Gene cluster conservation provides insight into cercosporin biosynthesis and extends production to the genus Colletotrichum.

Species in the genus Cercospora cause economically devastating diseases in sugar beet, maize, rice, soy bean, and other major food crops. Here, we sequenced the genome of the sugar beet pathogen Cercospora beticola and found it encodes 63 putative secondary metabolite gene clusters, including the cercosporin toxin biosynthesis (CTB) cluster. We show that the CTB gene cluster has experienced multiple duplications and horizontal transfers across a spectrum of plant pathogenic fungi, including the wide-host range Colletotrichum genus as well as the rice pathogen Magnaporthe oryzae Although cercosporin biosynthesis has been thought to rely on an eight-gene CTB cluster, our phylogenomic analysis revealed gene collinearity adjacent to the established cluster in all CTB cluster-harboring species. We demonstrate that the CTB cluster is larger than previously recognized and includes cercosporin facilitator protein, previously shown to be involved with cercosporin autoresistance, and four additional genes required for cercosporin biosynthesis, including the final pathway enzymes that install the unusual cercosporin methylenedioxy bridge. Lastly, we demonstrate production of cercosporin by Colletotrichum fioriniae, the first known cercosporin producer within this agriculturally important genus. Thus, our results provide insight into the intricate evolution and biology of a toxin critical to agriculture and broaden the production of cercosporin to another fungal genus containing many plant pathogens of important crops worldwide.

Treatment of potato tubers with the synthetic cytokinin 1-(α-ethylbenzyl)-3-nitroguanidine results in rapid termination of endodormancy and induction of transcripts associated with cell proliferation and growth.

Perennial plants undergo repression of meristematic activity in a process called dormancy. Dormancy is a complex metabolic process with implications for plant breeding and crop yield. Endodormancy, a specific subclass of dormancy, is characteristic of internal physiological mechanisms resulting in growth suppression. In this study, we examine transcriptional changes associated with the natural cessation of endodormancy in potato tuber meristems and in endodormant tubers treated with the cytokinin analog 1-(α-ethylbenzyl)-3-niroguanidine (NG), which terminates dormancy. RNA-sequencing was used to examine transcriptome changes between endodormant and non-dormant meristems from four different harvest years. A total of 35,091 transcripts were detected with 2132 differentially expressed between endodormant and non-dormant tuber meristems. Endodormant potato tubers were treated with the synthetic cytokinin NG and transcriptome changes analyzed using RNA-seq after 1, 4, and 7 days following NG exposure. A comparison of natural cessation of dormancy and NG-treated tubers demonstrated that by 4 days after NG exposure, potato meristems exhibited transcriptional profiles similar to the non-dormant state with elevated expression of multiple histones, a variety of cyclins, and other genes associated with proliferation and cellular replication. Three homologues encoding for CYCD3 exhibited elevated expression in both non-dormant and NG-treated potato tissues. These results suggest that NG terminates dormancy and induces expression cell cycle-associated transcripts within 4 days of treatment.

Increase in ACC oxidase levels and activities during paradormancy release of leafy spurge (Euphorbia esula) buds.

The plant hormone ethylene is known to affect various developmental processes including dormancy and growth. Yet, little information is available about the role of ethylene during paradormancy release in underground adventitious buds of leafy spurge. In this study, we examined changes in ethylene evolution and the ethylene biosynthetic enzyme ACC oxidase following paradormancy release (growth induction). Our results did not show an obvious increase in ethylene during bud growth. However, when buds were incubated with 1 mM ACC, ethylene levels were higher in growing than non-growing buds, suggesting that the levels of ACC oxidase increased in growing buds. Real-time qPCR indicated that the transcript of a Euphorbia esula ACC oxidase (Ee-ACO) increased up to threefold following growth induction. In addition, a 2.5- to 4-fold increase in ACO activity was observed 4 days after decapitation, and the Ee-ACO accounted for 40 % of the total ACO activity. Immunoblot analyses identified a 36-kD Ee-ACO protein that increased in expression during bud growth. This protein was highly expressed in leaves, moderately expressed in crown buds, stems and meristems, and weakly expressed in roots and flowers. Immunolocalization of Ee-ACO on growing bud sections revealed strong labeling of the nucleus and cytoplasm in cells at the shoot apical meristem and leaf primordia. An exception to this pattern occurred in cells undergoing mitosis, where labeling of Ee-ACO was negligible. Taken together, our results indicated an increase in the levels of Ee-ACO during paradormancy release of leafy spurge that was not correlated with an increase in ethylene synthesis.

Coupling calcium/calmodulin-mediated signaling and herbivore-induced plant response through calmodulin-binding transcription factor AtSR1/CAMTA3.

Calcium/calmodulin (Ca(2+)/CaM) has long been considered a crucial component in wound signaling pathway. However, very few Ca(2+)/CaM-binding proteins have been identified which regulate plant responses to herbivore attack/wounding stress. We have reported earlier that a family of Ca(2+)/CaM-binding transcription factors designated as AtSRs (also known as AtCAMTAs) can respond differentially to wounding stress. Further studies revealed that AtSR1/CAMTA3 is a negative regulator of plant defense, and Ca(2+)/CaM-binding to AtSR1 is indispensable for the suppression of salicylic acid (SA) accumulation and disease resistance. Here we report that Ca(2+)/CaM-binding is also critical for AtSR1-mediated herbivore-induced wound response. Interestingly, atsr1 mutant plants are more susceptible to herbivore attack than wild-type plants. Complementation of atsr1 mutant plants by overexpressing wild-type AtSR1 protein can effectively restore plant resistance to herbivore attack. However, when mutants of AtSR1 with impaired CaM-binding ability were overexpressed in atsr1 mutant plants, plant resistance to herbivore attack was not restored, suggesting a key role for Ca(2+)/CaM-binding in wound signaling. Furthermore, it was observed that elevated SA levels in atsr1 mutant plants have a negative impact on both basal and induced biosynthesis of jasmonates (JA). These results revealed that Ca(2+)/CaM-mediated signaling regulates plant response to herbivore attack/wounding by modulating the SA-JA crosstalk through AtSR1.

The sprout inhibitor 1,4-dimethylnaphthalene induces the expression of the cell cycle inhibitors KRP1 and KRP2 in potatoes.

The suppression of sprout growth is critical for the long-term storage of potato tubers. 1,4-Dimethylenapthlene (DMN) is a new class of sprout control agent but the metabolic mode of action for this compound has yet to be elucidated. Changes in transcriptional profiles of meristems isolated from potato tubers treated with the DMN were investigated using an Agilent 44 K 60-mer-oligo microarray. RNA was isolated from nondormant Russet Burbank meristems isolated from tubers treated with DMN for 3 days or activated charcoal as a control. RNA was used to develop probes that were hybridized against a microarray developed by the Potato Oligo Chip Initiative. Analysis of the array data was conducted in two stages: total array data was examined using a linear model and the software Limma and pathway analysis was conducted by linking the potato sequences to the Arabidopsis thaliana. DMN elicited a change in a number of transcripts associated with cold responses, water regulation, salt stress, and osmotic adjustment. DMN also resulted in a repression of cyclin or cyclin-like transcripts. DMN also resulted in a 50% decrease in thymidine incorporation suggesting a repression of the S phase of the cell cycle. Quantitative real-time polymerase chain reaction analysis demonstrated that DMN increased transcripts for the cell cycle inhibitors KRP1 and KRP2. We conclude the DMN results in alteration of genes associated with the maintenance of a G1/S phase block possibly through the induction of the cell cycle inhibitors KRP1 and KRP2.

Auxin and ABA act as central regulators of developmental networks associated with paradormancy in Canada thistle (Cirsium arvense).

Dormancy in underground vegetative buds of Canada thistle, an herbaceous perennial weed, allows escape from current control methods and contributes to its invasive nature. In this study, ~65 % of root sections obtained from greenhouse propagated Canada thistle produced new vegetative shoots by 14 days post-sectioning. RNA samples obtained from sectioned roots incubated 0, 24, 48, and 72 h at 25°C under 16:8 h light-dark conditions were used to construct four MID-tagged cDNA libraries. Analysis of in silico data obtained using Roche 454 GS-FLX pyrosequencing technologies identified molecular networks associated with paradormancy release in underground vegetative buds of Canada thistle. Sequencing of two replicate plates produced ~2.5 million ESTs with an average read length of 362 bases. These ESTs assembled into 67358 unique sequences (21777 contigs and 45581 singlets) and annotation against the Arabidopsis database identified 15232 unigenes. Among the 15232 unigenes, we identified processes enriched with transcripts involved in plant hormone signaling networks. To follow-up on these results, we examined hormone profiles in roots, which identified changes in abscisic acid (ABA) and ABA metabolites, auxins, and cytokinins post-sectioning. Transcriptome and hormone profiling data suggest that interaction between auxin- and ABA-signaling regulate paradormancy maintenance and release in underground adventitious buds of Canada thistle. Our proposed model shows that sectioning-induced changes in polar auxin transport alters ABA metabolism and signaling, which further impacts gibberellic acid signaling involving interactions between ABA and FUSCA3. Here we report that reduced auxin and ABA-signaling, in conjunction with increased cytokinin biosynthesis post-sectioning supports a model where interactions among hormones drives molecular networks leading to cell division, differentiation, and vegetative outgrowth.

Chemical inhibition of potato ABA-8'-hydroxylase activity alters in vitro and in vivo ABA metabolism and endogenous ABA levels but does not affect potato microtuber dormancy duration.

The effects of azole-type P450 inhibitors and two metabolism-resistant abscisic acid (ABA) analogues on in vitro ABA-8'-hydroxylase activity, in planta ABA metabolism, endogenous ABA content, and tuber meristem dormancy duration were examined in potato (Solanum tuberosum L. cv. Russet Burbank). When functionally expressed in yeast, three potato CYP707A genes were demonstrated to encode enzymatically active ABA-8'-hydroxylases with micromolar affinities for (+)-ABA. The in vitro activity of the three enzymes was inhibited by the P450 azole-type inhibitors ancymidol, paclobutrazol, diniconazole, and tetcyclasis, and by the 8'-acetylene- and 8'-methylene-ABA analogues, with diniconazole and tetcyclasis being the most potent inhibitors. The in planta metabolism of [(3)H](±)-ABA to phaseic acid and dihydrophaseic acid in tuber meristems was inhibited by diniconazole, tetcyclasis, and to a lesser extent by 8'-acetylene- and 8'-methylene-ABA. Continuous exposure of in vitro generated microtubers to diniconazole resulted in a 2-fold increase in endogenous ABA content and a decline in dihydrophaseic acid content after 9 weeks of development. Similar treatment with 8'-acetylene-ABA had no effects on the endogenous contents of ABA or phaseic acid but reduced the content of dihydrophaseic acid. Tuber meristem dormancy progression was determined ex vitro in control, diniconazole-, and 8'-acetylene-ABA-treated microtubers following harvest. Continuous exposure to diniconazole during microtuber development had no effects on subsequent sprouting at any time point. Continuous exposure to 8'-acetylene-ABA significantly increased the rate of microtuber sprouting. The results indicate that, although a decrease in ABA content is a hallmark of tuber dormancy progression, the decline in ABA levels is not a prerequisite for dormancy exit and the onset of tuber sprouting.

The metabolic and developmental roles of carotenoid cleavage dioxygenase4 from potato.

The factors that regulate storage organ carotenoid content remain to be fully elucidated, despite the nutritional and economic importance of this class of compound. Recent findings suggest that carotenoid pool size is determined, at least in part, by the activity of carotenoid cleavage dioxygenases. The aim of this study was to investigate whether Carotenoid Cleavage Dioxygenase4 (CCD4) activity affects potato (Solanum tuberosum) tuber carotenoid content. Microarray analysis revealed elevated expression of the potato CCD4 gene in mature tubers from white-fleshed cultivars compared with higher carotenoid yellow-fleshed tubers. The expression level of the potato CCD4 gene was down-regulated using an RNA interference (RNAi) approach in stable transgenic lines. Down-regulation in tubers resulted in an increased carotenoid content, 2- to 5-fold higher than in control plants. The increase in carotenoid content was mainly due to elevated violaxanthin content, implying that this carotenoid may act as the in vivo substrate. Although transcript level was also reduced in plant organs other than tubers, such as leaves, stems, and roots , there was no change in carotenoid content in these organs. However, carotenoid levels were elevated in flower petals from RNAi lines. As well as changes in tuber carotenoid content, tubers from RNAi lines exhibited phenotypes such as heat sprouting, formation of chain tubers, and an elongated shape. These results suggest that the product of the CCD4 reaction may be an important factor in tuber heat responses.

Apoplast proteome reveals that extracellular matrix contributes to multistress response in poplar.

BACKGROUND: Riverine ecosystems, highly sensitive to climate change and human activities, are characterized by rapid environmental change to fluctuating water levels and siltation, causing stress on their biological components. We have little understanding of mechanisms by which riverine plant species have developed adaptive strategies to cope with stress in dynamic environments while maintaining growth and development. RESULTS: We report that poplar (Populus spp.) has evolved a systems level "stress proteome" in the leaf-stem-root apoplast continuum to counter biotic and abiotic factors. To obtain apoplast proteins from P. deltoides, we developed pressure-chamber and water-displacement methods for leaves and stems, respectively. Analyses of 303 proteins and corresponding transcripts coupled with controlled experiments and bioinformatics demonstrate that poplar depends on constitutive and inducible factors to deal with water, pathogen, and oxidative stress. However, each apoplast possessed a unique set of proteins, indicating that response to stress is partly compartmentalized. Apoplast proteins that are involved in glycolysis, fermentation, and catabolism of sucrose and starch appear to enable poplar to grow normally under water stress. Pathogenesis-related proteins mediating water and pathogen stress in apoplast were particularly abundant and effective in suppressing growth of the most prevalent poplar pathogen Melampsora. Unexpectedly, we found diverse peroxidases that appear to be involved in stress-induced cell wall modification in apoplast, particularly during the growing season. Poplar developed a robust antioxidative system to buffer oxidation in stem apoplast. CONCLUSION: These findings suggest that multistress response in the apoplast constitutes an important adaptive trait for poplar to inhabit dynamic environments and is also a potential mechanism in other riverine plant species.

The sprout inhibitors chlorpropham and 1,4-dimethylnaphthalene elicit different transcriptional profiles and do not suppress growth through a prolongation of the dormant state.

Chlorpropham (CIPC) and 1,4-dimethylnapthalene (DMN) are used to control postharvest sprouting of potato tubers. CIPC alters microtubule structure and function resulting in inhibition of cell division. The mechanism of action of DMN is unknown but, because it is a natural product found in potato tubers, there is speculation that it inhibits sprout growth by prolonging the dormant state. To address this issue, the effects of CIPC and DMN on abscisic acid (ABA) content and gene expression in potato tuber meristems were determined and compared to those found in dormant and non-dormant meristems. Dormancy progression was accompanied by a dramatic decline in ABA content and the ABA levels in meristems isolated from CIPC- and DMN- treated tubers were identical to the levels found in nondormant meristems demonstrating that sprout repression is not a function of elevated ABA. Evaluation of transcriptional profiles using cDNA microarrays demonstrated that there were similarities between CIPC- and DMN- treated tuber tissues particularly in transcripts that encode phosphatases and proteins associated with oxygen-related metabolism. Despite these similarities, there were significant differences in transcript profiles derived from treatment with either CIPC or DMN and the dormant state. These results suggested the mechanisms-of -action of DMN and CIPC are distinct and not due to a prolongation of the normal dormant condition.

The qSD12 underlying gene promotes abscisic acid accumulation in early developing seeds to induce primary dormancy in rice.

Seeds acquire primary dormancy during their development and the phytohormone abscisic acid (ABA) is known to play a role in inducing the dormancy. qSD12 is a major seed dormancy quantitative trait locus (QTL) identified from weedy rice. This research was conducted to identify qSD12 candidate genes, isolate the candidates from weedy rice, and determine the relation of the dormancy gene to ABA. A fine mapping experiment, followed by marker-assisted progeny testing for selected recombinants, narrowed down qSD12 to a genomic region of <75 kb, where there are nine predicted genes including a cluster of six transposon/retrotransposon protein genes and three putative (a PIL5, a hypothetic protein, and a bHLH transcription factor) genes based on the annotated Nipponbare genome sequence. The PIL5 and bHLH genes are more likely to be the QTL candidate genes. A bacterial artificial chromosome (BAC) library equivalent to 8-9 times of the haploid genome size was constructed for the weedy rice. One of the two BAC contigs developed from the library covers the PIL5 to bHLH interval. A pair of lines different only in the QTL-containing region of <200 kb was developed as isogenic lines for the qSD12 dormancy and non-dormancy alleles. The dormant line accumulated much higher ABA in 10-day developing seeds than the non-dormant line. In the QTL-containing region there is no predicted gene that has been assigned to ABA biosynthetic or metabolic pathways. Thus, it is concluded that the qSD12 underlying gene promotes ABA accumulation in early developing seeds to induce primary seed dormancy.

Age-induced loss of wound-healing ability in potato tubers is partly regulated by ABA.

Wounding of potato (Solanum tuberosum L.) tubers induces the development of a suberized closing layer and wound periderm that resists desiccation and microbial invasion. Wound-healing ability declines with tuber age (storage period). The mechanism of loss in healing capacity with age is not known; however, upregulation of superoxide production, increased ABA biosynthesis and phenylalanine ammonia lyase (PAL) activity in response to wounding are processes critical to the development of a suberized closing layer and wound periderm. Therefore, the role of ABA in modulating the age-induced loss of wound-healing ability of tubers was examined. Non-wounded older tubers had 86% less ABA (dry matter basis) than younger tubers. PAL transcript increased in younger tubers within 24 h of wounding, but transcription was delayed by 5 days in older tubers. Wound-induced PAL activity increased more rapidly in younger than older tubers. ABA treatment increased PAL expression and activity in tissue from both ages of tubers and restored the 24 h transcription time line in older tubers. Moreover, ABA treatment of wounded older tubers enhanced their resistance to water vapor loss following a 6-day wound-healing period. Wound-induced accumulation of suberin poly(phenolic(s)) (SPP) and suberin poly(aliphatic(s)) (SPA) was measurably slower in older versus younger tubers. ABA treatment hastened SPP accumulation in older tubers to match that in younger tubers, but only enhanced SPA accumulations over the initial 4 days of healing. Age-induced loss of wound-healing ability is thus partly due to reduced ability to accumulate ABA and modulate the production of SPP through PAL in response to wounding and to dysfunction in the downstream signaling events that couple SPA biosynthesis and/or deposition to ABA. ABA treatment partly restored the healing ability of older tubers by enhancing the accumulation of SPP without restoring wound-induced superoxide forming ability to the level of younger tubers. The coupling of phenolic monomers into the poly(phenolic) domain of suberin was therefore not limited by the diminished wound-induced superoxide production of older tubers.

RUNX1-mutated families show phenotype heterogeneity and a somatic mutation profile unique to germline predisposed AML.

First reported in 1999, germline runt-related transcription factor 1 (RUNX1) mutations are a well-established cause of familial platelet disorder with predisposition to myeloid malignancy (FPD-MM). We present the clinical phenotypes and genetic mutations detected in 10 novel RUNX1-mutated FPD-MM families. Genomic analyses on these families detected 2 partial gene deletions, 3 novel mutations, and 5 recurrent mutations as the germline RUNX1 alterations leading to FPD-MM. Combining genomic data from the families reported herein with aggregated published data sets resulted in 130 germline RUNX1 families, which allowed us to investigate whether specific germline mutation characteristics (type, location) could explain the large phenotypic heterogeneity between patients with familial platelet disorder and different HMs. Comparing the somatic mutational signatures between the available familial (n = 35) and published sporadic (n = 137) RUNX1-mutated AML patients showed enrichment for somatic mutations affecting the second RUNX1 allele and GATA2. Conversely, we observed a decreased number of somatic mutations affecting NRAS, SRSF2, and DNMT3A and the collective genes associated with CHIP and epigenetic regulation. This is the largest aggregation and analysis of germline RUNX1 mutations performed to date, providing a unique opportunity to examine the factors underlying phenotypic differences and disease progression from FPD to MM.

Transcriptome analysis identifies novel responses and potential regulatory genes involved in seasonal dormancy transitions of leafy spurge (Euphorbia esula L.).

BACKGROUND: Dormancy of buds is a critical developmental process that allows perennial plants to survive extreme seasonal variations in climate. Dormancy transitions in underground crown buds of the model herbaceous perennial weed leafy spurge were investigated using a 23 K element cDNA microarray. These data represent the first large-scale transcriptome analysis of dormancy in underground buds of an herbaceous perennial species. Crown buds collected monthly from August through December, over a five year period, were used to monitor the changes in the transcriptome during dormancy transitions. RESULTS: Nearly 1,000 genes were differentially-expressed through seasonal dormancy transitions. Expected patterns of gene expression were observed for previously characterized genes and physiological processes indicated that resolution in our analysis was sufficient for identifying shifts in global gene expression. CONCLUSION: Gene ontology of differentially-expressed genes suggests dormancy transitions require specific alterations in transport functions (including induction of a series of mitochondrial substrate carriers, and sugar transporters), ethylene, jasmonic acid, auxin, gibberellic acid, and abscisic acid responses, and responses to stress (primarily oxidative and cold/drought). Comparison to other dormancy microarray studies indicated that nearly half of the genes identified in our study were also differentially expressed in at least two other plant species during dormancy transitions. This comparison allowed us to identify a particular MADS-box transcription factor related to the DORMANCY ASSOCIATED MADS-BOX genes from peach and hypothesize that it may play a direct role in dormancy induction and maintenance through regulation of FLOWERING LOCUS T.

Regulatory involvement of abscisic acid in potato tuber wound-healing.

Rapid wound-healing is crucial in protecting potato tubers from infection and dehydration. Wound-induced suberization and the accumulation of hydrophobic barriers to reduce water vapour conductance/loss are principal protective wound-healing processes. However, little is known about the cognate mechanisms that effect or regulate these processes. The objective of this research was to determine the involvement of abscisic acid (ABA) in the regulation of wound-induced suberization and tuber water vapour loss (dehydration). Analysis by liquid chromatography-mass spectrometry showed that ABA concentrations varied little throughout the tuber, but were slightly higher near the periderm and lowest in the pith. ABA concentrations increase then decrease during tuber storage. Tuber wounding induced changes in ABA content. ABA content in wound-healing tuber discs decreased after wounding, reached a minimum by 24 h, and then increased from the 3rd to the 7th day after wounding. Wound-induced ABA accumulations were reduced by fluridone (FLD); an inhibitor of de novo ABA biosynthesis. Wound-induced phenylalanine ammonia lyase activity was slightly reduced and the accumulation of suberin poly(phenolics) and poly(aliphatics) noticeably reduced in FLD-treated tissues. Addition of ABA to the FLD treatment restored phenylalanine ammonia lyase activity and suberization, unequivocally indicating that endogenous ABA is involved in the regulation of these wound-healing processes. Similar experiments showed that endogenous ABA is involved in the regulation of water vapour loss, a process linked to wax accumulation in wound-healing tubers. Rapid reduction of water vapour loss across the wound surface is essential in preventing desiccation and death of cells at the wound site; live cells are required for suberization. These results unequivocally show that endogenous ABA is involved in the regulation of wound-induced suberization and the processes that protect surface cells from water vapour loss and death by dehydration.

Cytoplasmic Immunoglobulin Light Chain Revelation and Interphase Fluorescence In Situ Hybridization in Myeloma.

The cytogenetic analysis of plasma cell myeloma (PCM) allows stratification of patients so that prognosis may be determined and appropriate therapeutic options can be discussed. Owing to the patchy nature of the disease in the bone marrow (BM), the low proliferative activity of plasma cells and the cryptic nature of some PCM-associated cytogenetic changes, karyotypic analysis in this disease should be augmented with targeted interphase fluorescence in situ hybridization (FISH). Immunofluorescent revelation of cytoplasmic immunoglobulin light chains, together with interphase FISH (cIg-FISH), allows the identification of plasma cells within a sample so that they may be scored preferentially. This is particularly useful in situations where there are only a small percentage of plasma cells in a sample. Where an underlying myeloid disease is suspected the cIg-FISH-negative cells can be scored separately. Two methods are provided in this chapter: the technique for cIg-FISH in fresh PCM BM samples and a procedure for use in fixed cytogenetics preparations.

Wounding induces changes in cytokinin and auxin content in potato tuber, but does not induce formation of gibberellins.

Cytokinin, auxin and gibberellin contents in resting and wound-responding potato tubers have not been fully determined and coordinated with wound-healing processes. Using a well-defined wound-healing model system, hormone content and expression of genes associated with hormone turnover were determined in tubers following wounding. Changes in hormone content were coordinated with: (I) formation and completion of the wound closing layer (0-5/6 days), and (II) initiation of phellogen and wound periderm formation (∼ 7 days). Quantifiable amounts of biologically active cytokinins (Z, DZ and IP) were not detected in resting or wound-responding tubers. However, the precursor IPA and catabolic product c-ZOG were found in small amounts in resting and wound-responding tubers. Wound-induced activation of cytokinin biosynthesis was suggested by an increase in t-ZR and c-ZR content at 0.5 days and large increases in IPA and c-ZR content by 3 days and throughout 7 days after wounding suggesting roles in II, but little or no role in I. Expression of key genes involved in cytokinin metabolism followed similar profiles with transcripts decreasing through 3 days and then increasing at 5-7 days after wounding. Both free IAA and IAA-Asp were present in resting tubers. While IAA-Asp was no longer present by 3 days after wounding, IAA content nearly doubled by 5 days and was more than 4-fold greater at 7 days compared to that in resting tuber (0 day) suggesting roles in II, but little or no role in I. Gibberellins were not present in quantifiable amounts in resting or wound-responding tubers. These results suggest that bio-active cytokinins are wound-induced, but their residency is temporal and highly regulated. The transient presence of active cytokinins and corresponding increases in IAA content strongly suggest their involvement in the regulation of wound periderm development. The absence of gibberellins indicates that they are not a regulatory component of wound-healing processes.

Chromatin remodeling in plant cell culture: patterns of DNA methylation and histone H3 and H4 acetylation vary during growth of asynchronous potato cell suspensions.

Changes in DNA cytosine methylation and core histone multi-acetylation were determined in cell suspension cultures of potato (Solanum tuberosum L. cv. Russet Burbank) during 15 days of in vitro culture. Cell subculture induced a transient 33% decrease in genome-wide 5-methylcytosine (5mC) content and a transient threefold increase in transcription rates that were most evident at 6 and 9 days after subculture, respectively. In contrast to the global reduction in 5mC content, subculture resulted in a transient twofold increase in 5mC levels within 5'-CCGG-3' sequences and no detectable change in 5'-CG-3' methylation. Multi-acetylation of histones H3.1, H3.2 and H4 rose 2-, 1.5- and 3-fold by 9, 9 and 12 days after subculture, respectively. All observed epigenetic changes were reset during aging of cell cultures. Inclusion of the histone deacetylase inhibitor trichostatin A (TSA) and/or the cytosine methylation inhibitor 5-azacytidine (5AC) in culture sequentially decreased genome-wide 5mC levels by approximately 25% at day 9, then decreased 5'-mCmCGG-3' by 30-50% and increased H3 and H4 multi-acetylation by 30-60% at day 15, compared to controls. Treatment with 5AC or TSA alone or in combination had no effect on RNA synthesis at day 9. At day 15, 5AC treatment remained ineffective, while de novo RNA synthesis was approximately twofold higher in cells grown in both inhibitors or in TSA alone. Collectively, these results demonstrate that in potato suspension cultures, rapid, reversible changes in 5mC levels precede regulatory post-translational acetylation of core histones, and suggest that interactions between these epigenetic processes appear to be necessary to power transcription and growth induction in potato cells.

Mechanism-based irreversible inhibitors of cytokinin dehydrogenase.

The effects of three N(6)-substituted aminopurine derivatives containing either allenic or acetylenic side-chains on in vitro and in vivo cytokinin dehydrogenase (CKX; EC 1.5.99.12) activities were determined. At concentrations < or = 100 microM, the acetylenic derivative (HA-2) had no effect on in vitro CKX activity. In contrast, the two allenic derivatives (HA-1, HA-8) inhibited in vitro CKX activity in a dose-dependent manner with 50% inhibition occurring at HA-1 and HA-8 concentrations of 9.0 and 0.4 microM (respectively). HA-8 inhibited the degradation of both the free bases and ribosides of N6-(2-isopentenyl)adenine and zeatin. Pretreatment with HA-8 inhibited CKX activity in both a time- and concentration-dependent manner. In contrast to the reversible phenylurea inhibitor N-(chloro-4-pyridyl)-N'-phenylurea, inhibition of CKX activity by HA-8 was not relieved by 24 h of dialysis. Both HA-1 and HA-8 (but not HA-2) inhibited the metabolism of exogenous [3H]-N(6)-(2-isopentenyl)adenosine in excised aseptic potato (Solanum tuberosum) leaves. These results demonstrate that HA-8 is a mechanism-based irreversible (suicide) inhibitor of CKX and indicate that it may be useful in determining the role of CKX in cytokinin homeostasis in planta.