Biosynthesis and antifungal activity of fungus-induced O-methylated flavonoids in maize.

Fungal infection of grasses, including rice (Oryza sativa), sorghum (Sorghum bicolor), and barley (Hordeum vulgare), induces the formation and accumulation of flavonoid phytoalexins. In maize (Zea mays), however, investigators have emphasized benzoxazinoid and terpenoid phytoalexins, and comparatively little is known about flavonoid induction in response to pathogens. Here, we examined fungus-elicited flavonoid metabolism in maize and identified key biosynthetic enzymes involved in the formation of O-methylflavonoids. The predominant end products were identified as two tautomers of a 2-hydroxynaringenin-derived compound termed xilonenin, which significantly inhibited the growth of two maize pathogens, Fusarium graminearum and Fusarium verticillioides. Among the biosynthetic enzymes identified were two O-methyltransferases (OMTs), flavonoid OMT 2 (FOMT2), and FOMT4, which demonstrated distinct regiospecificity on a broad spectrum of flavonoid classes. In addition, a cytochrome P450 monooxygenase (CYP) in the CYP93G subfamily was found to serve as a flavanone 2-hydroxylase providing the substrate for FOMT2-catalyzed formation of xilonenin. In summary, maize produces a diverse blend of O-methylflavonoids with antifungal activity upon attack by a broad range of fungi.

The maize heterotrimeric G protein ß subunit controls shoot meristem development and immune responses.

Heterotrimeric G proteins are important transducers of receptor signaling, functioning in plants with CLAVATA receptors in controlling shoot meristem size and with pathogen-associated molecular pattern receptors in basal immunity. However, whether specific members of the heterotrimeric complex potentiate cross-talk between development and defense, and the extent to which these functions are conserved across species, have not yet been addressed. Here we used CRISPR/Cas9 to knock out the maize G protein ß subunit gene (Gß) and found that the mutants are lethal, differing from those in Arabidopsis, in which homologous mutants have normal growth and fertility. We show that lethality is caused not by a specific developmental arrest, but by autoimmunity. We used a genetic diversity screen to suppress the lethal Gß phenotype and also identified a maize Gß allele with weak autoimmune responses but strong development phenotypes. Using these tools, we show that Gß controls meristem size in maize, acting epistatically with G protein α subunit gene (Gα), suggesting that Gß and Gα function in a common signaling complex. Furthermore, we used an association study to show that natural variation in Gß influences maize kernel row number, an important agronomic trait. Our results demonstrate the dual role of Gß in immunity and development in a cereal crop and suggest that it functions in cross-talk between these competing signaling networks. Therefore, modification of Gß has the potential to optimize the trade-off between growth and defense signaling to improve agronomic production.

Comparative proteomics combined with analyses of transgenic plants reveal ZmREM1.3 mediates maize resistance to southern corn rust.

Southern corn rust (SCR), which is a destructive disease caused by Puccinia polysora Underw. (P. polysora), commonly occurs in warm-temperate and tropical regions. To identify candidate proteins related to SCR resistance and characterize the molecular mechanisms underlying the maize-P. polysora interaction, a comparative proteomic analysis of susceptible and resistant maize lines was performed. Statistical analyses revealed 1489 differentially abundant proteins in the resistant line, as well as 1035 differentially abundant proteins in the susceptible line. After the P. polysora infection, the abundance of one remorin protein (ZmREM1.3) increased in the resistant genotype, but decreased in the susceptible genotype. Plant-specific remorins are important for responses to microbial infections as well as plant signalling processes. In this study, transgenic maize plants overexpressing ZmREM1.3 exhibited enhanced resistance to the biotrophic P. polysora. In contrast, homozygous ZmREM1.3 UniformMu mutant plants were significantly more susceptible to P. polysora than wild-type plants. Additionally, the ZmREM1.3-overexpressing plants accumulated more salicylic acid (SA) and jasmonic acid (JA). Moreover, the expression levels of defence-related genes were higher in ZmREM1.3-overexpressing maize plants than in non-transgenic control plants in response to the P. polysora infection. Overall, our results provide evidence that ZmREM1.3 positively regulates maize defences against P. polysora likely via SA/JA-mediated defence signalling pathways. This study represents the first large-scale proteomic analysis of the molecular mechanisms underlying the maize-P. polysora interaction. This is also the first report confirming the remorin protein family affects plant resistance to SCR.

Ethylene signaling regulates natural variation in the abundance of antifungal acetylated diferuloylsucroses and Fusarium graminearum resistance in maize seedling roots.

The production and regulation of defensive specialized metabolites play a central role in pathogen resistance in maize (Zea mays) and other plants. Therefore, identification of genes involved in plant specialized metabolism can contribute to improved disease resistance. We used comparative metabolomics to identify previously unknown antifungal metabolites in maize seedling roots, and investigated the genetic and physiological mechanisms underlying their natural variation using quantitative trait locus mapping and comparative transcriptomics approaches. Two maize metabolites, smilaside A (3,6-diferuloyl-3',6'-diacetylsucrose) and smiglaside C (3,6-diferuloyl-2',3',6'-triacetylsucrose), were identified that could contribute to maize resistance against Fusarium graminearum and other fungal pathogens. Elevated expression of an ethylene signaling gene, ETHYLENE INSENSITIVE 2 (ZmEIN2), co-segregated with a decreased smilaside A : smiglaside C ratio. Pharmacological and genetic manipulation of ethylene availability and sensitivity in vivo indicated that, whereas ethylene was required for the production of both metabolites, the smilaside A : smiglaside C ratio was negatively regulated by ethylene sensitivity. This ratio, rather than the absolute abundance of these two metabolites, was important for maize seedling root defense against F. graminearum. Ethylene signaling regulates the relative abundance of the two F. graminearum-resistance-related metabolites and affects resistance against F. graminearum in maize seedling roots.

Discovery, Biosynthesis and Stress-Related Accumulation of Dolabradiene-Derived Defenses in Maize.

Terpenoids are a major component of maize (Zea mays) chemical defenses that mediate responses to herbivores, pathogens, and other environmental challenges. Here, we describe the biosynthesis and elicited production of a class of maize diterpenoids, named dolabralexins. Dolabralexin biosynthesis involves the sequential activity of two diterpene synthases, ENT-COPALYL DIPHOSPHATE SYNTHASE (ZmAN2) and KAURENE SYNTHASE-LIKE4 (ZmKSL4). Together, ZmAN2 and ZmKSL4 form the diterpene hydrocarbon dolabradiene. In addition, we biochemically characterized a cytochrome P450 monooxygenase, ZmCYP71Z16, which catalyzes the oxygenation of dolabradiene to yield the epoxides 15,16-epoxydolabrene (epoxydolabrene) and 3ß-hydroxy-15,16-epoxydolabrene (epoxydolabranol). The absence of dolabradiene and epoxydolabranol in Zman2 mutants under elicited conditions confirmed the in vivo biosynthetic requirement of ZmAN2. Combined mass spectrometry and NMR experiments demonstrated that much of the epoxydolabranol is further converted into 3ß,15,16-trihydroxydolabrene (trihydroxydolabrene). Metabolite profiling of field-grown maize root tissues indicated that dolabralexin biosynthesis is widespread across common maize cultivars, with trihydroxydolabrene as the predominant diterpenoid. Oxidative stress induced dolabralexin accumulation and transcript expression of ZmAN2 and ZmKSL4 in root tissues, and metabolite and transcript accumulation were up-regulated in response to elicitation with the fungal pathogens Fusarium verticillioides and Fusarium graminearum Consistently, epoxydolabranol significantly inhibited the growth of both pathogens in vitro at 10 µg mL-1, while trihydroxydolabrene-mediated inhibition was specific to Fverticillioides These findings suggest that dolabralexins have defense-related roles in maize stress interactions and expand the known chemical space of diterpenoid defenses as genetic targets for understanding and ultimately improving maize resilience.

Selinene Volatiles Are Essential Precursors for Maize Defense Promoting Fungal Pathogen Resistance.

To ensure food security, maize (Zea mays) is a model crop for understanding useful traits underlying stress resistance. In contrast to foliar biochemicals, root defenses limiting the spread of disease remain poorly described. To better understand belowground defenses in the field, we performed root metabolomic profiling and uncovered unexpectedly high levels of the sesquiterpene volatile ß-selinene and the corresponding nonvolatile antibiotic derivative ß-costic acid. The application of metabolite-based quantitative trait locus mapping using biparental populations, genome-wide association studies, and near-isogenic lines enabled the identification of terpene synthase21 (ZmTps21) on chromosome 9 as a ß-costic acid pathway candidate gene. Numerous closely examined ß-costic acid-deficient inbred lines were found to harbor Zmtps21 pseudogenes lacking conserved motifs required for farnesyl diphosphate cyclase activity. For biochemical validation, a full-length ZmTps21 was cloned, heterologously expressed in Escherichia coli, and demonstrated to cyclize farnesyl diphosphate, yielding ß-selinene as the dominant product. Consistent with microbial defense pathways, ZmTps21 transcripts strongly accumulate following fungal elicitation. Challenged field roots containing functional ZmTps21 alleles displayed ß-costic acid levels over 100 µg g-1 fresh weight, greatly exceeding in vitro concentrations required to inhibit the growth of five different fungal pathogens and rootworm larvae (Diabrotica balteata). In vivo disease resistance assays, using ZmTps21 and Zmtps21 near-isogenic lines, further support the endogenous antifungal role of selinene-derived metabolites. Involved in the biosynthesis of nonvolatile antibiotics, ZmTps21 exists as a useful gene for germplasm improvement programs targeting optimized biotic stress resistance.

Abscisic acid promotes proteasome-mediated degradation of the transcription coactivator NPR1 in Arabidopsis thaliana.

Proteasome-mediated turnover of the transcription coactivator NPR1 is pivotal for efficient activation of the broad-spectrum plant immune responses known as localized acquired resistance (LAR) and systemic acquired resistance (SAR) in adjacent and systemic tissues, respectively, and requires the CUL3-based E3 ligase and its adaptor proteins, NPR3 and NPR4, which are receptors for the signaling molecule salicylic acid (SA). It has been shown that SA prevents NPR1 turnover under non-inducing and LAR/SAR-inducing conditions, but how cellular NPR1 homeostasis is maintained remains unclear. Here, we show that the phytohormone abscisic acid (ABA) and SA antagonistically influence cellular NPR1 protein levels. ABA promotes NPR1 degradation via the CUL3(NPR) (3/) (NPR) (4) complex-mediated proteasome pathway, whereas SA may protect NPR1 from ABA-promoted degradation through phosphorylation. Furthermore, we demonstrate that the timing and strength of SA and ABA signaling are critical in modulating NPR1 accumulation and target gene expression. Perturbing ABA or SA signaling in adjacent tissues alters the temporal dynamic pattern of NPR1 accumulation and target gene transcription. Finally, we show that sequential SA and ABA treatment leads to dynamic changes in NPR1 protein levels and target gene expression. Our results revealed a tight correlation between sequential SA and ABA signaling and dynamic changes in NPR1 protein levels and NPR1-dependent transcription in plant immune responses.

Arabidopsis Elongator subunit 2 positively contributes to resistance to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola.

The evolutionarily conserved Elongator complex functions in diverse biological processes including salicylic acid-mediated immune response. However, how Elongator functions in jasmonic acid (JA)/ethylene (ET)-mediated defense is unknown. Here, we show that Elongator is required for full induction of the JA/ET defense pathway marker gene PLANT DEFENSIN1.2 (PDF1.2) and for resistance to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola. A loss-of-function mutation in the Arabidopsis Elongator subunit 2 (ELP2) alters B. cinerea-induced transcriptome reprogramming. Interestingly, in elp2, expression of WRKY33, OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59 (ORA59), and PDF1.2 is inhibited, whereas transcription of MYC2 and its target genes is enhanced. However, overexpression of WRKY33 or ORA59 and mutation of MYC2 fail to restore PDF1.2 expression and B. cinerea resistance in elp2, suggesting that ELP2 is required for induction of not only WRKY33 and ORA59 but also PDF1.2. Moreover, elp2 is as susceptible as coronatine-insensitive1 (coi1) and ethylene-insensitive2 (ein2) to B. cinerea, indicating that ELP2 is an important player in B. cinerea resistance. Further analysis of the lesion sizes on the double mutants elp2 coi1 and elp2 ein2 and the corresponding single mutants revealed that the function of ELP2 overlaps with COI1 and is additive to EIN2 for B. cinerea resistance. Finally, basal histone acetylation levels in the coding regions of WRKY33, ORA59, and PDF1.2 are reduced in elp2 and a functional ELP2-GFP fusion protein binds to the chromatin of these genes, suggesting that constitutive ELP2-mediated histone acetylation may be required for full activation of the WRKY33/ORA59/PDF1.2 transcriptional cascade.

Elongator Plays a Positive Role in Exogenous NAD-Induced Defense Responses in Arabidopsis.

Extracellular NAD is emerging as an important signal molecule in animal cells, but its role in plants has not been well-established. Although it has been shown that exogenous NAD(+) activates defense responses in Arabidopsis, components in the exogenous NAD(+)-activated defense pathway remain to be fully discovered. In a genetic screen for mutants insensitive to exogenous NAD(+) (ien), we isolated a mutant named ien2. Map-based cloning revealed that IEN2 encodes ELONGATA3 (ELO3)/AtELP3, a subunit of the Arabidopsis Elongator complex, which functions in multiple biological processes, including histone modification, DNA (de)methylation, and transfer RNA modification. Mutations in the ELO3/AtELP3 gene compromise exogenous NAD(+)-induced expression of pathogenesis-related (PR) genes and resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326, and transgenic expression of the coding region of ELO3/AtELP3 in elo3/Atelp3 restores NAD(+) responsiveness to the mutant plants, demonstrating that ELO3/AtELP3 is required for exogenous NAD(+)-induced defense responses. Furthermore, mutations in genes encoding the other five Arabidopsis Elongator subunits (ELO2/AtELP1, AtELP2, ELO1/AtELP4, AtELP5, and AtELP6) also compromise exogenous NAD(+)-induced PR gene expression and resistance to P. syringae pv. maculicola ES4326. These results indicate that the Elongator complex functions as a whole in exogenous NAD(+)-activated defense signaling in Arabidopsis.

Reproducible growth of Brachypodium in EcoFAB 2.0 reveals that nitrogen form and starvation modulate root exudation.

Understanding plant-microbe interactions requires examination of root exudation under nutrient stress using standardized and reproducible experimental systems. We grew Brachypodium distachyon hydroponically in fabricated ecosystem devices (EcoFAB 2.0) under three inorganic nitrogen forms (nitrate, ammonium, and ammonium nitrate), followed by nitrogen starvation. Analyses of exudates with liquid chromatography-tandem mass spectrometry, biomass, medium pH, and nitrogen uptake showed EcoFAB 2.0's low intratreatment data variability. Furthermore, the three inorganic nitrogen forms caused differential exudation, generalized by abundant amino acids-peptides and alkaloids. Comparatively, nitrogen deficiency decreased nitrogen-containing compounds but increased shikimates-phenylpropanoids. Subsequent bioassays with two shikimates-phenylpropanoids (shikimic and p-coumaric acids) on soil bacteria or Brachypodium seedlings revealed their distinct capacity to regulate both bacterial and plant growth. Our results suggest that (i) Brachypodium alters exudation in response to nitrogen status, which can affect rhizobacterial growth, and (ii) EcoFAB 2.0 is a valuable standardized plant research tool.

Stressing the importance of plant specialized metabolites: omics-based approaches for discovering specialized metabolism in plant stress responses.

Plants produce a diverse range of specialized metabolites that play pivotal roles in mediating environmental interactions and stress adaptation. These unique chemical compounds also hold significant agricultural, medicinal, and industrial values. Despite the expanding knowledge of their functions in plant stress interactions, understanding the intricate biosynthetic pathways of these natural products remains challenging due to gene and pathway redundancy, multifunctionality of proteins, and the activity of enzymes with broad substrate specificity. In the past decade, substantial progress in genomics, transcriptomics, metabolomics, and proteomics has made the exploration of plant specialized metabolism more feasible than ever before. Notably, recent advances in integrative multi-omics and computational approaches, along with other technologies, are accelerating the discovery of plant specialized metabolism. In this review, we present a summary of the recent progress in the discovery of plant stress-related specialized metabolites. Emphasis is placed on the application of advanced omics-based approaches and other techniques in studying plant stress-related specialized metabolism. Additionally, we discuss the high-throughput methods for gene functional characterization. These advances hold great promise for harnessing the potential of specialized metabolites to enhance plant stress resilience in the future.

Maize Terpene Synthase 8 (ZmTPS8) Contributes to a Complex Blend of Fungal-Elicited Antibiotics.

In maize (Zea mays), fungal-elicited immune responses include the accumulation of terpene synthase (TPS) and cytochrome P450 monooxygenases (CYP) enzymes resulting in complex antibiotic arrays of sesquiterpenoids and diterpenoids, including α/ß-selinene derivatives, zealexins, kauralexins and dolabralexins. To uncover additional antibiotic families, we conducted metabolic profiling of elicited stem tissues in mapping populations, which included B73 × M162W recombinant inbred lines and the Goodman diversity panel. Five candidate sesquiterpenoids associated with a chromosome 1 locus spanning the location of ZmTPS27 and ZmTPS8. Heterologous enzyme co-expression studies of ZmTPS27 in Nicotiana benthamiana resulted in geraniol production while ZmTPS8 yielded α-copaene, δ-cadinene and sesquiterpene alcohols consistent with epi-cubebol, cubebol, copan-3-ol and copaborneol matching the association mapping efforts. ZmTPS8 is an established multiproduct α-copaene synthase; however, ZmTPS8-derived sesquiterpene alcohols are rarely encountered in maize tissues. A genome wide association study further linked an unknown sesquiterpene acid to ZmTPS8 and combined ZmTPS8-ZmCYP71Z19 heterologous enzyme co-expression studies yielded the same product. To consider defensive roles for ZmTPS8, in vitro bioassays with cubebol demonstrated significant antifungal activity against both Fusarium graminearum and Aspergillus parasiticus. As a genetically variable biochemical trait, ZmTPS8 contributes to the cocktail of terpenoid antibiotics present following complex interactions between wounding and fungal elicitation.

Inheritance of long staple fiber quality traits of Gossypium barbadense in G. hirsutum background using CSILs.

Gossypium hirsutum is a high yield cotton species that exhibits only moderate performance in fiber qualities. A promising but challenging approach to improving its phenotypes is interspecific introgression, the transfer of valuable traits or genes from the germplasm of another species such as G. barbadense, an important cultivated extra long staple cotton species. One set of chromosome segment introgression lines (CSILs) was developed, where TM-1, the genetic standard in G. hirsutum, was used as the recipient parent and the long staple cotton G. barbadense Hai7124 was used as the donor parent by molecular marker-assisted selection (MAS) in BC(5)S(1­4) and BC(4)S(1­3) generations. After four rounds of MAS, the CSIL population was comprised of 174 lines containing 298 introgressed segments, of which 86 (49.4%) lines had single introgressed segments. The total introgressed segment length covered 2,948.7 cM with an average length of 16.7 cM and represented 83.3% of tetraploid cotton genome. The CSILs were highly varied in major fiber qualities. By integrated analysis of data collected in four environments, a total of 43 additive quantitative trait loci (QTL) and six epistatic QTL associated with fiber qualities were detected by QTL IciMapping 3.0 and multi-QTL joint analysis. Six stable QTL were detected in various environments. The CSILs developed and the analyses presented here will enhance the understanding of the genetics of fiber qualities in long staple G. barbadense and facilitate further molecular breeding to improve fiber quality in Upland cotton.

Getting back to the grass roots: harnessing specialized metabolites for improved crop stress resilience.

Roots remain an understudied site of complex and important biological interactions mediating plant productivity. In grain and bioenergy crops, grass root specialized metabolites (GRSM) are central to key interactions, yet our basic knowledge of the chemical language remains fragmentary. Continued improvements in plant genome assembly and metabolomics are enabling large-scale advances in the discovery of specialized metabolic pathways as a means of regulating root-biotic interactions. Metabolomics, transcript coexpression analyses, forward genetic studies, gene synthesis and heterologous expression assays drive efficient pathway discoveries. Functional genetic variants identified through genome wide analyses, targeted CRISPR/Cas9 approaches, and both native and non-native overexpression studies critically inform novel strategies for bioengineering metabolic pathways to improve plant traits.

Differential Quantitative Requirements for NPR1 Between Basal Immunity and Systemic Acquired Resistance in Arabidopsis thaliana.

Non-expressor of pathogenesis-related (PR) genes1 (NPR1) is a key transcription coactivator of plant basal immunity and systemic acquired resistance (SAR). Two mutant alleles, npr1-1 and npr1-3, have been extensively used for dissecting the role of NPR1 in various signaling pathways. However, it is unknown whether npr1-1 and npr1-3 are null mutants. Moreover, the NPR1 transcript levels are induced two- to threefold upon pathogen infection or salicylic acid (SA) treatment, but the biological relevance of the induction is unclear. Here, we used molecular and biochemical approaches including quantitative PCR, immunoblot analysis, site-directed mutagenesis, and CRISPR/Cas9-mediated gene editing to address these questions. We show that npr1-3 is a potential null mutant, whereas npr1-1 is not. We also demonstrated that a truncated npr1 protein longer than the hypothesized npr1-3 protein is not active in SA signaling. Furthermore, we revealed that TGACG-binding (TGA) factors are required for NPR1 induction, but the reverse TGA box in the 5'UTR of NPR1 is dispensable for the induction. Finally, we show that full induction of NPR1 is required for basal immunity, but not for SAR, whereas sufficient basal transcription is essential for full-scale establishment of SAR. Our results indicate that induced transcript accumulation may be differentially required for different functions of a specific gene. Moreover, as npr1-1 is not a null mutant, we recommend that future research should use npr1-3 and potential null T-DNA insertion mutants for dissecting NPR1's function in various physiopathological processes.

Genetic elucidation of interconnected antibiotic pathways mediating maize innate immunity.

Specialized metabolites constitute key layers of immunity that underlie disease resistance in crops; however, challenges in resolving pathways limit our understanding of the functions and applications of these metabolites. In maize (Zea mays), the inducible accumulation of acidic terpenoids is increasingly considered to be a defence mechanism that contributes to disease resistance. Here, to understand maize antibiotic biosynthesis, we integrated association mapping, pan-genome multi-omic correlations, enzyme structure-function studies and targeted mutagenesis. We define ten genes in three zealexin (Zx) gene clusters that encode four sesquiterpene synthases and six cytochrome P450 proteins that collectively drive the production of diverse antibiotic cocktails. Quadruple mutants in which the ability to produce zealexins (ZXs) is blocked exhibit a broad-spectrum loss of disease resistance. Genetic redundancies ensuring pathway resiliency to single null mutations are combined with enzyme substrate promiscuity, creating a biosynthetic hourglass pathway that uses diverse substrates and in vivo combinatorial chemistry to yield complex antibiotic blends. The elucidated genetic basis of biochemical phenotypes that underlie disease resistance demonstrates a predominant maize defence pathway and informs innovative strategies for transferring chemical immunity between crops.

Multiple genes recruited from hormone pathways partition maize diterpenoid defences.

Duplication and divergence of primary pathway genes underlie the evolution of plant specialized metabolism; however, mechanisms partitioning parallel hormone and defence pathways are often speculative. For example, the primary pathway intermediate ent-kaurene is essential for gibberellin biosynthesis and is also a proposed precursor for maize antibiotics. By integrating transcriptional coregulation patterns, genome-wide association studies, combinatorial enzyme assays, proteomics and targeted mutant analyses, we show that maize kauralexin biosynthesis proceeds via the positional isomer ent-isokaurene formed by a diterpene synthase pair recruited from gibberellin metabolism. The oxygenation and subsequent desaturation of ent-isokaurene by three promiscuous cytochrome P450s and a new steroid 5α reductase indirectly yields predominant ent-kaurene-associated antibiotics required for Fusarium stalk rot resistance. The divergence and differential expression of pathway branches derived from multiple duplicated hormone-metabolic genes minimizes dysregulation of primary metabolism via the circuitous biosynthesis of ent-kaurene-related antibiotics without the production of growth hormone precursors during defence.

Functional Characterization of Two Class II Diterpene Synthases Indicates Additional Specialized Diterpenoid Pathways in Maize (Zea mays).

As a major staple food, maize (Zea mays) is critical to food security. Shifting environmental pressures increasingly hamper crop defense capacities, causing expanded harvest loss. Specialized labdane-type diterpenoids are key components of maize chemical defense and ecological adaptation. Labdane diterpenoid biosynthesis most commonly requires the pairwise activity of class II and class I diterpene synthases (diTPSs) that convert the central precursor geranylgeranyl diphosphate into distinct diterpenoid scaffolds. Two maize class II diTPSs, ANTHER EAR 1 and 2 (ZmAN1/2), have been previously identified as catalytically redundant ent-copalyl diphosphate (CPP) synthases. ZmAN1 is essential for gibberellin phytohormone biosynthesis, whereas ZmAN2 is stress-inducible and governs the formation of defensive kauralexin and dolabralexin diterpenoids. Here, we report the biochemical characterization of the two remaining class II diTPSs present in the maize genome, COPALYL DIPHOSPHATE SYNTHASE 3 (ZmCPS3) and COPALYL DIPHOSPHATE SYNTHASE 4 (ZmCPS4). Functional analysis via microbial co-expression assays identified ZmCPS3 as a (+)-CPP synthase, with functionally conserved orthologs occurring in wheat (Triticum aestivum) and numerous dicot species. ZmCPS4 formed the unusual prenyl diphosphate, 8,13-CPP (labda-8,13-dien-15-yl diphosphate), as verified by mass spectrometry and nuclear magnetic resonance. As a minor product, ZmCPS4 also produced labda-13-en-8-ol diphosphate (LPP). Root gene expression profiles did not indicate an inducible role of ZmCPS3 in maize stress responses. By contrast, ZmCPS4 showed a pattern of inducible gene expression in roots exposed to oxidative stress, supporting a possible role in abiotic stress responses. Identification of the catalytic activities of ZmCPS3 and ZmCPS4 clarifies the first committed reactions controlling the diversity of defensive diterpenoids in maize, and suggests the existence of additional yet undiscovered diterpenoid pathways.

An apoplastic peptide activates salicylic acid signalling in maize.

Localized control of cell death is crucial for the resistance of plants to pathogens. Papain-like cysteine proteases (PLCPs) regulate plant defence to drive cell death and protection against biotrophic pathogens. In maize (Zea mays), PLCPs are crucial in the orchestration of salicylic acid (SA)-dependent defence signalling. Despite this central role in immunity, it remains unknown how PLCPs are activated, and which downstream signals they induce to trigger plant immunity. Here, we discover an immune signalling peptide, Z. mays immune signalling peptide 1 (Zip1), which is produced after salicylic acid (SA) treatment. In vitro studies demonstrate that PLCPs are required to release bioactive Zip1 from its propeptide precursor. Conversely, Zip1 treatment strongly elicits SA accumulation in leaves. Moreover, transcriptome analyses revealed that Zip1 and SA induce highly overlapping transcriptional changes. Consequently, Zip1 promotes the infection of the necrotrophic fungus Botrytis cinerea, while it reduces virulence of the biotrophic fungus Ustilago maydis. Thus, Zip1 represents the previously missing signal that is released by PLCPs to activate SA defence signalling.

Molecular marker assisted selection and pyramiding of two QTLs for fiber strength in upland cotton.

Based on two major QTLs that control high fiber strength which originated from an elite fiber germ-plasm line 7235 (Gossypium hiusutum L.), the efficiency of molecular marker-assisted selection (MAS) was investigated using two populations from pedigree selection and modified backcrossing pyramiding developed for the breeding purpose. Simian 3 (SM3), a widely planted variety in the Yangtze River Valley, and 7235 were used as parents to develop the two populations. In the two major QTLs for fiber strength from 7235, QTLfs-1 could explain more than 30% of the phenotypic variation (PV) in the (7235 x TM-1) F2 population. QTLfs-2 was at first identified in another super quality fiber line HS427-10 from (HS427-10 x TM-1) F2 population with 12.5% of PV explanation,which was further also identified in 7235 line but was non-allelic with QTLfs-1. The result of molecular marker-assisted selection for fiber strength showed that the genetic effect of the QTLfs-1 was stable under different environmental conditions, and its molecular marker-assisted selection showed significant selective efficiency among breeding populations with different genetic backgrounds. QTLfs-2 also showed high selective efficiency in advanced generation populations though its effect was a little lower than the former. When QTLfs-1 was selected simultaneously with 2 molecular markers with known genetic distance, the selection efficiency for the fiber strength was greatly increased. The pyramiding for two QTLs that control high fiber strength by MAS greatly improved the selection efficiency for cotton fiber strength. This report provides a successful example of MAS pyramiding for QTL for favorable traits in breeding programs.