Eric Craft

GEOCHEM-EZ: a chemical speciation program with greater power and flexibility

GEOCHEM-EZ is a multi-functional chemical speciation program, designed to replace GEOCHEM-PC, which can only be used on DOS consoles. Chemical speciation programs, such as GEOCHEM and GEOCHEM-PC, have been excellent tools for scientists designing appropriate solutions for their experiments. GEOCHEM-PC is widely used in plant nutrition and soil and environmental chemistry research to perform equilibrium speciation computations, allowing the user to estimate solution ion activities and to consider simple complexes and solid phases. As helpful as GEOCHEM-PC has been to scientists, the consensus was that the program was not very user friendly, was difficult to learn and to troubleshoot, and suffered from several functional weaknesses. To enhance the usability and to address the problems found in GEOCHEM-PC, we upgraded the program with a Java graphical interface, added Help files, and improved its power and function, allowing it to run on any computer that supports Windows XP, Vista or Windows 7.

Transport properties of members of the ZIP family in plants and their role in Zn and Mn homeostasis

A better understanding of the role of the Arabidopsis ZIP family of micronutrient transporters is necessary in order to advance our understanding of plant Zn, Fe, Mn, and Cu homeostasis. In the current study, the 11 Arabidopsis ZIP family members not yet well characterized were first screened for their ability to complement four yeast mutants defective in Zn, Fe, Mn, or Cu uptake. Six of the Arabidopsis ZIP genes complemented a yeast Zn uptake-deficient mutant, one was able partially to complement a yeast Fe uptake-deficient mutant, six ZIP family members complemented an Mn uptake-deficient mutant, and none complemented the Cu uptake-deficient mutant. AtZIP1 and AtZIP2 were then chosen for further study, as the preliminary yeast and in planta analysis suggested they both may be root Zn and Mn transporters. In yeast, AtZIP1 and AtZIP2 both complemented the Zn and Mn uptake mutants, suggesting that they both may transport Zn and/or Mn. Expression of both genes is localized to the root stele, and AtZIP1 expression was also found in the leaf vasculature. It was also found that AtZIP1 is a vacuolar transporter, while AtZIP2 is localized to the plasma membrane. Functional studies with Arabidopsis AtZIP1 and AtZIP2 T-DNA knockout lines suggest that both transporters play a role in Mn (and possibly Zn) translocation from the root to the shoot. AtZIP1 may play a role in remobilizing Mn from the vacuole to the cytoplasm in root stellar cells, and may contribute to radial movement to the xylem parenchyma. AtZIP2, on the other hand, may mediate Mn (and possibly Zn) uptake into root stellar cells, and thus also may contribute to Mn/Zn movement in the stele to the xylem parenchyma, for subsequent xylem loading and transport to the shoot.

Development of a Novel Aluminum Tolerance Phenotyping Platform Used for Comparisons of Cereal Aluminum Tolerance and Investigations into Rice Aluminum Tolerance Mechanisms

The genetic and physiological mechanisms of aluminum (Al) tolerance have been well studied in certain cereal crops, and Al tolerance genes have been identified in sorghum (Sorghum bicolor) and wheat (Triticum aestivum). Rice (Oryza sativa) has been reported to be highly Al tolerant; however, a direct comparison of rice and other cereals has not been reported, and the mechanisms of rice Al tolerance are poorly understood. To facilitate Al tolerance phenotyping in rice, a high-throughput imaging system and root quantification computer program was developed, permitting quantification of the entire root system, rather than just the longest root. Additionally, a novel hydroponic solution was developed and optimized for Al tolerance screening in rice and compared with the Yoshidas rice solution commonly used for rice Al tolerance studies. To gain a better understanding of Al tolerance in cereals, comparisons of Al tolerance across cereal species were conducted at four Al concentrations using seven to nine genetically diverse genotypes of wheat, maize (Zea mays), sorghum, and rice. Rice was significantly more tolerant than maize, wheat, and sorghum at all Al concentrations, with the mean Al tolerance level for rice found to be 2- to 6-fold greater than that in maize, wheat, and sorghum. Physiological experiments were conducted on a genetically diverse panel of more than 20 rice genotypes spanning the range of rice Al tolerance and compared with two maize genotypes to determine if rice utilizes the well-described Al tolerance mechanism of root tip Al exclusion mediated by organic acid exudation. These results clearly demonstrate that the extremely high levels of rice Al tolerance are mediated by a novel mechanism, which is independent of root tip Al exclusion.

High‐throughput two‐dimensional root system phenotyping platform facilitates genetic analysis of root growth and development

High-throughput phenotyping of root systems requires a combination of specialized techniques and adaptable plant growth, root imaging and software tools. A custom phenotyping platform was designed to capture images of whole root systems, and novel software tools were developed to process and analyse these images. The platform and its components are adaptable to a wide range root phenotyping studies using diverse growth systems (hydroponics, paper pouches, gel and soil) involving several plant species, including, but not limited to, rice, maize, sorghum, tomato and Arabidopsis. The RootReader2D software tool is free and publicly available and was designed with both user-guided and automated features that increase flexibility and enhance efficiency when measuring root growth traits from specific roots or entire root systems during large-scale phenotyping studies. To demonstrate the unique capabilities and high-throughput capacity of this phenotyping platform for studying root systems, genome-wide association studies on rice (Oryza sativa) and maize (Zea mays) root growth were performed and root traits related to aluminium (Al) tolerance were analysed on the parents of the maize nested association mapping (NAM) population.

OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signaling and Redistribution of Iron and Cadmium in Arabidopsis

This work identifies a physiological substrate and a physiological function of the Arabidopsis oligopeptide transporter, OPT3, in iron (Fe) homeostasis, provides a mechanistic explanation of the role of OPT3 in systemic Fe signaling, and uncovers an aspect of crosstalk between Fe homeostasis and cadmium partitioning. Iron is essential for both plant growth and human health and nutrition. Knowledge of the signaling mechanisms that communicate iron demand from shoots to roots to regulate iron uptake as well as the transport systems mediating iron partitioning into edible plant tissues is critical for the development of crop biofortification strategies. Here, we report that OPT3, previously classified as an oligopeptide transporter, is a plasma membrane transporter capable of transporting transition ions in vitro. Studies in Arabidopsis thaliana show that OPT3 loads iron into the phloem, facilitates iron recirculation from the xylem to the phloem, and regulates both shoot-to-root iron signaling and iron redistribution from mature to developing tissues. We also uncovered an aspect of crosstalk between iron homeostasis and cadmium partitioning that is mediated by OPT3. Together, these discoveries provide promising avenues for targeted strategies directed at increasing iron while decreasing cadmium density in the edible portions of crops and improving agricultural productivity in iron deficient soils.

COPT6 Is a Plasma Membrane Transporter That Functions in Copper Homeostasis in Arabidopsis and Is a Novel Target of SQUAMOSA Promoter-binding Protein-like 7

Background: Copper uptake is tightly regulated to prevent deficiency while avoiding toxicity. Results: AtCOPT6 localizes to the plasma membrane, is regulated by copper availability, interacts with itself and AtCOPT1, and regulates response to copper limitation and excess. Conclusion: AtCOPT6 is a novel SPL7 target that functions in copper homeostasis in Arabidopsis. Significance: Identification and characterization of copper transporters are crucial for understanding of copper homeostasis. Among the mechanisms controlling copper homeostasis in plants is the regulation of its uptake and tissue partitioning. Here we characterized a newly identified member of the conserved CTR/COPT family of copper transporters in Arabidopsis thaliana, COPT6. We showed that COPT6 resides at the plasma membrane and mediates copper accumulation when expressed in the Saccharomyces cerevisiae copper uptake mutant. Although the primary sequence of COPT6 contains the family conserved domains, including methionine-rich motifs in the extracellular N-terminal domain and a second transmembrane helix (TM2), it is different from the founding family member, S. cerevisiae Ctr1p. This conclusion was based on the finding that although the positionally conserved Met106 residue in the TM2 of COPT6 is functionally essential, the conserved Met27 in the N-terminal domain is not. Structure-function studies revealed that the N-terminal domain is dispensable for COPT6 function in copper-replete conditions but is important under copper-limiting conditions. In addition, COPT6 interacts with itself and with its homolog, COPT1, unlike Ctr1p, which interacts only with itself. Analyses of the expression pattern showed that although COPT6 is expressed in different cell types of different plant organs, the bulk of its expression is located in the vasculature. We also show that COPT6 expression is regulated by copper availability that, in part, is controlled by a master regulator of copper homeostasis, SPL7. Finally, studies using the A. thaliana copt6-1 mutant and plants overexpressing COPT6 revealed its essential role during copper limitation and excess.

Characterization of the high affinity Zn transporter from Noccaea caerulescens, NcZNT1, and dissection of its promoter for its role in Zn uptake and hyperaccumulation

• In this paper, we conducted a detailed analysis of the ZIP family transporter, NcZNT1, in the zinc (Zn)/cadmium (Cd) hyperaccumulating plant species, Noccaea caerulescens, formerly known as Thlaspi caerulescens. NcZNT1 was previously suggested to be the primary root Zn/Cd uptake transporter. Both a characterization of NcZNT1 transport function in planta and in heterologous systems, and an analysis of NcZNT1 gene expression and NcZNT1 protein localization were carried out. • We show that NcZNT1 is not only expressed in the root epidermis, but also is highly expressed in the root and shoot vasculature, suggesting a role in long-distance metal transport. Also, NcZNT1 was found to be a plasma membrane transporter that mediates Zn but not Cd, iron (Fe), manganese (Mn) or copper (Cu) uptake into plant cells. • Two novel regions of the NcZNT1 promoter were identified which may be involved in both the hyperexpression of NcZNT1 and its ability to be regulated by plant Zn status. • In conclusion, we demonstrate here that NcZNT1 plays a role in Zn and not Cd uptake from the soil, and based on its strong expression in the root and shoot vasculature, could be involved in long-distance transport of Zn from the root to the shoot via the xylem.

Evolving technologies for growing, imaging and analyzing 3D root system architecture of crop plants

A plants ability to maintain or improve its yield under limiting conditions, such as nutrient deficiency or drought, can be strongly influenced by root system architecture (RSA), the three-dimensional distribution of the different root types in the soil. The ability to image, track and quantify these root system attributes in a dynamic fashion is a useful tool in assessing desirable genetic and physiological root traits. Recent advances in imaging technology and phenotyping software have resulted in substantive progress in describing and quantifying RSA. We have designed a hydroponic growth system which retains the three-dimensional RSA of the plant root system, while allowing for aeration, solution replenishment and the imposition of nutrient treatments, as well as high-quality imaging of the root system. The simplicity and flexibility of the system allows for modifications tailored to the RSA of different crop species and improved throughput. This paper details the recent improvements and innovations in our root growth and imaging system which allows for greater image sensitivity (detection of fine roots and other root details), higher efficiency, and a broad array of growing conditions for plants that more closely mimic those found under field conditions.

Quantitative iTRAQ Proteomics Revealed Possible Roles for Antioxidant Proteins in Sorghum Aluminum Tolerance

Aluminum (Al) toxicity inhibits root growth and limits crop yields on acid soils worldwide. However, quantitative information is scarce on protein expression profiles under Al stress in crops. In this study, we report on the identification of potential Al responsive proteins from root tips of Al sensitive BR007 and Al tolerant SC566 sorghum lines using a strategy employing iTRAQ and 2D-liquid chromatography (LC) coupled to MS/MS (2D-LC-MS/MS). A total of 771 and 329 unique proteins with abundance changes of >1.5 or <0.67-fold were identified in BR007 and SC566, respectively. Protein interaction and pathway analyses indicated that proteins involved in the antioxidant system were more abundant in the tolerant line than in the sensitive one after Al treatment, while opposite trends were observed for proteins involved in lignin biosynthesis. Higher levels of ROS accumulation in root tips of the sensitive line due to decreased activity of antioxidant enzymes could lead to higher lignin production and hyper-accumulation of toxic Al in cell walls. These results indicated that activities of peroxidases and the balance between production and consumption of ROS could be important for Al tolerance and lignin biosynthesis in sorghum.

Arabidopsis Pollen Fertility Requires the Transcription Factors CITF1 and SPL7 that Regulate Copper Delivery to Anthers and Jasmonic Acid Synthesis

Transcription factors CITF1 and SPL7 regulate the delivery of a micronutrient copper to anthers, thereby influencing fertility, and link copper homeostasis and the jasmonic acid metabolic pathway. A deficiency of the micronutrient copper (Cu) leads to infertility and grain/seed yield reduction in plants. How Cu affects fertility, which reproductive structures require Cu, and which transcriptional networks coordinate Cu delivery to reproductive organs is poorly understood. Using RNA-seq analysis, we showed that the expression of a gene encoding a novel transcription factor, CITF1 (Cu-DEFICIENCY INDUCED TRANSCRIPTION FACTOR1), was strongly upregulated in Arabidopsis thaliana flowers subjected to Cu deficiency. We demonstrated that CITF1 regulates Cu uptake into roots and delivery to flowers and is required for normal plant growth under Cu deficiency. CITF1 acts together with a master regulator of copper homeostasis, SPL7 (SQUAMOSA PROMOTER BINDING PROTEIN LIKE7), and the function of both is required for Cu delivery to anthers and pollen fertility. We also found that Cu deficiency upregulates the expression of jasmonic acid (JA) biosynthetic genes in flowers and increases endogenous JA accumulation in leaves. These effects are controlled in part by CITF1 and SPL7. Finally, we show that JA regulates CITF1 expression and that the JA biosynthetic mutant lacking the CITF1- and SPL7-regulated genes, LOX3 and LOX4, is sensitive to Cu deficiency. Together, our data show that CITF1 and SPL7 regulate Cu uptake and delivery to anthers, thereby influencing fertility, and highlight the relationship between Cu homeostasis, CITF1, SPL7, and the JA metabolic pathway.