AtIAR1 is a Zn transporter that regulates auxin metabolism in Arabidopsis thaliana.

Root growth in Arabidopsis is inhibited by exogenous auxin-amino acid conjugates, and mutants resistant to one such conjugate [indole-3-acetic acid (IAA)-Ala] map to a gene (AtIAR1) that is a member of a metal transporter family. Here, we test the hypothesis that AtIAR1 controls the hydrolysis of stored conjugated auxin to free auxin through zinc transport. AtIAR1 complements a yeast mutant sensitive to zinc, but not manganese- or iron-sensitive mutants, and the transporter is predicted to be localized to the endoplasmic reticulum/Golgi in plants. A previously identified Atiar1 mutant and a non-expressed T-DNA mutant both exhibit altered auxin metabolism, including decreased IAA-glucose conjugate levels in zinc-deficient conditions and insensitivity to the growth effect of exogenous IAA-Ala conjugates. At a high concentration of zinc, wild-type plants show a novel enhanced response to root growth inhibition by exogenous IAA-Ala which is disrupted in both Atiar1 mutants. Furthermore, both Atiar1 mutants show changes in auxin-related phenotypes, including lateral root density and hypocotyl length. The findings therefore suggest a role for AtIAR1 in controlling zinc release from the secretory system, where zinc homeostasis plays a key role in regulation of auxin metabolism and plant growth regulation.

BRUTUS-LIKE (BTSL) E3 ligase-mediated fine-tuning of Fe regulation negatively affects Zn tolerance of Arabidopsis.

The mineral micronutrients zinc (Zn) and iron (Fe) are essential for plant growth and human nutrition, but interactions between the homeostatic networks of these two elements are not fully understood. Here we show that loss of function of BTSL1 and BTSL2, which encode partially redundant E3 ubiquitin ligases that negatively regulate Fe uptake, confers tolerance to Zn excess in Arabidopsis thaliana. Double btsl1 btsl2 mutant seedlings grown on high Zn medium accumulated similar amounts of Zn in roots and shoots to the wild type, but suppressed the accumulation of excess Fe in roots. RNA-sequencing analysis showed that roots of mutant seedlings had relatively higher expression of genes involved in Fe uptake (IRT1, FRO2, and NAS) and in Zn storage (MTP3 and ZIF1). Surprisingly, mutant shoots did not show the transcriptional Fe deficiency response which is normally induced by Zn excess. Split-root experiments suggested that within roots the BTSL proteins act locally and downstream of systemic Fe deficiency signals. Together, our data show that constitutive low-level induction of the Fe deficiency response protects btsl1 btsl2 mutants from Zn toxicity. We propose that BTSL protein function is disadvantageous in situations of external Zn and Fe imbalances, and formulate a general model for Zn-Fe interactions in plants.

Countering elevated CO2 induced Fe and Zn reduction in Arabidopsis seeds.

Growth at increased concentrations of CO2 induces a reduction in seed zinc (Zn) and iron (Fe). Using Arabidopsis thaliana, we investigated whether this could be mitigated by reducing the elevated CO2 -induced decrease in transpiration. We used an infrared imaging-based screen to isolate mutants in At1g08080 that encodes ALPHA CARBONIC ANHYDRASE 7 (ACA7). aca7 mutant alleles display wild-type (WT) responses to abscisic acid (ABA) and light but are compromised in their response to elevated CO2 . ACA7 is expressed in guard cells. When aca7 mutants are grown at 1000 ppm CO2 they exhibit higher transpiration and higher seed Fe and Zn content than WT grown under the same conditions. Our data show that by increasing transpiration it is possible to partially mitigate the reduction in seed Fe and Zn content when Arabidopsis is grown at elevated CO2 .

Interplay of Plasma Membrane and Vacuolar Ion Channels, Together with BAK1, Elicits Rapid Cytosolic Calcium Elevations in Arabidopsis during Aphid Feeding.

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 localized plant calcium elevations around the feeding sites of M. persicae, and by monitoring aphid feeding behavior 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 defense coreceptor BRASSINOSTEROID INSENSITIVE-ASSOCIATED KINASE1 (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 CHANNEL1 (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.

Cytokinin-dependent regulatory module underlies the maintenance of zinc nutrition in rice.

Zinc (Zn) deficiency is a critical problem in human nutrition. Rice is the main source of calories for nearly half the world's population but has the shortcoming, from a nutritional perspective, of being low in Zn and other essential nutrients. Here we performed analyses with cytokinin-related mutants and transgenic lines to provide unequivocal evidence that cytokinins have a key role in controlling Zn status in plants. Transporters responsible for Zn uptake and chelators for the internal transport of Zn were strictly controlled by cytokinins. Moreover, cytokinin metabolism was regulated in a highly dynamic way in response to Zn status, which allows rice to adapt to heterogeneous Zn availability. Subsequently, fine-tuning of cytokinin metabolism by root-specific expression of a cytokinin degradation enzyme was able to improve both Zn nutrient and yield traits. Importantly, X-ray fluorescence imaging revealed that the increased Zn was broadly distributed from the aleurone layer to the inner endosperm. These findings show that metabolic control of cytokinin could provide the key to breeding Zn-enriched rice.

Using membrane transporters to improve crops for sustainable food production.

With the global population predicted to grow by at least 25 per cent by 2050, the need for sustainable production of nutritious foods is critical for human and environmental health. Recent advances show that specialized plant membrane transporters can be used to enhance yields of staple crops, increase nutrient content and increase resistance to key stresses, including salinity, pathogens and aluminium toxicity, which in turn could expand available arable land.

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

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.

A Model for Developing Clinical Analytics Capacity: Closing the Loops on Outcomes to Optimize Quality.

Closed Loop Analytics© is receiving growing interest in healthcare as a term referring to information technology, local data and clinical analytics working together to generate evidence for improvement. The Closed Loop Analytics model consists of three loops corresponding to the decision-making levels of an organization and the associated data within each loop - Patients, Protocols, and Populations. The authors propose that each of these levels should utilize the same ecosystem of electronic health record (EHR) and enterprise data warehouse (EDW) enabled data, in a closed-loop fashion, with that data being repackaged and delivered to suit the analytic and decision support needs of each level, in support of better outcomes.

Standards for plant synthetic biology: a common syntax for exchange of DNA parts.

Inventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease-mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering.

Mother trees, altruistic fungi, and the perils of plant personification.

There are growing doubts about the true role of the common mycorrhizal networks (CMN or wood wide web) connecting the roots of trees in forests. We question the claims of a substantial carbon transfer from 'mother trees' to their offspring and nearby seedlings through the CMN. Recent reviews show that evidence for the 'mother tree concept' is inconclusive or absent. The origin of this concept seems to stem from a desire to humanize plant life but can lead to misunderstandings and false interpretations and may eventually harm rather than help the commendable cause of preserving forests. Two recent books serve as examples: The Hidden Life of Trees and Finding the Mother Tree.

Metal selectivity determinants in a family of transition metal transporters.

Metal tolerance proteins (MTPs) are plant members of the cation diffusion facilitator (CDF) transporter family involved in cellular metal homeostasis. Members of the CDF family are ubiquitously found in all living entities and show principal selectivity for Zn(2+), Mn(2+), and Fe(2+). Little is known regarding metal selectivity determinants of CDFs. We identified a novel cereal member of CDFs in barley, termed HvMTP1, that localizes to the vacuolar membrane. Unlike its close relative AtMTP1, which is highly selective for Zn(2+), HvMTP1 exhibits selectivity for both Zn(2+) and Co(2+) as assessed by its ability to suppress yeast mutant phenotypes for both metals. Expression of HvMTP1/AtMTP1 chimeras in yeast revealed a five-residue sequence within the AtMTP1 N-segment of the His-rich intracytoplasmic loop that confines specificity to Zn(2+). Furthermore, mutants of AtMTP1 generated through random mutagenesis revealed residues embedded within transmembrane domain 3 that additionally specify the high degree of Zn(2+) selectivity. We propose that the His-rich loop, which might play a role as a zinc chaperone, determines the identity of the metal ions that are transported. The residues within transmembrane domain 3 can also influence metal selectivity, possibly through conformational changes induced at the cation transport site located within the membrane or at the cytoplasmic C-terminal domain.

Zinc in plants: Integrating homeostasis and biofortification.

Zinc plays many essential roles in life. As a strong Lewis acid that lacks redox activity under environmental and cellular conditions, the Zn2+ cation is central in determining protein structure and catalytic function of nearly 10% of most eukaryotic proteomes. While specific functions of zinc have been elucidated at a molecular level in a number of plant proteins, wider issues abound with respect to the acquisition and distribution of zinc by plants. An important challenge is to understand how plants balance between Zn supply in soil and their own nutritional requirement for zinc, particularly where edaphic factors lead to a lack of bioavailable zinc or, conversely, an excess of zinc that bears a major risk of phytotoxicity. Plants are the ultimate source of zinc in the human diet, and human Zn deficiency accounts for over 400 000 deaths annually. Here, we review the current understanding of zinc homeostasis in plants from the molecular and physiological perspectives. We provide an overview of approaches pursued so far in Zn biofortification of crops. Finally, we outline a "push-pull" model of zinc nutrition in plants as a simplifying concept. In summary, this review discusses avenues that can potentially deliver wider benefits for both plant and human Zn nutrition.

Diffusion and bulk flow of amino acids mediate calcium waves in plants.

In plants, a variety of stimuli trigger long-range calcium signals that travel rapidly along the vasculature to distal tissues via poorly understood mechanisms. Here, we use quantitative imaging and analysis to demonstrate that traveling calcium waves are mediated by diffusion and bulk flow of amino acid chemical messengers. We propose that wounding triggers release of amino acids that diffuse locally through the apoplast, activating the calcium-permeable channel GLUTAMATE RECEPTOR-LIKE 3.3 as they pass. Over long distances through the vasculature, the wound-triggered dynamics of a fluorescent tracer show that calcium waves are likely driven by bulk flow of a channel-activating chemical. We observed that multiple stimuli trigger calcium waves with similar dynamics, but calcium waves alone cannot initiate all systemic defense responses, suggesting that mobile chemical messengers are a core component of complex systemic signaling in plants.

The vacuolar Ca2+-activated channel TPC1 regulates germination and stomatal movement.

Cytosolic free calcium ([Ca2+]cyt) is a ubiquitous signalling component in plant cells. Numerous stimuli trigger sustained or transient elevations of [Ca2+]cyt that evoke downstream stimulus-specific responses. Generation of [Ca2+]cyt signals is effected through stimulus-induced opening of Ca2+-permeable ion channels that catalyse a flux of Ca2+ into the cytosol from extracellular or intracellular stores. Many classes of Ca2+ current have been characterized electrophysiologically in plant membranes. However, the identity of the ion channels that underlie these currents has until now remained obscure. Here we show that the TPC1 ('two-pore channel 1') gene of Arabidopsis thaliana encodes a class of Ca2+-dependent Ca2+-release channel that is known from numerous electrophysiological studies as the slow vacuolar channel. Slow vacuolar channels are ubiquitous in plant vacuoles, where they form the dominant conductance at micromolar [Ca2+]cyt. We show that a tpc1 knockout mutant lacks functional slow vacuolar channel activity and is defective in both abscisic acid-induced repression of germination and in the response of stomata to extracellular calcium. These studies unequivocally demonstrate a critical role of intracellular Ca2+-release channels in the physiological processes of plants.

Zinc biofortification of cereals: problems and solutions.

The goal of biofortification is to develop plants that have an increased content of bioavailable nutrients in their edible parts. Cereals serve as the main staple food for a large proportion of the world population but have the shortcoming, from a nutrition perspective, of being low in zinc and other essential nutrients. Major bottlenecks in plant biofortification appear to be the root-shoot barrier and--in cereals--the process of grain filling. New findings demonstrate that the root-shoot distribution of zinc is controlled mainly by heavy metal transporting P1B-ATPases and the metal tolerance protein (MTP) family. A greater understanding of zinc transport is important to improve crop quality and also to help alleviate accumulation of any toxic metals.

A proteomic and phosphoproteomic analysis of Oryza sativa plasma membrane and vacuolar membrane.

Proteomic and phosphoproteomic analyses of rice shoot and root tonoplast-enriched and plasma membrane-enriched membrane fractions were carried out to look at tissue-specific expression, and to identify putative regulatory sites of membrane transport proteins. Around 90 unique membrane proteins were identified, which included primary and secondary transporters, ion channels and aquaporins. Primary H(+) pumps from the AHA family showed little isoform specificity in their tissue expression pattern, whereas specific isoforms of the Ca(2+) pump ECA/ACA family were expressed in root and shoot tissues. Several ABC transporters were detected, particularly from the MDR and PDR subfamilies, which often showed expression in either roots or shoots. Ammonium transporters were expressed in root, but not shoot, tissue. Large numbers of sugar transporters were expressed, particularly in green tissue. The occurrence of phosphorylation sites in rice transporters such as AMT1;1 and PIP2;6 agrees with those previously described in other species, pointing to conserved regulatory mechanisms. New phosphosites were found in many transporters, including H(+) pumps and H(+):cation antiporters, often at residues that are well conserved across gene families. Comparison of root and shoot tissue showed that phosphorylation of AMT1;1 and several further transporters may be tissue dependent.

Getting the most out of publicly available T-DNA insertion lines.

In the course of several different projects, we came to realize that there is a significant amount of untapped potential in the publicly available T-DNA insertion lines. In addition to the GABI-Kat lines, which were designed specifically for activation tagging, lines from the SAIL and FLAGdb collections are also useful for this purpose. As well as the 35S promoter chosen for activation tagging in GABI-Kat lines, we found that the 1'2' bidirectional promoter is capable of activating expression of flanking genomic sequences in both GABI-Kat and SAIL lines. Thus these lines have added potential for activation tagging. We also show that these lines are capable of generating antisense transcripts and so have the potential to be used for suppression (loss/reduction of function) studies. By virtue of weak terminator sequences in some T-DNA constructs, transcript read-through from selectable markers is also possible, which again has the potential to be exploited in activation/suppression studies. Finally, we show that, by selecting and characterizing lines in which the T-DNA insertions are present specifically within introns of a target gene, an allelic series of mutants with varying levels of reduced expression can be generated, due to differences in efficiency of intron splicing. Taken together, our analyses demonstrate that there is a wealth of untapped potential within existing insertion lines for studies on gene function, and the effective exploitation of these resources is discussed.

Identification of novel proteins and phosphorylation sites in a tonoplast enriched membrane fraction of Arabidopsis thaliana.

Plant vacuoles play essential roles in many physiological processes, particularly in mineral nutrition, turgor provision and cellular signalling. The vacuolar membrane, the tonoplast, contains many membrane transporters that are critical in the execution of these processes. However, although increasing knowledge is available about the identity of proteins involved in these processes very little is known about the regulation of tonoplast transporters. By studying the phosphoproteome of tonoplast-enriched membranes, we identified 66 phosphorylation sites on 58 membrane proteins. Amongst these, 31 sites were identified in 28 membrane transporters of various families including tonoplast anion transporters of the CLC family, potassium transporters of the KUP family, tonoplast sugar transporters and ABC transporters. In a number of cases, the detected sites were well conserved across isoforms of one family pointing to common mechanisms of regulation. In other cases, isoform-unique sites were present, suggesting regulatory mechanisms tailored to the function of individual proteins. These results provide the basis for future studies to elucidate the mechanistic regulation of tonoplast membrane transporters.