Plasticity of bud outgrowth varies at cauline and rosette nodes in Arabidopsis thaliana.

Shoot branching is a complex mechanism in which secondary shoots grow from buds that are initiated from meristems established in leaf axils. The model plant Arabidopsis (Arabidopsis thaliana) has a rosette leaf growth pattern in the vegetative stage. After flowering initiation, the main stem elongates with the top leaf primordia developing into cauline leaves. Meristems in Arabidopsis initiate in the axils of rosette or cauline leaves, giving rise to rosette or cauline buds, respectively. Plasticity in the process of shoot branching is regulated by resource and nutrient availability as well as by plant hormones. However, few studies have attempted to test whether cauline and rosette branching are subject to the same plasticity. Here, we addressed this question by phenotyping cauline and rosette branching in three Arabidopsis ecotypes and several Arabidopsis mutants with varied shoot architectures. Our results showed no negative correlation between cauline and rosette branch numbers in Arabidopsis, demonstrating that there is no tradeoff between cauline and rosette bud outgrowth. Through investigation of the altered branching pattern of flowering pathway mutants and Arabidopsis ecotypes grown in various photoperiods and light regimes, we further elucidated that the number of cauline branches is closely related to flowering time. The number of rosette branches has an enormous plasticity compared with cauline branches and is influenced by genetic background, flowering time, light intensity, and temperature. Our data reveal different levels of plasticity in the regulation of branching at rosette and cauline nodes, and promote a framework for future branching analyses.

Combining conventional QTL analysis and whole-exome capture-based bulk-segregant analysis provides new genetic insights into tuber sprout elongation and dormancy release in a diploid potato population.

Tuber dormancy and sprouting are commercially important potato traits as long-term tuber storage is necessary to ensure year-round availability. Premature dormancy release and sprout growth in tubers during storage can result in a significant deterioration in product quality. In addition, the main chemical sprout suppressant chlorpropham has been withdrawn in Europe, necessitating alternative approaches for controlling sprouting. Breeding potato cultivars with longer dormancy and slower sprout growth is a desirable goal, although this must be tempered by the needs of the seed potato industry, where dormancy break and sprout vigour are required for rapid emergence. We have performed a detailed genetic analysis of tuber sprout growth using a diploid potato population derived from two highly heterozygous parents. A dual approach employing conventional QTL analysis allied to a combined bulk-segregant analysis (BSA) using a novel potato whole-exome capture (WEC) platform was evaluated. Tubers were assessed for sprout growth in storage at six time-points over two consecutive growing seasons. Genetic analysis revealed the presence of main QTL on five chromosomes, several of which were consistent across two growing seasons. In addition, phenotypic bulks displaying extreme sprout growth phenotypes were subjected to WEC sequencing for performing BSA. The combined BSA and WEC approach corroborated QTL locations and served to narrow the associated genomic regions, while also identifying new QTL for further investigation. Overall, our findings reveal a very complex genetic architecture for tuber sprouting and sprout growth, which has implications both for potato and other root, bulb and tuber crops where long-term storage is essential.

Nitrate modulates stem cell dynamics in Arabidopsis shoot meristems through cytokinins.

The shoot apical meristem (SAM) is responsible for the generation of all the aerial parts of plants. Given its critical role, dynamical changes in SAM activity should play a central role in the adaptation of plant architecture to the environment. Using quantitative microscopy, grafting experiments, and genetic perturbations, we connect the plant environment to the SAM by describing the molecular mechanism by which cytokinins signal the level of nutrient availability to the SAM. We show that a systemic signal of cytokinin precursors mediates the adaptation of SAM size and organogenesis rate to the availability of mineral nutrients by modulating the expression of WUSCHEL, a key regulator of stem cell homeostasis. In time-lapse experiments, we further show that this mechanism allows meristems to adapt to rapid changes in nitrate concentration, and thereby modulate their rate of organ production to the availability of mineral nutrients within a few days. Our work sheds light on the role of the stem cell regulatory network by showing that it not only maintains meristem homeostasis but also allows plants to adapt to rapid changes in the environment.

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.

How to build an effective research network: lessons from two decades of the GARNet plant science community.

Successful collaborative research is dependent on excellent ideas and innovative experimental approaches, as well as the provision of appropriate support networks. Collaboration requires venues, infrastructures, training facilities, and, perhaps most importantly, a sustained commitment to work together as a community. These activities do not occur without significant effort, yet can be facilitated and overseen by the leadership of a research network that has a clearly defined role to help build resources for their community. Over the past 20 years, this is a role that the UKRI-BBSRC-funded GARNet network has played in the support of the UK curiosity-driven, discovery-led plant science research community. This article reviews the lessons learnt by GARNet in the hope that they can inform the practical implementation of current and future research networks.

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.

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.

Cell-Type-Specific Cytokinin Distribution within the Arabidopsis Primary Root Apex.

Cytokinins (CKs) play a crucial role in many physiological and developmental processes at the levels of individual plant components (cells, tissues, and organs) and by coordinating activities across these parts. High-resolution measurements of intracellular CKs in different plant tissues can therefore provide insights into their metabolism and mode of action. Here, we applied fluorescence-activated cell sorting of green fluorescent protein (GFP)-marked cell types, combined with solid-phase microextraction and an ultra-high-sensitivity mass spectrometry (MS) method for analysis of CK biosynthesis and homeostasis at cellular resolution. This method was validated by series of control experiments, establishing that protoplast isolation and cell sorting procedures did not greatly alter endogenous CK levels. The MS-based method facilitated the quantification of all the well known CK isoprenoid metabolites in four different transgenic Arabidopsis thaliana lines expressing GFP in specific cell populations within the primary root apex. Our results revealed the presence of a CK gradient within the Arabidopsis root tip, with a concentration maximum in the lateral root cap, columella, columella initials, and quiescent center cells. This distribution, when compared with previously published auxin gradients, implies that the well known antagonistic interactions between the two hormone groups are cell type specific.

An histidine covalent receptor and butenolide complex mediates strigolactone perception.

Strigolactone plant hormones control plant architecture and are key players in both symbiotic and parasitic interactions. They contain an ABC tricyclic lactone connected to a butenolide group, the D ring. The DWARF14 (D14) strigolactone receptor belongs to the superfamily of α/ß-hydrolases, and is known to hydrolyze the bond between the ABC lactone and the D ring. Here we characterized the binding and catalytic functions of RAMOSUS3 (RMS3), the pea (Pisum sativum) ortholog of rice (Oryza sativa) D14 strigolactone receptor. Using new profluorescent probes with strigolactone-like bioactivity, we found that RMS3 acts as a single-turnover enzyme that explains its apparent low enzymatic rate. We demonstrated the formation of a covalent RMS3-D-ring complex, essential for bioactivity, in which the D ring was attached to histidine 247 of the catalytic triad. These results reveal an undescribed mechanism of plant hormone reception in which the receptor performs an irreversible enzymatic reaction to generate its own ligand.

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.

Cucurbit extrafascicular phloem has strong negative impacts on aphids and is not a preferred feeding site.

Cucurbits have long been known to possess two types of phloem: fascicular (FP) within vascular bundles and extrafascicular phloem (EFP) surrounding vascular bundles and scattered through the cortex. Recently, their divergent composition was revealed, with FP having high sugar content consistent with conventional phloem, but EFP having much lower sugar levels and a very different proteome. However, the evolutionary advantages of possessing both FP and EFP have remained unclear. Here, we present four lines of quantitative evidence that together support the hypothesis that FP represents a typical phloem and is an attractive diet for aphids, whereas aphids avoid feeding on EFP. First, aphid stylet track endings were more abundant near the abaxial FP element of minor veins, suggesting a feeding preference for FP over EFP. Second, sugar profiles from stylet exudates were wholly consistent with FP origins, further supporting preference for FP and avoidance of EFP. Third, supplementation of EFP exudate into artificial diets confirmed an aversion to EFP in choice experiments. Finally, EFP exudate had negative effects on aphid performance. On the basis of aphids' inability to thrive on EFP, we conclude that EFP is atypical and perhaps should not be classed as a phloem system.

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.

The pea TCP transcription factor PsBRC1 acts downstream of Strigolactones to control shoot branching.

The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated. The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application. The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes. The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine. The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth. However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth. In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant. Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1. This phenotype indicates a localized regulation of branching at these nodes specific to pea.

Divergent metabolome and proteome suggest functional independence of dual phloem transport systems in cucurbits.

Cucurbitaceous plants (cucurbits) have long been preferred models for studying phloem physiology. However, these species are unusual in that they possess two different phloem systems, one within the main vascular bundles [fascicular phloem (FP)] and another peripheral to the vascular bundles and scattered through stem and petiole cortex tissues [extrafascicular phloem (EFP)]. We have revisited the assumption that the sap released after shoot incision originates from the FP, and also investigated the long-standing question of why the sugar content of this sap is ~30-fold less than predicted for requirements of photosynthate delivery. Video microscopy and phloem labeling experiments unexpectedly reveal that FP very quickly becomes blocked upon cutting, whereas the extrafascicular phloem bleeds for extended periods. Thus, all cucurbit phloem sap studies to date have reported metabolite, protein, and RNA composition and transport in the relatively minor extrafascicular sieve tubes. Using tissue dissection and direct sampling of sieve tube contents, we show that FP in fact does contain up to 1 M sugars, in contrast to low-millimolar levels in the EFP. Moreover, major phloem proteins in sieve tubes of FP differ from those that predominate in the extrafascicular sap, and include several previously uncharacterized proteins with little or no homology to databases. The overall compositional differences of the two phloem systems strongly indicate functional isolation. On this basis, we propose that the fascicular phloem is largely responsible for sugar transport, whereas the extrafascicular phloem may function in signaling, defense, and transport of other metabolites.

IPT9, a cis-zeatin cytokinin biosynthesis gene, promotes root growth.

Cytokinin and auxin are plant hormones that coordinate many aspects of plant development. Their interactions in plant underground growth are well established, occurring at the levels of metabolism, signaling, and transport. Unlike many plant hormone classes, cytokinins are represented by more than one active molecule. Multiple mutant lines, blocking specific parts of cytokinin biosynthetic pathways, have enabled research in plants with deficiencies in specific cytokinin-types. While most of these mutants have confirmed the impeding effect of cytokinin on root growth, the ipt29 double mutant instead surprisingly exhibits reduced primary root length compared to the wild type. This mutant is impaired in cis-zeatin (cZ) production, a cytokinin-type that had been considered inactive in the past. Here we have further investigated the intriguing ipt29 root phenotype, opposite to known cytokinin functions, and the (bio)activity of cZ. Our data suggest that despite the ipt29 short-root phenotype, cZ application has a negative impact on primary root growth and can activate a cytokinin response in the stele. Grafting experiments revealed that the root phenotype of ipt29 depends mainly on local signaling which does not relate directly to cytokinin levels. Notably, ipt29 displayed increased auxin levels in the root tissue. Moreover, analyses of the differential contributions of ipt2 and ipt9 to the ipt29 short-root phenotype demonstrated that, despite its deficiency on cZ levels, ipt2 does not show any root phenotype or auxin homeostasis variation, while ipt9 mutants were indistinguishable from ipt29. We conclude that IPT9 functions may go beyond cZ biosynthesis, directly or indirectly, implicating effects on auxin homeostasis and therefore influencing plant growth.

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.