Genes Regulating Differentiation at the Shoot Apex of Fax (Linum Usitatissimum)

Abstract

Fiber harvested from flax phloem tissue is a renewable resource with promising uses in eco- friendly composites. Most molecular and cellular research to date has focused on later stages of fiber differentiation including the development of the fiber cell wall. On the other hand, the molecular mechanisms that govern specification of fibers are largely unknown. All phloem fibers in flax are formed during primary growth. Therefore transcription factors enriched in the shoot apices are likely to govern fiber identity, and therefore fiber yield. In this study, I used RNA-Seq to compare the gene expression in the apical region (AR) of the shoot apex which contained the apical-most 0.5mm of the stem and basal region (BR), which contained the entire stem except for the apical-most 1 cm. AR included the SAM and its immediate derivatives whereas BR represented all stem and vascular tissues at later stages of differentiation. The RNA-Seq study identified 349 putative transcription factors that are preferentially expressed in the AR including 18 MYBs and nine NACs. MYBs and NACs have been revealed to be required for the vascular cell identity in other species. A total of 240 putative MYBs and 182 predicted NAC domain genes were identified within the whole-genome sequence of flax. Phylogenetic analysis of the flax NAC gene family revealed that two distinct subfamilies were largely expanded. Flax had a higher proportion of R2R3-MYB than most of other sequenced plant species. Analysis of the expression data in public database indicated that the majority of LusMYBs and LusNACs were expressed in wide range of tissues with low expression level while a few others were particularly abundant in some specific tissues. Transcript expression profiling of the LusNACs in the VNS subfamily in 12 different flax tissues suggested that LusNAC28 and LusNAC125 were highly expressed in developing fibers. A previously uncharacterized Arabidopsis gene, At3g05980, encodes a predicted protein of 245 amino acids (27.6 kDa). This protein does not contain any annotated domains, and its predicted secondary structure consists mostly of disordered coils. It has one closely-related paralog in Arabidopsis, At5g19340. Homologs of At3g05980 are found in all eudicots examined, but not in any other taxa. There are four highly conserved amino acid motifs within the protein. Using qRT- PCR and GUS reporter assays, I found that transcripts of At3g05980 were highly expressed in immature embryos and the micropylar endosperm, as well as petals, and apices of shoots and roots, and atrichoblasts. Transcripts were highly induced by cold treatment, but not by other stress or hormone treatments. These results were consistent with expression patterns previously reported in public databases. I produced loss-of-function (LOF) mutants of this gene, using CRISPR/Cas9- mediated gene editing, as well as overexpression (OX) lines using the 35S-CaMV promoter. LOF lines were morphologically indistinguishable from wild-type, but OX lines had minor defects, including cotyledon epinasty, and slight shortening of both plant height and silique length. Neither LOF nor OX differed from WT in tolerance to freezing. In the absence of cold-treatment, LOF mutants had increased transcript abundance of the stress- and cold-responsive gene RD29, compared to WT, but expression patterns of five other cold-responsive genes were largely unchanged in LOF, compared to wild-type, both before and during cold treatment. Translational fusions of At3g05980 with fluorescent proteins were localized to peroxisomes. However, assays of peroxisomal function, including dark growth of seedlings, and sensitivity to 2, 4-DB and IBA, were similar between LOF, OX, and WT. Furthermore, fatty acid profiling of seeds did not show any difference between the genotypes. Thus, At3g05980 encodes a eudicot-specific, peroxisomaly localized protein with transcripts that are cold-inducible, and enriched in specific tissues (particularly rapidly growing tissues), but this gene does not appear to be required for normal morphology, peroxisomal function, or cold tolerance responses. The immediate future task will be to examine phenotypes in double mutants of both At3g05980 and it paralog At5g19340.