R. J. Neil Emery

Disruption of OPR7 and OPR8 Reveals the Versatile Functions of Jasmonic Acid in Maize Development and Defense

Maize OPR7 and OPR8 are redundant paralogs responsible for JA biosynthesis. Double mutation in both genes revealed diverse roles of JA in developmental and defense processes, including regulation of sex determination and female organ outgrowth, anthocyanin pigmentation, leaf senescence, and immunity against insects and pathogens. Here, multiple functions of jasmonic acid (JA) in maize (Zea mays) are revealed by comprehensive analyses of JA-deficient mutants of the two oxo-phytodienoate reductase genes, OPR7 and OPR8. Single mutants produce wild-type levels of JA in most tissues, but the double mutant opr7 opr8 has dramatically reduced JA in all organs tested. opr7 opr8 displayed strong developmental defects, including formation of a feminized tassel, initiation of female reproductive buds at each node, and extreme elongation of ear shanks; these defects were rescued by exogenous JA. These data provide evidence that JA is required for male sex determination and suppression of female reproductive organ biogenesis. Moreover, opr7 opr8 exhibited delayed leaf senescence accompanied by reduced ethylene and abscisic acid levels and lack of anthocyanin pigmentation of brace roots. Remarkably, opr7 opr8 is nonviable in nonsterile soil and under field conditions due to extreme susceptibility to a root-rotting oomycete (Pythium spp), demonstrating that these genes are necessary for maize survival in nature. Supporting the importance of JA in insect defense, opr7 opr8 is susceptible to beet armyworm. Overall, this study provides strong genetic evidence for the global roles of JA in maize development and immunity to pathogens and insects.

Sucrose non-fermenting kinase 1 (SnRK1) coordinates metabolic and hormonal signals during pea cotyledon growth and differentiation.

Seed development passes through developmental phases such as cell division, differentiation and maturation: each have specific metabolic demands. The ubiquitous sucrose non-fermenting-like kinase (SnRK1) coordinates and adjusts physiological and metabolic demands with growth. In protoplast assays sucrose deprivation and hormone supplementation, such as with auxin and abscisic acid (ABA), stimulate SnRK1-promoter activity. This indicates regulation by nutrients: hormonal crosstalk under conditions of nutrient demand and cell proliferation. SnRK1-repressed pea (Pisum sativum) embryos show lower cytokinin levels and deregulation of cotyledonary establishment and growth, together with downregulated gene expression related to cell proliferation, meristem maintenance and differentiation, leaf formation, and polarity. This suggests that at early stages of seed development SnRK1 regulates coordinated cotyledon emergence and growth via cytokinin-mediated auxin transport and/or distribution. Decreased ABA levels and reduced gene expression, involved in ABA-mediated seed maturation and response to sugars, indicate that SnRK1 is required for ABA synthesis and/or signal transduction at an early stage. Metabolic profiling of SnRK1-repressed embryos revealed lower levels of most organic and amino acids. In contrast, levels of sugars and glycolytic intermediates were higher or unchanged, indicating decreased carbon partitioning into subsequent pathways such as the tricarbonic acid cycle and amino acid biosynthesis. It is hypothesized that SnRK1 mediates the responses to sugar signals required for early cotyledon establishment and patterning. As a result, later maturation and storage activity are strongly impaired. Changes observed in SnRK1-repressed pea seeds provide a framework for how SnRK1 communicates nutrient and hormonal signals from auxins, cytokinins and ABA to control metabolism and development.

Pea PR 10.1 is a ribonuclease and its transgenic expression elevates cytokinin levels

The constitutive expression of a cDNA encoding a pea (Pisum sativum L.) PR 10 protein in Brassica napus leading to an enhancement of germination under saline conditions has been previously reported. In order to understand the biochemical function of this pea PR 10 protein, its cDNA has been expressed in Escherichia coli and the recombinant protein purified to homogeneity. Ribonuclease activity of the recombinant pea PR 10 protein has been demonstrated for the first time using an in-solution as well as an in-gel RNA degradation assay. Furthermore, in order to characterize the changes brought about as a result of the constitutive expression of the pea PR 10 cDNA in B. napus, we have measured the endogenous concentrations of several phytohormones. Increased cytokinin and, decreased abscisic acid (ABA) were observed in 7-day-old transgenic seedlings whereas no significant changes in the concentrations of gibberellin (GA) or indoleacetic acid (IAA) were observed at this stage of growth and development. The potential role(s) of PR 10 proteins with RNase activity and elevated cytokinins during plant stress responses as well as the possible relationship between PR 10 protein and changes in cytokinin concentrations are discussed.

Ustilago maydis Produces Cytokinins and Abscisic Acid for Potential Regulation of Tumor Formation in Maize

The infection of maize (Zea mays) by the basidiomycete fungus Ustilago maydis leads to common smut of corn characterized by the production of tumors in susceptible aboveground plant tissues. LC-(ES)MS/MS profiles of abscisic acid (ABA) and 12 different cytokinins (CKs) were determined for infected and uninfected maize tissues over a time course following fungal exposure. Samples were taken at points corresponding to the appearance of disease symptoms. Axenic cultures of haploid and dikaryon forms of U. maydis were also profiled. This study confirmed the capability of Ustilago maydis to synthesize CKs, ABA, and auxin (IAA). It also provided evidence for the involvement of CK and ABA in the U. maydis-maize infection process. Significant quantities of CKs and ABA were detected from axenic cultures of U. maydis as was IAA. CKs and ABA levels were elevated in leaves and stems of maize after infection; notable was the high level of cis-zeatin 9-riboside. Variation among hormone profiles of maize tissues was observed at different time points during infection and between infections with nonpathogenic haploid and pathogenic dikaryon strains. This suggested that CKs and ABA accumulate and are likely metabolized in maize tissue infected with U. maydis. Because U. maydis produced these phytohormones at significant levels, it is possible that the fungal pathogen is a source of these compounds in infected tissue. This is the first study to confirm the production of CKs and document the production of ABA by U. maydis. This study also established an involvement of these phytohormones and a possible functional role for ABA in U. maydis infection of maize.

A Laser Dissection-RNAseq Analysis Highlights the Activation of Cytokinin Pathways by Nod Factors in the Medicago truncatula Root Epidermis.

Nod factors induce massive reprogramming of gene expression in the root epidermis, including the CRE1 cytokinin pathway which leads to both positive and negative regulation of nodulation. Nod factors (NFs) are lipochitooligosaccharidic signal molecules produced by rhizobia, which play a key role in the rhizobium-legume symbiotic interaction. In this study, we analyzed the gene expression reprogramming induced by purified NF (4 and 24 h of treatment) in the root epidermis of the model legume Medicago truncatula. Tissue-specific transcriptome analysis was achieved by laser-capture microdissection coupled to high-depth RNA sequencing. The expression of 17,191 genes was detected in the epidermis, among which 1,070 were found to be regulated by NF addition, including previously characterized NF-induced marker genes. Many genes exhibited strong levels of transcriptional activation, sometimes only transiently at 4 h, indicating highly dynamic regulation. Expression reprogramming affected a variety of cellular processes, including perception, signaling, regulation of gene expression, as well as cell wall, cytoskeleton, transport, metabolism, and defense, with numerous NF-induced genes never identified before. Strikingly, early epidermal activation of cytokinin (CK) pathways was indicated, based on the induction of CK metabolic and signaling genes, including the CRE1 receptor essential to promote nodulation. These transcriptional activations were independently validated using promoter:β-glucuronidase fusions with the MtCRE1 CK receptor gene and a CK response reporter (TWO COMPONENT SIGNALING SENSOR NEW). A CK pretreatment reduced the NF induction of the EARLY NODULIN11 (ENOD11) symbiotic marker, while a CK-degrading enzyme (CYTOKININ OXIDASE/DEHYDROGENASE3) ectopically expressed in the root epidermis led to increased NF induction of ENOD11 and nodulation. Therefore, CK may play both positive and negative roles in M. truncatula nodulation.

Gall-inducing Pachypsylla celtidis (Psyllidae) infiltrate hackberry trees with high concentrations of phytohormones

Abstract In order to identify phytohormones involved in the initiation and maintenance of galls on the hackberry tree, Celtis occidentalis (Ulmaceae), in the presence of the insect Pachypsylla celtidis (Psyllidae); endogenous levels of cytokinins (CKs) and abscisic acid (ABA) were measured in the tissues of leaves, galls, and larval insects using liquid-chromatography-tandem-mass-spectrometry. The CKs isopentenyl adenine, isopentenyl adenosine, trans-zeatin and cis-zeatin, were extremely concentrated in insect larvae compared to surrounding tissue of leaves and galls. ABA concentrations in the insects were also relatively high at about 3268 pmol•g-1 fresh mass – approximately 25 times higher than in leaves, and 17 times higher than in galls. This represents a novel case of high concentrations of ABA being found in a larval insect. These findings indicate that manipulation of CK and ABA are involved in the initiation and maintenance of hackberry galls in the presence of larval P. celtidis.

Concurrent profiling of indole-3-acetic acid, abscisic acid, and cytokinins and structurally related purines by high-performance-liquid-chromatography tandem electrospray mass spectrometry.

BackgroundCytokinins (CKs) are a group of plant growth regulators that are involved in several plant developmental processes. Despite the breadth of knowledge surrounding CKs and their diverse functions, much remains to be discovered about the full potential of CKs, including their relationship with the purine salvage pathway, and other phytohormones. The most widely used approach to query unknown facets of CK biology utilized functional genomics coupled with CK metabolite assays and screening of CK associated phenotypes. There are numerous different types of assays for determining CK quantity, however, none of these methods screen for the compendium of metabolites that are necessary for elucidating all roles, including purine salvage pathway enzymes in CK metabolism, and CK cross-talk with other phytohormones. Furthermore, all published analytical methods have drawbacks ranging from the required use of radiolabelled compounds, or hazardous derivatization reagents, poor sensitivity, lack of resolution between CK isomers and lengthy run times.ResultsIn this paper, a method is described for the concurrent extraction, purification and analysis of several CKs (freebases, ribosides, glucosides, nucleotides), purines (adenosine monophosphate, inosine, adenosine, and adenine), indole-3-acetic acid, and abscisic acid from hundred-milligram (mg) quantities of Arabidopsis thaliana leaf tissue. This method utilizes conventional Bieleski solvents extraction, solid phase purification, and is unique because of its diverse range of detectable analytes, and implementation of a conventional HPLC system with a fused core column that enables good sensitivity without the requirement of a UHPLC system. Using this method we were able to resolve CKs about twice as fast as our previous method. Similarly, analysis of adenosine, indole-3-acetic acid, and abscisic acid, was comparatively rapid. A further enhancement of the method was the utilization of a QTRAP 5500 mass analyzer, which improved upon several aspects of our previous analytical method carried out on a Quattro mass analyzer. Notable improvements included much superior sensitivity, and number of analytes detectable within a single run. Limits of detection ranged from 2 pM for (9G)Z to almost 750 pM for indole-3-acetic acid.ConclusionsThis method is well suited for functional genomics platforms tailored to understanding CK metabolism, CK interrelationships with purine recycling and associated hormonal cross-talk.

ABA inhibits germination but not dormancy release in mature imbibed seeds of Lolium rigidum Gaud

Dormancy release in imbibed annual ryegrass (Lolium rigidum Gaud.) seeds is promoted in the dark but inhibited in the light. The role of abscisic acid (ABA) in inhibition of dormancy release was found to be negligible, compared with its subsequent effect on germination of dormant and non-dormant seeds. Inhibitors of ABA metabolism had the expected effects on seed germination but did not influence ABA concentration, suggesting that they act upon other (unknown) factors regulating dormancy. Although gibberellin (GA) synthesis was required for germination, the influence of exogenous GA on both germination and dormancy release was minor or non-existent. Embryo ABA concentration was the same following treatments to promote (dark stratification) and inhibit (light stratification) dormancy release; exogenous ABA had no effect on this process. However, the sensitivity of dark-stratified seeds to ABA supplied during germination was lower than that of light-stratified seeds. Therefore, although ABA definitely plays a role in the germination of annual ryegrass seeds, it is not the major factor mediating inhibition of dormancy release in imbibed seeds.

A Crucial Role for Cytokinins in Pea ABR17-mediated Enhanced Germination and Early Seedling Growth of Arabidopsis thaliana under Saline and Low-temperature Stresses

The role of cytokinins (CKs) in mediating the previously observed ABR17-mediated enhancement of germination of Arabidopsis thaliana under salinity and low-temperature stresses has been evaluated. We determined the endogenous concentrations of CK in the three transgenic and wild-type seedlings, which indicated that the transgenic seedlings had higher endogenous concentrations of CK. Furthermore, the relative levels of expression of ABR17 cDNA and the primary CK response gene, ARR5, were evaluated in the transgenic and wild-type seedlings by quantitative real-time polymerase chain reaction (RT-PCR). Our results indicated that two of the three independently derived transgenic plants possessed higher levels of ABR17 transcripts, which correlated well with increased ARR5 expression, further supporting a possible role for CK in mediating the observed phenomenon. In addition, the exogenous application of various CKs on the germination of wild-type A. thaliana under these abiotic stress conditions enhanced its germination. Finally, the ribonuclease (RNase) activity of the pea ABR17 protein was also demonstrated after expression of its cDNA in Escherichia coli, purification of the recombinant protein, and in vitro RNase assays. Our findings are discussed within the context of ABR17-mediated enhancement of endogenous CK concentrations, the involvement of CKs in germination under abiotic stress, as well as the role of the RNase activity of ABR17 protein in mediating the observed effects.

Interactions of temperature and light quality on phytohormone-mediated elongation of Helianthus annuus hypocotyls

Two important environmental signals, shade light, where the red/far-red (R/FR) light ratio is reduced, and elevated temperatures can each promote shoot growth. We examined their interactions using hypocotyl elongation of young sunflower (Helianthus annuus) seedlings, and we did this in the context of a possible hormonal mechanism for the growth increases that were induced by each environmental signal. Seedlings were subjected to combinations of six different temperatures (10, 15, 20, 25, 30 and 35°C) and four R/FR ratios (normal at 1.2 and reduced at 0.9, 0.6 and 0.3). Hypocotyl length was significantly increased by each of elevated temperature and FR enrichment. The magnitude of elongation induced by FR enrichment (low R/FR ratios) was dependent on temperature, with maximal effects of FR enrichment being seen at 20°C. Hypocotyl tissue concentrations of four endogenous gibberellins (GAs) and abscisic acid (ABA) were measured using the stable isotope dilution method. Hypocotyl ethylene evolution was also assessed. Thus, hypocotyl growth in both normal and shade light is highly dependent on temperature, with the most significant increases in FR-induced growth occurring at 20 and 25°C. A causal involvement of endogenous hormones, especially the GAs, in the growth that is induced by elevated temperatures, as well as in FR-induced growth, is strongly implied, with temperature being the stronger signal.