Sexual and Apogamous Species of Woodferns Show Different Protein and Phytohormone Profiles

The gametophyte of ferns reproduces either by sexual or asexual means. In the latter, apogamy represents a peculiar case of apomixis, in which an embryo is formed from somatic cells. A proteomic and physiological approach was applied to the apogamous fern Dryopteris affinis ssp. affinis and its sexual relative D. oreades. The proteomic analysis compared apogamous vs. female gametophytes, whereas the phytohormone study included, in addition to females, three apogamous stages (filamentous, spatulate, and cordate). The proteomic profiles revealed a total of 879 proteins and, after annotation, different regulation was found in 206 proteins of D. affinis and 166 of its sexual counterpart. The proteins upregulated in D. affinis are mostly associated to protein metabolism (including folding, transport, and proteolysis), ribosome biogenesis, gene expression and translation, while in the sexual counterpart, they account largely for starch and sucrose metabolism, generation of energy and photosynthesis. Likewise, ultra-performance liquid chromatography-tandem spectrometry (UHPLC-MS/MS) was used to assess the levels of indol-3-acetic acid (IAA); the cytokinins: 6-benzylaminopurine (BA), trans-Zeatine (Z), trans-Zeatin riboside (ZR), dyhidrozeatine (DHZ), dyhidrozeatin riboside (DHZR), isopentenyl adenine (iP), isopentenyl adenosine (iPR), abscisic acid (ABA), the gibberellins GA3 and GA4, salicylic acid (SA), and the brassinosteroids: brassinolide (BL) and castasterone (CS). IAA, the cytokinins Z, ZR, iPR, the gibberellin GA4, the brassinosteoids castasterone, and ABA accumulated more in the sexual gametophyte than in the apogamous one. When comparing the three apogamous stages, BA and SA peaked in filamentous, GA3 and BL in spatulate and DHRZ in cordate gametophytes. The results point to the existence of large metabolic differences between apogamous and sexual gametophytes, and invite to consider the fern gametophyte as a good experimental system to deepen our understanding of plant reproduction.

Structural, biochemical and functional analyses of tRNA-monooxygenase enzyme MiaE from Pseudomonas putida provide insights into tRNA/MiaE interaction

MiaE (2-methylthio-N6-isopentenyl-adenosine37-tRNA monooxygenase) is a unique non-heme diiron enzyme that catalyzes the O2-dependent post-transcriptional allylic hydroxylation of a hypermodified nucleotide 2-methylthio-N6-isopentenyl-adenosine (ms2i6A37) at position 37 of selected tRNA molecules to produce 2-methylthio-N6–4-hydroxyisopentenyl-adenosine (ms2io6A37). Here, we report the in vivo activity, biochemical, spectroscopic characterization and X-ray crystal structure of MiaE from Pseudomonas putida. The investigation demonstrates that the putative pp-2188 gene encodes a MiaE enzyme. The structure shows that Pp-MiaE consists of a catalytic diiron(III) domain with a four alpha-helix bundle fold. A docking model of Pp-MiaE in complex with tRNA, combined with site directed mutagenesis and in vivo activity shed light on the importance of an additional linker region for substrate tRNA recognition. Finally, krypton-pressurized Pp-MiaE experiments, revealed the presence of defined O2 site along a conserved hydrophobic tunnel leading to the diiron active center.

Phytohormone production by the arbuscular mycorrhizal fungus Rhizophagus irregularis

Arbuscular mycorrhizal symbiosis is a mutualistic interaction between most land plants and fungi of the glomeromycotina subphylum. The initiation, development and regulation of this symbiosis involve numerous signalling events between and within the symbiotic partners. Among other signals, phytohormones are known to play important roles at various stages of the interaction. During presymbiotic steps, plant roots exude strigolactones which stimulate the fungus, and favour the initiation of symbiosis. At later stages, different plant hormone classes can act as positive or negative regulators of the interaction. Although the fungus is known to reciprocally emit regulatory signals, its potential contribution to the phytohormonal pool has received little attention, and has so far only been addressed by indirect assays. In this study, using mass spectrometry, we analyzed phytohormones released into the medium by germinated spores of the arbuscular mycorrhizal fungus Rhizophagus irregularis. We detected the presence of a cytokinin (isopentenyl-adenosine) and an auxin (indole-acetic acid). In addition, we identified a gibberellin (gibberellic acid 4) in spore extracts. We also used gas chromatography to show that R. irregularis produces ethylene from methionine and the α-keto γ-methylthiobutyric acid pathway. These results highlight the possibility for AM fungi to use phytohormones to interact with their host plants, or to regulate their own development.

Salt Stress-induced Injury is Associated with Hormonal Alteration in Kentucky Bluegrass

Plant hormones play an important role in plant adaptation to abiotic stress, but hormonal responses of cool-season turfgrass species to salt stress are not well documented. This study was carried out to investigate the responses of hormones to salt stress and examine if salt stress-induced injury was associated with hormonal alteration in kentucky bluegrass (KBG, Poa pratensis L.). The grass was grown in a growth chamber for 6 weeks and then subjected to salt stress (170 mm NaCl) for 28 days. Salt stress caused cell membrane damage, resulting in photosynthetic rate (Pn), chlorophyll (Chl), and turf quality decline in KBG. Salt stress increased leaf abscisic acid (ABA) and ABA/cytokinin (CK) ratio; reduced trans-zeatin riboside (ZR), isopentenyl adenosine (iPA), and indole-3-acetic acid (IAA), but did not affect gibberellin A4 (GA4). On average, salt stress reduced ZR by 67.4% and IAA by 58.6%, whereas it increased ABA by 398.5%. At the end of the experiment (day 28), turf quality, Pn, and stomatal conductance (gs) were negatively correlated with ABA and ABA/CK ratio, but positively correlated with ZR, iPA, and IAA. Electrolyte leakage (EL) was positively correlated with ABA and ABA/CK and negatively correlated with ZR, iPA, IAA, and GA4. GA4 was also positively correlated with turf quality and gs. The results of this study suggest that salt stress-induced injury of the cell membrane and photosynthetic function may be associated with hormonal alteration and imbalance in KBG.

The levels of hormone and carbohydrate in autumn and non-autumn flowering tree peonies

Mornya, P. M. P. and Cheng, F. 2011. The levels of hormone and carbohydrate in autumn and non-autumn flowering tree peonies. Can. J. Plant Sci. 91: 991–998. This study analyzed the levels of gibberellic acid (GA3), indole-3-acetic acid (IAA), cytokinin (CTK) and abscisic acid (ABA) hormones using high-performance liquid chromatography, along with the levels of sucrose, reducing sugar and starch carbohydrates by spectrophotometer during induction, initiation and differentiation stages of bud development in autumn (AFP) and non-autumn (NAFP) flowering tree peony cultivars exhibiting variations in flowering pattern. The experimental design was a randomized complete block with three replications. The variation in flowering pattern between AFP and NAFP was largely influenced by differences in GA3, IAA and CTK levels at different bud developmental stages. The flower formation cycle was completed earlier in AFP than in NAFP, hence flowering was twice annually. Cytokinin, particularly N6-(Δ2-isopentenyl)-adenosine (iPA), could be a critical hormone in autumn flowering of tree peony, as its differences in levels between AFP and NAFP remained significant across bud developmental stages. However, only GA3 had negative differences across bud developmental stages, indicating that GA3 levels were consistently higher in NAFP than in AFP, but the reverse holds true for CTK. The differences in GA3, IAA and CTK between AFP and NAFP were significant (P<0.05) for at least two-thirds of the bud developmental stages. Carbohydrates may not significantly influence the flowering pattern of tree peonies. Autumn flowering in tree peonies could therefore be achieved by regulating GA3, IAA and CTK levels, particularly at the induction and initiation stages of bud development to facilitate the completion of the floral formation cycle, well in advance of bud dormancy period. The findings of this study could lay the scientific basis for genetic engineering of flowering pattern of tree peonies.

Cytokinin production by plant growth promoting rhizobacteria and selected mutants

One of the proposed mechanisms by which rhizobacteria enhance plant growth is through the production of plant growth regulators. Five plant growth promoting rhizobacterial (PGPR) strains produced the cytokinin dihydrozeatin riboside (DHZR) in pure culture. Cytokinin production by Pseudomonas fluorescens G20–18, a rifampicin-resistant mutant (RIF), and two TnphoA-derived mutants (CNT1, CNT2), with reduced capacity to synthesize cytokinins, was further characterized in pure culture using immunoassay and thin layer chromatography. G20–18 produced higher amounts of three cytokinins, isopentenyl adenosine (IPA), trans-zeatin ribose (ZR), and DHZR than the three mutants during stationary phase. IPA was the major metabolite produced, but the proportion of ZR and DHZR accumulated by CNT1 and CNT2 increased with time. No differences were observed between strain G20–18 and the mutants in the amounts of indole acetic acid synthesized, nor were gibberellins detected in supernatants of any of the strains. Addition of 10–5 M adenine increased cytokinin production in 96- and 168-h cultures of strain G20–18 by approximately 67%. G20–18 and the mutants CNT1 and CNT2 may be useful for determination of the role of cytokinin production in plant growth promotion by PGPR.Key words: cytokinins, plant growth regulation, Pseudomonas fluorescens, rhizobacteria, plant growth promoting rhizobacteria (PGPR).