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1.  Hormone crosstalk in plants 
Journal of Experimental Botany  2015;66(16):4853-4854.
PMCID: PMC4513929
2.  The co-chaperone p23 controls root development through the modulation of auxin distribution in the Arabidopsis root meristem 
Journal of Experimental Botany  2015;66(16):5113-5122.
p23 co-chaperones play a key role in the root meristem maintenance via regulation of auxin signalling and the consequent balance between cell differentiation and division rate at the transition zone.
Homologues of the p23 co-chaperone of HSP90 are present in all eukaryotes, suggesting conserved functions for this protein throughout evolution. Although p23 has been extensively studied in animal systems, little is known about its function in plants. In the present study, the functional characterization of the two isoforms of p23 in Arabidopsis thaliana is reported, suggesting a key role of p23 in the regulation of root development. Arabidopsis p23 mutants, for either form, show a short root length phenotype with a reduced meristem length. In the root meristem a low auxin level associated with a smaller auxin gradient was observed. A decrease in the expression levels of PIN FORMED PROTEIN (PIN)1, PIN3, and PIN7, contextually to an inefficient polar localization of PIN1, was detected. Collectively these results suggest that both Arabidopsis p23 isoforms are required for root growth, in particular in the maintenance of the root meristem, where the proteins are located.
PMCID: PMC4513928  PMID: 26163704
Arabidopsis; auxin; p23-chaperone; pin formed protein (PIN); polar auxin transport; root growth.
3.  Brassinazole resistant 1 (BZR1)-dependent brassinosteroid signalling pathway leads to ectopic activation of quiescent cell division and suppresses columella stem cell differentiation 
Journal of Experimental Botany  2015;66(15):4835-4849.
Brassinosteroids (BRs) lead to ectopic activation of quiescent centre division as well as modulation of the columella stem cells differentiation in Arabidopsis roots in a BR concentration- and BZR1-/BES1-dependent manner.
Previous publications have shown that BRI1 EMS suppressor 1 (BES1), a positive regulator of the brassinosteroid (BR) signalling pathway, enhances cell divisions in the quiescent centre (QC) and stimulates columella stem cell differentiation. Here, it is demonstrated that BZR1, a BES1 homologue, also promotes cell divisions in the QC, but it suppresses columella stem cell differentiation, opposite to the action of BES1. In addition, BR and its BZR1-mediated signalling pathway are shown to alter the expression/subcellular distribution of pin-formed (PINs), which may result in changes in auxin movement. BR promotes intense nuclear accumulation of BZR1 in the root tip area, and the binding of BZR1 to the promoters of several root development-regulating genes, modulating their expression in the root stem cell niche area. These BZR1-mediated signalling cascades may account for both the ectopic activation of QC cell divisions as well as the suppression of the columella stem cell differentiation. They could also inhibit auxin-dependent distal stem cell differentiation by antagonizing the auxin/WOX5-dependent pathway. In conclusion, BZR1-/BES1-mediated BR signalling pathways show differential effects on the maintenance of root apical meristem activities: they stimulate ectopic QC division while they show opposite effects on the differentiation of distal columella stem cells in a BR concentration- and BZR1-/BES1-dependent manner.
PMCID: PMC4507784  PMID: 26136267
Auxin; brassinosteroid; BZR1; columella stem cell; PINs; quiescent centre (QC); root apical meristem; root development.
4.  Strigolactones as an auxiliary hormonal defence mechanism against leafy gall syndrome in Arabidopsis thaliana  
Journal of Experimental Botany  2015;66(16):5123-5134.
To antagonize the developmental process initiated by Rhodococcus fascians and in response to the bacterial cytokinins, Arabidopsis activates its strigolactone response, partially suppressing shoot branching in the rosette.
Leafy gall syndrome is the consequence of modified plant development in response to a mixture of cytokinins secreted by the biotrophic actinomycete Rhodococcus fascians. The similarity of the induced symptoms with the phenotype of plant mutants defective in strigolactone biosynthesis and signalling prompted an evaluation of the involvement of strigolactones in this pathology. All tested strigolactone-related Arabidopsis thaliana mutants were hypersensitive to R. fascians. Moreover, treatment with the synthetic strigolactone mixture GR24 and with the carotenoid cleavage dioxygenase inhibitor D2 illustrated that strigolactones acted as antagonistic compounds that restricted the morphogenic activity of R. fascians. Transcript profiling of the MORE AXILLARY GROWTH1 (MAX1), MAX2, MAX3, MAX4, and BRANCHED1 (BRC1) genes in the wild-type Columbia-0 accession and in different mutant backgrounds revealed that upregulation of strigolactone biosynthesis genes was triggered indirectly by the bacterial cytokinins via host-derived auxin and led to the activation of BRC1 expression, inhibiting the outgrowth of the newly developing shoots, a typical hallmark of leafy gall syndrome. Taken together, these data support the emerging insight that balances are critical for optimal leafy gall development: the long-lasting biotrophic interaction is possible only because the host activates a set of countermeasures—including the strigolactone response—in reaction to bacterial cytokinins to constrain the activity of R. fascians.
PMCID: PMC4513927  PMID: 26136271
Apical dominance; Gram-positive phytopathogen; witches’ broom.
5.  Auxin-driven patterning with unidirectional fluxes 
Journal of Experimental Botany  2015;66(16):5083-5102.
The key modes of auxin-driven patterning— the formation of convergence points and canals—depend on whether auxin efflux and influx feed back on polar auxin transport synergistically or antagonistically.
The plant hormone auxin plays an essential role in the patterning of plant structures. Biological hypotheses supported by computational models suggest that auxin may fulfil this role by regulating its own transport, but the plausibility of previously proposed models has been questioned. We applied the notion of unidirectional fluxes and the formalism of Petri nets to show that the key modes of auxin-driven patterning—the formation of convergence points and the formation of canals—can be implemented by biochemically plausible networks, with the fluxes measured by dedicated tally molecules or by efflux and influx carriers themselves. Common elements of these networks include a positive feedback of auxin efflux on the allocation of membrane-bound auxin efflux carriers (PIN proteins), and a modulation of this allocation by auxin in the extracellular space. Auxin concentration in the extracellular space is the only information exchanged by the cells. Canalization patterns are produced when auxin efflux and influx act antagonistically: an increase in auxin influx or concentration in the extracellular space decreases the abundance of efflux carriers in the adjacent segment of the membrane. In contrast, convergence points emerge in networks in which auxin efflux and influx act synergistically. A change in a single reaction rate may result in a dynamic switch between these modes, suggesting plausible molecular implementations of coordinated patterning of organ initials and vascular strands predicted by the dual polarization theory.
PMCID: PMC4513925  PMID: 26116915
Petri nets; auxin-driven patterning; canalization; convergence point formation; dual polarization; modulated feedback; polar auxin transport; unidirectional flux.
6.  Effects of PHENYLALANINE AMMONIA LYASE (PAL) knockdown on cell wall composition, biomass digestibility, and biotic and abiotic stress responses in Brachypodium  
Journal of Experimental Botany  2015;66(14):4317-4335.
Reducing the function of PAL, the first enzyme in the phenylpropanoid pathway, in Brachypodium distachyon alters cell wall composition, increases fungal susceptibility, but minimally affects caterpillar herbivory and abiotic stress tolerance.
The phenylpropanoid pathway in plants synthesizes a variety of structural and defence compounds, and is an important target in efforts to reduce cell wall lignin for improved biomass conversion to biofuels. Little is known concerning the trade-offs in grasses when perturbing the function of the first gene family in the pathway, PHENYLALANINE AMMONIA LYASE (PAL). Therefore, PAL isoforms in the model grass Brachypodium distachyon were targeted, by RNA interference (RNAi), and large reductions (up to 85%) in stem tissue transcript abundance for two of the eight putative BdPAL genes were identified. The cell walls of stems of BdPAL-knockdown plants had reductions of 43% in lignin and 57% in cell wall-bound ferulate, and a nearly 2-fold increase in the amounts of polysaccharide-derived carbohydrates released by thermochemical and hydrolytic enzymic partial digestion. PAL-knockdown plants exhibited delayed development and reduced root growth, along with increased susceptibilities to the fungal pathogens Fusarium culmorum and Magnaporthe oryzae. Surprisingly, these plants generally had wild-type (WT) resistances to caterpillar herbivory, drought, and ultraviolet light. RNA sequencing analyses revealed that the expression of genes associated with stress responses including ethylene biosynthesis and signalling were significantly altered in PAL knocked-down plants under non-challenging conditions. These data reveal that, although an attenuation of the phenylpropanoid pathway increases carbohydrate availability for biofuel, it can adversely affect plant growth and disease resistance to fungal pathogens. The data identify notable differences between the stress responses of these monocot pal mutants versus Arabidopsis (a dicot) pal mutants and provide insights into the challenges that may arise when deploying phenylpropanoid pathway-altered bioenergy crops.
PMCID: PMC4493789  PMID: 26093023
Bioenergy; ferulic acid; Fusarium; grass; herbivory; lignin; phenylpropanoid; saccharification; tyrosine ammonia lyase; ultraviolet light.
7.  Using quantitative PCR with retrotransposon-based insertion polymorphisms as markers in sugarcane 
Journal of Experimental Botany  2015;66(14):4239-4250.
qPCR-RBIP was used to examine the dosage of particular markers and evolutionary history in Saccharum and the related genera, Erianthus and Miscanthus. It also differentiated between S. spontaneum and S. officinarum.
Sugarcane is the main source of the world’s sugar and is becoming increasingly important as a source of biofuel. The highly polyploid and heterozygous nature of the sugarcane genome has meant that characterization of the genome has lagged behind that of other important crops. Here we developed a method using a combination of quantitative PCR with a transposable marker system to score the relative number of alleles with a transposable element (TE) present at a particular locus. We screened two genera closely related to Saccharum (Miscanthus and Erianthus), wild Saccharum, traditional cultivars, and 127 modern cultivars from Brazilian and Australian breeding programmes. We showed how this method could be used in various ways. First, we showed that the method could be extended to be used as part of a genotyping system. Secondly, the history of insertion and timing of the three TEs examined supports our current understanding of the evolution of the Saccharum complex. Thirdly, all three TEs were found in only one of the two main lineages leading to the modern sugarcane cultivars and are therefore the first TEs identified that could potentially be used as markers for Saccharum spontaneum.
PMCID: PMC4493790  PMID: 26093024
Marker; polyploid; real-time PCR; retrotransposon-based insertion polymorphism; sugarcane; transposable element.
8.  Contrasting allelic distribution of CO/Hd1 homologues in Miscanthus sinensis from the East Asian mainland and the Japanese archipelago 
Journal of Experimental Botany  2015;66(14):4227-4237.
Homologues of CONSTANS/Heading date 1 were cloned from Miscanthus sinensis and named MsiHd1. MsiHd1a in most Japanese accessions contained non-functional alleles, whereas Asian mainland accessions harboured only functional alleles.
The genus Miscanthus is a perennial C4 grass native to eastern Asia and is a promising candidate bioenergy crop for cool temperate areas. Flowering time is a crucial factor governing regional and seasonal adaptation; in addition, it is also a key target trait for extending the vegetative phase to improve biomass potential. Homologues of CONSTANS (CO)/Heading date 1(Hd1) were cloned from Miscanthus sinensis and named MsiHd1. Sequences of MsiHd1 homologues were compared among 24 wild M. sinensis accessions from Japan, 14 from China, and three from South Korea. Two to five MsiHd1 alleles in each accession were identified, suggesting that MsiHd1 consists of at least three loci in the Miscanthus genome. Verifying the open reading frame in MsiHd1, they were classified as putative functional alleles without mutations or non-functional alleles caused by indels. The Neighbor–Joining tree indicated that one of the multiple MsiHd1 loci is a pseudogene locus without any functional alleles. The pseudogene locus was named MsiHd1b, and the other loci were considered to be part of the MsiHd1a multi-locus family. Interestingly, in most Japanese accessions 50% or more of the MsiHd1a alleles were non-functional, whereas accessions from the East Asian mainland harboured only functional alleles. Five novel miniature inverted transposable elements (MITEs) (MsiMITE1–MsiMITE5) were observed in MsiHd1a/b. MsiMITE1, detected in exon 1 of MsiHd1a, was only observed in Japanese accessions and its revertant alleles derived from retransposition were predominantly in Chinese accessions. These differences in MsiHd1a show that the dependency on functional MsiHd1a alleles is different between accessions from the East Asian mainland and Japan.
PMCID: PMC4493791  PMID: 26089536
Bioenergy; flowering time gene; gene duplication; geographical allelic differentiation; Miscanthus sinensis; MITEs.
9.  New candidate genes for the fine regulation of the colour of grapes 
Journal of Experimental Botany  2015;66(15):4427-4440.
The integration of metabolic, molecular marker, and transcriptomic data from a segregating grapevine progeny provides novel insights into the genetic control of anthocyanin content and composition in ripe berries.
In the last decade, great progress has been made in clarifying the main determinants of anthocyanin accumulation in grape berry skin. However, the molecular details of the fine variation among cultivars, which ultimately contributes to wine typicity, are still not completely understood. To shed light on this issue, the grapes of 170 F1 progeny from the cross ‘Syrah’×’Pinot Noir’ were characterized at the mature stage for the content of 15 anthocyanins during four growing seasons. This huge data set was used in combination with a dense genetic map to detect genomic regions controlling the anthocyanin pathway both at key enzymatic points and at particular branches. Genes putatively involved in fine tuning the global regulation of anthocyanin biosynthesis were identified by exploring the gene predictions in the QTL (quantitative trait locus) confidence intervals and their expression profile during berry development in offspring with contrasting anthocyanin accumulation. New information on some aspects which had scarcely been investigated so far, such as anthocyanin transport into the vacuole, or completely neglected, such as acylation, is provided. These genes represent a valuable resource in grapevine molecular-based breeding programmes to improve both fruit and wine quality and to tailor wine sensory properties according to consumer demand.
PMCID: PMC4507754  PMID: 26071528
Anthocyanin; berry; candidate gene; metabolic profiling; microarray; quantitative trait locus; Vitis vinifera.
10.  Regulation of flavonol content and composition in (Syrah×Pinot Noir) mature grapes: integration of transcriptional profiling and metabolic quantitative trait locus analyses 
Journal of Experimental Botany  2015;66(15):4441-4453.
Novel candidate genes for the fine regulation of flavonol content in ripe berries are identified through integration of transcriptional profiling and metabolic QTL analyses of a segregating grapevine progeny.
Flavonols are a ubiquitous class of flavonoids that accumulate preferentially in flowers and mature berries. Besides their photo-protective function, they play a fundamental role during winemaking, stabilizing the colour by co-pigmentation with anthocyanins and contributing to organoleptic characteristics. Although the general flavonol pathway has been genetically and biochemically elucidated, the genetic control of flavonol content and composition at harvest is still not clear. To this purpose, the grapes of 170 segregating F1 individuals from a ‘Syrah’×’Pinot Noir’ population were evaluated at the mature stage for the content of six flavonol aglycons in four seasons. Metabolic data in combination with genetic data enabled the identification of 16 mQTLs (metabolic quantitative trait loci). For the first time, major genetic control by the linkage group 2 (LG 2)/MYBA region on flavonol variation, in particular of tri-hydroxylated flavonols, is demonstrated. Moreover, seven regions specifically associated with the fine control of flavonol biosynthesis are identified. Gene expression profiling of two groups of individuals significantly divergent for their skin flavonol content identified a large set of differentially modulated transcripts. Among these, the transcripts coding for MYB and bZIP transcription factors, methyltranferases, and glucosyltranferases specific for flavonols, proteins, and factors belonging to the UV-B signalling pathway and co-localizing with the QTL regions are proposed as candidate genes for the fine regulation of flavonol content and composition in mature grapes.
PMCID: PMC4507773  PMID: 26071529
Berry; flavonols; candidate gene; metabolic profiling; microarray; quantitative trait loci; segregating population; Vitis vinifera.
12.  Tricho- and atrichoblast cell files show distinct PIN2 auxin efflux carrier exploitations and are jointly required for defined auxin-dependent root organ growth 
Journal of Experimental Botany  2015;66(16):5103-5112.
PIN2 shows distinct trafficking and vacuolar turnover in neighbouring tricho- and atrichoblast cell files. Differential abundance of PIN2 in the root epidermis could have developmental importance for root gravitropism.
The phytohormone auxin is a vital growth regulator in plants. In the root epidermis auxin steers root organ growth. However, the mechanisms that allow adjacent tissues to integrate growth are largely unknown. Here, the focus is on neighbouring epidermal root tissues to assess the integration of auxin-related growth responses. The pharmacologic, genetic, and live-cell imaging approaches reveal that PIN2 auxin efflux carriers are differentially controlled in tricho- and atrichoblast cells. PIN2 proteins show lower abundance at the plasma membrane of trichoblast cells, despite showing higher rates of intracellular trafficking in these cells. The data suggest that PIN2 proteins display distinct cell-type-dependent trafficking rates to the lytic vacuole for degradation. Based on this insight, it is hypothesized that auxin-dependent processes are distinct in tricho- and atrichoblast cells. Moreover, genetic interference with epidermal patterning supports this assumption and suggests that tricho- and atrichoblasts have distinct importance for auxin-sensitive root growth and gravitropic responses.
PMCID: PMC4513926  PMID: 26041320
Atrichoblast; auxin; epidermal patterning; PIN2; trafficking; trichoblast.
13.  Nitrogen deficiency in barley (Hordeum vulgare) seedlings induces molecular and metabolic adjustments that trigger aphid resistance 
Journal of Experimental Botany  2015;66(12):3639-3655.
Nitrogen deficiency induces extensive metabolic adjustment in barley leaves mediated largely by sugar signalling and receptor-like kinase cascades that overlap with biotic stress pathways to induce aphid resistance.
Agricultural nitrous oxide (N2O) pollution resulting from the use of synthetic fertilizers represents a significant contribution to anthropogenic greenhouse gas emissions, providing a rationale for reduced use of nitrogen (N) fertilizers. Nitrogen limitation results in extensive systems rebalancing that remodels metabolism and defence processes. To analyse the regulation underpinning these responses, barley (Horedeum vulgare) seedlings were grown for 7 d under N-deficient conditions until net photosynthesis was 50% lower than in N-replete controls. Although shoot growth was decreased there was no evidence for the induction of oxidative stress despite lower total concentrations of N-containing antioxidants. Nitrogen-deficient barley leaves were rich in amino acids, sugars and tricarboxylic acid cycle intermediates. In contrast to N-replete leaves one-day-old nymphs of the green peach aphid (Myzus persicae) failed to reach adulthood when transferred to N-deficient barley leaves. Transcripts encoding cell, sugar and nutrient signalling, protein degradation and secondary metabolism were over-represented in N-deficient leaves while those associated with hormone metabolism were similar under both nutrient regimes with the exception of mRNAs encoding proteins involved in auxin metabolism and responses. Significant similarities were observed between the N-limited barley leaf transcriptome and that of aphid-infested Arabidopsis leaves. These findings not only highlight significant similarities between biotic and abiotic stress signalling cascades but also identify potential targets for increasing aphid resistance with implications for the development of sustainable agriculture.
PMCID: PMC4463806  PMID: 26038307
Cross-tolerance; kinase cascades; metabolite profiles; Myzus persicae; nitrogen limitation; oxidative stress; sugar signalling.
14.  SCF E3 ligase PP2-B11 plays a positive role in response to salt stress in Arabidopsis  
Journal of Experimental Botany  2015;66(15):4683-4697.
AtPP2-B11, an F-box protein, enhances the salt stress tolerance by regulating AnnAt1 expression, repressing reactive oxygen species production, and disrupting Na+ homeostasis in Arabidopsis.
Skp1–Cullin–F-box (SCF) E3 ligases are essential to the post-translational regulation of many important factors involved in cellular signal transduction. In this study, we identified an F-box protein from Arabidopsis thaliana, AtPP2-B11, which was remarkably induced with increased duration of salt treatment in terms of both transcript and protein levels. Transgenic Arabidopsis plants overexpressing AtPP2-B11 exhibited obvious tolerance to high salinity, whereas the RNA interference line was more sensitive to salt stress than wild-type plants. Isobaric tag for relative and absolute quantification analysis revealed that 4311 differentially expressed proteins were regulated by AtPP2-B11 under salt stress. AtPP2-B11 could upregulate the expression of annexin1 (AnnAt1) and function as a molecular link between salt stress and reactive oxygen species accumulation in Arabidopsis. Moreover, AtPP2-B11 influenced the expression of Na+ homeostasis genes under salt stress, and the AtPP2-B11 overexpressing lines exhibited lower Na+ accumulation. These results suggest that AtPP2-B11 functions as a positive regulator in response to salt stress in Arabidopsis.
PMCID: PMC4507775  PMID: 26041321
AnnAt1; E3 AtPP2-B11; ligases; Na+ homeostasis; ROS.
15.  Role of chromatin in water stress responses in plants 
Journal of Experimental Botany  2013;65(10):2785-2799.
As sessile organisms, plants are exposed to environmental stresses throughout their life. They have developed survival strategies such as developmental and morphological adaptations, as well as physiological responses, to protect themselves from adverse environments. In addition, stress sensing triggers large-scale transcriptional reprogramming directed at minimizing the deleterious effect of water stress on plant cells. Here, we review recent findings that reveal a role of chromatin in water stress responses. In addition, we discuss data in support of the idea that chromatin remodelling and modifying enzymes may be direct targets of stress signalling pathways. Modulation of chromatin regulator activity by these signaling pathways may be critical in minimizing potential trade-offs between growth and stress responses. Alterations in the chromatin organization and/or in the activity of chromatin remodelling and modifying enzymes may furthermore contribute to stress memory. Mechanistic insight into these phenomena derived from studies in model plant systems should allow future engineering of broadly drought-tolerant crop plants that do not incur unnecessary losses in yield or growth.
PMCID: PMC4110454  PMID: 24302754
ABA; chromatin; drought; epigenetics; genome accessibility; water stress.
16.  Identification of regulatory network hubs that control lipid metabolism in Chlamydomonas reinhardtii  
Journal of Experimental Botany  2015;66(15):4551-4566.
Characterization of regulatory networks in Chlamydomonas reinhardtii led to the identification of regulatory hubs that control the repatterning of cellular metabolism that leads to triacylglycerol accumulation in microalgae.
Microalgae-based biofuels are promising sources of alternative energy, but improvements throughout the production process are required to establish them as economically feasible. One of the most influential improvements would be a significant increase in lipid yields, which could be achieved by altering the regulation of lipid biosynthesis and accumulation. Chlamydomonas reinhardtii accumulates oil (triacylglycerols, TAG) in response to nitrogen (N) deprivation. Although a few important regulatory genes have been identified that are involved in controlling this process, a global understanding of the larger regulatory network has not been developed. In order to uncover this network in this species, a combined omics (transcriptomic, proteomic and metabolomic) analysis was applied to cells grown in a time course experiment after a shift from N-replete to N-depleted conditions. Changes in transcript and protein levels of 414 predicted transcription factors (TFs) and transcriptional regulators (TRs) were monitored relative to other genes. The TF and TR genes were thus classified by two separate measures: up-regulated versus down-regulated and early response versus late response relative to two phases of polar lipid synthesis (before and after TAG biosynthesis initiation). Lipidomic and primary metabolite profiling generated compound accumulation levels that were integrated with the transcript dataset and TF profiling to produce a transcriptional regulatory network. Evaluation of this proposed regulatory network led to the identification of several regulatory hubs that control many aspects of cellular metabolism, from N assimilation and metabolism, to central metabolism, photosynthesis and lipid metabolism.
PMCID: PMC4507760  PMID: 26022256
Biofuel; Chlamydomonas reinhardtii; metabolomics; network analysis; proteomics; regulatory hubs; RNA-seq; transcription factors; transcriptional regulators.
17.  CYCD3 D-type cyclins regulate cambial cell proliferation and secondary growth in Arabidopsis  
Journal of Experimental Botany  2015;66(15):4595-4606.
Arabidopsis CyclinD3 genes are revealed as central regulators of cambial cell proliferation and vascular development, which constitutes part of a novel mechanism controlling secondary growth and radial organ size.
A major proportion of plant biomass is derived from the activity of the cambium, a lateral meristem responsible for vascular tissue formation and radial organ enlargement in a process termed secondary growth. In contrast to our relatively good understanding of the regulation of primary meristems, remarkably little is known concerning the mechanisms controlling secondary growth, particularly how cambial cell divisions are regulated and integrated with vascular differentiation. A genetic loss-of-function approach was used here to reveal a rate-limiting role for the Arabidopsis CYCLIN D3 (CYCD3) subgroup of cell-cycle genes in the control of cambial cell proliferation and secondary growth, providing conclusive evidence of a direct link between the cell cycle and vascular development. It is shown that all three CYCD3 genes are specifically expressed in the cambium throughout vascular development. Analysis of a triple loss-of-function CYCD3 mutant revealed a requirement for CYCD3 in promoting the cambial cell cycle since mutant stems and hypocotyls showed a marked reduction in diameter linked to reduced mitotic activity in the cambium. Conversely, loss of CYCD3 provoked an increase in xylem cell size and the expression of differentiation markers, showing that CYCD3 is required to restrain the differentiation of xylem precursor cells. Together, our data show that tight control of cambial cell division through developmental- and cell type-specific regulation of CYCD3 is required for normal vascular development, constituting part of a novel mechanism controlling organ growth in higher plants.
PMCID: PMC4507761  PMID: 26022252
Arabidopsis; cambium; cell cycle; cell division; cell expansion; cyclin D3; inflorescence stem; organ size; secondary growth; vascular development; xylem.
18.  Quantitative trait locus mapping of deep rooting by linkage and association analysis in rice 
Journal of Experimental Botany  2015;66(15):4749-4757.
Major quantitative trait loci (QTLs) of rice deep rooting were identified by combined linkage-based and linage disequilibrium-based QTL mapping in this study.
Deep rooting is a very important trait for plants’ drought avoidance, and it is usually represented by the ratio of deep rooting (RDR). Three sets of rice populations were used to determine the genetic base for RDR. A linkage mapping population with 180 recombinant inbred lines and an association mapping population containing 237 rice varieties were used to identify genes linked to RDR. Six quantitative trait loci (QTLs) of RDR were identified as being located on chromosomes 1, 2, 4, 7, and 10. Using 1 019 883 single-nucleotide polymorphisms (SNPs), a genome-wide association study of the RDR was performed. Forty-eight significant SNPs of the RDR were identified and formed a clear peak on the short arm of chromosome 1 in a Manhattan plot. Compared with the shallow-rooting group and the whole collection, the deep-rooting group had selective sweep regions on chromosomes 1 and 2, especially in the major QTL region on chromosome 2. Seven of the nine candidate SNPs identified by association mapping were verified in two RDR extreme groups. The findings from this study will be beneficial to rice drought-resistance research and breeding.
PMCID: PMC4507776  PMID: 26022253
Drought avoidance; genome-wide association study (GWAS); quantitative trait locus (QTL); ratio of deep rooting (RDR); rice; root architecture; selective sweep.
19.  Characterization, localization, and seasonal changes of the sucrose transporter FeSUT1 in the phloem of Fraxinus excelsior  
Journal of Experimental Botany  2015;66(15):4807-4819.
In Fraxinus excelsior the sucrose transporter FeSUT1 is located in the phloem and involved in apoplastic sucrose loading and retrieval. The expression of FeSUT1 is higher under low sucrose conditions.
Trees are generally assumed to be symplastic phloem loaders. A typical feature for most wooden species is an open minor vein structure with symplastic connections between mesophyll cells and phloem cells, which allow sucrose to move cell-to-cell through the plasmodesmata into the phloem. Fraxinus excelsior (Oleaceae) also translocates raffinose family oligosaccharides in addition to sucrose. Sucrose concentration was recently shown to be higher in the phloem sap than in the mesophyll cells. This suggests the involvement of apoplastic steps and the activity of sucrose transporters in addition to symplastic phloem-loading processes. In this study, the sucrose transporter FeSUT1 from F. excelsior was analysed. Heterologous expression in baker’s yeast showed that FeSUT1 mediates the uptake of sucrose. Immunohistochemical analyses revealed that FeSUT1 was exclusively located in phloem cells of minor veins and in the transport phloem of F. excelsior. Further characterization identified these cells as sieve elements and possibly ordinary companion cells but not as intermediary cells. The localization and expression pattern point towards functions of FeSUT1 in phloem loading of sucrose as well as in sucrose retrieval. FeSUT1 is most likely responsible for the observed sucrose gradient between mesophyll and phloem. The elevated expression level of FeSUT1 indicated an increased apoplastic carbon export activity from the leaves during spring and late autumn. It is hypothesized that the importance of apoplastic loading is high under low-sucrose conditions and that the availability of two different phloem-loading mechanisms confers advantages for temperate woody species like F. excelsior.
PMCID: PMC4507781  PMID: 26022258
Immunolocalization; Fraxinus excelsior; heterologous expression; phloem loading; sucrose transporter.
20.  The paralogous R3 MYB proteins CAPRICE, TRIPTYCHON and ENHANCER OF TRY AND CPC1 play pleiotropic and partly non-redundant roles in the phosphate starvation response of Arabidopsis roots 
Journal of Experimental Botany  2015;66(15):4821-4834.
RNA sequencing of mutants defective in the expression of three paralogous MYB transcription factors revealed pleiotropic roles and dynamic shifts in the function of the proteins upon exposure to phosphate starvation.
Phosphate (Pi) deficiency alters root hair length and frequency as a means of increasing the absorptive surface area of roots. Three partly redundant single R3 MYB proteins, CAPRICE (CPC), ENHANCER OF TRY AND CPC1 (ETC1) and TRIPTYCHON (TRY), positively regulate the root hair cell fate by participating in a lateral inhibition mechanism. To identify putative targets and processes that are controlled by these three transcription factors (TFs), we conducted transcriptional profiling of roots from Arabidopsis thaliana wild-type plants, and cpc, etc1 and try mutants grown under Pi-replete and Pi-deficient conditions using RNA-seq. The data show that in an intricate interplay between the three MYBs regulate several developmental, physiological and metabolic processes that are putatively located in different tissues. When grown on media with a low Pi concentration, all three TFs acquire additional functions that are related to the Pi starvation response, including transition metal transport, membrane lipid remodelling, and the acquisition, uptake and storage of Pi. Control of gene activity is partly mediated through the regulation of potential antisense transcripts. The current dataset extends the known functions of R3 MYB proteins, provides a suite of novel candidates with critical function in root hair development under both control and Pi-deficient conditions, and challenges the definition of genetic redundancy by demonstrating that environmental perturbations may confer specific functions to orthologous proteins that could have similar roles under control conditions.
PMCID: PMC4507782  PMID: 26022254
Gene regulation; genetic redundancy; phosphate starvation; RNA-seq; root hairs; transcriptional profiling.
21.  Changing the spatial pattern of TFL1 expression reveals its key role in the shoot meristem in controlling Arabidopsis flowering architecture 
Journal of Experimental Botany  2015;66(15):4769-4780.
Plants carefully control where and when flowers are made through activators and repressors. We show that spatially the shoot meristem is key in responding to the repressors of flowering TFL1.
Models for the control of above-ground plant architectures show how meristems can be programmed to be either shoots or flowers. Molecular, genetic, transgenic, and mathematical studies have greatly refined these models, suggesting that the phase of the shoot reflects different genes contributing to its repression of flowering, its vegetativeness (‘veg’), before activators promote flower development. Key elements of how the repressor of flowering and shoot meristem gene TFL1 acts have now been tested, by changing its spatiotemporal pattern. It is shown that TFL1 can act outside of its normal expression domain in leaf primordia or floral meristems to repress flower identity. These data show how the timing and spatial pattern of TFL1 expression affect overall plant architecture. This reveals that the underlying pattern of TFL1 interactors is complex and that they may be spatially more widespread than TFL1 itself, which is confined to shoots. However, the data show that while TFL1 and floral genes can both act and compete in the same meristem, it appears that the main shoot meristem is more sensitive to TFL1 rather than floral genes. This spatial analysis therefore reveals how a difference in response helps maintain the ‘veg’ state of the shoot meristem.
PMCID: PMC4507777  PMID: 26019254
Architecture; expression; flowering; identity; meristem; TFL1.
22.  Cytokinin as a positional cue regulating lateral root spacing in Arabidopsis  
Journal of Experimental Botany  2015;66(15):4759-4768.
Cytokinin synthesis gene expression patterns and mutant phenotypes show the relevance of cytokinins in generating a paracrine signal to regulate lateral root spacing, which is important in shaping root system architecture.
The root systems of plants have developed adaptive architectures to exploit soil resources. The formation of lateral roots (LRs) contributes to root system architecture. Roots of plants with a lower cytokinin status form LR primordia (LRP) in unusually close proximity, indicating a role for the hormone in regulating the positioning of LRs along the main root axis. Data obtained from cytokinin-synthesis mutants of Arabidopsis thaliana combined with gene expression analysis indicate that cytokinin synthesis by IPT5 and LOG4 occurring early during LRP initiation generates a local cytokinin signal abbreviating LRP formation in neighbouring pericycle cells. In addition, IPT3, IPT5, and IPT7 contribute to cytokinin synthesis in the vicinity of existing LRP, thus suppressing initiation of new LRs. Interestingly, mutation of CYP735A genes required for trans-zeatin biosynthesis caused strong defects in LR positioning, indicating an important role for this cytokinin metabolite in regulating LR spacing. Further it is shown that cytokinin and a known regulator of LR spacing, the receptor-like kinase ARABIDOPSIS CRINKLY4 (ACR4), operate in a non-hierarchical manner but might exert reciprocal control at the transcript level. Taken together, the results suggest that cytokinin acts as a paracrine hormonal signal in regulating root system architecture.
PMCID: PMC4507779  PMID: 26019251
Arabidopsis thaliana; cytokinin; lateral root; lateral root spacing; root branching; root system architecture.
23.  Arabidopsis seed-specific vacuolar aquaporins are involved in maintaining seed longevity under the control of ABSCISIC ACID INSENSITIVE 3  
Journal of Experimental Botany  2015;66(15):4781-4794.
Arabidopsis tonoplast intrinsic protein TIP3;1 and TIP3;2 are shown to play a role in seed longevity under the transcriptional control of ABI3.
The tonoplast intrinsic proteins TIP3;1 and TIP3;2 are specifically expressed during seed maturation and localized to the seed protein storage vacuole membrane. However, the function and physiological roles of TIP3s are still largely unknown. The seed performance of TIP3 knockdown mutants was analysed using the controlled deterioration test. The tip3;1/tip3;2 double mutant was affected in seed longevity and accumulated high levels of hydrogen peroxide compared with the wild type, suggesting that TIP3s function in seed longevity. The transcription factor ABSCISIC ACID INSENSITIVE 3 (ABI3) is known to be involved in seed desiccation tolerance and seed longevity. TIP3 transcript and protein levels were significantly reduced in abi3-6 mutant seeds. TIP3;1 and TIP3;2 promoters could be activated by ABI3 in the presence of abscisic acid (ABA) in Arabidopsis protoplasts. TIP3 proteins were detected in the protoplasts transiently expressing ABI3 and in ABI3-overexpressing seedlings when treated with ABA. Furthermore, ABI3 directly binds to the RY motif of the TIP3 promoters. Therefore, seed-specific TIP3s may help maintain seed longevity under the expressional control of ABI3 during seed maturation and are members of the ABI3-mediated seed longevity pathway together with small heat shock proteins and late embryo abundant proteins.
PMCID: PMC4507774  PMID: 26019256
ABI3; Arabidopsis; hydrogen peroxide; seed longevity; TIP3.
24.  Effect of herbicide resistance endowing Ile-1781-Leu and Asp-2078-Gly ACCase gene mutations on ACCase kinetics and growth traits in Lolium rigidum  
Journal of Experimental Botany  2015;66(15):4711-4718.
Impaired ACCase enzyme kinetics associated with a particular herbicide resistance mutation is associated with a reduction in plant growth.
The rate of herbicide resistance evolution in plants depends on fitness traits endowed by alleles in both the presence and absence (resistance cost) of herbicide selection. The effect of two Lolium rigidum spontaneous homozygous target-site resistance-endowing mutations (Ile-1781-Leu, Asp-2078-Gly) on both ACCase activity and various plant growth traits have been investigated here. Relative growth rate (RGR) and components (net assimilation rate, leaf area ratio), resource allocation to different organs, and growth responses in competition with a wheat crop were assessed. Unlike plants carrying the Ile-1781-Leu resistance mutation, plants homozygous for the Asp-2078-Gly mutation exhibited a significantly lower RGR (30%), which translated into lower allocation of biomass to roots, shoots, and leaves, and poor responses to plant competition. Both the negligible and significant growth reductions associated, respectively, with the Ile-1781-Leu and Asp-2078-Gly resistance mutations correlated with their impact on ACCase activity. Whereas the Ile-1781-Leu mutation showed no pleiotropic effects on ACCase kinetics, the Asp-2078-Gly mutation led to a significant reduction in ACCase activity. The impaired growth traits are discussed in the context of resistance costs and the effects of each resistance allele on ACCase activity. Similar effects of these two particular ACCase mutations on the ACCase activity of Alopecurus myosuroides were also confirmed.
PMCID: PMC4507778  PMID: 26019257
ACCase activity; Alopecurus myosuroides; competition; resistance cost; resistance mutation; RGR.
25.  Synchrotron-based X-ray absorption near-edge spectroscopy imaging for laterally resolved speciation of selenium in fresh roots and leaves of wheat and rice 
Journal of Experimental Botany  2015;66(15):4795-4806.
Using synchrotron-based XANES imaging, in situ laterally resolved speciation of Se in hydrated tissues was obtained, which assists in understanding the Se uptake, translocation, and transformation in fresh plants.
Knowledge of the distribution of selenium (Se) species within plant tissues will assist in understanding the mechanisms of Se uptake and translocation, but in situ analysis of fresh and highly hydrated plant tissues is challenging. Using synchrotron-based fluorescence X-ray absorption near-edge spectroscopy (XANES) imaging to provide laterally resolved data, the speciation of Se in fresh roots and leaves of wheat (Triticum aestivum L.) and rice (Oryza sativa L.) supplied with 1 μM of either selenate or selenite was investigated. For plant roots exposed to selenate, the majority of the Se was efficiently converted to C-Se-C compounds (i.e. methylselenocysteine or selenomethionine) as selenate was transported radially through the root cylinder. Indeed, even in the rhizodermis which is exposed directly to the bulk solution, only 12–31% of the Se was present as uncomplexed selenate. The C-Se-C compounds were probably sequestered within the roots, whilst much of the remaining uncomplexed Se was translocated to the leaves—selenate accounting for 52–56% of the total Se in the leaves. In a similar manner, for plants exposed to selenite, the Se was efficiently converted to C-Se-C compounds within the roots, with only a small proportion of uncomplexed selenite observed within the outer root tissues. This resulted in a substantial decrease in translocation of Se from the roots to leaves of selenite-exposed plants. This study provides important information for understanding the mechanisms responsible for the uptake and subsequent transformation of Se in plants.
PMCID: PMC4507780  PMID: 26019258
Fluorescence-XANES imaging; laterally resolved speciation; selenium uptake; speciation; transformation; translocation.

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