The interaction of O3 and CO2 concentration, exposure timing and duration on stem rust severity on winter wheat variety 'Coker 9553'.

Wheat rusts, elevated ozone (O3), and carbon dioxide (CO2) are simultaneously impacting wheat production worldwide, but their interactions are not well understood. This study investigated whether near-ambient O3 is suppressive or conducive to stem rust (Sr) of wheat, considering the interactions with ambient and elevated CO2. Winter wheat variety 'Coker 9553' (Sr-susceptible; O3 sensitive) was inoculated with Sr (race QFCSC) following pre-treatment with four different concentrations of O3 (CF, 50, 70, and 90 ppbv) at ambient CO2 levels. Gas treatments were continued during the development of disease symptoms. Disease severity, measured as percent sporulation area (PSA), significantly increased relative to the CF control only under near-ambient O3 conditions (50 ppbv) in the absence of O3-induced foliar injury. Disease symptoms at higher O3 exposures (70 and 90 ppbv) were similar to or less than the CF control. When Coker 9553 was inoculated with Sr while exposed to CO2 (400; 570 ppmv) and O3 (CF; 50 ppbv) in four different combinations, and seven combinations of exposure timing and duration, PSA significantly increased only under continuous treatment with O3 for six weeks or pre-inoculation treatment for three weeks, suggesting that O3-predisposes wheat to the disease rather than enhancing disease post-inoculation. O3 singly and in combination with CO2 increased PSA on flag leaves of adult Coker 9553 plants while elevated CO2 alone had little effect on PSA. These findings show that sub-symptomatic O3 conditions are conducive to stem rust, contradicting the current consensus that biotrophic pathogens are suppressed by elevated O3. This suggests that sub-symptomatic O3 stress may enhance rust diseases in wheat-growing regions.

A Rapid Alkalinization Factor-like Peptide EaF82 Impairs Tapetum Degeneration during Pollen Development through Induced ATP Deficiency.

In plants, the timely degeneration of tapetal cells is essential for providing nutrients and other substances to support pollen development. Rapid alkalinization factors (RALFs) are small, cysteine-rich peptides known to be involved in various aspects of plant development and growth, as well as defense against biotic and abiotic stresses. However, the functions of most of them remain unknown, while no RALF has been reported to involve tapetum degeneration. In this study, we demonstrated that a novel cysteine-rich peptide, EaF82, isolated from shy-flowering 'Golden Pothos' (Epipremnum aureum) plants, is a RALF-like peptide and displays alkalinizing activity. Its heterologous expression in Arabidopsis delayed tapetum degeneration and reduced pollen production and seed yields. RNAseq, RT-qPCR, and biochemical analyses showed that overexpression of EaF82 downregulated a group of genes involved in pH changes, cell wall modifications, tapetum degeneration, and pollen maturation, as well as seven endogenous Arabidopsis RALF genes, and decreased proteasome activity and ATP levels. Yeast two-hybrid screening identified AKIN10, a subunit of energy-sensing SnRK1 kinase, as its interacting partner. Our study reveals a possible regulatory role for RALF peptide in tapetum degeneration and suggests that EaF82 action may be mediated through AKIN10 leading to the alteration of transcriptome and energy metabolism, thereby causing ATP deficiency and impairing pollen development.

Alteration of the alkaloid profile in genetically modified tobacco reveals a role of methylenetetrahydrofolate reductase in nicotine N-demethylation.

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme of the tetrahydrofolate (THF)-mediated one-carbon (C1) metabolic network. This enzyme catalyzes the reduction of 5,10-methylene-THF to 5-methyl-THF. The latter donates its methyl group to homocysteine, forming methionine, which is then used for the synthesis of S-adenosyl-methionine, a universal methyl donor for numerous methylation reactions, to produce primary and secondary metabolites. Here, we demonstrate that manipulating tobacco (Nicotiana tabacum) MTHFR gene (NtMTHFR1) expression dramatically alters the alkaloid profile in transgenic tobacco plants by negatively regulating the expression of a secondary metabolic pathway nicotine N-demethylase gene, CYP82E4. Quantitative real-time polymerase chain reaction and alkaloid analyses revealed that reducing NtMTHFR expression by RNA interference dramatically induced CYP82E4 expression, resulting in higher nicotine-to-nornicotine conversion rates. Conversely, overexpressing NtMTHFR1 suppressed CYP82E4 expression, leading to lower nicotine-to-nornicotine conversion rates. However, the reduced expression of NtMTHFR did not affect the methionine and S-adenosyl-methionine levels in the knockdown lines. Our finding reveals a new regulatory role of NtMTHFR1 in nicotine N-demethylation and suggests that the negative regulation of CYP82E4 expression may serve to recruit methyl groups from nicotine into the C1 pool under C1-deficient conditions.

Identification of a Mg-protoporphyrin IX monomethyl ester cyclase homologue, EaZIP, differentially expressed in variegated Epipremnum aureum 'Golden Pothos' is achieved through a unique method of comparative study using tissue regenerated plants.

Variegated plants provide a valuable tool for studying chloroplast biogenesis by allowing direct comparison between green and white/yellow sectors within the same leaf. While variegated plants are abundant in nature, the mechanism of leaf variegation remains largely unknown. Current studies are limited to a few mutants in model plant species, and are complicated by the potential for cross-contamination during dissection of leaf tissue into contrasting sectors. To overcome these obstacles, an alternative approach was explored using tissue-culture techniques to regenerate plantlets from unique sectors. Stable green and pale yellow plants were developed from a naturally variegated Epipremnum aureum 'Golden Pothos'. By comparing the gene expression between green and pale yellow plants using suppression subtractive hybridization in conjunction with homologous sequence search, nine down-regulated and 18 up-regulated genes were identified in pale yellow plants. Transcript abundance for EaZIP (Epipremnum aureum leucine zipper), a nuclear gene homologue of tobacco NTZIP and Arabidopsis CHL27, was reduced more than 4000-fold in qRT-PCR analysis. EaZIP encodes the Mg-protoporphyrin IX monomethyl ester cyclase, one of the key enzymes in the chlorophyll biosynthesis pathway. Examination of EaZIP expression in naturally variegated 'Golden Pothos' confirmed that EaZIP transcript levels were correlated with leaf chlorophyll contents, suggesting that this gene plays a major role in the loss of chlorophyll in the pale yellow sectors of E. aureum 'Golden Pothos'. This study further suggests that tissue-culture regeneration of plantlets from different coloured sectors of variegated leaves can be used to investigate the underlying mechanisms of variegation.