The John Innes Centre Publications Repository contains details of all publications resulting from our researchers.

The repository also includes Open Access publications, which can be identified by the icons found on search results.

 Green open access publications are marked by the PDF icon. Click on the publication title, or the PDF icon, and read a pre-print PDF version of the publication.  Gold open access publications have the gold open padlock icon. You can read the full version of these papers on the publishing journal’s website without a subscription. 

The creation of this publications repository was funded by BBSRC.

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Sun S. K., Chen Y., Che J., Konishi N., Tang Z., Miller A. J., Ma J. F., Zhao F. J. (2018)

Decreasing arsenic accumulation in rice by overexpressing OsNIP1;1 and OsNIP3;3 through disrupting arsenite radial transport in roots.

New Phytologist

Publisher's version: 10.1111/nph.15190

ID: 58731

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Rice is a major dietary source of the toxic metalloid arsenic. Reducing arsenic accumulation in rice grain is important for food safety. We generated transgenic rice overexpressing two aquaporin genes, OsNIP1;1 and OsNIP3;3, under the control of a maize ubiquitin promoter or the rice OsLsi1 promoter, and tested the effect on arsenite uptake and translocation. OsNIP1;1 and OsNIP3;3 were highly permeable to arsenite in Xenopus oocyte assays. Both transporters were localized at the plasma membrane. Knockout of either gene had little effect on arsenite uptake or translocation. Overexpression of OsNIP1;1 or OsNIP3;3 in rice did not affect arsenite uptake but decreased root-to-shoot translocation of arsenite and shoot arsenic concentration markedly. The overexpressed OsNIP1;1 and OsNIP3;3 proteins were localized in all root cells without polarity. Expression of OsNIP1;1 driven by the OsLsi1 promoter produced similar effects. When grown in two arsenic-contaminated paddy soils, overexpressing lines contained significantly lower arsenic concentration in rice grain than the wild-type without compromising plant growth or the accumulation of essential nutrients. Overexpression of OsNIP1;1 or OsNIP3;3 provides a route for arsenite to leak out of the stele, thus restricting arsenite loading into the xylem. This strategy is effective in reducing arsenic accumulation in rice grain.

Capstaff N. M., Miller A. J. (2018)

Improving the Yield and Nutritional Quality of Forage Crops

Frontiers in Plant Science (9)

Publisher's version: 10.3389/fpls.2018.00535

ID: 58534

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Despite being some of the most important crops globally, there has been limited research on forages when compared with cereals, fruits, and vegetables. This review summarizes the literature highlighting the significance of forage crops, the current improvements and some of future directions for improving yield and nutritional quality. We make the point that the knowledge obtained from model plant and grain crops can be applied to forage crops. The timely development of genomics and bioinformatics together with genome editing techniques offer great scope to improve forage crops. Given the social, environmental and economic importance of forage across the globe and especially in poorer countries, this opportunity has enormous potential to improve food security and political stability.

Helliwell J. R., Strurrock C. J., Mairhofer S., Craigon J., Ashton R. W., Miller A. J., Whalley W. R., Mooney S. J. (2017)

The emergent rhizosphere: imaging the development of the porous architecture at the root-soil interface

Scientific Reports (Published online) Published online

Publisher's version: 10.1038/s41598-017-14904-w

ID: 57692

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The rhizosphere is the zone of soil influenced by a plant root and is critical for plant health and nutrient acquisition. All below ground resources must pass through this dynamic zone prior to their capture by plant roots. However, researching the undisturbed rhizosphere has proved very challenging. Here we compare the temporal changes to the intact rhizosphere pore structure during the emergence of a developing root system in different soils. High resolution X-ray Computed Tomography (CT) was used to quantify the impact of root development on soil structural change, at scales relevant to individual micro-pores and aggregates (µm). A comparison of micro-scale structural evolution in homogenously packed soils highlighted the impacts of a penetrating root system in changing the surrounding porous architecture and morphology. Results indicate the structural zone of influence of a root can be more localised than previously reported (µm scale rather than mm scale). With time, growing roots significantly alter the soil physical environment in their immediate vicinity through reducing root-soil contact and crucially increasing porosity at the root-soil interface and not the converse as has often been postulated. This ‘rhizosphere pore structure’ and its impact on associated dynamics are discussed


Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Vincent T. R., Avramova M., Canham J., Higgins P., Bilkey N., Mugford S. T., Pitino M., Toyota M., Gilroy S., Miller A. J., Hogenhout S., Sanders D. (2017)

Interplay of plasma membrane and vacuolar ion channels, together with BAK1, elicits rapid cytosolic calcium elevations in Arabidopsis during aphid feeding.

Plant Cell (Epub ahead of print) Epub ahead of print

Publisher's version: 10.1105/tpc.17.00136

ID: 56585

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A transient rise in cytosolic calcium ion concentration is one of the main signals used by plants in perception of their environment. The role of calcium in the detection of abiotic stress is well documented; however, its role during biotic interactions remains unclear. Here, we use a fluorescent calcium biosensor (GCaMP3) in combination with the green peach aphid (Myzus persicae) as a tool to study Arabidopsis thaliana calcium dynamics in vivo and in real time during a live biotic interaction. We demonstrate rapid and highly-localised plant calcium elevations around the feeding sites of M. persicae, and by monitoring aphid feeding behaviour electrophysiologically we demonstrate that these elevations correlate with aphid probing of epidermal and mesophyll cells. Furthermore, we dissect the molecular mechanisms involved, showing that interplay between the plant defence co-receptor BRASSINOSTEROID INSENSITIVE-ASSOCIATED KINASE 1 (BAK1), the plasma membrane ion channels GLUTAMATE RECEPTOR-LIKE 3.3 and 3.6 (GLR3.3 and GLR3.6) and the vacuolar ion channel TWO-PORE CHANNEL 1 (TPC1) mediate these calcium elevations. Consequently, we identify a link between plant perception of biotic threats by BAK1, cellular calcium entry mediated by GLRs, and intracellular calcium release by TPC1 during a biologically relevant interaction.

Chen Y., Sun S. K., Tang Z., Liu G., Moore K. L., Maathuis F. J. M., Miller A. J., McGrath S. P., Zhao F. J. (2017)

The Nodulin 26-like intrinsic membrane protein OsNIP3;2 is involved in arsenite uptake by lateral roots in rice.

Journal of Experimental Botany (Epub ahead of print) Epub ahead of print

Publisher's version: 10.1093/jxb/erx165

ID: 56488

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Previous studies have shown that the Nodulin 26-like intrinsic membrane protein (NIP) Lsi1 (OsNIP2;1) is involved in arsenite [As(III)] uptake in rice (Oryza sativa). However, the role of other rice NIPs in As(III) accumulation in planta remains unknown. In the present study, we investigated the role OsNIP3;2 in As(III) uptake in rice. When expressed in Xenopus laevis oocytes, OsNIP3;2 showed a high transport activity for As(III). Quantitative real-time RT-PCR showed that the expression of OsNIP3;2 was suppressed by 5 µM As(III), but enhanced by 20 and 100 µM As(III). Transgenic rice plants expressing OsNIP3;2pro-GUS showed that the gene was predominantly expressed in the lateral roots and the stele region of the primary roots. Transient expression of OsNIP3;2:GFP fusion protein in rice protoplasts showed that the protein was localized in the plasma membrane. Knockout of OsNIP3;2 significantly decreased As concentration in the roots, but had little effect on shoot As concentration. Synchrotron microfocus X-ray fluorescence showed decreased As accumulation in the stele of the lateral roots in the mutants compared with wild-type. Our results indicate that OsNIP3;2 is involved in As(III) uptake by lateral roots, but its contribution to As accumulation in the shoots is limited.

Fan X., Naz M., Fan X., Xuan W., Miller A. J., Xu G. (2017)

Plant nitrate transporters: from gene function to application.

Journal of Experimental Botany (68) 2463-2475

Publisher's version: 10.1093/jxb/erx011

ID: 56995

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We summarize nitrate transporters and discuss their potential in breeding for improved nitrogen use efficiency and yield.

Menguer P., Vincent T., Miller A. J., Brown J. K. M., Vincze E., Borg S., Holm P. B., Sanders D., Podar D. (2017)

Improving zinc accumulation in barley endosperm using HvMTP1, a transition metal transporter.

Plant Biotechnology Journal (Plant Biotechnol J. 2017 Apr 24. doi: 10.1111/pbi.12749. [Epub ahead of print]) doi: 10.1111/pbi.12749

Publisher's version: 10.1111/pbi.12749

ID: 56109

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Zinc (Zn) is essential for all life forms, including humans. It is estimated that around two billion people are deficient in their Zn intake. Human dietary Zn intake relies heavily on plants, which in many developing countries consists mainly of cereals. The inner part of cereal grain, the endosperm, is the part that is eaten after milling but contains only a quarter of the total grain Zn. Here we present results demonstrating that endosperm Zn content can be enhanced through expression of a transporter responsible for vacuolar Zn accumulation in cereals. The barley (Hordeum vulgare) vacuolar Zn transporter HvMTP1 was expressed under the control of the endosperm-specific D-hordein promoter. Transformed plants exhibited no significant change in growth but had higher total grain Zn concentration, as measured by ICP-OES, compared to parental controls. Compared with Zn, transformants had smaller increases in concentrations of Cu and Mn but not Fe. Staining grain cross-sections with the Zn-specific stain DTZ revealed a significant enhancement of Zn accumulation in the endosperm of two of three transformed lines, a result confirmed by ICP-OES in the endosperm of dissected grain. Synchrotron X-ray fluorescence analysis of longitudinal grain sections demonstrated a redistribution of grain Zn from aleurone to endosperm. We argue that this proof-of-principle study provides the basis of a strategy for biofortification of cereal endosperm with Zn. This article is protected by copyright. All rights reserved.

Kenzhebayeva S. S., Doktyrbay G., Capstaff N., Sarsu F., Omirbekova N. Z. H., Eilam T., Tashenev D. K., Miller A. J. (2017)

Searching a spring wheat mutation resource for correlations between yield, grain size, and quality parameters

Journal of Crop Improvement (31 (tbc)) 1-20

Publisher's version: 10.1080/15427528.2016.1276990

ID: 55621

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To broaden genetic variation, an irradiated wheat (Triticum aestivum L.) M5 population was generated in the background of spring wheat cv. Almaken. This resource was used to measure components of productivity, including grain number and grain weight (GW) per main spike, GW per plant (GWP), 1000-grain weight (TGW), grain size and grain shape, and some quality parameters. Some mutant lines, mostly in the 200-Gy-dosed germplasm, had 2–4 times higher grain iron and zinc concentrations and 7–11% higher protein content relative to the parent line. Some irradiated lines had significantly larger TGW, and grain area (GA), length, and width than the parent, cv. Almaken. The largest GA and grain length (GL) were 30–40% greater than those of the parent. Correlations for Zn concentration versus GA = 0.191, p ˂ 0.01, grain protein content (GPC) versus GA = 0.128, p ˂ 0.05, GPC versus GL = 0.113, p ˂ 0.05, and GPC versus grain width = 0.191, p˂0.001 were observed in 200 Gy-dosed mutants. In 100 Gy-dosed mutants, correlations for Fe concentration versus GWP = 0.302, p ˂ 0.001 and Fe concentration versus TGW = 0.153, p ˂ 0.01 were found. The mutant lines showed the capacity to biofortify wheat grain without negatively impacting on crop productivity and this population offers promising donors for improving grain parameters such as GA, length, and width and quality. The data presented showed how the genetic variation generated through radiation could be used to test the linkage between various important grain parameters.

Murray J. D., Liu C. W., Chen Y., Miller A. J. (2016)

Nitrogen sensing in legumes.

Journal of Experimental Botany (Advance Access) 1-8

Publisher's version: doi:10.1093/jxb/erw405

ID: 55447

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Legumes fix atmospheric nitrogen (N) in a symbiotic relationship with bacteria. For this reason, although legume crops can be low yielding and less profitable when compared with cereals, they are frequently included in crop rotations. Grain legumes form only a minor part of most human diets, and legume crops are greatly underutilized. Food security and soil fertility could be significantly improved by greater grain legume usage and increased improvement of a range of grain legumes. One limitation for the use of legumes as a source of N input into agricultural systems is the fact that the formation of N-fixing nodules is suppressed when soils are replete with n. In this review, we report what is known about this process and how soil N supply might be sensed and feed back to regulate nodulation.

Princi M. P., Lupini A., Longo C., Miller T., Sunseri F., Abenavoli M. R. (2016)

Long- and short-term effects of boron excess to root form and function in two tomato genotypes.

Plant physiology and biochemistry : PPB (109) 9-19

Publisher's version: 10.1016/j.plaphy.2016.08.023

ID: 56204

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Boron (B) is an essential plant nutrient, but when present in excess it is toxic. Morphological measurements were made to assess the impact of B toxicity on the growth of two different tomato hybrids, Losna and Ikram. Contrasting long and short-term B responses in these tomato hybrids, were observed. Losna showed less toxicity symptoms, maintaining higher growth and showing much less B content in both root and shoot tissues compared to Ikram. Root morphological differences did not explain the tolerance between the two hybrids. Under excess B supply, a significant inhibition on net nitrate uptake rate was observed in Ikram, but not in Losna. This effect may be explained by a decrease of nitrate transporter transcripts in Ikram, which was not measured in Losna. There was a different pattern of B transporter expression in two tomatoes and this can explain the contrasting tolerance observed. Indeed, Losna may be able to exclude or efflux B resulting in less accumulation in the shoot. Particularly, SlBOR4 expression showed significant differences between the tomato hybrids, with higher expression in Losna explaining the improved B-tolerance.

Hu R., Qiu D., Chen Y., Miller A. J., Fan X., Pan X., Zhang M. (2016)

Knock-down of a tonoplast localized low-affinity nitrate transporter OsNPF7.2 affects rice growth under high nitrate supply

Frontiers in Plant Science (7) 1-13

Publisher's version: 10.3389/fpls.2016.01529

ID: 55217

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The large nitrate transporter 1/peptide transporter family (NPF) has been shown to transport diverse substrates, including nitrate, amino acids, peptides, phytohormones, and glucosinolates. However, the rice (Oryza sativa) root-specific family member OsNPF7.2 has not been functionally characterized. Here, our data show that OsNPF7.2 is a tonoplast localized low-affinity nitrate transporter, that affects rice growth under high nitrate supply. Expression analysis showed that OsNPF7.2 was mainly expressed in the elongation and maturation zones of roots, especially in the root sclerenchyma, cortex and stele. It was also induced by high concentrations of nitrate. Subcellular localization analysis showed that OsNPF7.2 was localized on the tonoplast of large and small vacuoles. Heterologous expression in Xenopus laevis oocytes suggested that OsNPF7.2 was a low-affinity nitrate transporter. Knock-down of OsNPF7.2 retarded rice growth under high concentrations of nitrate. Therefore, we deduce that OsNPF7.2 plays a role in intracellular allocation of nitrate in roots, and thus influences rice growth under high nitrate supply.

Foyer C. H., Lam H. M., Nguyen H. T., Kadambot H. M. S., Varshney R. K., Colmer T. D., Cowling W., Bramley H., Mori T. A., Hodgson J. M., Cooper J. W., Miller A. J., Kunert K., Vorster J., Cullis C., Ozga J. A., Wahlqvist M. L., Liang Y., Shou H., Shi K., Yu J., Fodor N., Kaiser B. N., Wong F. K., Valliyodan B., Considine M. J. (2016)

Neglecting legumes has compromised human health and sustainable food production

Nature Plants (2 ) 16112

Publisher's version: 10.1038/nplants.2016.112

ID: 55056

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The United Nations declared 2016 as the International Year of Pulses (grain legumes) under the banner 'nutritious seeds for a sustainable future'.  A second green revolution is required to ensure food and nutritional security in the face of global climate change.  Grain legumes provide an unparalleled solution to this problem because of their inherent capacity for symbiotic atmospheric nitrogen fixation, which provides economically sustainable advantages for farming.  In addition, a legume-rich diet has health benefits for humans and livestock alike.  However, grain legumes form only a minor part of most current human diets, and legume crops are greatly under-used.  Food security and soil fertility could be significantly improved by greater grain legume usage and increased improvement of a range of grain legumes.  The current lack of coordianted focus on grain legumes has compormised human helath, nutritional security and sustainable food production.

Fan X., Tang Z., Tan Y., Zhang Y., Luo B., Yang M., Lian X., Shen Q., Miller A. J., Xu G. (2016)

Overexpression of a pH-sensitive nitrate transporter in rice increases crop yields.

Proceedings of the National Academy of Sciences of the United States of America (113) 7118-23

Publisher's version: 10.1073/pnas.1525184113

ID: 53382

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Cellular pH homeostasis is fundamental for life, and all cells adapt to maintain this balance. In plants, the chemical form of nitrogen supply, nitrate and ammonium, is one of the cellular pH dominators. We report that the rice nitrate transporter OsNRT2.3 is transcribed into two spliced isoforms with a natural variation in expression ratio. One splice form, OsNRT2.3b is located on the plasma membrane, is expressed mainly in the phloem, and has a regulatory motif on the cytosolic side that acts to switch nitrate transport activity on or off by a pH-sensing mechanism. High OsNRT2.3b expression in rice enhances the pH-buffering capacity of the plant, increasing N, Fe, and P uptake. In field trials, increased expression of OsNRT2.3b improved grain yield and nitrogen use efficiency (NUE) by 40%. These results indicate that pH sensing by the rice nitrate transporter OsNRT2.3b is important for plant adaption to varied N supply forms and can provide a target for improving NUE.

Chen Y., Ma J., Miller A. J., Luo B., Wang M., Zhu Z., Ouwerkerk P. B. F. (2016)

OsCHX14 is Involved in the K+ Homeostasis in Rice (Oryza sativa) Flowers

Plant & Cell Physiology (57 (7)) 1530-1543

Publisher's version: 10.1093/pcp/pcw088

ID: 55219

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Previously we showed in the osjar1 mutants that the lodicule senescence which controls the closing of rice flowers was delayed. This resulted in florets staying open longer when compared with the wild type. The gene OsJAR1 is silenced in osjar1 mutants and is a key member of the jasmonic acid (JA) signaling pathway. We found that K concentrations in lodicules and flowers of osjar1-2 were significantly elevated compared with the wild type, indicating that K+ homeostasis may play a role in regulating the closure of rice flowers. The cation/H+ exchanger (CHX) family from rice was screened for potential K+ transporters involved as many members of this family in Arabidopsis were exclusively or preferentially expressed in flowers. Expression profiling confirmed that among 17 CHX genes in rice, OsCHX14 was the only member that showed an expression polymorphism, not only in osjar1 mutants but also in RNAi (RNA interference) lines of OsCOI1, another key member of the JA signaling pathway. This suggests that the expression of OsCHX14 is regulated by the JA signaling pathway. Green fluorescent protein (GFP)-tagged OsCHX14 protein was preferentially localized to the endoplasmic reticulum. Promoter–β-glucuronidase (GUS) analysis of transgenic rice revealed that OsCHX14 is mainly expressed in lodicules and the region close by throughout the flowering process. Characterization in yeast and Xenopus laevis oocytes verified that OsCHX14 is able to transport K+, Rb+ and Cs+ in vivo. Our data suggest that OsCHX14 may play an important role in K+ homeostasis during flowering in rice.

Lupini A., Mercanti F., Araniti F., Miller A. J., Sunseri F., Abenavoli M. R. (2016)

NAR2.1/NRT2.1 functional interaction with NO3- and H+ fluxes in high-affinity nitrate transport in maize root regions.

Plant Physiology and Biochemistry (102) 107-114

Publisher's version: doi:10.1016/j.plaphy.2016.02.022

ID: 53306

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Spatial and temporal fluctuations in nitrate (NO3-) availability are very common in agricultural soils. Therefore, understanding the molecular and physiological mechanisms involved in regulating NO3- uptake in regions along the primary root is important for improving the NO3- uptake efficiency (NUpE) in crops. Different regions of maize primary root, named R1, R2 and R3, NO3- starved for 3 days, were exposed to 50 μM NO3-. Electrophysiological measurements (membrane potential and H+ and NO3- fluxes) and NPF6.3, NRT2.1, NAR2.1, MHA1, MHA3 and MHA4 gene expression analyses were carried out. The results confirmed variable spatial and temporal patterns in both NO3- and H+ fluxes and gene expression along the primary maize root. A significant correlation (P = 0.0023) between nitrate influx and gene transcript levels was observed only when NAR2.1 and NRT2.1 co-expression were considered together, showing for the first time the NRT2.1/NAR2.1 functional interaction in nitrate uptake along the root axis.Taken together these results suggest differing roles among the primary root regions, in which the apical part seem to be involved to sensing and signaling in contrast with the basal root which appears to be implicate in nitrate acquisition.

Castro-Rodríguez V., Assaf-Casals I., Pérez-Tienda J., Fan X., Avila C., Miller A. J., Cánovas F. M. (2015)

Deciphering the molecular basis of ammonium uptake and transport in maritime pine.

Plant Cell & Environment (39) 16691682

Publisher's version: 10.1111/pce.12692

ID: 52505

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Ammonium is the predominant form of inorganic nitrogen in the soil of coniferous forests. Despite the ecological and economic importance of conifers, the molecular basis of ammonium uptake and transport in this group of gymnosperms is largely unknown. In this study, we describe the functional characterization of members of the AMT gene family in Pinus pinaster: PpAMT1.1, PpAMT1.2 and PpAMT1.3 (subfamily 1) and PpAMT2.1 and PpAMT2.3 (subfamily 2). Our phylogenetic analysis indicates that in conifers, all members of the AMT1 subfamily evolved from a common ancestor that is evolutionarily related to the ancient PpAMT1.2 gene. Individual AMT genes are developmentally- and nutritionally-regulated and their transcripts are specifically distributed in different organs. PpAMT1.3 was predominantly expressed in the roots, particularly during N starvation and mycorrhizal interaction whereas PpAMT2.3 was preferentially expressed in lateral roots. Immunolocalization studies of roots with varied nitrogen availability revealed that PpAMT1 and PpAMT2 proteins play complementary roles in the uptake of external ammonium. Heterologous expression in yeast and Xenopus oocytes revealed that the AMT genes encode functional transporters with different kinetics and with different capacities for ammonium transport. Our results provide new insights on how nitrogen is acquired and transported in conifers. This article is protected by copyright. All rights reserved.

Chen Y., Moore K. L., Miller A. J., McGrath S. P., Ma J. F., Zhao F. J. (2015)

The role of nodes in arsenic storage and distribution in rice

Journal of Experimental Botany (66) 3717-3724

Publisher's version: 10.1093/jxb/erv164

ID: 49596

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Knowledge of arsenic (As) accumulation in rice (Oryza sativa L.) is important for minimizing As transfer to the food chain. The aim of this study was to investigate the role of rice nodes in As storage and distribution. Synchrotron μX-ray fluorescence (μ-XRF) was used to map As distribution in the top node and internode of a lsi2 mutant defective in silicon/arsenite efflux carrier and its wild-type (WT) grown in soil. Lsi2 expression in different tissues during grain filling was investigated by quantitative RT-PCR. Arsenite or dimethylarsinic acid (DMA) was supplied to excised panicles to investigate the roles of Lsi2 and phytochelatins (PC) in As distribution. μ-XRF mapping revealed As storage in the phloem of different vascular bundles in the top node and internode. Soil-grown plants of lsi2 had markedly decreased As accumulation in the phloem compared with the WT. Lsi2 was strongly expressed, not only in the roots but also in the nodes. When excised panicles were exposed to As(III), the lsi2 mutant distributed more As to the node and flag leaf but less As to the grain compared with the WT, while there was no significant difference in DMA distribution. Inhibition of PC synthesis by l-buthionine-sulphoximine decreased As(III) deposition in the top node but increased As accumulation in the grain and flag leaf. The results suggest that rice nodes serve as a filter restricting As(III) distribution to the grain. Furthermore, Lsi2 plays a role in As(III) distribution in rice nodes and phytochelatins are important compounds for As(III) storage in the nodes.

Xia X., Fan X., Wei J., Feng H., Qu H., Xie D., Miller A. J., Xu G. (2014)

Rice nitrate transporter OsNPF2.4 functions in low-affinity acquisition and long-distance transport

Journal of Experimental Botany (66) 317-331

Publisher's version: 10.1093/jxb/eru425

ID: 48638

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Plant proteins belonging to the NPF (formerly NRT1/PTR) family are well represented in every genome and function in transporting a wide variety of substrates. In this study, we showed that rice OsNPF2.4 is located in the plasma membrane and is expressed mainly in the epidermis, xylem parenchyma, and phloem companion cells. Functional analysis in oocytes showed that OsNPF2.4 is a pH-dependent, low-affinity NO–3 transporter. Short-term 15NO–3 influx rate, long-term NO–3 acquisition by root, and upward transfer from root to shoot were decreased by disruption of OsNPF2.4 and increased by OsNPF2.4 overexpression under high NO–3 supply. Moreover, the redistribution of NO–3 in the mutants in comparison with the wild type from the oldest leaf to other organs, particularly to N-starved roots, was dramatically changed. Knockout of OsNPF2.4 decreased rice growth and potassium (K) concentration in xylem sap, root, culm, and sheath, but increased the shoot:root ratio of tissue K under higher NO–3. We conclude that OsNPF2.4 functions in acquisition and long-distance transport of NO–3, and that altering its expression has an indirect effect on K recycling between the root and shoot.


A partner protein, NAR2, is essential for high-affinity nitrate transport of the NRT2 protein in plants. However, the NAR2 motifs that interact with NRT2s for their plasma membrane (PM) localization and nitrate transporter activity have not been functionally characterized. In this study, OsNAR2.1 mutations with different carbon (C)-terminal deletions and nine different point mutations in the conserved regions of NAR2 homologs in plants were generated to explore the essential motifs involved in the interaction with OsNRT2.3a. Screening using the membrane yeast two-hybrid system and Xenopus oocytes for nitrogen-15 ((15) N) uptake demonstrated that either R100G or D109N point mutations impaired the OsNAR2.1 interaction with OsNRT2.3a. Western blotting and visualization using green fluorescent protein fused to either the N- or C-terminus of OsNAR2.1 indicated that OsNAR2.1 is expressed in both the PM and cytoplasm. The split-yellow fluorescent protein (YFP)/BiFC analyses indicated that OsNRT2.3a was targeted to the PM in the presence of OsNAR2.1, while either R100G or D109N mutation resulted in the loss of OsNRT2.3a-YFP signal in the PM. Based on these results, arginine 100 and aspartic acid 109 of the OsNAR2.1 protein are key amino acids in the interaction with OsNRT2.3a, and their interaction occurs in the PM but not cytoplasm.

Helliwell J. R., Miller A. J., Whalley W. R., Mooney S. J., Sturrock C. J. (2014)

Quantifying the impact of microbes on soil structural development and behaviour in wet soils

Soil Biology & Biochemistry (74) 138-147

Publisher's version: 10.1016/j.soilbio.2014.03.009

ID: 46715

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There is evidence that microbial populations play an important role in altering soil pore geometry, but a full understanding of how this affects subsequent soil behaviour and function is still unclear. In particular the role of microorganisms in soil structural evolution and its consequence for pore morphological development is lacking. Using a combination of bio-chemical measurements and X-ray Computed Tomography (CT) imaging, a temporal comparison of microscale soil structural development in contrasting soil environments was made. The aim was to quantify the effect of microbial activity in the absence of other features likely to cause soil deformation (e.g. earthworms, roots etc.) on soil structural development in wet soils, defined by changes in the soil porous architecture i.e. pore connectivity, pore shape and pore volume during a 24 week period. Three contrasting soil textures were examined and changes compared between field soil, sterilised soil and a glucose enhanced soil treatment. Our results indicate that soil biota can significantly alter their microhabitat by changing soil pore geometry and connectivity, primarily through localised gaseous release. This demonstrates the ability of microorganisms to modify soil structure, and may help reveal the scope by which the microbial-rich rhizosphere can locally influence water and nutrient delivery to plant roots.

Miller A. J. (2014)

Plant Mineral Nutrition.

eLS (N/A) N/A

Publisher's version: 10.1002/9780470015902.a0023717

ID: 48223

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Several inorganic minerals are essential for plant growth and these are usually obtained by roots from the soil. Availability of minerals in the soil is determined by the physical and chemical characteristics of the soil. Plants can directly influence nutrient availability around the root surface; this zone is called the rhizosphere. Plants adjust root architecture and exudation according to their nutrient requirements and under deficiency these changes can be a marker for nutrient status. Nutrients are taken up from the soil using plasma]membrane located transporter proteins and excess is stored in the cell vacuole or converted into polymerised storage forms. For crops it is essential to match nutrient supply to demand throughout the growth season to obtain the maximum yield. These nutrient storage forms can be used as agricultural indicators of crop nutrient status and the potential for fertilizer leaching losses. Membrane transporters provide a gateway for nutrient entry into plants, but the selectivity of these filters can breakdown when chemically similar minerals are present at very high concentrations. The minerals may not be essential for growth, but they can enter plant cells and cause toxicity.

Wang E., Yu N., Asma Bano S., Liu C., Miller A. J., Cousins D., Zhang X., Ratet P., Tadege M., Mysore K. S., Downie J. A., Murray J. D., Oldroyd G. E. D., Schultze M. (2014)

A H+-ATPase That Energizes Nutrient Uptake during Mycorrhizal Symbioses in Rice and Medicago truncatula[

Plant Cell (26) 1818-1830

Publisher's version: 10.1105/tpc.113.120527

ID: 47297

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Most plant species form symbioses with arbuscular mycorrhizal (AM) fungi, which facilitate the uptake of mineral nutrients such as phosphate from the soil. Several transporters, particularly proton-coupled phosphate transporters, have been identified on both the plant and fungal membranes and contribute to delivering phosphate from fungi to plants. The mechanism of nutrient exchange has been studied in plants during mycorrhizal colonization, but the source of the electrochemical proton gradient that drives nutrient exchange is not known. Here, we show that plasma membrane H+-ATPases that are specifically induced in arbuscule-containing cells are required for enhanced proton pumping activity in membrane vesicles from AM-colonized roots of rice (Oryza sativa) and Medicago truncatula. Mutation of the H+-ATPases reduced arbuscule size and impaired nutrient uptake by the host plant through the mycorrhizal symbiosis. Overexpression of the H+-ATPase Os-HA1 increased both phosphate uptake and the plasma membrane potential, suggesting that this H+-ATPase plays a key role in energizing the periarbuscular membrane, thereby facilitating nutrient exchange in arbusculated plant cells.


Wheat, like many other staple cereals, contains low levels of the essential micronutrients iron and zinc. Up to two billion people worldwide suffer from iron and zinc deficiencies, particularly in regions with predominantly cereal-based diets. Although wheat flour is commonly fortified during processing, an attractive and more sustainable solution is biofortification, which requires developing new varieties of wheat with inherently higher iron and zinc content in their grains. Until now most studies aimed at increasing iron and zinc content in wheat grains have focused on discovering natural variation in progenitor or related species. However, recent developments in genomics and transformation have led to a step change in targeted research on wheat at a molecular level. We discuss promising approaches to improve iron and zinc content in wheat using knowledge gained in model grasses. We explore how the latest resources developed in wheat, including sequenced genomes and mutant populations, can be exploited for biofortification. We also highlight the key research and practical challenges that remain in improving iron and zinc content in wheat.

Feng H., Xia X., Fan X., Xu G., Miller A. J. (2013)

Optimizing plant transporter expression in Xenopus oocytes.

Plant Methods (9) 48

Publisher's version: 10.1186/1746-4811-9-48

ID: 46115

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Rapid improvements in DNA synthesis technology are revolutionizing gene cloning and the characterization of their encoded proteins. Xenopus laevis oocytes are a commonly used heterologous system for the expression and functional characterization of membrane proteins. For many plant proteins, particularly transporters, low levels of expression can limit functional activity in these cells making it difficult to characterize the protein. Improvements in synthetic DNA technology now make it quick, easy and relatively cheap to optimize the codon usage of plant cDNAs for Xenopus. We have tested if this optimization process can improve the functional activity of a two-component plant nitrate transporter assayed in oocytes.

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