWL-1 (At1g31720) from ATTED-II represented as MR value. (DOCX)

S3 Table. Top 300 Arabidopsis co-expressed genes for MWL-2 (At4g19370) from ATTED-II represented as MR value. (DOCX)

S4 Table. Structural cell wall carbohydrates and lignin content from MWL-1 and MWL-2 overexpression, single and double knockout lines compared to its respective wildtype (WT) control. (DOCX)

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