Transcriptome and phytohormone changes associated with ethylene-induced onion bulb dormancy.

Control of dormancy and sprouting in onion bulbs is commercially important for postharvest management. Although ethylene application is sometimes used to extend dormancy, the underlying mechanisms regulating dormancy transition remain unclear. Since the sprout leaves emerge from the bulb baseplate, we used this tissue to assess the impact of ethylene treatment and storage time on the hormone profile and the transcriptome. Reads from 30 libraries were assembled and annotated, with 94,840 unigenes retained after filtering. The de novo transcriptome assembly was of high quality and continuity (N50: 1809 bp, GC content: 36.21 %), and was used to analyse differential expression and Gene Onotologies. Across two years, applied ethylene resulted in delayed dormancy break and reduced post-dormancy sprout vigour. Ethylene supplementation enhanced endogenous ethylene production and caused a transient climacteric-like increase in respiration. Significant changes in hormone and associated transcript profiles occurred through storage and in response to ethylene. In particular, abscisic acid (ABA) and its metabolite phaseic acid (PA) increased under ethylene during the longer dormancy period; however, cytokinin increases observed during storage appeared largely independent of ethylene treatment. Several hormone-related transcripts showed differential expression over time and/or in response to ethylene. Expression of ethylene biosynthesis (ACO), receptor (EIN4) and transcription factor (EIL3) genes were modified by ethylene, as were ABA biosynthesis genes such NCED, and cytokinin biosynthesis genes such as LOG and CKX. We conclude that ethylene substantially modifies expression of genes in several phytohormone pathways, and some of these changes may underlie the dormancy-extending effects of exogenous ethylene.

Speciation in Howea Palms Occurred in Sympatry, Was Preceded by Ancestral Admixture, and Was Associated with Edaphic and Phenological Adaptation.

Howea palms are viewed as one of the most clear-cut cases of speciation in sympatry. The sister species Howea belmoreana and H. forsteriana are endemic to the oceanic Lord Howe Island, Australia, where they have overlapping distributions and are reproductively isolated mainly by flowering time differences. However, the potential role of introgression from Australian mainland relatives had not previously been investigated, a process that has recently put other examples of sympatric speciation into question. Furthermore, the drivers of flowering time-based reproductive isolation remain unclear. We sequenced an RNA-seq data set that comprehensively sampled Howea and their closest mainland relatives (Linospadix, Laccospadix), and collected detailed soil chemistry data on Lord Howe Island to evaluate whether secondary gene flow had taken place and to examine the role of soil preference in speciation. D-statistics analyses strongly support a scenario whereby ancestral Howea hybridized frequently with its mainland relatives, but this only occurred prior to speciation. Expression analysis, population genetic and phylogenetic tests of selection, identified several flowering time genes with evidence of adaptive divergence between the Howea species. We found expression plasticity in flowering time genes in response to soil chemistry as well as adaptive expression and sequence divergence in genes pleiotropically linked to soil adaptation and flowering time. Ancestral hybridization may have provided the genetic diversity that promoted their subsequent adaptive divergence and speciation, a process that may be common for rapid ecological speciation.

A member of the TERMINAL FLOWER 1/CENTRORADIALIS gene family controls sprout growth in potato tubers.

Potato tuber bud dormancy break followed by premature sprouting is a major commercial problem which results in quality losses and decreased tuber marketability. An approach to controlling premature tuber sprouting is to develop potato cultivars with a longer dormancy period and/or reduced rate of sprout growth. Our recent studies using a potato diploid population have identified several quantitative trait loci (QTLs) that are associated with tuber sprout growth. In the current study, we aim to characterize a candidate gene associated with one of the largest effect QTLs for rapid tuber sprout growth on potato chromosome 3. Underlying this QTL is a gene encoding a TERMINAL FLOWER 1/CENTRORADIALIS homologue (PGSC0003DMG400014322). Here, we use a transgenic approach to manipulate the expression level of the CEN family member in a potato tetraploid genotype (cv. Désirée). We demonstrate a clear effect of manipulation of StCEN expression, with decreased expression levels associated with an increased rate of sprout growth, and overexpressing lines showing a lower rate of sprout growth than controls. Associated with different levels of StCEN expression were different levels of abscisic acid and cytokinins, implying a role in controlling the levels of plant growth regulators in the apical meristem.

Arbuscular mycorrhizal fungi promote coexistence and niche divergence of sympatric palm species on a remote oceanic island.

Microbes can have profound effects on their hosts, driving natural selection, promoting speciation and determining species distributions. However, soil-dwelling microbes are rarely investigated as drivers of evolutionary change in plants. We used metabarcoding and experimental manipulation of soil microbiomes to investigate the impact of soil and root microbes in a well-known case of sympatric speciation, the Howea palms of Lord Howe Island (Australia). Whereas H. forsteriana can grow on both calcareous and volcanic soils, H. belmoreana is restricted to, but more successful on, volcanic soil, indicating a trade-off in adaptation to the two soil types. We suggest a novel explanation for this trade-off. Arbuscular mycorrhizal fungi (AMF) are significantly depleted in H. forsteriana on volcanic soil, relative to both H. belmoreana on volcanic soil and H. forsteriana on calcareous soil. This is mirrored by the results of survival experiments, where the sterilization of natural soil reduces Howea fitness in every soil-species combination except H. forsteriana on volcanic soil. Furthermore, AMF-associated genes exhibit evidence of divergent selection between Howea species. These results show a mechanism by which divergent adaptation can have knock-on effects on host-microbe interactions, thereby reducing interspecific competition and promoting the coexistence of plant sister species.

Comparative proteomics of cucurbit phloem indicates both unique and shared sets of proteins.

Cucurbits are well-studied models for phloem biology but unusually possess both fascicular phloem (FP) within vascular bundles and additional extrafascicular phloem (EFP). Although the functional differences between the two systems are not yet clear, sugar analysis and limited protein profiling have established that FP and EFP have divergent compositions. Here we report a detailed comparative proteomics study of FP and EFP in two cucurbits, pumpkin and cucumber. We re-examined the sites of exudation by video microscopy, and confirmed that in both species, the spontaneous exudate following tissue cutting derives almost exclusively from EFP. Comparative gel electrophoresis and mass spectrometry-based proteomics of exudates, sieve element contents and microdissected stem tissues established that EFP and FP profiles are highly dissimilar, and that there are also species differences. Searches against cucurbit databases enabled identification of more than 300 FP proteins from each species. Few of the detected proteins (about 10%) were shared between the sieve element contents of FP and EFP, and enriched Gene Ontology categories also differed. To explore quantitative differences in the proteomes, we developed multiple reaction monitoring methods for cucumber proteins that are representative markers for FP or EFP and assessed exudate composition at different times after tissue cutting. Based on failure to detect FP markers in exudate samples, we conclude that FP is blocked very rapidly and therefore makes a minimal contribution to the exudates. Overall, the highly divergent contents of FP and EFP indicate that they are substantially independent vascular compartments.

Cryptic virulence and avirulence alleles revealed by controlled sexual recombination in pea aphids.

Although aphids are worldwide crop pests, little is known about aphid effector genes underlying virulence and avirulence. Here we show that controlling the genetics of both aphid and host can reveal novel recombinant genotypes with previously undetected allelic variation in both virulence and avirulence functions. Clonal F1 progeny populations were derived from reciprocal crosses and self-matings between two parental genotypes of pea aphid (Acyrthosiphon pisum) differing in virulence on a Medicago truncatula host carrying the RAP1 and RAP2 resistance genes. These populations showed Mendelian segregation consistent with aphid performance being controlled largely by a dominant virulence allele derived from only one parent. Altered segregation ratios on near-isogenic host genotypes differing in the region carrying RAP1 were indicative of additional heritable functions likely related to avirulence genes originating from both parents. Unexpectedly, some virulent F1 progeny were recovered from selfing of an avirulent parent, suggesting a reservoir of cryptic alleles. Host chlorosis was associated with virulence, whereas necrotic hypersensitive-like response was not. No maternal inheritance was found for any of these characteristics, ruling out sex-linked, cytoplasmic, and endosymbiotic factors. Our results demonstrate the tractability of dissecting the genetic basis of pest-host resistance mechanisms and indicate that the annual sexual cycle in aphids may lead to frequent novel genotypes with both increased and decreased virulence. Availability of genomes for both pest and host can facilitate definition of cognate gene-for-gene relationships, potentially leading to selection of crop genotypes with multiple resistance traits.

Conditional Auxin Response and Differential Cytokinin Profiles in Shoot Branching Mutants.

Strigolactone (SL), auxin, and cytokinin (CK) are hormones that interact to regulate shoot branching. For example, several ramosus (rms) branching mutants in pea (Pisum sativum) have SL defects, perturbed xylem CK levels, and diminished responses to auxin in shoot decapitation assays. In contrast with the last of these characteristics, we discovered that buds on isolated nodes (explants) of rms plants instead respond normally to auxin. We hypothesized that the presence or absence of attached roots would result in transcriptional and hormonal differences in buds and subtending stem tissues, and might underlie the differential auxin response. However, decapitated plants and explants both showed similar up-regulation of CK biosynthesis genes, increased CK levels, and down-regulation of auxin transport genes. Moreover, auxin application counteracted these trends, regardless of the effectiveness of auxin at inhibiting bud growth. Multivariate analysis revealed that stem transcript and CK changes were largely associated with decapitation and/or root removal and auxin response, whereas bud transcript profiles related more to SL defects. CK clustering profiles were indicative of additional zeatin-type CKs in decapitated stems being supplied by roots and thus promoting bud growth in SL-deficient genotypes even in the presence of added auxin. This difference in CK content may explain why rms buds on explants respond better to auxin than those on decapitated plants. We further conclude that rapid changes in CK status in stems are auxin dependent but largely SL independent, suggesting a model in which auxin and CK are dominant regulators of decapitation-induced branching, whereas SLs are more important in intact plants.

A purine nucleoside phosphorylase in Solanum tuberosum L. (potato) with specificity for cytokinins contributes to the duration of tuber endodormancy.

StCKP1 (Solanum tuberosum cytokinin riboside phosphorylase) catalyses the interconversion of the N9-riboside form of the plant hormone CK (cytokinin), a subset of purines, with its most active free base form. StCKP1 prefers CK to unsubstituted aminopurines. The protein was discovered as a CK-binding activity in extracts of tuberizing potato stolon tips, from which it was isolated by affinity chromatography. The N-terminal amino acid sequence matched the translation product of a set of ESTs, enabling a complete mRNA sequence to be obtained by RACE-PCR. The predicted polypeptide includes a cleavable signal peptide and motifs for purine nucleoside phosphorylase activity. The expressed protein was assayed for purine nucleoside phosphorylase activity against CKs and adenine/adenosine. Isopentenyladenine, trans-zeatin, dihydrozeatin and adenine were converted into ribosides in the presence of ribose 1-phosphate. In the opposite direction, isopentenyladenosine, trans-zeatin riboside, dihydrozeatin riboside and adenosine were converted into their free bases in the presence of Pi. StCKP1 had no detectable ribohydrolase activity. Evidence is presented that StCKP1 is active in tubers as a negative regulator of CKs, prolonging endodormancy by a chill-reversible mechanism.

Heavy traffic in the fast lane: long-distance signalling by macromolecules.

The two major vascular conduits in plants, the xylem and phloem, theoretically provide opportunities for the long-distance translocation of almost any type of water-borne molecule. This review focuses on the signalling functions conveyed by the movement of macromolecules. Here, a signal is defined as the communication of information from source to destination, where it modifies development, physiology or defence through altered gene expression or by direct influences on other cellular processes. Xylem and phloem sap both contain diverse classes of proteins; in addition, phloem contains many full-length and small RNA species. Only a few of these mobile molecules have proven functions in signalling. The transduction of signals typically depends on connection to appropriate signalling pathways. Incoming protein signals require specific detection systems, generally via receptors. Mobile RNAs require either the translation or presence of a homologous target. Given that phloem sieve elements are enucleate and lack translation machinery, RNA function requires subsequent unloading at least into adjacent companion cells. The binding of RNA by proteins in ribonucleoprotein complexes enables the translocation of some signals, with evidence for both sequence-specific and size-specific binding. Several examples of long-distance macromolecular signalling are highlighted, including the FT protein signal which regulates flowering time and other developmental switches.

Grafting as a research tool.

Grafting as a means to connect different plant tissues has been enormously useful in many studies of long-distance signalling and transport in relation to regulation of development and physiology. There is an almost infinite number of pairwise graft combinations that can be tested, typically between two different genotypes and/or between plants previously exposed to different environmental treatments. Grafting experiments are especially powerful for unambiguous demonstration of spatial separation of source and target, including genetic complementation of mutant phenotypes across a graft union, direct detection of transmitted molecules in receiving tissue or vascular sap, and activation or suppression of molecular targets due to signal transmission. Although grafting has a long history in research, only in the past decade has it been applied extensively to the Arabidopsis model. This chapter compares the main Arabidopsis grafting methods now available and describes seedling grafting in detail. Information is also provided on grafting of other common research model species, together with outlines of some successful applications.

Arabidopsis auxin mutants are compromised in systemic acquired resistance and exhibit aberrant accumulation of various indolic compounds.

Systemic acquired resistance is a widespread phenomenon in the plant kingdom that confers heightened and often enduring immunity to a range of diverse pathogens. Systemic immunity develops through activation of plant disease resistance protein signaling networks following local infection with an incompatible pathogen. The accumulation of the phytohormone salicylic acid in systemically responding tissues occurs within days after a local immunizing infection and is essential for systemic resistance. However, our knowledge of the signaling components underpinning signal perception and the establishment of systemic immunity are rudimentary. Previously, we showed that an early and transient increase in jasmonic acid in distal responding tissues was central to effective establishment of systemic immunity. Based upon predicted transcriptional networks induced in naive Arabidopsis (Arabidopsis thaliana) leaves following avirulent Pseudomonas syringae challenge, we show that a variety of auxin mutants compromise the establishment of systemic immunity. Linking together transcriptional and targeted metabolite studies, our data provide compelling evidence for a role of indole-derived compounds, but not auxin itself, in the establishment and maintenance of systemic immunity.

Feedback regulation of xylem cytokinin content is conserved in pea and Arabidopsis.

Increased-branching mutants of garden pea (Pisum sativum; ramosus [rms]) and Arabidopsis (Arabidopsis thaliana; more axillary branches) were used to investigate control of cytokinin export from roots in relation to shoot branching. In particular, we tested the hypothesis that regulation of xylem sap cytokinin is dependent on a long-distance feedback signal moving from shoot to root. With the exception of rms2, branching mutants from both species had greatly reduced amounts of the major cytokinins zeatin riboside, zeatin, and isopentenyl adenosine in xylem sap compared with wild-type plants. Reciprocally grafted mutant and wild-type Arabidopsis plants gave similar results to those observed previously in pea, with xylem sap cytokinin down-regulated in all graft combinations possessing branched shoots, regardless of root genotype. This long-distance feedback mechanism thus appears to be conserved between pea and Arabidopsis. Experiments with grafted pea plants bearing two shoots of the same or different genotype revealed that regulation of root cytokinin export is probably mediated by an inhibitory signal. Moreover, the signaling mechanism appears independent of the number of growing axillary shoots because a suppressed axillary meristem mutation that prevents axillary meristem development at most nodes did not abolish long-distance regulation of root cytokinin export in rms4 plants. Based on double mutant and grafting experiments, we conclude that RMS2 is essential for long-distance feedback regulation of cytokinin export from roots. Finally, the startling disconnection between cytokinin content of xylem sap and shoot tissues of various rms mutants indicates that shoots possess powerful homeostatic mechanisms for regulation of cytokinin levels.

Rhythmic emission of floral volatiles from Rosa damascena semperflorens cv. 'Quatre Saisons'.

The control of rhythmic emission of floral volatiles emitted from Rosa damascena semperflorens cv. 'Quatre Saisons' throughout floral development under various light regimes was studied. 2-Phenylethanol was the major volatile emitted in addition to monoterpenols, oxidised monoterpenols, monoterpenes and aromatic compounds. All detected volatiles were emitted rhythmically, with maximum peaks coinciding 8-10 h into a 12-h photoperiod. For some compounds a secondary, nocturnal peak was apparent. The primary and secondary maxima both occurred at approximately 24-h intervals. Rhythms appeared to be regulated endogenously: rhythmic emission continued upon exposure to continuous light or continuous darkness, and a phase shift in emission was induced upon inversion of the photoperiod. Additionally, emission continued after flower excision. A similar profile of free volatiles was stored within the floral tissue, together with glycosidic forms of 2-phenylethanol (>99% beta-D-glucoside), benzyl alcohol, citronellol and geraniol. Regression analysis indicated a significant decrease in glycosylated 2-phenylethanol through the photoperiod. These results suggest that glycosylated volatiles stored within petals may be a source of rhythmically emitted volatiles.

Effects of nitrogen supply on xylem cytokinin delivery, transpiration and leaf expansion of pea genotypes differing in xylem-cytokinin concentration.

The rms2 and rms4 pea (Pisum sativum L.) branching mutants have higher and lower xylem-cytokinin concentration, respectively, relative to wild type (WT) plants. These genotypes were grown at two levels of nitrogen (N) supply for 18-20 d to determine whether or not xylem-cytokinin concentration (X-CK) or delivery altered the transpiration and leaf growth responses to N deprivation. Xylem sap was collected by pressurising de-topped root systems. As sap-flow rate increased, X-CK declined in WT and rms2, but did not change in rms4. When grown at 5.0 mm N, X-CKs of rms2 and rms4 were 36% higher and 6-fold lower, respectively, than WT at sap-flow rates equivalent to whole-plant transpiration. Photoperiod cytokinin (CK) delivery rates (the product of transpiration and X-CK) decreased more than 6-fold in rms4. Growth of plants at 0.5 mm N had negligible (< 10%) effects on transpiration rates expressed on a leaf area basis in WT and rms4, but decreased transpiration rates of rms2. The low-N treatment decreased leaf expansion by 20-25% and expanding leaflet N concentration by 15%. These changes were similar in all genotypes. At sap-flow rates equivalent to whole-plant transpiration, the low N treatment decreased X-CK in rms2 but had no discernible effect in WT and rms4. Since the low N treatment decreased transpiration of all genotypes, photoperiod CK delivery rates also decreased in all genotypes. The similar leaf growth response of all genotypes to N deprivation despite differences in both absolute and relative X-CKs and deliveries suggests that shoot N status is more important in regulating leaf expansion than xylem-supplied cytokinins. The decreased X-CK and transpiration rate of rms2 following N deprivation suggests that changes in xylem-supplied CKs may modify water use.

Micrografting techniques for testing long-distance signalling in Arabidopsis.

Grafting in species other than Arabidopsis has generated persuasive evidence for long-distance signals involved in many plant processes, including regulation of flowering time and shoot branching. Hitherto, such approaches in Arabidopsis have been hampered by the lack of suitable grafting techniques. Here, a range of micrografting methods for young Arabidopsis seedlings are described. The simplest configuration was a single-hypocotyl graft, constructed with or without a supporting collar, allowing tests of root-shoot communication. More complex two-shoot grafts were also constructed, enabling tests of shoot-shoot communication. Integrity of grafts and absence of adventitious roots on scions were assessed using plants constitutively expressing a GUS gene as one graft partner. Using the max1 (more axillary growth) and max3 increased branching mutants, it was shown that a wild-type (WT) rootstock was able to inhibit rosette branching of mutant shoots. In two-shoot grafts with max1 and WT shoots on a max1 rootstock, the mutant shoot branched profusely, but the WT one did not. In two-shoot grafts with max1 and WT shoots on a WT rootstock, neither shoot exhibited increased branching. The results mirror those previously demonstrated in equivalent grafting experiments with the ramosus mutants in pea, and are consistent with the concept that a branching signal is capable of moving from root to shoot, but not from shoot to shoot. These grafting procedures will be valuable for revealing genes associated with many other long-distance signalling pathways, including flowering, systemic resistance and abiotic stress responses.