Fitness costs associated with acetyl‐coenzyme A carboxylase mutations endowing herbicide resistance in American sloughgrass (Beckmannia syzigachne Steud.)

Abstract Weed resistance to herbicide can be conferred by gene mutations, and some mutations can cause pleiotropic effects in some cases. We investigated the pleiotropic effects associated with five specific ACCase mutations (Ile1781Leu, Trp2027Cys, Ile2041Asn, Asp2078Gly, and Gly2096Ala) on the plant growth, seed production, and resource competitiveness in American sloughgrass. Resistant plants (M/M) homozygous for specific ACCase mutation and susceptible wild‐type plants (W/W) were derived from single heterozygous mother plant (M/W) by genotyping. Plant growth assay and neighborhood experiments were performed to quantify variation between M/M plants and W/W plants. The Ile1781Leu mutation resulted in slight increases in plant growth in pure stands and improved resource competitiveness under low‐competition conditions in pot experiments, but no clear variation was observed under high competitive pressure or field conditions. During competition with wheat plants under field conditions, American sloughgrass plants containing Ile2041Asn ACCase exhibited a significantly lower (12.5%) aboveground biomass but no distinct differences in seed production or resource competitiveness. No significant detrimental pleiotropic effects associated with Gly2096Ala were detected in American sloughgrass. The Trp2027Cys mutation distinctly reduced seed production, especially under high competitive pressure, but did not significantly alter plant growth. The Asp2078Gly mutation consistently reduced not only plant growth and seed production but also resource competitiveness. Synthesis. The Trp2027Cys and Asp2078Gly mutations led to significant fitness costs, which may reduce the frequency of resistance alleles and reduce the propagation speed of resistant weeds in the absence of ACCase inhibitor herbicides. The Ile1781Leu, Ile2041Asn, and Gly2096Ala mutations displayed no obvious fitness costs or displayed very small fitness penalties, which would likely have no effect on the establishment of resistant weeds in the field.

However, these resistance-associated fitness costs are usually difficult to predict, and their expression depends on weed species and particular mutant alleles. For example, Ile1781Leu ACCase improves plant growth in foxtail millet (Setaria italica) (Wang, Picard, Tian, & Darmency, 2010) but not in A. myosuroides (Menchari et al., 2008).
American sloughgrass (Beckmannia syzigachne Steud.), a member of the Poaceae family, is a widespread and severely harmful weed in wheat (Triticum aestivum L.) and oilseed rape (Brassica napus L.) fields in southern China. Many American sloughgrass populations have evolved ACCase inhibitor resistance, and various ACCase mutations have been identified in those resistant populations (Li, Du, Liu, Yuan, & Wang, 2014;Pan et al., 2015;Tang, Zhou, Zhang, & Chen, 2015). However, the effects of those ACCase mutations on the fitness-related growth, seed production, and resource competitiveness of American sloughgrass have never been evaluated. In this study, to compare precisely the variation in fitness of resistant plants versus susceptible plants sharing a common genetic background, pairwise segregating resistant and susceptible progenies were generated from each original field population; each resistant genotype progeny was individually homozygous for the Ile1781Leu, Trp2027Cys, Ile2041Asn, Asp2078Gly, or Gly2096Ala mutation. The pleiotropic effects of those ACCase mutations on the fitness of this weed were subsequently assessed ( Figure 1).

| Plant materials
Four field-evolved resistant American sloughgrass populations were collected at different locations in southern China and were analyzed by ACCase genotyping and sequencing.
Five ACCase mutations Ile1781Leu, Trp2027Cys, Ile2041Asn, Asp2078Gly, and Gly2096Al were observed. Two segregating genotype progenies were derived from a single mother plant that contained the specific heterozygous ACCase (M/W) mutation and no other known ACCase mutation. For each mutation, pairwise comparisons were performed between each M/M progenies and its corresponding W/W ones; both two genotype progenies were derived from the same original progenitor and shared a common genetic background, except for ACCase mutation of interest. Each mother plant was cultivated appropriately and isolated within a pollen-proof enclosure during the flowering phase. After they ripened, F1-progeny seeds from each mother plant were collected.
Ten homozygous mutant (M/M) and ten wild-type (W/W) ACCase plants in the F1 progeny were identified, and each group of ten mother plants was cultivated within a pollen-proof enclosure to produce F2 seeds that were used for subsequent experiments (Table 1). And those progenies were the same ones used to assess cross-resistance patterns associated with homozygous ACCase

K E Y W O R D S
ACCase mutation, competition, evolution of resistance, fitness cost, plant growth, resistance mutant alleles (Du et al., 2016). Ten plants of each F2 segregating progenies were randomly selected for ACCase sequencing, and all plants were confirmed to be homozygous for the specific mutant or wild-type allele.

| Seed germination and growth
The seeds of the segregating F2 progenies were germinated in 9-cm Petri dishes that contained two layers of filter paper soaked with 5 ml of distilled water; germination occurred in a growth chamber under a constant temperature of 10°C and a 12-hr photoperiod (11,000 lx). After germination, the 1-cm-long seedlings were transplanted to plastic pots containing a mix of 50% sandy loam and 50% nursing media.

| Assessment of plant growth
The relative growth rate (RGR) and its net assimilation rate (NAR) and leaf area ratio (LAR) components were analyzed for the homozygous mutant and wild-type plants derived from the same single original population. The evaluation of growth traits involved classic and combined growth analyses (Hunt, 1982;Poorter, 1989).
With respect to the classic growth analysis experiments, uniform-size seedlings from each genotype progeny were transplanted to individual plastic pots (20.5 cm diameter × 40 cm height) that contained the abovementioned substrate, after which the seedlings were grown in a greenhouse under day/night temperatures of 25/15 ± 5°C and natural sunlight. The plants were regularly watered and rearranged to randomize environmental differences within the greenhouse. The aboveground biomass of 10 plants from each progeny was collected for each date at 30 days and 50 days after the seedlings were transplanted. The leaf area was measured with a leaf area meter (Yaxin-1242, Beijing Ya Xin Li Technology Co., Ltd., Beijing), after which the plants were harvested and then dried at 82°C for 3 days. The photosynthetic rates (µmol CO 2 m −2 s −1 ) were estimated at 30 days and 50 days before the plants were cut (CIRAS-3, PP Systems, Amesbury, MA). Photosynthesis was measured at a CO 2 density of 400-410 μmol/mol under natural light at 25°C.
With respect to the combined growth analysis experiments, uniform-size seedlings from each progeny were transplanted to plastic pots (40 pots for each progeny, 5 plants per pot, 20.5 cm diameter × 40 cm height) and cultivated under the abovementioned conditions. The leaf area and aboveground biomass were estimated at 25,29,33,37,41,45,49,53, and 57 days after the seedlings were transplanted. Four pots (20 plants) were selected per harvest.

| Assessment of resource competitiveness against wheat (reproductive stage) in pots
A plant's response to environmental resources is related to its ability to persist regardless of competitor presence. A neighborhood design was used to compare the reproductive biomass between mutant and susceptible plants derived from the same field germinated American sloughgrass and wheat seeds were simultaneously transplanted to pots and cultivated outdoors during the grown season. The pots were arranged in a completely randomized block design and were rearranged regularly. Before the wheat plants were harvested, the aboveground biomass and seeds of American sloughgrass and wheat plants were collected; each treatment consisted of four replicates.

| Assessment of resource competitiveness against wheat in the field
The seeds of each genotype progeny were germinated as described above. The seedlings were transplanted to multipot plates and grown in a glasshouse. When the seedlings grew to 3 cm, forty uniform-size plants (10 plants per block × 4 replicates) from each progeny were transplanted between wheat rows (17 cm apart) and spaced 30 cm apart within each row. Cultural practices in line with local conventional practices were performed, with the exceptions that no herbicides were applied during the whole grown season and that other grassy weeds were removed by hand. After the American sloughgrass plants matured, their aboveground biomass and seeds were collected. Four replicates were included per treatment in a fully randomized design.

| Statistical analysis and regression analysis
To assess plant growth assessment, the RGR and its components were determined using an unbiased formula. The variance of the RGR was evaluated in accordance with the following formula: where w 1 and w 2 represent the plant dry weight at harvest times t 1 and t 2 . One-way analysis of variance (ANOVA) in conjunction with Tukey's honestly significant difference (HSD) test (α = 0.05) was performed to compare RGR and photosynthesis estimates between the mutant and susceptible progenies derived from the same original field population.
The combined growth analysis was performed using a splined cubic polynomial model to describe the comparison of time trends in the classically derived RGR, NAR, and LAR (Hunt & Evans, 1980;Poorter, 1989) as follows: where y is the RGR, NAR, or LAR of the plant; x is the time; y 0 is the y value when x = 0; and a, b, and c are the rates of increase at different times.
The per unit size competitiveness of each progeny against wheat in pots was analyzed using a hyperbolic non-linear model (Vila-Aiub et al., 2009a) as follows: where y represents the seed production of American sloughgrass at wheat density or biomass x, a denotes the seed production of American sloughgrass in the absence of wheat (x = 0), and b is the slope of the regression. Steep slopes denote weak resource competitiveness.
In the field experiments, differences in plant height, aboveground biomass, and seed production between the mutant and susceptible genotypes of each group were compared using Tukey's HSD test (α = 0.05).

| Assessment of plant growth
The results of the classic growth analysis during a 20-day period (30-50 days after transplanting (DAT)) are shown in (2) y = y 0 + ax + bx 2 + cx 3

| Assessment of resource competitiveness against wheat in pots
The responses of each mutant and corresponding wild-type target plant to resource pressure under increasing wheat competition were compared. Target American sloughgrass seed production and above-

| Assessment of resource competitiveness against wheat in the field
To simulate a realistic growth environment, plants from ten derived genotype progenies were transplanted to the field to compete with wheat plants. The plant height, biomass, seed production, and number of tillers of each progeny were measured.
Due to the competition with wheat, the effective number of tillers of most plants ranged from 1 to 2, so the tiller numbers of each genotype were not compared. The significant differences in plant height, biomass, and seed production of each genotype are shown in Figure 4.
As the field experiment conditions were worse than potting experiment conditions, the American sloughgrass biomass and seed production were lower under field conditions than under potting conditions. When comparing among the five wild-type genotypes,  (Figure 4).
The biomass of the 2041M/M plants was 12.5% lower than that of the 2041W/W plants, but no significant differences in seed production or plant height were observed (Figure 4).

| Comparison of plant growth
Herbicide resistance endowed by target site mutations in weeds often adversely affects their growth and fitness (Vila-Aiub et al., 2009b;Yu et al., 2010). This negative influence may be due to a mutant gene that alters the target enzyme catalytic activity; the mutant gene either reduces the affinity of the target enzyme for its substrate or alters the feedback inhibition of the target enzyme, which in turn causes the enzyme to catalyze the synthesis of its product in excessive or inadequate amounts. In resistant L. rigidum plants whose resistance is conferred by ACCase mutations, the ACCase Asp2078Gly mutation can lead to decreases in the RGR and the NAR by 30% and 38%, respectively, during the vegetative growth stage. It is reasonable that the degradation of ACCase Raphanus raphanistrum (Li et al., 2013) and Avena fatua (Lehnhoff, Keith, Dyer, Peterson, & Menalled, 2013). In this study, the photosynthetic rate contributed little to the variation in fitness of resistant American sloughgrass plants.

| Estimation of ecological fitness
It is generally known that environmental factors can strongly affect plant growth (Yu, Ahmad-Hamdani, Han, Christoffers, & Powles, 2013), and the ecological fitness costs of plants may be more evident when plants are growing under stressful environmental conditions (Yang, Dong, Li, & Moss, 2007), such as predation (Gassmann, 2005), disease (Brown, 2003), and/or resource competition. If the ability of herbicide-resistant weeds to acquire resources diminishes, ecological fitness costs may be more readily F I G U R E 4 Comparisons of plant height, biomass, and seed production among each genotype. Significant differences were calculated between mutant genotypes and their corresponding wild-type genotypes TA B L E 3 Comparisons of plant height, biomass, and seed production among each wild-type American sloughgrass genotype in the field experiment  (Purrington & Bergelson, 1997).
In this study, pot and field experiments were conducted to evaluate the variation in ecological fitness between resistant and susceptible weeds derived from the same field population. The American sloughgrass biomass and seed production decreased significantly as the density of neighboring wheat plants increased. and F5 field populations is more accurate proof of this phenomenon (Wang et al., 2010). It is likely difficult to predict the fitness costs associated with resistance alleles on a case-by-case basis.
Moreover, the pleiotropic effects associated with resistance alleles are multitudinous, so it is important to use a range of evaluations throughout the whole plant life cycle and under some competitive pressures to detect small variations in fitness.
Asp2078Gly ACCase generally results in fitness costs in plant growth and resource competitiveness (Menchari et al., 2008;Vila-Aiub, Yu et al., 2015). Homozygous Asp2078Gly mutations could clearly reduce American sloughgrass biomass and seed production. This result is consistent with those of most reports. One explanation for fitness costs associated with Asp2078Gly is that this mutant allele may reduce enzyme activity (Vila-Aiub, Yu et al., 2015). Impaired ACCase activity can cause a shortage of lipids available for plant growth and is correlated with impaired resource competitiveness in plants homozygous for the Asp2078Gly mutation (Vila-Aiub, Yu et al., 2015).
To date, only one published study has evaluated the ecological fitness costs associated with the Trp2027Cys mutation in Avena sterilis (Papapanagiotou et al., 2015), and no definitive ecological fitness costs were detected. In our study, the homozygous Trp2027Cys mutation could lead to a significant decrease in seed production; in addition, as the wheat planting density increased, the decrease in seed production became more pronounced. When the density of the wheat plants was 480 plants per m 2 , the seed production decreased by 66.5%. In other words, plants containing Trp2027Cys ACCase showed a distinctly weaker competitive response to increasing wheat density, although no significant variation in plant growth was observed. In our previous study, the Trp2027Cys ACCase did not significantly affect seed germination (Du et al., 2017). Throughout the whole American sloughgrass growth period, fitness costs associated with the Trp2027Cys ACCase were detected only during the reproductive stage. This impaired trait may lead to an incompatibility with the natural ecosystem in the field and reduce the risk of spreading in cropping systems in the absence of herbicides.
We found that the Ile2041Asn mutation caused no obvious variation in responses to resource competition in the pot experiment.
Interestingly, when growing under competition with wheat plants in the field, the plants with the Ile2041Asn allele exhibited reduced (12.5%) aboveground biomass, but no evident difference in seed production or plant height was observed in the field experiment. These results agree with similar results reported for A. myosuroides growing in competition with wheat plants in the field: The Ile2041Asn mutation caused no obvious fitness costs in terms of plant growth, seed production, and plant height (Menchari et al., 2008). In our study, no pleiotropic effects associated with Gly2096Ala ACCase were observed. Presumably, the Ile2041Asn or Gly2096Ala ACCase mutation had little effect on the reproduction of American sloughgrass in the field. In summary, to accurately estimate fitness costs associated with specific ACCase mutations conferring herbicide resistance to American sloughgrass, mutant plants (individually homozygous for the Ile1781Leu, Trp2027Cys, Ile2041Asn, Asp2078Gly, or Gly2096Ala mutation) and wild-type plants were generated from single mother plants that were individually heterozygous for the specific ACCase mutations, and several key parameters of the life cycle of this grassy weed were measured, including those parameters under conditions of resource competition pressure. The Trp2027Cys and Asp2078Gly mutations led to significant fitness costs, which may reduce the frequency of resistance alleles and reduce the propagation speed of resistant weeds in the absence of ACCase inhibitor herbicides. The Ile1781Leu, Ile2041Asn, and Gly2096Ala mutations displayed no obvious fitness costs or displayed very small fitness penalties, which would likely have no effect on the establishment of resistant weeds in the field.