Update:
We have isolated RNA from the resistant and
susceptible Palmer amaranth plants and sent the samples for RNA-sequencing. Briefly, to
analyze the transcriptome, we sequenced RNA from 3 plants each of resistant and susceptible
populations of Palmer amaranth. Third and 4th young leaf were collected from individual plant 6
hours after 2,4-D and mesotrione treatment, separately and at the same time from the non-treated
plants. Total RNA was extracted, and the samples were sent to Azenta Lifesciences for RNA
sequencing. Paired-end reads obtained from RNA-seq analysis were trimmed and mapped
against Amaranthus hypochondriacus reference transcriptome., which is the closet to Palmer
amaranth available in the database. A pairwise differential gene expression analysis was
performed using DEseq2 package in R studio. Probable gene function for differentially
expressed genes were annotated with reference to Arabidopsis thaliana genome. Following
alignment and differential expression with DESeq2, Filtering parameters were for the adjusted p
value = < 0.05 and log2 fold change > 2.0 and false discovery rate (FDR) was < 0.05. We
identified 97 differentially expressed genes in the resistant plants when compared to susceptible
without any treatment. From our previous research, the Palmer amaranth population used in this
research exhibits metabolic resistance to 2,4-D and mesotrione. Therefore, we hypothesize that
the genes related to herbicide metabolism may have differential regulation or be highly
expressed in the non-treated samples of resistant plants compared to the susceptible plants. Our
data suggest that out of 97 differentially expressed genes, there are 3 GST (glutathione-Stransferases) and 2 CYPs (cytochrome P450 enzymes) up regulated in the resistant plants
compared to the susceptible plants. The GSTs and P450s are known to metabolize multiple
herbicides in plants. Additionally, the data of transcriptome of resistant and susceptible plants at
6 hours after treatment with 2,4-D and mesotrione suggest that about 104 and 123 genes
differentially expressed respectively in resistant plants when compare susceptible plants.
Moreover, we have identified certain common GSTs and CYPs overexpressed constitutively in
the resistant plants with both 2,4-D and mesotrione application and without any treatment. These
results are important to assess if these specific GST or P450 enzyme activity predisposes Palmer
amaranth to evolve resistance to multiple herbicides. Research is in progress to assess the
differentially expressed genes between resistant and susceptible plants in response to other
herbicide treatments as well
View uploaded report 
A Palmer amaranth population with resistance to six herbicides (6-way) was identified in KS. This Palmer amaranth population was collected from a 45-year old tillage study maintained in continuous sorghum. A variety of herbicides were used in the research trial, with 2,4-D and atrazine used most frequently. However, when plants from this population were studied in the greenhouse, they survived applications of group 2 herbicides Glean (chlorsulfuron), Harmony (thifensulfuron), Beyond (imazamox), Pursuit (imazethapyr); group 14 herbicides Cobra (lactofen) and Flexstar (fomesafen); the group 27 herbicides Callisto (mesotrione) and Laudis (tembotrione), metribuzin (group 5); and glyphosate (group 9); as well as atrazine (group 5) and 2,4-D (group 4). The only herbicides in the study that provided 100% control were Liberty (glufosinate) and Gramoxone (paraquat). Such multiple herbicide resistant Palmer amaranth populations are spreading rapidly posing a serious threat for sustainable soybean production. In some scenarios growers are losing herbicide options to manage this weed. Although use of herbicides still offers an effective weed management strategy, such strategy needs to be complemented with long term sustainable biological-based strategies. The 6-way resistant Palmer amaranth converts the herbicides to inactive forms before the plant can be killed, often due to the activity of two groups of enzymes: cytochrome P450s (P450) and glutathione S-transfersases (GSTs). These enzymes provide selectivity to many of the herbicides used in crops. We hypothesized that the activity of these enzymes may be elevated in the 6-way resistant Palmer amaranth. In Year-1 of this project we proposed genetic and molecular methods to assess the activity of herbicide degrading enzymes in this Palmer amaranth. In the results of Year-I research indicated that certain genes (cytochrome P450) involved in herbicide degradation show increased activity in the resistant plants but not in the susceptible plants. These results were achieved after treatment with two herbicides (2,4-D and mesotrione). Currently experiments are in progress to assess the activity of herbicide degrading enzymes in
Year-2 funding from KSC, we are currently investigating the possibility of any novel enzymes that may be involved in multiple herbicide degradation. Additionally, to explore biological-based weed control strategies, in the Year-1 of this project, we have also conducted experiments to optimize protocols to generate Palmer amaranth plants via tissue culture (generating multiple plants from either a single young leaf or stem cutting or any other plant parts), so we can use this protocol for advanced molecular biology techniques (gene-editing) to reduce the aggressive growth habits of this weed to make it less competitive. Using young leaf or stem cuttings of Palmer amaranth, we were able to produce callus tissue which is needed for regeneration of multiple plants in tissue culture. The callus needs to be grown on a medium with appropriate growth hormones, so the plant regeneration will occur. Year-2 of this project is primarily focused on characterization of the herbicide degrading enzymes including P450 or GST. The outcome of this research will demonstrate the greatest threat associated with multiple resistance in weeds and importantly, that a single resistance mechanism can provide resistance to multiple herbicide groups. While mixing and rotation herbicides with multiple, effective modes of action can slow the evolution of resistance, cross-resistance associated with multiple resistance greatly reduces the effectiveness of this strategy. Minimizing the weed seed bank and adopting alternative management strategies is essential to protect the value of existing and future herbicides.