Structural basis for tRNA methylthiolation by the radical SAM enzyme MiaB.

Numerous post-transcriptional modifications of transfer RNAs have vital roles in translation. The 2-methylthio-N6-isopentenyladenosine (ms2i6A) modification occurs at position 37 (A37) in transfer RNAs that contain adenine in position 36 of the anticodon, and serves to promote efficient A:U codon-anticodon base-pairing and to prevent unintended base pairing by near cognates, thus enhancing translational fidelity1-4. The ms2i6A modification is installed onto isopentenyladenosine (i6A) by MiaB, a radical S-adenosylmethionine (SAM) methylthiotransferase. As a radical SAM protein, MiaB contains one [Fe4S4]RS cluster used in the reductive cleavage of SAM to form a 5'-deoxyadenosyl 5'-radical, which is responsible for removing the C2 hydrogen of the substrate5. MiaB also contains an auxiliary [Fe4S4]aux cluster, which has been implicated6-9 in sulfur transfer to C2 of i6A37. How this transfer takes place is largely unknown. Here we present several structures of MiaB from Bacteroides uniformis. These structures are consistent with a two-step mechanism, in which one molecule of SAM is first used to methylate a bridging µ-sulfido ion of the auxiliary cluster. In the second step, a second SAM molecule is cleaved to a 5'-deoxyadenosyl 5'-radical, which abstracts the C2 hydrogen of the substrate but only after C2 has undergone rehybridization from sp2 to sp3. This work advances our understanding of how enzymes functionalize inert C-H bonds with sulfur.

Posttranscriptional modifications at the 37th position in the anticodon stem-loop of tRNA: structural insights from MD simulations.

Transfer RNA (tRNA) is the most diversely modified RNA. Although the strictly conserved purine position 37 in the anticodon stem-loop undergoes modifications that are phylogenetically distributed, we do not yet fully understand the roles of these modifications. Therefore, molecular dynamics simulations are used to provide molecular-level details for how such modifications impact the structure and function of tRNA. A focus is placed on three hypermodified base families that include the parent i6A, t6A, and yW modifications, as well as derivatives. Our data reveal that the hypermodifications exhibit significant conformational flexibility in tRNA, which can be modulated by additional chemical functionalization. Although the overall structure of the tRNA anticodon stem remains intact regardless of the modification considered, the anticodon loop must rearrange to accommodate the bulky, dynamic hypermodifications, which includes changes in the nucleotide glycosidic and backbone conformations, and enhanced or completely new nucleobase-nucleobase interactions compared to unmodified tRNA or tRNA containing smaller (m1G) modifications at the 37th position. Importantly, the extent of the changes in the anticodon loop is influenced by the addition of small functional groups to parent modifications, implying each substituent can further fine-tune tRNA structure. Although the dominant conformation of the ASL is achieved in different ways for each modification, the molecular features of all modified tRNA drive the ASL domain to adopt the functional open-loop conformation. Importantly, the impact of the hypermodifications is preserved in different sequence contexts. These findings highlight the likely role of regulating mRNA structure and translation.

Cyclanilide Induces Lateral Bud Outgrowth by Modulating Cytokinin Biosynthesis and Signalling Pathways in Apple Identified via Transcriptome Analysis.

Cyclanilide (CYC), a plant growth regulator, is a potent shoot branching agent in apple. However, its mechanism remains unclear. The current study revealed that CYC treatment resulted in massive reprogramming of the axillary bud transcriptome, implicating several hormones in the response. We observed a marked increase (approximately 2-fold) in the level of zeatin riboside and a significant decrease (approximately 2-fold) in the level of abscisic acid (ABA). Zeatin metabolism gene cytokinin (CTK) oxidase 1 (CKX 1) was down-regulated at 168 h after CYC treatment compared with the control. Weighted gene co-expression network analysis of differentially expressed genes demonstrated the turquoise module clusters exhibited the highest positive correlation with zeatin riboside (r = 0.92) and the highest negative correlation with ABA (r = -0.8). A total of 37 genes were significantly enriched in the plant hormone signal transduction pathway in the turquoise module. Among them, the expressions of CTK receptor genes WOODEN LEG and the CTK type-A response regulators genes ARR3 and ARR9 were up-regulated. ABA signal response genes protein phosphatase 2C genes ABI2 and ABI5 were down-regulated in lateral buds after CYC treatment at 168 h. In addition, exogenous application of 6-benzylaminopurine (6-BA, a synthetic type of CTK) and CYC enhanced the inducing effect of CYC, whereas exogenous application of lovastatin (a synthetic type of inhibitor of CTK biosynthesis) or ABA and CYC weakened the promoting effect of CYC. These results collectively revealed that the stimulation of bud growth by CYC might involve CTK biosynthesis and signalling, including genes CKX1 and ARR3/9, which provided a direction for further study of the branching promoting mechanism of CYC.

Transcriptome Analysis Reveals Multiple Genes and Complex Hormonal-Mediated Interactions with PEG during Adventitious Root Formation in Apple.

Adventitious root (AR) formation is a bottleneck for the mass propagation of apple rootstocks, and water stress severely restricts it. Different hormones and sugar signaling pathways in apple clones determine AR formation under water stress, but these are not entirely understood. To identify them, GL-3 stem cuttings were cultured on polyethylene glycol (PEG) treatment. The AR formation was dramatically decreased compared with the PEG-free control (CK) cuttings by increasing the endogenous contents of abscisic acid (ABA), zeatin riboside (ZR), and methyl jasmonate (JA-me) and reducing the indole-3-acetic acid (IAA) and gibberellic acid 3 (GA3) contents. We performed a transcriptomic analysis to identify the responses behind the phenotype. A total of 3204 differentially expressed genes (DEGs) were identified between CK and PEG, with 1702 upregulated and 1502 downregulated genes. Investigation revealed that approximately 312 DEGs were strongly enriched in hormone signaling, sugar metabolism, root development, and cell cycle-related pathways. Thus, they were selected for their possible involvement in adventitious rooting. However, the higher accumulation of ABA, ZR, and JA-me contents and the upregulation of their related genes, as well as the downregulation of sugar metabolism-related genes, lead to the inhibition of ARs. These results indicate that AR formation is a complicated biological process chiefly influenced by multiple hormonal signaling pathways and sugar metabolism. This is the first study to demonstrate how PEG inhibits AR formation in apple plants.

Cytokinin Perception in Ancient Plants beyond Angiospermae.

Cytokinins (CKs) control many plant developmental processes and responses to environmental cues. Although the CK signaling is well understood, we are only beginning to decipher its evolution. Here, we investigated the CK perception apparatus in early-divergent plant species such as bryophyte Physcomitrium patens, lycophyte Selaginella moellendorffii, and gymnosperm Picea abies. Of the eight CHASE-domain containing histidine kinases (CHKs) examined, two CHKs, PpCHK3 and PpCHK4, did not bind CKs. All other CHK receptors showed high-affinity CK binding (KD of nM range), with a strong preference for isopentenyladenine over other CK nucleobases in the moss and for trans-zeatin over cis-zeatin in the gymnosperm. The pH dependences of CK binding for these six CHKs showed a wide range, which may indicate different subcellular localization of these receptors at either the plasma- or endoplasmic reticulum membrane. Thus, the properties of the whole CK perception apparatuses in early-divergent lineages were demonstrated. Data show that during land plant evolution there was a diversification of the ligand specificity of various CHKs, in particular, the rise in preference for trans-zeatin over cis-zeatin, which indicates a steadily increasing specialization of receptors to various CKs. Finally, this distinct preference of individual receptors to different CK versions culminated in vascular plants, especially angiosperms.

Mechanism of Allium Crops Bulb Enlargement in Response to Photoperiod: A Review.

The photoperiod marks a varied set of behaviors in plants, including bulbing. Bulbing is controlled by inner signals, which can be stimulated or subdued by the ecological environment. It had been broadly stated that phytohormones control the plant development, and they are considered to play a significant part in the bulb formation. The past decade has witnessed significant progress in understanding and advancement about the photoperiodic initiation of bulbing in plants. A noticeable query is to what degree the mechanisms discovered in bulb crops are also shared by other species and what other qualities are also dependent on photoperiod. The FLOWERING LOCUS T (FT) protein has a role in flowering; however, the FT genes were afterward reported to play further functions in other biological developments (e.g., bulbing). This is predominantly applicable in photoperiodic regulation, where the FT genes seem to have experienced significant development at the practical level and play a novel part in the switch of bulb formation in Alliums. The neofunctionalization of FT homologs in the photoperiodic environments detects these proteins as a new class of primary signaling mechanisms that control the growth and organogenesis in these agronomic-related species. In the present review, we report the underlying mechanisms regulating the photoperiodic-mediated bulb enlargement in Allium species. Therefore, the present review aims to systematically review the published literature on the bulbing mechanism of Allium crops in response to photoperiod. We also provide evidence showing that the bulbing transitions are controlled by phytohormones signaling and FT-like paralogues that respond to independent environmental cues (photoperiod), and we also show that an autorelay mechanism involving FT modulates the expression of the bulbing-control gene. Although a large number of studies have been conducted, several limitations and research gaps have been identified that need to be addressed in future studies.

N6 -Isopentenyladenosine in RNA Determines the Cleavage Site of Endonuclease Deoxyribozymes.

RNA-cleaving deoxyribozymes can serve as selective sensors and catalysts to examine the modification state of RNA. However, site-specific endonuclease deoxyribozymes that selectively cleave post-transcriptionally modified RNA are extremely rare and their specificity over unmodified RNA is low. We report that the native tRNA modification N6 -isopentenyladenosine (i6 A) strongly enhances the specificity and has the power to reconfigure the active site of an RNA-cleaving deoxyribozyme. Using in vitro selection, we identified a DNA enzyme that cleaves i6 A-modified RNA at least 2500-fold faster than unmodified RNA. Another deoxyribozyme shows unique and unprecedented behaviour by shifting its cleavage site in the presence of the i6 A RNA modification. Together with deoxyribozymes that are strongly inhibited by i6 A, these results highlight that post-transcriptional RNA modifications modulate the catalytic activity of DNA in various intricate ways.

Chemical Deprenylation of N6 -Isopentenyladenosine (i6 A) RNA.

N6 -isopentenyladenosine (i6 A) is an RNA modification found in cytokinins, which regulate plant growth/differentiation, and a subset of tRNAs, where it improves the efficiency and accuracy of translation. The installation and removal of this modification is mediated by prenyltransferases and cytokinin oxidases, and a chemical approach to selective deprenylation of i6 A has not been developed. We show that a selected group of oxoammonium cations function as artificial deprenylases to promote highly selective deprenylation of i6 A in nucleosides, oligonucleotides, and live cells. Importantly, other epigenetic modifications, amino acid residues, and natural products were not affected. Moreover, a significant phenotype difference in the Arabidopsis thaliana shoot and root development was observed with incubation of the cation. These results establish these small organic molecules as direct chemical regulators/artificial deprenylases of i6 A.

Gene ssfg_01967 (miaB) for tRNA modification influences morphogenesis and moenomycin biosynthesis in Streptomyces ghanaensis ATCC14672.

Streptomyces ghanaensis ATCC14672 is remarkable for its production of phosphoglycolipid compounds, moenomycins, which serve as a blueprint for the development of a novel class of antibiotics based on inhibition of peptidoglycan glycosyltransferases. Here we employed mariner transposon (Tn) mutagenesis to find new regulatory genes essential for moenomycin production. We generated a library of 3000 mutants which were screened for altered antibiotic activity. Our focus centred on a single mutant, HIM5, which accumulated lower amounts of moenomycin and was impaired in morphogenesis as compared to the parental strain. HIM5 carried the Tn insertion within gene ssfg_01967 for putative tRNA (N6-isopentenyl adenosine(37)-C2)-methylthiotransferase, or MiaB, and led to a reduced level of thiomethylation at position 37 in the anticodon of S. ghanaensis transfer ribonucleic acid (tRNA). It is likely that the mutant phenotype of HIM5 stems from the way in which ssfg_01967::Tn influences translation of the rare leucine codon UUA in several genes for moenomycin production and life cycle progression in S. ghanaensis. This is the first report showing that quantitative changes in tRNA modification status in Streptomyces have physiological consequences.

Noninvasive diagnosis of TRIT1-related mitochondrial disorder by measuring i6 A37 and ms2 i6 A37 modifications in tRNAs from blood and urine samples.

Subsets of mitochondrial transfer RNA (tRNA) contain the N6 -isopentenyladenosine (i6 A) or 2-methylthio-N6 -isopentenyladenosine (ms2 i6 A) modification at position A37, which is adjacent to an anticodon. These modifications are essential for efficient protein translation in mitochondria and contribute to energy metabolism. The first step in i6 A and ms2 i6 A modifications is catalyzed by tRNA isopentenyltransferase, which is encoded by the TRIT1 gene. Herein, we report a girl with a developmental delay, frequent episodes of seizures induced by febrile illness, and myoclonic epilepsy who had compound heterozygous missense mutations in TRIT1. A mass spectrometry analysis of RNA nucleoside obtained from the subject's peripheral blood and urine showed a marked decrease in both i6 A and ms2 i6 A modifications. These results suggest that the mitochondrial disorder was caused by defective tRNA isopentenylation arising from a loss-of-function mutation in TRIT1. Furthermore, the present observations suggest that noninvasive biochemical analysis using peripheral blood and urine samples are sufficient for the diagnosis of TRIT1-related disorders, making muscle biopsy for the direct measurement of oxidative phosphorylation unnecessary. Such biochemical analyses before the start of antiepileptic medications would be beneficial to avoid hepatotoxicity in patients with possible mitochondrial disorders.

Cdk5rap1-mediated 2-methylthio-N6-isopentenyladenosine modification is absent from nuclear-derived RNA species.

2-Methylthio-N6-isopentenyl modification of adenosine (ms2i6A) is an evolutionally conserved modification that is found in transfer RNAs (tRNAs). We have recently shown that Cdk5 regulatory subunit-associated protein 1 (Cdk5rap1) specifically converts i6A to ms2i6A at position A37 of four mitochondrial DNA-encoded tRNAs, and that the modification regulates efficient mitochondrial translation and energy metabolism in mammals. Curiously, a previous study reported that ms2i6A is present abundantly in nuclear-derived RNA species such as microRNAs, but not in tRNA fractions. To fully understand the molecular property of ms2i6A, the existence of non-canonical ms2i6A must be carefully validated. In the present study, we examined ms2i6A in total RNA purified from human and murine ρ0 cells, in which mitochondrial DNA-derived tRNAs were completely depleted. The ms2i6A was not detected in these cells at all. We generated a monoclonal antibody against ms2i6A and examined ms2i6A in murine RNAs using the antibody. The anti-ms2i6A antibody only reacted with the tRNA fractions and not in other RNA species. Furthermore, immunocytochemistry analysis using the antibody showed the predominant localization of ms2i6A in mitochondria and co-localization with the mitochondrial elongation factor Tu. Taken together, we propose that ms2i6A is a mitochondrial tRNA-specific modification and is absent from nuclear-encoded RNA species.

Antibody-mediated modulation of cytokinins in tobacco: organ-specific changes in cytokinin homeostasis.

Cytokinins comprise a group of phytohormones with an organ-specific mode of action. Although the mechanisms controlling the complex networks of cytokinin metabolism are partially known, the role of individual cytokinin types in the maintenance of cytokinin homeostasis remains unclear. Utilizing the overproduction of single-chain Fv antibodies selected for their ability to bind trans-zeatin riboside and targeted to the endoplasmic reticulum, we post-synthetically modulated cytokinin ribosides, the proposed transport forms of cytokinins. We observed asymmetric activity of cytokinin biosynthetic genes and cytokinin distribution in wild-type tobacco seedlings with higher cytokinin abundance in the root than in the shoot. Antibody-mediated modulation of cytokinin ribosides further enhanced the relative cytokinin abundance in the roots and induced cytokinin-related phenotypes in an organ-specific manner. The activity of cytokinin oxidase/dehydrogenase in the roots was strongly up-regulated in response to antibody-mediated formation of the cytokinin pool in the endoplasmic reticulum. However, we only detected a slight decrease in the root cytokinin levels. In contrast, a significant decrease of cytokinins occurred in the shoot. We suggest the roots as the main site of cytokinin biosynthesis in tobacco seedlings. Conversely, cytokinin levels in the shoot seem to depend largely on long-range transport of cytokinin ribosides from the root and their subsequent metabolic activation.

Grain development and endogenous hormones in summer maize (Zea mays L.) submitted to different light conditions.

Low light is a type of abiotic stress that seriously affects plant growth and production efficiency. We investigated the response mechanisms of summer maize to low light by measuring the changes in endogenous hormones in the grains and during grain filling in summer maize at different light intensities to provide a theoretical basis for the production and management of summer maize under light stress. We applied different light treatments in a field experiment as follows: S, shading from tassel stage (VT) to maturity stage (R6); CK, natural lighting in the field; and L, increasing light from VT to R6. The shading level was 60%, and the maximum illumination intensity of the increasing light treatment on cloudy days was 1600-1800 µmol m-2 s-1. Compared with the control, shading significantly increased the grain abscisic acid (ABA) content at 5-20 days after pollination and decreased the indole acetic acid (IAA), zeatin riboside (ZR), and gibberellin (GA) contents (P < 0.05). The grain-filling rate decreased under shading conditions. Meanwhile, the grain volume, grain weight, and yield all decreased; the yields in 2013 and 2014 decreased by 61 and 60%, respectively. The grain IAA, ZR, and GA contents were increased by increasing light. The grain ABA content at 5-20 days after pollination did not significantly differ from that of CK (P < 0.05). After 20 days after pollination, the ABA content decreased, the grain-filling rate and the filling duration increased, and the yield increased. However, shading after anthesis increased the grain ABA content and reduced the IAA, ZR, and GA contents. Grain growth and development were inhibited, and the yield decreased. The grain ABA content decreased; the IAA, ZR, and GA contents increased; and the yield increased after increasing light. The results indicate that different light intensities regulated the levels of grains endogenous hormones, which influenced the grain-filling rate and duration, and consequently, regulated grain weight and yield.

The modified base isopentenyladenosine and its derivatives in tRNA.

Base 37 in tRNA, 3'-adjacent to the anticodon, is occupied by a purine base that is thought to stabilize codon recognition by stacking interactions on the first Watson-Crick base pair. If the first codon position forms an A.U or U.A base pair, the purine is likely further modified in all domains of life. One of the first base modifications found in tRNA is N6-isopentenyl adenosine (i6A) present in a fraction of tRNAs in bacteria and eukaryotes, which can be further modified to 2-methyl-thio-N6-isopentenyladenosine (ms2i6A) in a subset of tRNAs. Homologous tRNA isopentenyl transferase enzymes have been identified in bacteria (MiaA), yeast (Mod5, Tit1), roundworm (GRO-1), and mammals (TRIT1). In eukaryotes, isopentenylation of cytoplasmic and mitochondrial tRNAs is mediated by products of the same gene. Accordingly, a patient with homozygous mutations in TRIT1 has mitochondrial disease. The role of i6A in a subset of tRNAs in gene expression has been linked with translational fidelity, speed of translation, skewed gene expression, and non-sense suppression. This review will not cover the action of i6A as a cytokinin in plants or the potential function of Mod5 as a prion in yeast.

Physiological and molecular analysis on root growth associated with the tolerance to aluminum and drought individual and combined in Tibetan wild and cultivated barley.

MAIN CONCLUSION: The drought-stimulated gene expression of NCED, SUS, and KS - DHN and ABA signal cross-talk with other phytohormones maintains barley root growth under drought stress at pH 4.0 plus polyethylene glycol plus aluminum. Aluminum (Al) toxicity and drought are two major factors that limit barley production. In this work, the individual and combined effects of Al/acid and polyethylene glycol (PEG 6000) induced drought stress that suppressed root growth and caused oxidative damage as characterized by increased H2O2 and O2(.-) accumulation. The wild-barley genotypes, XZ5 and XZ29, exhibited a higher tolerance than the two cultivars Dayton (Al tolerant) and Tadmor (drought tolerant) under combined stress (pH 4.0 + PEG + Al). The oxidative damage induced by PEG was more severe at pH 4.0 than at pH 6.0. In XZ29, the highest root secretion of malate and citrate was recorded, and the least Al uptake in the four genotypes. In XZ5, a peak accumulation of ABA and minor synthesis of zeatin riboside and ethylene were found being essential in maintaining primary root elongation and root hair development. PEG-induced drought stress repressed Al uptake in root tips, with a lower increase in callose formation and HvMATE (Hordeum vulgare multidrug and toxic compound exudation) expression compared to Al-induced callose production. Stress by pH 4.0 + PEG + Al up-regulated 9-cis-epoxycarotenoid dioxygenase (NCED) which is involved in ABA biosynthesis. Such treatment stimulated the regulation of ABA-dependent genes sucrose synthase (SUS) and KS-type dehydrin (KS-DHN) in root tips. Our results suggest that the tolerance ranking to pH 4.0 + PEG + Al stress in Tibetan wild barley by gene expression is closely correlated to physiological indices. The results show that acclimatisation to pH 4.0 + PEG + Al stress involves specific responses in XZ5 and XZ29. The present study provides insights into the effects of Al/acid and drought combined stress on the abundance of physiological indices in the roots of barley varieties.

Methylated Cytokinins from the Phytopathogen Rhodococcus fascians Mimic Plant Hormone Activity.

Cytokinins (CKs), a class of phytohormones that regulate plant growth and development, are also synthesized by some phytopathogens to disrupt the hormonal balance and to facilitate niche establishment in their hosts. Rhodococcus fascians harbors the fasciation (fas) locus, an operon encoding several genes homologous to CK biosynthesis and metabolism. This pathogen causes unique leafy gall symptoms reminiscent of CK overproduction; however, bacterial CKs have not been clearly correlated with the severe symptoms, and no virulence-associated unique CKs or analogs have been identified. Here, we report the identification of monomethylated N(6)-(∆(2)-isopentenyl)adenine and dimethylated N(6)-(∆(2)-isopentenyl)adenine (collectively, methylated cytokinins [MeCKs]) from R. fascians. MeCKs were recognized by a CK receptor and up-regulated type-A ARABIDOPSIS THALIANA RESPONSE REGULATOR genes. Treatment with MeCKs inhibited root growth, a hallmark of CK action, whereas the receptor mutant was insensitive. MeCKs were retained longer in planta than canonical CKs and were poor substrates for a CK oxidase/dehydrogenase, suggesting enhanced biological stability. MeCKs were synthesized by S-adenosyl methionine-dependent methyltransferases (MT1 and MT2) that are present upstream of the fas genes. The best substrate for methylation was isopentenyl diphosphate. MT1 and MT2 catalyzed distinct methylation reactions; only the MT2 product was used by FAS4 to synthesize monomethylated N(6)-(∆(2)-isopentenyl)adenine. The MT1 product was dimethylated by MT2 and used as a substrate by FAS4 to produce dimethylated N(6)-(∆(2)-isopentenyl)adenine. Chemically synthesized MeCKs were comparable in activity. Our results strongly suggest that MeCKs function as CK mimics and play a role in this plant-pathogen interaction.

H2O2 and ABA signaling are responsible for the increased Na+ efflux and water uptake in Gossypium hirsutum L. roots in the non-saline side under non-uniform root zone salinity.

Non-uniform root salinity increases the Na(+)efflux, water use, and growth of the root in non-saline side, which may be regulated by some form of signaling induced by the high-salinity side. However, the signaling and its specific function have remained unknown. Using a split-root system to simulate a non-uniform root zone salinity in Gossypium hirsutum L., we showed that the up-regulated expression of sodium efflux-related genes (SOS1, SOS2, PMA1, and PMA2) and water uptake-related genes (PIP1 and PIP2) was possibly involved in the elevated Na(+) efflux and water use in the the roots in the non-saline side. The increased level of indole acetic acid (IAA) in the non-saline side was the likely cause of the increased root growth. Also, the abscisic acid (ABA) and H2O2 contents in roots in the non-saline side increased, possibly due to the increased expression of their key biosynthesis genes, NCED and RBOHC, and the decreased expression of ABA catabolic CYP707A genes. Exogenous ABA added to the non-saline side induced H2O2 generation by up-regulating the RBOHC gene, but this was decreased by exogenous fluridone. Exogenous H2O2 added to the non-saline side reduced the ABA content by down-regulating NCED genes, which can be induced by diphenylene iodonium (DPI) treatment in the non-saline side, suggesting a feedback mechanism between ABA and H2O2.Both exogenous ABA and H2O2 enhanced the expression of SOS1, PIP1;7 ,PIP2;2, and PIP2;10 genes, but these were down-regulated by fluridone and DPI, suggesting that H2O2 and ABA are important signals for increasing root Na(+) efflux and water uptake in the roots in the non-saline side.

Changes in transcription of cytokinin metabolism and signalling genes in grape (Vitis vinifera L.) berries are associated with the ripening-related increase in isopentenyladenine.

BACKGROUND: Cytokinins are known to play an important role in fruit set and early fruit growth, but their involvement in later stages of fruit development is less well understood. Recent reports of greatly increased cytokinin concentrations in the flesh of ripening kiwifruit (Actinidia deliciosa (A. Chev.) C.F. Liang & A.R. Ferguson) and grapes (Vitis vinifera L.) have suggested that these hormones are implicated in the control of ripening-related processes. RESULTS: A similar pattern of isopentenyladenine (iP) accumulation was observed in the ripening fruit of several grapevine cultivars, strawberry (Fragaria ananassa Duch.) and tomato (Solanum lycopersicum Mill.), suggesting a common, ripening-related role for this cytokinin. Significant differences in maximal iP concentrations between grapevine cultivars and between fruit species might reflect varying degrees of relevance or functional adaptations of this hormone in the ripening process. Grapevine orthologues of five Arabidopsis (Arabidopsis thaliana L.) gene families involved in cytokinin metabolism and signalling were identified and analysed for their expression in developing grape berries and a range of other grapevine tissues. Members of each gene family were characterised by distinct expression profiles during berry development and in different grapevine organs, suggesting a complex regulation of cellular cytokinin activities throughout the plant. The post-veraison-specific expression of a set of biosynthesis, activation, perception and signalling genes together with a lack of expression of degradation-related genes during the ripening phase were indicative of a local control of berry iP concentrations leading to the observed accumulation of iP in ripening grapes. CONCLUSIONS: The transcriptional analysis of grapevine genes involved in cytokinin production, degradation and response has provided a possible explanation for the ripening-associated accumulation of iP in grapes and other fruit. The pre- and post-veraison-specific expression of different members from each of five gene families suggests a highly complex and finely-tuned regulation of cytokinin concentrations and response to different cytokinin species at particular stages of fruit development. The same complexity and specialisation is also reflected in the distinct expression profiles of cytokinin-related genes in other grapevine organs.

MicroRNA156: a potential graft-transmissible microRNA that modulates plant architecture and tuberization in Solanum tuberosum ssp. andigena.

MicroRNA156 (miR156) functions in maintaining the juvenile phase in plants. However, the mobility of this microRNA has not been demonstrated. So far, only three microRNAs, miR399, miR395, and miR172, have been shown to be mobile. We demonstrate here that miR156 is a potential graft-transmissible signal that affects plant architecture and tuberization in potato (Solanum tuberosum). Under tuber-noninductive (long-day) conditions, miR156 shows higher abundance in leaves and stems, whereas an increase in abundance of miR156 has been observed in stolons under tuber-inductive (short-day) conditions, indicative of a photoperiodic control. Detection of miR156 in phloem cells of wild-type plants and mobility assays in heterografts suggest that miR156 is a graft-transmissible signal. This movement was correlated with changes in leaf morphology and longer trichomes in leaves. Overexpression of miR156 in potato caused a drastic phenotype resulting in altered plant architecture and reduced tuber yield. miR156 overexpression plants also exhibited altered levels of cytokinin and strigolactone along with increased levels of LONELY GUY1 and StCyclin D3.1 transcripts as compared with wild-type plants. RNA ligase-mediated rapid amplification of complementary DNA ends analysis validated SQUAMOSA PROMOTER BINDING-LIKE3 (StSPL3), StSPL6, StSPL9, StSPL13, and StLIGULELESS1 as targets of miR156. Gel-shift assays indicate the regulation of miR172 by miR156 through StSPL9. miR156-resistant SPL9 overexpression lines exhibited increased miR172 levels under a short-day photoperiod, supporting miR172 regulation via the miR156-SPL9 module. Overall, our results strongly suggest that miR156 is a phloem-mobile signal regulating potato development.

Antiangiogenic effects of N6-isopentenyladenosine, an endogenous isoprenoid end product, mediated by AMPK activation.

N6-isopentenyladenosine (iPA), an end product of the mevalonate pathway with an isopentenyl chain, is already known to exert a suppressor effect against various tumors. In this work, we investigated whether iPA also directly interferes with the angiogenic process, which is fundamental to tumor growth and progression. To this end, using human umbilical vein endothelial cells (HUVECs) as a suitable in vitro model of angiogenesis, we evaluated their viability, proliferation, migration, invasion, tube formation in response to iPA, and molecular mechanisms involved. Data were corroborated in mice by using a gel plug assay. iPA dose- and time-dependently inhibited all the neoangiogenesis stages, with an IC50 of 0.98 µM. We demonstrated for the first time, by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS), that iPA was monophosphorylated into 5'-iPA-monophosphate (iPAMP) by the adenosine kinase (ADK) inside the cells. iPAMP is the active form that inhibits angiogenesis through the direct activation of AMP-kinase (AMPK). Indeed, all effects were completely reversed by pretreatment with 5-iodotubercidin (5-Itu), an ADK inhibitor. The isoprenoid intermediate isopentenyl pyrophosphate (IPP), which shares the isopentenyl moiety with iPA, was ineffective in the inhibition of angiogenesis, thus showing that the iPA structure is specific for the observed effects. In conclusion, iPA is a novel AMPK activator and could represent a useful tool for the treatment of diseases where excessive neoangiogenesis is the underlying pathology.