Diversity of α‐tubulin transcripts in Lolium rigidum

Abstract

BACKGROUND Tubulin, the target site of dinitroaniline herbicides, is encoded by small gene families in plants. To better characterize the mechanisms of target‐site resistance to dinitroaniline herbicides in the globally important weedy species Lolium rigidum, attempts were made to amplify and sequence α‐tubulin transcripts. RESULTS Four α‐tubulin isoforms (TUA1, TUA2, TUA3 and TUA4) were identified in L. rigidum. Variations in the number and sequence of transcripts encoding these α‐tubulin proteins were found in individuals from the two L. rigidum populations examined. Within and among populations, differences in the 5′‐ and 3′‐untranslated regions of cDNA in TUA3 and TUA4 were identified. Furthermore, a novel double mutation, Arg‐390‐Cys+Asp‐442‐Glu, in the TUA3 transcript was identified and has the potential to confer dinitroaniline resistance. CONCLUSION This research reveals the complexity of the α‐tubulin gene family in individuals/populations of the cross‐pollinated weedy species L. rigidum, and highlights the need for better understanding of the molecular architecture of tubulin gene families for detecting resistance point mutations. Although TUA4 is a commonly expressed α‐tubulin isoform containing most frequently reported resistance mutations, other mutant tubulin isoforms may also have a role in conferring dinitroaniline resistance.

Dinitroaniline Herbicide Resistance and Mechanisms in Weeds

“…Target-site resistance in the obligatory cross-pollinated L. rigidum is more complicated when compared with selfpollinated weed species, mostly due to Lolium genetic diversity. At least four α-tubulin isoforms (named TUA1-TUA4) have been identified in plants from two L. rigidum populations, with resistance-endowing mutations occurring largely in TUA3 and TUA4, according to our sequencing results in several resistant populations (Chen et al, 2018b(Chen et al, , 2020bChu et al, 2018). Moreover, the same α-tubulin isoform from a single plant can be encoded by transcripts with differences only in untranslated regions (UTR), and thus adding another layer of complexity to tubulin gene sequencing and cloning (Chen et al, 2020b).…”

“…At least four α-tubulin isoforms (named TUA1-TUA4) have been identified in plants from two L. rigidum populations, with resistance-endowing mutations occurring largely in TUA3 and TUA4, according to our sequencing results in several resistant populations (Chen et al, 2018b(Chen et al, , 2020bChu et al, 2018). Moreover, the same α-tubulin isoform from a single plant can be encoded by transcripts with differences only in untranslated regions (UTR), and thus adding another layer of complexity to tubulin gene sequencing and cloning (Chen et al, 2020b). In addition, there exists a substantial amount of synonymous mutation among α-tubulin transcripts encoding the same isoform from different populations, which challenges the success of PCR amplification by using a single primer pair (Chen et al, 2020b).…”

“…Similarly in E. indica, four β-tubulin and three α-tubulin isoforms have been identified (Waldin et al, 1992;Yamamoto et al, 1998;Yamamoto and Baird, 1999), whereas in Setaria viridis, two αand two β-tubulin genes have been isolated (Waldin et al, 1992;Délye et al, 2004). In cross-pollinated L. rigidum, there are at least four α-tubulin isoforms, TUA1, TUA2, TUA3, and TUA4, but the number of transcripts coding for each isoform varies among individual/population (Chen et al, 2020b).…”

“…Moreover, the same α-tubulin isoform from a single plant can be encoded by transcripts with differences only in untranslated regions (UTR), and thus adding another layer of complexity to tubulin gene sequencing and cloning (Chen et al, 2020b). In addition, there exists a substantial amount of synonymous mutation among α-tubulin transcripts encoding the same isoform from different populations, which challenges the success of PCR amplification by using a single primer pair (Chen et al, 2020b). This discovery suggests care should be taken when attempting to definitively determine the presence or absence of TSR mutations if the herbicide target is derived from a multiple gene family, even in a diploid species (i.e., it is similar to herbicides that target single genes in polypoid species).…”

“…More than two resistance alleles can exist in a single plant due to presence of multiple gene copies or transcripts. For instance, a plant homozygous for the Val-202-Phe mutant alleles in TUA4 also has the Arg-390-Cys + Asp-442-Glu resistance allele in TUA3 (Chen et al, 2020b). Nevertheless, the frequency of resistance mutations varies even among individuals of a population.…”