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Grain size is one of the most frequently selected traits during domestication and modern breeding. The continued discovery and characterization of new genes and alleles in controlling grain size are important in safeguarding the food supply for the world's growing population. Previously, a small grain size was observed in a rice restorer line 'Fuhui212', while the underlying genetic factors controlling this trait were unknown. In this study, by combining QTL mapping, variant effect prediction, and complementation experiments, we recovered a novel allele RGA1-FH that explains most of the phenotypic changes. The RGA1-FH allele contains an A-to-T splicing site variant that disrupts the normal function of RGA1. While population analysis suggests extremely strong artificial selection in maintaining a functional allele of RGA1, our study is the first, to the best of our knowledge, to prove that a dysfunctional RGA1 allele can also be beneficial in real agricultural production. Future breeding programs would benefit from paying more attention to the rational utilization of those overlooked 'unfavored' alleles.

Plants are thought to lack an early segregating germline and often retain both asexual and sexual reproduction, both of which may allow somatic mutations to enter the gametes or clonal progeny, and thereby impact plant evolution. It is yet unclear how often these somatic mutations occur during plant development and what proportion is transmitted to their sexual or cloned offspring. Asexual "seedless" propagation has contributed greatly to the breeding in many fruit crops, such as citrus, grapes and bananas. Whether plants in these lineages experience substantial somatic mutation accumulation is unknown. To estimate the somatic mutation accumulation and inheritance among a clonal population of plant, here we assess somatic mutation accumulation in Musa basjoo, a diploid banana wild relative, using 30 whole-genome resequenced samples collected from five structures, including leaves, sheaths, panicle, roots and underground rhizome connecting three clonal individuals. We observed 18.5 high proportion de novo somatic mutations on average between each two adjacent clonal suckers, equivalent to ~ 2.48 × 10 -8 per site per asexual generation, higher than the per site per sexual generation rates (< 1 × 10 -8 ) reported in Arabidopsis and peach. Interestingly, most of these inter-ramet somatic mutations were shared simultaneously in different tissues of the same individual with a high level of variant allele fractions, suggesting that these somatic mutations arise early in ramet development and that each individual may develop only from a few apical stem cells. These results thus suggest substantial mutation accumulation in a wild relative of banana. Our work reveals the significance of somatic mutation in Musa basjoo genetics variations and contribute to the trait improvement breeding of bananas and other asexual clonal crops., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The introduction of frameshifting non-3n indels enables the identification of gene-trait associations. However, it has been hypothesised that recovery of the original reading frame owing to usage of non-canonical splice forms could cause rescue. To date there is very little evidence for organism-level rescue by such a mechanism and it is unknown how commonly indels induce, or are otherwise associated with, frame-restoring splice forms. We perform CRISPR/Cas9 editing of randomly selected loci in rice to investigate these issues. We find that the majority of loci have a frame-restoring isoform. Importantly, three quarters of these isoforms are not seen in the absence of the indels, consistent with indels commonly inducing novel isoforms. This is supported by analysis in the context of NMD knockdowns. We consider in detail the two top rescue candidates, in wax deficient anther 1 (wda1) and brittle culm (bc10), finding that organismal-level rescue in both cases is strong but owing to different splice modification routes. More generally, however, as frame-restoring isoforms are low abundance and possibly too disruptive, such rescue we suggest to be the rare exception, not the rule. Nonetheless, assuming that indels commonly induce frame-restoring isoforms, these results emphasize the need to examine RNA level effects of non-3n indels and suggest that multiple non-3n indels in any given gene are advisable to probe a gene's trait associations., Competing Interests: The authors have declared that no competing interests exist.

Whole genome duplication (WGD) in plants is typically followed by genomic downsizing, where large portions of the new genome are lost. Whether this downsizing is accompanied by increased or decreased evolutionary rates of the remaining genes is poorly known, not least because homeolog pairings are often obscured by chromosomal rearrangement. Here, we use the newly published genome from a sedge, namely Kobresia littledalei, and CRISPR/Cas-9 editing to investigate how the Rho WGD event 70 million years ago (MYA) affected transcription factor evolutionary rates, fates, and function in rice (Oryza sativa) and sorghum (Sorghum bicolor). We focus on the 30-member DNA-binding with one zinc finger (Dof) transcription factor family in both crops due to their agronomic importance. Using the known speciation dates of rice from Kobresia (97 MYA) and sorghum (50 MYA), we find that rates of amino acid substitution in the critical Dof domain region were over twofold higher during the 20-million-year period following the WGD than before or afterward. Through comparison of synteny blocks, we report that at least 11% of Dof genes were purged from 70 to 50 MYA, while only 6% have been lost in the most recent 50-million-year interval. CRISPR/Cas9 editing revealed widespread fitness-related defects in flowering and lack of redundancy of paired members, as well as significant differences in expression between gene pairs. Together these findings demonstrate the strength of Dof genes as a model for deep evolutionary study and offer one of the most detailed portraits yet of the Rho WGD impact on a gene lineage., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

In contrast to common meiotic gene conversion, mitotic gene conversion, because it is so rare, is often ignored as a process influencing allelic diversity. We show that if there is a large enough number of premeiotic cell divisions, as seen in many organisms without early germline sequestration, such as plants, this is an unsafe position. From examination of 1.1 million rice plants, we determined that the rate of mitotic gene conversion events, per mitosis, is 2 orders of magnitude lower than the meiotic rate. However, owing to the large number of mitoses between zygote and gamete and because of long mitotic tract lengths, meiotic and mitotic gene conversion can be of approximately equivalent importance in terms of numbers of markers converted from zygote to gamete. This holds even if we assume a low number of premeiotic cell divisions (approximately 40) as witnessed in Arabidopsis. A low mitotic rate associated with long tracts is also seen in yeast, suggesting generality of results. For species with many mitoses between each meiotic event, mitotic gene conversion should not be overlooked., Competing Interests: The authors have declared that no competing interests exist.

Comparative genomic analysis within Asian cultivated rice (Oryza sativa L.) populations has greatly enriched our knowledge regarding rice domestication and the divergence of the indica and japonica subspecies, while study on genomic regions associated with improvement within the indica subspecies is still limited. Here, through combined investigation of 2,429 indica cultivar genomes from public sequencing projects, we depict the improvement of modern indica rice in China. We identify three subgroups within indica populations: two geographically distinct, historical subgroups indica I (Ind&#95;I) and indica III (Ind&#95;III) and a modern subgroup indica II (Ind&#95;II). The modern indica subgroup Ind&#95;II shows admixture of the other two subgroups and enrichment of alleles that had been low-frequency in the other two subgroups. The Chinese indica cultivars exhibit a strong subgroup component change from Ind&#95;I to Ind&#95;II in the 1980s. Through haplotype-based comparative analysis, we detect 187 regions associated with separation of Ind&#95;II compared to Ind&#95;I or Ind&#95;III. Within those regions we find strong representation of beneficial agricultural production-related alleles in Ind&#95;II and a positive correlation between grain yield and number of differentiated haplotypes. Phenotypic features of long and slender grain, small tiller angle and decreased flowering time were detected for Ind&#95;II. Through haplotype-based comparative analysis between rice subpopulations and subspecies, we find differentiated haplotypes not only from indica itself but also from japonica and aus, suggesting that introgression from other rice sub-populations has substantially contributed to modern indica rice breeds. These results help clarify the evolutionary landscape of modern indica rice in China and provide useful targets for future improvement., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Although SQUAMOSA promoter-binding-like (SPL) transcription factors are important regulators of development in rice (Oryza sativa), prior assessments of the SPL family have been limited to single genes. A functional comparison across the full gene family in standardized genetic backgrounds has not been reported previously. Here, we demonstrate that the SPL gene family in rice is enriched due to the most recent whole genome duplication (WGD). Notably, 10 of 19 rice SPL genes (52%) cluster in four units that have persisted for at least 50 million years. We show that SPL gene grouping and retention following WGD is widespread in angiosperms, suggesting the conservatism and importance of this gene arrangement. We used Cas9 editing to generate transformation lines for all 19 SPL genes in a common set of backgrounds, and found that knockouts of 14 SPL genes exhibited defects in plant height, 10 exhibited defects in panicle size, and nine had altered grain lengths. We observed subfunctionalization of genes in the paleoduplicated pairs, but little evidence of neofunctionalization. Expression of OsSPL3 was negatively correlated with that of its closest neighbor in its synteny group, OsSPL4, and its sister paired gene, OsSPL12, in the opposing group. Nucleotide diversity was lower in eight of the nine singleton genes in domesticated rice, relative to wild rice, whereas the reverse was true for the paired genes. Together, these results provide functional information on eight previously unexamined OsSPL family members and suggest that paleoduplicate pair redundancy benefits plant survival and innovation., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

The "Green Revolution" that dramatically reduced cultivar heights and sharply boosted rice production mid-century was achieved in large part through introgression of defective alleles of Semi-Dwarf 1 (SD1), which encodes a GA20ox oxidase involved in the final steps of the synthesis of bioactive gibberellin in rice. Here, we ask whether converting the defective sd1 version in a modern semi-dwarf cultivar back to wild-type SD1 in situ recovers ancestral plant traits, and more broadly, what it reveals about pleiotropic effects of this gene. We assess these effects of SD1 restoration in three independent recombinant lines recovered from F 2 progeny of a cross between 93-11 and PA64s. We then used RNA-seq to dissect gene network changes that accompanied SD1 restoration. We report that this in situ restoration of wild-type SD1 nearly doubles plant height, increases total grain yield per panicle, and elongates the second-leaf length. Comparison of expression profiles reveals changes in key nodes of the gibberellin pathway, such as OsKO1 and OsGA2ox3, and more broadly in genes related to metabolic networks, defense response, and catabolic processes. Two JA-induced genes, RIR1b and OsPR1b, are extremely down-regulated after SD1 restoration, suggesting that SD1 restoration alters the balance between GA and JA to plant growth, at the cost of degrading the defense response. This in situ approach at the SD1 locus also provides a model example that is applicable to other systems and will further understanding of gene networks underlying high-yield traits in crops., Competing Interests: Declaration of Competing Interest All the authors have no conflicts of interest to declare., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Gibberellin (GA) functions as an essential natural regulator of growth and development in plants. For each step of the GA metabolic pathway, different copy numbers can be found in different species, as is the case with the 13 genes across four enzymatic steps in rice ( Oryza sativa ). A common view is that such gene duplication creates homologs that buffer organisms against loss-of-function (LOF) mutations. Therefore, knockouts of any single homolog might be expected to have little effect. To test this question, we generated clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) knockouts for these homologs and measured effects on growth and reproduction. Surprisingly, we report here that there is consistently one or more essential gene at each enzymatic step, for which LOF mutation induces death or sterility-suggesting that the GA pathway does not have a redundancy route and that each gene family is essential for GA metabolism. In most of these genes from the same gene family, we observed defects in plant height and infertility, suggesting that the duplicated members retain functions related to GA synthesis or degradation. We identified both subfunctionalization of the three recently diversified homologs OsKO1 , OsKO2 , and OsKO5 and neofunctionalization in OsKO3 and OsKO4 Thus, although the function of each step is conserved, the evolution of duplicates in that step is diversified. Interestingly, the CRISPR/Cas9 lines at the SD1 locus were typically sterile, whereas the natural sd1 mutants, related to the "Green Revolution" in rice, show normal setting rates. Collectively, our results identify candidates for control of GA production and provide insight into the evolution of four critical gene families in plants., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Key Message: We have isolated several Osiaa23 rice mutants with different knockout genotypes, resulting in different phenotypes, which suggested that different genetic backgrounds or mutation types influence gene function. The Auxin/Indole-3-Acetic Acid (Aux/IAA) gene family performs critical roles in auxin signal transduction in plants. In rice, the gene OsIAA23 (Os06t0597000) is known to affect development of roots and shoots, but previous knockouts in OsIAA23 have been sterile and difficult for research continuously. Here, we isolate new Osiaa23 mutants using the CRISPR/Cas9 system in japonica (Wuyunjing24) and indica (Kasalath) rice, with extensive genome re-sequencing to confirm the absence of off-target effects. In Kasalath, mutants with a 13-amino acid deletion showed profoundly greater dwarfing, lateral root developmental disorder, and fertility deficiency, relative to mutants with a single amino acid deletion, demonstrating that those 13 amino acids in Kasalath are essential to gene function. In Wuyunjing24, we predicted that mutants with a single base-pair frameshift insertion would experience premature termination and strong phenotypic defects, but instead these lines exhibited negligible phenotypic difference and normal fertility. Through RNA-seq, we show here that new mosaic transcripts of OsIAA23 were produced de novo, which circumvented the premature termination and thereby preserved the wild-type phenotype. This finding is a notable demonstration in plants that mutants can mask loss of function CRISPR/Cas9 editing of the target gene through de novo changes in alternative splicing.

Recombination during meiosis plays an important role in genome evolution by reshuffling existing genetic variations into fresh combinations with the possibility of recovery of lost ancestral genotypes. While crossover (CO) events have been well studied, gene conversion events (GCs), which represent non-reciprocal information transfer between chromosomes, are poorly documented and difficult to detect due to their relatively small converted tract size. Here, we document these GC events and their phenotypic effects at an important locus in rice containing the SD1 gene, where multiple defective alleles contributed to the semi-dwarf phenotype of rice in the 'Green Revolution' of the 1960s. Here, physical separation of two defects allows recombination to generate the wild-type SD1 gene, for which plant height can then be used as a reporter. By screening 18 000 F 2 progeny from a cross between two semi-dwarf cultivars that carry these different defective alleles, we detected 24 GC events, indicating a conversion rate of ~3.3 × 10 -4 per marker per generation in a single meiotic cycle in rice. Furthermore, our data show that indels and single-nucleotide polymorphisms (SNPs) do not differ significantly in GC rates, at least at the SD1 locus. Our results provide strong evidence that GC by itself can regain an ancestral phenotype that was lost through mutation. This GC detection approach is likely to be broadly applicable to natural or artificial alleles of other phenotype-related functional genes, which are abundant in other plant genomes., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Herbivory-induced responses in plants can both negatively affect subsequently colonizing herbivores and mitigate the effect of herbivory on the host. However, it is still less known whether plants exhibit specific responses to specialist and generalist herbivores in non-secondary metabolite traits and how specificity to specialists and generalists differs between invasive and native plant populations. We exposed an invasive plant, Alternanthera philoxeroides , to Agasicles hygrophila (Coleoptera, Chrysomelidae; specialist), Spodoptera litura (Lepidoptera, Noctuidae; generalist), manual clipping, or application of exogenous jasmonic acid and examined both the specificity of elicitation in traits of fitness (e.g., aboveground biomass), morphology (e.g., root:shoot ratio), and chemistry (e.g., C/N ratio and lignin), and specificity of effect on the subsequent performance of A. hygrophila and S. litura . Then, we assessed variation of the specificity between invasive and native populations (USA and Argentina, respectively). The results showed S. litura induced higher branching intensity and specific leaf area but lower C/N ratio than A. hygrophila , whereas A. hygrophila induced higher trichome density than S. litura . The negative effect of induction on subsequent larval growth was greater for S. litura than for A. hygrophila . Invasive populations had a weaker response to S. litura than to A. hygrophila in triterpenoid saponins and C/N ratio, while native populations responded similarly to these two herbivores. The specific effect on the two herbivores feeding on induced plants did not vary between invasive and native populations. Overall, we demonstrate specificity of elicitation to specialist and generalist herbivores in non-secondary metabolite traits, and that the generalist is more susceptible to induction than the specialist. Furthermore, chemical responses specific to specialist and generalist herbivores only exist in the invasive populations, consistent with an evolutionary change in specificity in the invasive populations.

The phyllosphere supports a tremendous diversity of microbes and other organisms. However, little is known about the colonization and survival of pathogenic and beneficial bacteria alone or together in the phyllosphere across the whole plant life-cycle under herbivory, which hinders our ability to understand the role of phyllosphere bacteria on plant performance. We addressed these questions in experiments using four genetically and biogeographically diverse accessions of Arabidopsis thaliana , three ecologically important bacterial strains ( Pseudomonas syringae DC3000, Xanthomonas campestris , both pathogens, and Bacillus cereus , plant beneficial) under common garden conditions that included fungus gnats ( Bradysia spp.). Plants supported greater abundance of B. cereus over either pathogenic strain in the phyllosphere under such greenhouse conditions. However, the Arabidopsis accessions performed much better (i.e., early flowering, biomass, siliques, and seeds per plant) in the presence of pathogenic bacteria rather than in the presence of the plant beneficial B. cereus . As a group, the plants inoculated with any of the three bacteria ( Pst DC3000, Xanthomonas , or Bacillus ) all had a higher fitness than uninoculated controls under these conditions. These results suggest that the plants grown under the pressure of different natural enemies, such as pathogens and an herbivore together perform relatively better, probably because natural enemies induce host defense against each other. However, in general, a positive impact of Bacillus on plant performance under herbivory may be due to its plant-beneficial properties. In contrast, bacterial species in the mixture (all three together) performed poorer than as monocultures in their total abundance and host plant growth promotion, possibly due to negative interspecific interactions among the bacteria. However, bacterial species richness linearly promoted seed production in the host plants under these conditions, suggesting that natural enemies diversity may be beneficial from the host perspective. Collectively, these results highlight the importance of bacterial community composition on plant performance and bacterial abundance in the phyllosphere.

The phyllosphere (comprising the leaf surface and interior) is one of the world's largest microbial habitats and is host to an abundant and diverse array of bacteria. Nonetheless, the degree to which bacterial communities are benign, harmful, or beneficial to plants in situ is unknown. We tested the hypothesis that the net effect of reducing bacterial abundance and diversity would vary substantially among host species (from harmful to beneficial) and this would be strongly mediated by soil resource availability. To test this, we monitored tree seedling growth responses to commercial antibiotics among replicated resource supply treatments (N, P, K) in a tropical forest in Panama for 29 months. We applied either antibiotics or control water to replicated seedlings of five common tree species (Alseis blackiana, Desmopsis panamensis, Heisteria concinna, Sorocea affinis, and Tetragastris panamensis). These antibiotic treatments significantly reduced both the abundance and diversity of bacteria epiphytically as well as endophytically. Overall, the effect of antibiotics on performance was highly host specific. Applying antibiotics increased growth for three species by as much as 49% (Alseis, Heisteria, and Tetragastris), decreased growth for a fourth species by nearly 20% (Sorocea), and had no impact on a fifth species (Desmopsis). Perhaps more importantly, the degree to which foliar bacteria were harmful or not varied with soil resource supply. Specifically, applying antibiotics had no effect when potassium was added but increased growth rate by almost 40% in the absence of potassium. Alternatively, phosphorus enrichment caused the effect of bacteria to switch from being primarily beneficial to harmful or vice versa, but this depended entirely on the presence or absence of nitrogen enrichment (i.e., important and significant interactions). Our results are the first to demonstrate that the net effect of reducing the abundance and diversity of bacteria can have very strong positive and negative effects on seedling performance. Moreover, these effects were clearly mediated by soil resource availability. Though speculative, we suggest that foliar bacteria may interact with soil fertility to comprise an important, yet cryptic dimension of niche differentiation, which can have important implications for species coexistence., (© 2016 by the Ecological Society of America.)

Treatment of plants with exogenous salicylic acid (SA) improves resistance to many bacterial pathogens, but can suppress resistance to insect herbivores. While plants vary naturally in constitutive SA, whether such differences are predictive of resistance to insect herbivores has not been studied previously. We examined the possible role of this endogenous SA in structuring the interactions between the cabbage white butterfly, Pieris rapae, and ten hosts in the mustard family (Brassicaceae). Because P. rapae has multiple generations that utilize different hosts across the year, we included five spring-flowering mustards and five summer-flowering mustards that co-occur in ruderal habitats in upstate New York. Under common garden conditions, the spring flowering mustards (Capsella bursa-pastoris, Draba verna, Cardamine impatiens, Barbarea vulgaris, and Arabidopsis thaliana) were significantly more resistant to P. rapae, supporting 42 % less herbivory (P = 0.015) and 64 % lower relative growth rates (P = 0.007), relative to the summer flowering mustards (Sisymbrium altissimum, Brassica nigra, Sinapis arvense, Lepidium campestre, and Arabis canadensis). Leaf total constitutive SA explained significant variation in larval herbivory (R (2)  = 75.3 %, P = 0.007) and relative growth rates (R (2)  = 59.4 %, P = 0.043). The three species with the lowest levels of constitutive SA (Capsella bursa-pastoris, Draba verna, and Cardamine impatiens) were the most resistant to larvae. Barbarea vulgaris and Arabis canadensis were notable exceptions, exhibiting high SA concentrations and intermediate resistance to P. rapae. These results suggest a curvilinear relationship between leaf constitutive SA and the herbivory by P. rapae, and they provide some insight into the ecology and possible management of this economically important crop pest.

Salicylic acid (SA) occupies a key role as a hormone central to both plant resistance to bacterial pathogens and tolerance of abiotic stresses. Plants at high elevation experience colder temperatures and elevated UV levels. While it has been predicted that SA concentrations will be higher in plants from high elevation populations, few studies have addressed this question. Here, we asked how concentrations of SA vary in natural populations of Arabidopsis thaliana collected across an elevational gradient on the Iberian Peninsula. In a series of common garden experiments, we found that constitutive SA concentrations were highest in genotypes from the low elevation populations. This result was in the opposite direction from our prediction and is an exception to the general finding that phenolic compounds increase with increasing elevation. These data suggest that high constitutive SA is not associated with resistance to cold temperatures in these plants. Furthermore, we also found that leaf constitutive camalexin concentrations, an important defense against some bacterial and fungal enemies, were highest in the low elevation populations, suggesting that pathogen pressures may be important. Further examination of this elevational cline will likely provide additional insights into the interplay between phenolic compounds and biotic and abiotic stress.

Plants have been shown previously to perceive bacteria on the leaf surface and respond by closing their stomata. The virulent bacterial pathogen Pseudomonas syringae pv tomato DC3000 (PstDC3000) responds by secreting a virulence factor, coronatine, which blocks the functioning of guard cells and forces stomata to reopen. After it is inside the leaf, PstDC3000 has been shown to up-regulate abscisic acid (ABA) signaling and thereby suppress salicylic acid-dependent resistance. Some wild plants exhibit resistance to PstDC3000, but the mechanisms by which they achieve this resistance remain unknown. Here, we used genome-wide association mapping to identify an ATP-dependent binding cassette transporter gene (ATP-dependent binding cassette transporter G family member16) in Arabidopsis (Arabidopsis thaliana) that contributes to wild plant resistance to PstDC3000. Through microarray analysis and β-glucuronidase reporter lines, we showed that the gene is up-regulated by ABA, bacterial infection, and coronatine. We also used a green fluorescent protein fusion protein and found that transporter is more likely to localize on plasma membranes than in cell walls. Transferred DNA insertion lines exhibited consistent defective tolerance of exogenous ABA and reduced resistance to infection by PstDC3000. Our conclusion is that ATP-dependent binding cassette transporter G family member16 is involved in ABA tolerance and contributes to plant resistance against PstDC3000. This is one of the first examples, to our knowledge, of ATP-dependent binding cassette transporter involvement in plant resistance to infection by a bacterial pathogen. It also suggests a possible mechanism by which plants reduce the deleterious effects of ABA hijacking during pathogen attack. Collectively, these results improve our understanding of basal resistance in Arabidopsis and offer unique ABA-related targets for improving the innate resistance of plants to bacterial infection., (© 2014 American Society of Plant Biologists. All Rights Reserved.)

Optimal defense theory predicts that induction of defensive secondary metabolites in plants will be inversely correlated with constitutive expression of those compounds. Here, we asked whether camalexin, an important defense against fungal and bacterial pathogens, support this prediction in structured natural populations of Arabidopsis thaliana from the Iberian Peninsula. In common garden experiments, we found that genotypes from the VIE population constitutively hyper-accumulated camalexin. Camalexin concentrations were not induced significantly when plants were exposed to a temperature of 10°C for 48h. However, they were induced when plants were exposed to 48h of infection by the virulent bacterial pathogen, Pseudomonas syringae pv. tomato DC3000. Genotypes from the VIE population with the hyper-accumulation of camalexin were significantly more resistant to bacterial growth. Induction of camalexin was negatively correlated with constitutive camalexin concentrations following log transformation and two different corrections for autocorrelation, thus supporting the tradeoff predicted by optimal defense theory. Constitutive overexpression of camalexin was not explained by the only known natural genetic polymorphism at the Accelerated Cell Death 6, ACD6, locus. Collectively, the results support an important role of camalexin in defense against P. syringae as well as significant structured variation in defense levels within wild populations., (Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Natural plant hybrid zones have been described as 'sinks' and 'centers of biodiversity' for herbivores and fungal pathogens. Jasmonic acid is known to be a critical signaling molecule for defense against these enemies. Does inhibition of jasmonic acid perhaps contribute to the susceptibility of hybrid plants to attack by herbivores? Here, we discuss recent evidence that plant immune system incompatibilities are likely to downregulate jasmonic acid (JA)-dependent responses through their effect on expression of the salicylic acid (SA)-dependent pathway. Because these hybrid immune incompatibilities are a function of environmental temperatures, they suggest a dependency between attack rates in hybrid zones and environmental conditions. Hybrid zones in colder environments, for example at higher elevations or latitudes, are more likely to exhibit elevated SA, suppressed jasmonic acid-dependent defenses, and lower resistance to herbivores and other necrotrophic pathogens., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Although pioneered by human geneticists as a potential solution to the challenging problem of finding the genetic basis of common human diseases, genome-wide association (GWA) studies have, owing to advances in genotyping and sequencing technology, become an obvious general approach for studying the genetics of natural variation and traits of agricultural importance. They are particularly useful when inbred lines are available, because once these lines have been genotyped they can be phenotyped multiple times, making it possible (as well as extremely cost effective) to study many different traits in many different environments, while replicating the phenotypic measurements to reduce environmental noise. Here we demonstrate the power of this approach by carrying out a GWA study of 107 phenotypes in Arabidopsis thaliana, a widely distributed, predominantly self-fertilizing model plant known to harbour considerable genetic variation for many adaptively important traits. Our results are dramatically different from those of human GWA studies, in that we identify many common alleles of major effect, but they are also, in many cases, harder to interpret because confounding by complex genetics and population structure make it difficult to distinguish true associations from false. However, a-priori candidates are significantly over-represented among these associations as well, making many of them excellent candidates for follow-up experiments. Our study demonstrates the feasibility of GWA studies in A. thaliana and suggests that the approach will be appropriate for many other organisms.

Plants can defend themselves against a wide array of enemies, from microbes to large animals, yet there is great variability in the effectiveness of such defences, both within and between species. Some of this variation can be explained by conflicting pressures from pathogens with different modes of attack. A second explanation comes from an evolutionary 'tug of war', in which pathogens adapt to evade detection, until the plant has evolved new recognition capabilities for pathogen invasion. If selection is, however, sufficiently strong, susceptible hosts should remain rare. That this is not the case is best explained by costs incurred from constitutive defences in a pest-free environment. Using a combination of forward genetics and genome-wide association analyses, we demonstrate that allelic diversity at a single locus, ACCELERATED CELL DEATH 6 (ACD6), underpins marked pleiotropic differences in both vegetative growth and resistance to microbial infection and herbivory among natural Arabidopsis thaliana strains. A hyperactive ACD6 allele, compared to the reference allele, strongly enhances resistance to a broad range of pathogens from different phyla, but at the same time slows the production of new leaves and greatly reduces the biomass of mature leaves. This allele segregates at intermediate frequency both throughout the worldwide range of A. thaliana and within local populations, consistent with this allele providing substantial fitness benefits despite its marked impact on growth.

The ascorbic acid (AA)-deficient Arabidopsis thaliana vtc1-1 mutant exhibits increased resistance to the virulent bacterial pathogen Pseudomonas syringae. This response correlates with heightened levels of salicylic acid (SA), which induces antimicrobial pathogenesis-related (PR) proteins. To determine if SA-mediated, enhanced disease resistance is a general phenomenon of AA deficiency, to elucidate the signal that stimulates SA synthesis, and to identify the biosynthetic pathway through which SA accumulates, we studied the four AA-deficient vtc1-1, vtc2-1, vtc3-1, and vtc4-1 mutants. We also studied double mutants defective in the AA-biosynthetic gene VTC1 and the SA signaling pathway genes PAD4, EDS5, and NPR1, respectively. All vtc mutants were more resistant to P. syringae than the wild type. With the exception of vtc4-1, this correlated with constitutively upregulated H(2)O(2), SA, and messenger RNA levels of PR genes. Double mutants exhibited decreased SA levels and enhanced susceptibility to P. syringae compared with the wild type, suggesting that vtc1-1 requires functional PAD4, EDS5, and NPR1 for SA biosynthesis and pathogen resistance. We suggest that AA deficiency causes constitutive priming through a buildup of H(2)O(2) that stimulates SA accumulation, conferring enhanced disease resistance in vtc1-1, vtc2-1, and vtc3-1, whereas vtc4-1 might be sensitized to H(2)O(2) and SA production after infection.

Plants use signaling pathways involving salicylic acid, jasmonic acid, and ethylene to defend against pathogen and herbivore attack. Many defense response genes involved in these signaling pathways have been characterized, but little is known about the selective pressures they experience. A representative set of 27 defense response genes were resequenced in a worldwide set of 96 Arabidopsis thaliana accessions, and patterns of single nucleotide polymorphisms (SNPs) were evaluated in relation to an empirical distribution of SNPs generated from either 876 fragments or 236 fragments with >400 bp coding sequence (this latter set was selected for comparisons with coding sequences) distributed across the genomes of the same set of accessions. Defense response genes have significantly fewer protein variants, display lower levels of nonsynonymous nucleotide diversity, and have fewer nonsynonymous segregating sites. The majority of defense response genes appear to be experiencing purifying selection, given the dearth of protein variation in this set of genes. Eight genes exhibit some evidence of partial selective sweeps or transient balancing selection. These results therefore provide a strong contrast to the high levels of balancing selection exhibited by genes at the upstream positions in these signaling pathways.

Glucosinolates, trichomes, nitrogen, and carbon are not distributed uniformly through the canopies of mustards. In this study, we asked whether glucosinolate concentrations and trichome densities in two sympatric mustards, Brassica kaber and B. nigra, are highest in tissues of greatest value to the plant. We also asked whether nitrogen or carbon content is the stronger predictor of tissue value, and what fraction of each resource is incorporated in glucosinolates. To quantify tissue values, we removed three equal-area fractions (lower, middle, and upper) from the canopies of B. kaber and B. nigra in the greenhouse, as well as whole canopies of naturally growing B. nigra in the field, at two times during growth and measured reductions in their performance relative to controls. We also measured trichome density in both experiments, as well as glucosinolate, nitrogen, and carbon concentrations for the equal-area fractions in the greenhouse. We found that upper leaves had the highest glucosinolate concentrations, trichome densities, and tissue values. Furthermore, young plants in the field had higher trichome densities and tissue values than did older plants. Collectively, these data provide strong support for optimal defense theory and are among the first such evidence for glucosinolates and for physical defenses. The positive relationship between trichome density and tissue value was strong even after we accounted for the effects of leaf expansion. While nitrogen and carbon have both received attention as currencies for trade-offs, our data suggest that nitrogen concentration is a significantly better predictor of tissue value in these two mustard species. Interestingly, <1% of the nitrogen or carbon in leaves was incorporated in glucosinolates, which may explain why glucosinolates lack a consistent response to nitrogen fertilization.

Terrestrial plants serve as large and diverse habitats for a wide range of pathogenic and nonpathogenic microbes, yet these communities are not well described and little is known about the effects of plant defense on microbial communities in nature. We designed a field experiment to determine how variation in two plant defense signaling pathways affects the size, diversity, and composition of the natural endophytic and epiphytic bacterial communities of Arabidopsis thaliana. To do this, we provide an initial characterization of these bacterial communities in one population in southwestern Michigan, United States, and we compare these two communities among A. thaliana mutants deficient in salicylic acid (SA) and jasmonic acid (JA) signaling defense pathways, controls, and plants with artificially elevated levels of defense. We identified 30 distinct bacterial groups on A. thaliana that differ in colony morphology and 16S rRNA sequence. We show that induction of SA-mediated defenses reduced endophytic bacterial community diversity, whereas plants deficient in JA-mediated defenses experienced greater epiphytic bacterial diversity. Furthermore, there was a positive relationship between total community size and diversity, indicating that relatively susceptible plants should, in general, harbor higher bacterial diversity. This experiment provides novel information about the ecology of bacteria on A. thaliana and demonstrates that variation in two specific plant-signaling defense pathways can influence bacterial diversity on plants.

Given the substantial costs of plant defenses against pathogens, there should be corresponding benefits that prevent resistance from being lost in natural plant populations. Here, we present evidence that systemic acquired resistance (SAR) benefits plants attacked by pathogenic bacteria in nature. In a large field experiment, we found that Arabidopsis thaliana treated with salicylic acid exhibited reduced titers of bacteria in their leaves and elevated fitness relative to controls. Most common members of the culturable bacterial community suffered this decrease, consistent with the role of SAR as a broad spectrum defense. We found no evidence of negative interactions between SAR and jasmonate-dependent resistance. Plants treated with jasmonic acid received significantly lower insect damage to their siliques, but exhibited no differences in bacterial growth or fitness relative to controls. Collectively, these data suggest a likely role of pathogenic bacteria in the maintenance of SAR, but not jasmonate-dependent resistance, in nature.

The role of salicylic acid (SA) in plant responses to pathogens has been well documented, but its direct and indirect effects on plant responses to insects are not so well understood. We examined the effects of SA, alone and in combination with jasmonic acid (JA), on the performance of the generalist herbivore, Spodoptera exigua, in wild-type and mutant Arabidopsis thaliana genotypes that varied genetically in their ability to mount SA- and JA-mediated defence responses. In one experiment, growth of S. exigua larvae was highest on the Wassilewskija wild-type, intermediate on the Columbia wild-type and the JA-deficient fad mutant, and lowest on the nim1-1 and jar1-mutants, which are defective in the SA and JA pathways, respectively. Activity of guaiacol peroxidase, polyphenoloxidase, n-acetylglucosaminidase, and trypsin inhibitor varied by genotype but did not correlate with insect performance. SA treatment increased growth of S. exigua larvae by approximately 35% over all genotypes, but had no discernable effect on activities of the four defence proteins. In a second experiment, growth of S. exigua was highest across treatments on the cep1 mutant, a constitutive expressor of high SA levels and systemic acquired resistance, and lowest on the fad mutant, which is JA-deficient. JA treatment generally increased activity of all four defence proteins, increased total glucosinolate levels and reduced insect growth by approximately 25% over all genotypes. SA generally inhibited expression of JA-induced resistance to S. exigua when both hormones were applied simultaneously. Across genotypes and treatments, larval mass was negatively correlated with the activity of trypsin inhibitor and polyphenoloxidase and with total glucosinolate levels, and insect damage was negatively correlated with the activity of polyphenoloxidase. SA had little effect on the induction of defence protein activity by JA. However, SA attenuated the induction of glucosinolates by JA and therefore may explain better the interactive effects of SA and JA on insect performance. This study illustrates that direct and indirect cross-effects of SA on resistance to S. exigua can occur in A. thaliana. Effects of SA may be mediated through effects on plant defence chemistry or other aspects of the suitability of foliage for insect feeding and growth.

Leaf trichomes protect plants from attack by insect herbivores and are often induced following damage. Hormonal regulation of this plant induction response has not been previously studied. In a series of experiments, we addressed the effects of artificial damage, jasmonic acid, salicylic acid, and gibberellin on induction of trichomes in Arabidopsis. Artificial damage and jasmonic acid caused significant increases in trichome production of leaves. The jar1-1 mutant exhibited normal trichome induction following treatment with jasmonic acid, suggesting that adenylation of jasmonic acid is not necessary. Salicylic acid had a negative effect on trichome production and consistently reduced the effect of jasmonic acid, suggesting negative cross-talk between the jasmonate and salicylate-dependent defense pathways. Interestingly, the effect of salicylic acid persisted in the nim1-1 mutant, suggesting that the Npr1/Nim1 gene is not downstream of salicylic acid in the negative regulation of trichome production. Last, we found that gibberellin and jasmonic acid had a synergistic effect on the induction of trichomes, suggesting important interactions between these two compounds.

Resistance genes (R-genes) act as an immune system in plants by recognizing pathogens and inducing defensive pathways. Many R-gene loci are present in plant genomes, presumably reflecting the need to maintain a large repertoire of resistance alleles. These loci also often segregate for resistance and susceptibility alleles that natural selection has maintained as polymorphisms within a species for millions of years. Given the obvious advantage to an individual of being disease resistant, what prevents these resistance alleles from being driven to fixation by natural selection? A cost of resistance is one potential explanation; most models require a lower fitness of resistant individuals in the absence of pathogens for long-term persistence of susceptibility alleles. Here we test for the presence of a cost of resistance at the RPM1 locus of Arabidopsis thaliana. Results of a field experiment comparing the fitness of isogenic strains that differ in the presence or absence of RPM1 and its natural promoter reveal a large cost of RPM1, providing the first evidence that costs contribute to the maintenance of an ancient R-gene polymorphism.

Plants often respond to attack by insect herbivores and necrotrophic pathogens with induction of jasmonate-dependent resistance traits, but respond to attack by biotrophic pathogens with induction of salicylate-dependent resistance traits. To assess the degree to which the jasmonate- and salicylate-dependent pathways interact, we compared pathogenesis-related protein activity and bacterial performance in four mutant Arabidopsis thaliana lines relative to their wild-type backgrounds. We found that two salicylate-dependent pathway mutants (cep1, nim1-1) exhibited strong effects on the growth of the generalist biotrophic pathogen, Pseudomonas syringae pv. tomato, whereas two jasmonate-dependent pathway mutants (fad3-2fad7-2fad8, jar1-1) did not. Leaf peroxidase and exochitinase activity were negatively correlated with bacterial growth, whereas leaf polyphenol oxidase activity and trypsin inhibitor concentration were not. Interestingly, leaf total glucosinolate concentration was positively correlated with bacterial growth. In the same experiment, we also found that application of jasmonic acid generally increased leaf peroxidase activity and trypsin inhibitor concentration in the mutant lines. However, the cep1 mutant, shown previously to overexpress salicylic acid, exhibited no detectable biological or chemical responses to jasmonic acid, suggesting that high levels of salicylic acid may have inhibited a plant response. In a second experiment, we compared the effect of jasmonic acid and/or salicylic acid on two ecotypes of A. thaliana. Application of salicylic acid to the Wassilewskija ecotype decreased bacterial growth. However, this effect was not observed when both salicylic acid and jasmonic acid were applied, suggesting that jasmonic acid negated the beneficial effect of salicylic acid. Collectively, our results confirm that the salicylate-dependent pathway is more important than the jasmonate-dependent pathway in determining growth of P. syringae pv. tomato in A. thaliana, and suggest important negative interactions between these two major defensive pathways in the Wassilewskija ecotype. In contrast, the Columbia ecotype exhibited little evidence of negative interactions between the two pathways, suggesting intraspecific variability in how these pathways interact in A. thaliana.

Black mustard, Brassica nigra, is highly variable in both constitutive resistance and induction response following damage by herbivores. A focal population from Ithaca, New York, was used to test the following two predictions of optimal defense theory: (1) that allocation to resistance will reduce plant performance in the absence of herbivores; and (2) that induction response will be negatively correlated with constitutive resistance. The experiment consisted of a half-sib mating design with 47 paternal families and four dams per sire, fully crossed with a damage treatment consisting of 25% leaf removal by the cabbage white butterfly, Pieris rapae, when plants had four leaves. Leaf trichome density, sinigrin concentration, and glucobrassicin concentration were 38%, 19%, and 16% higher, respectively, for the seventh leaf of damaged plants. Paternal families did not vary significantly in their induction response. Narrow-sense heritabilities were h(S)(2) = 0.51, 0.76, and 0.50 for constitutive leaf trichome density, sinigrin concentration, and glucobrassicin concentration, respectively. Positive genetic correlations were found between glucobrassicin concentration and days to first flower, suggesting a genetic cost of resistance. Induction responses were negatively correlated with constitutive allocation for leaf trichome density and sinigrin concentration. The results were therefore consistent with optimal defense theory, offering modest evidence for both predictions.

Plant species differ broadly in their responses to an elevated CO 2 atmosphere, particularly in the extent of nitrogen dilution of leaf tissue. Insect herbivores are often limited by the availability of nutrients, such as nitrogen, in their host plant tissue and may therefore respond differentially on different plant species grown in CO 2 -enriched environments. We reared gyspy moth larvae (Lymantria dispar) in situ on seedlings of yellow birch (Betula allegheniensis) and gray birch (B. populifolia) grown in an ambient (350 ppm) or elevated (700 ppm) CO 2 atmosphere to test whether larval responses in the elevated CO 2 atmosphere were species-dependent. We report that female gypsy moths (Lymantria dispar) reared on gray birch (Betula populifolia) achieved similar pupal masses on plants grown at an ambient or an elevated CO 2 concentration. However, on yellow birch (B. allegheniensis), female pupal mass was 38% smaller on plants in the elevated-CO 2 atmosphere. Larval mortality was significantly higher on yellow birch than gray birch, but did not differ between the CO 2 treatments. Relative growth rate declined more in the elevated CO 2 atmosphere for larvae on yellow birch than for those on gray birch. In preference tests, larvae preferred ambient over elevated CO 2 -grown leaves of yellow birch, but showed no preference between gray birch leaves from the two CO 2 atmospheres. This differential response of gypsy moths to their host species corresponded to a greater decline in leaf nutritional quality in the elevated CO 2 atmosphere in yellow birch than in gray birch. Leaf nitrogen content of yellow birch dropped from 2.68% to 1.99% while that of gray birch leaves only declined from 3.23% to 2.63%. Meanwhile, leaf condensed tannin concentration increased from 8.92% to 11.45% in yellow birch leaves while gray birch leaves only increased from 10.72% to 12.34%. Thus the declines in larval performance in a future atmosphere may be substantial and host-species-specific.

Elevated CO 2 may increase dry mass production of canopies directly through increasing net assimilation rate of leaves and also indirectly through increasing leaf area index (LAI). We studied the effects of CO 2 elevation on canopy productivity and development in monospecific and mixed (1:1) stands of two co-occurring C 3 annual species, Abutilon theophrasti, and Ambrosia artemisiifolia. The stands were established in the glasshouse with two CO 2 levels (360 and 700 μl/l) under natural light conditions. The planting density was 100 per m 2 and LAI increased up to 2.6 in 53 days of growth. Root competition was excluded by growing each plant in an individual pot. However, interference was apparent in the amount of photons absorbed by the plants and in photon absorption per unit leaf area. Greater photon absorption by Abutilon in the mixed stand was due to different canopy structures: Abutilon distributed leaves in the upper layers in the canopy while Ambrosia distributed leaves more to the lower layers. CO 2 elevation did not affect the relative performance and light interception of the two species in mixed stands. Total aboveground dry mass was significantly increased with CO 2 elevation, while no significant effects on leaf area development were observed. CO 2 elevation increased dry mass production by 30-50%, which was mediated by 35-38% increase in the net assimilation rate (NAR) and 37-60% increase in the nitrogen use efficiency (NUE, net assimilation rate per unit leaf nitrogen). Since there was a strong overall correlation between LAI and aboveground nitrogen and no significant difference was found in the regression of LAI against aboveground nitrogen between the two CO 2 levels, we hypothesized that leaf area development was controlled by the amount of nitrogen taken up from the soil. This hypothesis suggests that the increased LAI with CO 2 elevation observed by several authors might be due to increased uptake of nitrogen with increased root growth.