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Clathrin plays a critical role in clathrin-mediated endocytosis (CME) in plants, and it is required for autophagy in mammals. However, the functional interconnection of clathrin with autophagy has not been firmly established in plants. Here, we demonstrate that loss of function of clathrin light chain (CLC) subunit 2 and 3 results in salicylic acid (SA)- and H2O2-dependent accelerated senescence and activated defense responses in Arabidopsis, which are hallmarks of the autophagy-related gene (ATG) mutants. Similar toatgmutants, theclc2-1clc3-1double mutant has enhanced sensitivity to both carbon and nitrogen starvation and enhanced resistance to biotrophic bacterial and fungal pathogens. In addition, the autophagy flux was significantly reduced in the roots ofclc2-1clc3-1mutant plants relative to Col-0 plants under carbon starvation conditions. Furthermore, our Yeast-2-hybrid (Y2H) and Luciferase complementation assays showed that CLC2 directly interacted with ATG8h and ATG8i. Mutations within the unique ATG8-interacting motif (AIM) of CLC2 as well as at the LIR/AIM-docking site (LDS) of ATG8h abolished the interaction between CLC2 and ATG8h. As anticipated, both GFP-ATG8h/GFP-ATG8i and CLC2 were subjected to autophagic degradation in the vacuoles. Together, our data revealed that the accelerated senescence and activated immune responses observed in Arabidopsisclc2-1clc3-1mutant plants result from impaired autophagy, and CLC2 participates in autophagy through direct interactions with ATG8h and ATG8i in an AIM1- and LDS-dependent manner. Our results unveil a previously unidentified link between the function of CLCs and autophagy.

Increasing populations and temperatures are expected to escalate food demands beyond production capacities, and the development of maize lines with better performance under heat stress is desirable. Here, we report that constitutive ectopic expression of a heterologous glutaredoxin S17 from Arabidopsis thaliana (AtGRXS17) can provide thermotolerance in maize through enhanced chaperone activity and modulation of heat stress-associated gene expression. The thermotolerant maize lines had increased protection against protein damage and yielded a sixfold increase in grain production in comparison to the non-transgenic counterparts under heat stress field conditions. The maize lines also displayed thermotolerance in the reproductive stages, resulting in improved pollen germination and the higher fidelity of fertilized ovules under heat stress conditions. Our results present a robust and simple strategy for meeting rising yield demands in maize and, possibly, other crop species in a warming global environment.

Obesity is often associated with metabolic dysregulation and oxidative stress with the latter serving as a possible unifying link between obesity and cardiovascular complications. Glutaredoxins (Grxs) comprise one of the major antioxidant systems in the heart. Although Grx3 has been shown to act as an endogenous negative regulator of cardiac hypertrophy and heart failure, its metabolic impact on cardiac function in diet-induced obese (DIO) mice remains largely unknown. In the present study, analysis of Grx3 expression indicated that Grx3 protein levels, but not mRNA levels, were significantly increased in the hearts of DIO mice. Cardiac-specific Grx3 deletion (Grx3 CKO) mice were viable and grew indistinguishably from their littermates after being fed a high fat diet (HFD) for one month, starting at 2 months of age. After being fed with a HFD for 8 months (starting at 2 months of age); however, Grx3 CKO DIO mice displayed left ventricular systolic dysfunction with a significant decrease in ejection fraction and fractional shortening that was associated with heart failure. ROS production was significantly increased in Grx3 CKO DIO cardiomyocytes compared to control cells. Gene expression analysis revealed a significant decline in the level of transcripts corresponding to genes associated with processes such as fatty acid uptake, mitochondrial fatty acid transport and oxidation, and citrate cycle in Grx3 CKO DIO mice compared to DIO controls. In contrast, an increase in the level of transcripts corresponding to genes associated with glucose uptake and utilization were found in Grx3 CKO DIO mice compared to DIO controls. Taken together, these findings indicate that Grx3 may play a critical role in redox balance and as a metabolic switch in cardiomyocytes contributing to the development and progression of heart failure.

Heat stress can have detrimental effects on plant growth and development. However, the mechanisms by which the plant is able to perceive changes in ambient temperature, transmit this information, and initiate a temperature-induced response are not fully understood. Previously, we showed that heterologous expression of an Arabidopsis thaliana L. monothiol glutaredoxin AtGRXS17 enhances thermotolerance in various crops, while disruption of AtGRXS17 expression caused hypersensitivity to permissive temperature. In this study, we extend our investigation into the effect of AtGRXS17 and heat stress on plant growth and development. Although atgrxs17 plants were found to exhibit a slight decrease in hypocotyl elongation, shoot meristem development, and root growth compared to wild-type when grown at 22°C, these growth phenotypic differences became more pronounced when growth temperatures were raised to 28°C. Transcriptome analysis revealed significant changes in genome-wide gene expression in atgrxs17 plants compared to wild-type under conditions of heat stress. The expression of genes related to heat stress factors, auxin response, cellular communication, and abiotic stress were altered in atgrxs17 plants in response to heat stress. Overall, our findings indicate that AtGRXS17 plays a critical role in controlling the transcriptional regulation of plant heat stress response pathways.

Considering the widespread occurrence of oxalate in nature and its broad impact on a host of organisms, it is surprising that so little is known about the turnover of this important acid. In plants, oxalate oxidase is the most well-studied enzyme capable of degrading oxalate, but not all plants possess this activity. Recently, acyl-activating enzyme 3 (AAE3), encoding an oxalyl-CoA synthetase, was identified in Arabidopsis. This enzyme has been proposed to catalyze the first step in an alternative pathway of oxalate degradation. Since this initial discovery, this enzyme and proposed pathway have been found to be important to other plants and yeast as well. In this study, we identify, in Arabidopsis, an oxalyl-CoA decarboxylase (AtOXC) that is capable of catalyzing the second step in this proposed pathway of oxalate catabolism. This enzyme breaks down oxalyl-CoA, the product of AtAAE3, into formyl-CoA and CO2. AtOXC:GFP localization suggested that this enzyme functions within the cytosol of the cell. An Atoxc knock-down mutant showed a reduction in the ability to degrade oxalate into CO2. This reduction in AtOXC activity resulted in an increase in the accumulation of oxalate and the enzyme substrate, oxalyl-CoA. Size exclusion studies suggest that the enzyme functions as a dimer. Computer modeling of the AtOXC enzyme structure identified amino acids of predicted importance in co-factor binding and catalysis. Overall, these results suggest that AtOXC catalyzes the second step in this alternative pathway of oxalate catabolism.

Chickpea (Cicer arietinum L.) is the second most important grain legume worldwide. Recent advances in the sequencing of the chickpea genome has provided a new and valuable resource to aid efforts in gene discovery and crop trait improvement. Technical difficulties in stable chickpea transgenics and the lack of a transient expression system for rapid analysis of gene expression and function; however, has limited the usefulness of this genomic resource. As a step toward alleviating this limitation, we report here the development of a simple and efficient transient gene expression protocol. Using leaves from chickpea seedlings, we have established a procedure that enables the generation of large quantities of vital chickpea protoplasts within only a few hours. In addition, we have optimized a PEG-calcium-mediated transfection method to efficiently deliver exogenous DNA into the chickpea protoplast. The current study is the first to present a detailed step-by-step procedures for protoplast isolation, evaluation, transfection, and application in chickpea. In addition, we optimize the transfection efficiency which has not been previously reported. Our protoplast transfection approach provides a platform that will allow rapid high-throughput screening and systematic characterization of gene expression and function. Knowledge gained through such studies will benefit current efforts to improve chickpea production and quality.•Modified enzymatic digestion solution for higher yield and viability.•Optimize transfection of chickpea protoplasts., Graphical abstract Image, graphical abstract

Iron (Fe) is a mineral nutrient and a metal cofactor essential for plants. Iron limitation can have detrimental effects on plant growth and development, while excess iron inside plant cells leads to oxidative damage. As a result, plants have evolved complex regulatory networks to respond to fluctuations in cellular iron concentrations. The mechanisms that regulate these responses however, are not fully understood. Heterologous expression of an Arabidopsis thaliana monothiol glutaredoxin S17 (GRXS17) suppresses the over-accumulation of iron in the Saccharomyces cerevisiae Grx3/Grx4 mutant and disruption of GRXS17 causes plant sensitivity to exogenous oxidants and iron deficiency stress. GRXS17 may act as an important regulator in the plant’s ability to respond to iron deficiency stress and maintain redox homeostasis. Here, we extend this investigation by analyzing iron-responsive gene expression of the Fer-like iron deficiency-induced transcription factor (FIT) network (FIT, IRT1, FRO1, and FRO2) and the bHLH transcription factor POPEYE (PYE) network (PYE, ZIF1, FRO3, NAS4, and BTS) in GRXS17 KO plants and wildtype controls grown under iron sufficiency and deficiency conditions. Our findings suggest that GRXS17 is required for tolerance to iron deficiency, and plays a negative regulatory role under conditions of iron sufficiency.

The Acyl-Activating Enzyme (AAE) 3 gene encodes an oxalyl-CoA synthetase that catalyzes the conversion of oxalate to oxalyl-CoA in a CoA and ATP-dependent manner. Although the biochemical activity of AAE3 has been established, its biological role in plant growth and development remains unclear. To advance our understanding of the role of AAE3 in plant growth and development, we report here the characterization of two Medicago truncatula AAE3 (Mtaae3) mutants. Characterization of a Mtaae3 RNAi mutant revealed an accumulation of calcium oxalate crystals and increased seed permeability. These phenotypes were also exhibited in the Arabidopsis aae3 (Ataae3) mutants. Unlike the Ataae3 mutants, the Mtaae3 RNAi mutant did not show a reduction in vegetative growth, decreased seed germination, or increased seed calcium concentration. In an effort to clarify these phenotypic differences, a Mtaae3 Tnt1 mutant was identified and characterized. This Mtaae3 Tnt1 mutant displayed reduced vegetative growth, decreased seed germination, and increased seed calcium concentration as well as an accumulation of calcium oxalate crystals and increased seed permeability as found in Ataae3. Overall, the results presented here show the importance of AAE3 in the growth and development of plants. In addition, this study highlights the ability to separate specific growth and development phenotypes based on the level of AAE3 gene expression.

MAPK signaling pathways play critical roles in plant immunity. Here, we silenced multiple genes encoding MAPKs using virus-induced gene silencing mediated by Bean pod mottle virus to identify MAPK genes involved in soybean (Glycine max) immunity. Surprisingly, a strong hypersensitive response (HR) cell death was observed when soybean MAPK KINASE KINASE1 (GmMEKK1), a homolog of Arabidopsis (Arabidopsis thaliana) MEKK1, was silenced. The HR was accompanied by the overaccumulation of defense signaling molecules, salicylic acid (SA) and hydrogen peroxide. Genes involved in primary metabolism, translation/transcription, photosynthesis, and growth/development were down-regulated in GmMEKK1-silenced plants, while the expression of defense-related genes was activated. Accordingly, GmMEKK1-silenced plants were more resistant to downy mildew (Peronospora manshurica) and Soybean mosaic virus compared with control plants. Silencing GmMEKK1 reduced the activation of GmMPK6 but enhanced the activation of GmMPK3 in response to flg22 peptide. Unlike Arabidopsis MPK4, GmMPK4 was not activated by either flg22 or SA. Interestingly, transient overexpression of GmMEKK1 in Nicotiana benthamiana also induced HR. Our results indicate that GmMEKK1 plays both positive and negative roles in immunity and appears to differentially activate downstream MPKs by promoting GmMPK6 activation but suppressing GmMPK3 activation in response to flg22. The involvement of GmMPK4 kinase activity in cell death and in flg22- or SA-triggered defense responses in soybean requires further investigation.

Pleurotus eryngii contains bioactive compounds that can activate the immune system. Here we report the identification, purification, and functional characterization of the bioactive P. eryngii protein (PEP) 1b. PEP 1b was discovered to be a 21.9 kDa protein with the ability to induce the M1-polarization of the macrophage cell line RAW 264.7 cells. Biochemical measurements showed that PEP 1b stimulated nitric oxide (NO), IL-1β, IL-6 and TNF-α production and regulated inducible NO synthase. Phosphorylation and inhibitor studies revealed that PEP 1b promoted the translocation of NF-kB from the cytosol to the nucleus allowing the induction of target gene expression and NO production. The phosphorylation of JNK and ERK1/2 was found to be necessary for NO production. Each phosphorylation pathway was found to require a Toll-like receptor (TLR) 4 as a prerequisite for PEP 1b-induced NO production. This study suggests that PEP 1b is an immunomodulatory protein that can boost cellular immune responses through the activation of the TLR4-NF-κB and MAPK signaling pathways.

Our ability to genetically manipulate microbial systems is often hampered by the availability of genetic tools. Thus, there is a need for the continued expansion of our molecular tool box. In support of this expansion, this study reports the design, construction, and validation of a new bicistronic shuttle vector series, pDUO, for the dual expression of genes in different hosts. Each vector was designed and constructed to contain two araC-pBAD inducible promoter systems for tight control over gene expression. Each araC-pBAD promoter precedes a ribosomal binding site and a multicloning site (MCS). The 5' end of MCS1 contains a sequence encoding an affinity HIS-tag N-terminus and MCS2 terminates with a sequence encoding an affinity S-tag C-terminus for one-step purification of recombinant proteins encoded by the inserted genes. Both MCS are followed by an rrnBT1 and T2 transcriptional terminator sequence. Each vector in this series also contains a PBR322 and pRO1600-derived replicon to support replication in different host bacteria along with one of four different selectable markers. The functionality of the pDUO vector series was validated through the dual expression of oxalate biosynthetic component (obc) 1 and mrfp in Escherichia coli and Pseudomonas fluorescens. It is anticipated that this new vector series will facilitate functional studies as well as the engineering of bacterial strains for biotechnological purposes.

Growing evidence suggests that redox‐sensitive proteins including glutaredoxins (Grxs) can protect cardiac muscle cells from oxidative stress‐induced damage. Mammalian Grx3 has been shown to be critical in regulating cellular redox states. However, how Grx3 affects cardiac function by modulating reactive oxygen species (ROS) signaling remains unknown. In this study, we found that the expression of Grx3 in the heart is decreased during aging. To assess the physiological role of Grx3 in the heart, we generated mice in which Grx3 was conditionally deleted in cardiomyocytes (Grx3 conditional knockout (CKO) mice). Grx3 CKO mice were viable and grew indistinguishably from their littermates at young age. No difference in cardiac function was found comparing Grx3 CKO mice and littermate controls at this age. However, by the age of 12 months, Grx3 CKO mice exhibited left ventricular hypertrophy with a significant decrease in ejection fraction and fractional shortening along with a significant increase of ROS production in cardiomyocytes compared to controls. Deletion of Grx3 also impaired Ca2+ handling, caused enhanced sarcoplasmic reticulum (SR) calcium (Ca2+) leak, and decreased SR Ca2+ uptake. Furthermore, enhanced ROS production and alteration of Ca2+ handling in cardiomyocytes occurred, prior to cardiac dysfunction in young mice. Therefore, our findings demonstrate that Grx3 is an important factor in regulating cardiac hypertrophy and heart failure by modulating both cellular redox homeostasis and Ca2+ handling in the heart.

The objective was to determine the contribution of A. auricular polysaccharides (AAP) in modulating the composition and diversity of the intestinal microbe in mice. AAP was extracted from A. auricula freeze-dried powder and different amounts of AAP (40, 80, 160 mg AAP/kg body weight) were administered to 6 week-old male ICR mice by gavage feeding over a five-week period. AAP feeding changed the intestinal environment in the mice. The pH value of the intestinal compartments decreased while SCFAs concentrations increased in AAP-fed groups, in a dose dependent manner, compared to the controls (P

β-Carotene is a natural fat-soluble antioxidant, while the poor oral bioavailability of β-carotene restricts its application for functional foods. To overcome this barrier, a stable oil-in-water emulsion was structured to effectively deliver β-carotene. In this study, soy protein isolate (SPI)-Pleurotus eryngii polysaccharide (PEP) conjugate was prepared by Maillard reaction under controlled wet-heating conditions. β-Carotene was encapsulated in SPI-PEP conjugate-stabilized emulsion and its gastrointestinal behavior and antioxidant activity were assessed in vitro. The results showed that such conjugate with high molecular weight was mainly formed through PEP covalently binding to lysine and cysteine of SPI via C–N. Changes in spatial structure of SPI-PEP conjugate resulted from its decreased tryptophan fluorescence intensity and unfolded protein secondary structure. A decreased surface hydrophobicity and flat surface morphology of SPI-PEP conjugate contributed to its increased water solubility and emulsibility. Additionally, lipid digestion and the formation of mixed micelles were promoted in SPI-PEP conjugate-stabilized emulsion due to its strong steric repulsion, resulting in an improved bioavailability of β-carotene. Such improvement was favorable for β-carotene to relieve tert-butyl hydroperoxide-induced oxidative stress by decreasing reactive oxygen species production and enhancing antioxidant enzyme activities in Caco-2 cells. Our study suggested that Maillard-type protein-polysaccharide conjugates-stabilized emulsions had a potential industry application for the delivery of fat-soluble nutrients.

Iron (Fe) is a mineral nutrient and a metal cofactor essential for plants. Iron limitation can have detrimental effects on plant growth and development, while excess iron inside plant cells leads to oxidative damage. As a result, plants have evolved complex regulatory networks to respond to fluctuations in cellular iron concentrations. The mechanisms that regulate these responses however, are not fully understood. Heterologous expression of an Arabidopsis thaliana monothiol glutaredoxin S17 (GRXS17) suppresses the over-accumulation of iron in the Saccharomyces cerevisiae Grx3/Grx4 mutant and disruption of GRXS17 causes plant sensitivity to exogenous oxidants and iron deficiency stress. GRXS17 may act as an important regulator in the plant's ability to respond to iron deficiency stress and maintain redox homeostasis. Here, we extend this investigation by analyzing iron-responsive gene expression of the Fer-like iron deficiency-induced transcription factor (FIT) network (FIT, IRT1, FRO1, and FRO2) and the bHLH transcription factor POPEYE (PYE) network (PYE, ZIF1, FRO3, NAS4, and BTS) in GRXS17 KO plants and wildtype controls grown under iron sufficiency and deficiency conditions. Our findings suggest that GRXS17 is required for tolerance to iron deficiency, and plays a negative regulatory role under conditions of iron sufficiency.

Abiotic stresses are a major factor limiting crop growth and productivity. The Arabidopsis thaliana glutaredoxin S17 (AtGRXS17) gene has conserved functions in plant tolerance to heat and chilling stress in Arabidopsis and, when expressed ectopically, in tomato. Here, we report that ectopic expression of AtGRXS17 in tomato also enhanced tolerance to drought and oxidative stress. AtGRXS17-expressing tomato plants contained twice the shoot water content compared to wild-type plants under water limiting conditions. This enhanced drought tolerance correlated with a higher maximal photosynthetic efficiency of photosystem II (Fv/Fm). Ectopic AtGRXS17-expression was concomitant with the expression of Solanum lycopersicum catalase 1 (SlCAT1) and mitigated defects in the growth of primary roots in response to methyl viologen exposure. In addition, AtGRXS17 expression was found to prolong elevated expression levels of the Solanum lycopersicum ABA-responsive element binding protein 1 (SlAREB1) during drought stress. The findings demonstrate that expression of AtGRXS17 can simultaneously improve the tolerance of tomato, and possibly other agriculturally important crops, to drought, heat, and chilling stresses.

Glutaredoxins (GRXs) modulate redox-dependent signaling pathways and have emerged as key mediators in plant responses to environmental stimuli. Here we report that RNAi-mediated suppression of Oryza sativa GRXS17 (OsGRXS17) improved drought tolerance in rice. Gene expression studies showed that OsGRXS17 was present throughout the plant and that transcript abundance increased in response to drought stress and abscisic acid (ABA) treatment. Localization studies, utilizing GFP-OsGRXS17 fusion proteins, indicated that OsGRXS17 resides in both the cytoplasm and the nuclear envelope. Under drought stress conditions, rice plants with reduced OsGRXS17 expression showed lower rates of water loss and stomatal conductance, higher relative water content, and enhanced survival compared to wild-type controls. Further characterization of the OsGRXS17 down-regulated plants revealed an elevation in H2O2 production within the guard cells, increased sensitivity to ABA, and a reduction in stomatal apertures. The findings demonstrate a critical link between OsGRXS17, the modulation of guard cell H2O2 concentrations, and stomatal closure, expanding our understanding of the mechanisms governing plant responses to drought.

Iron (Fe) is an essential mineral nutrient and a metal cofactor required for many proteins and enzymes involved in the processes of DNA synthesis, respiration, and photosynthesis. Iron limitation can have detrimental effects on plant growth and development. Such effects are mediated, at least in part, through the generation of reactive oxygen species (ROS). Thus, plants have evolved a complex regulatory network to respond to conditions of iron limitations. However, the mechanisms that couple iron deficiency and oxidative stress responses are not fully understood. Here, we report the discovery that an Arabidopsis thaliana monothiol glutaredoxin S17 (AtGRXS17) plays a critical role in the plants ability to respond to iron deficiency stress and maintain redox homeostasis. In a yeast expression assay, AtGRXS17 was able to suppress the iron accumulation in yeast ScGrx3/ScGrx4 mutant cells. Genetic analysis indicated that plants with reduced AtGRXS17 expression were hypersensitive to iron deficiency and showed increased iron concentrations in mature seeds. Disruption of AtGRXS17 caused plant sensitivity to exogenous oxidants and increased ROS production under iron deficiency. Addition of reduced glutathione rescued the growth and alleviates the sensitivity of atgrxs17 mutants to iron deficiency. These findings suggest AtGRXS17 helps integrate redox homeostasis and iron deficiency responses.

Oxalic acid is produced by a variety of organisms ranging from simple microbes to complex animals. This acid has been proposed to fulfill various physiological and pathological functions which vary between organisms. In bacteria from the Burkholderia genus, oxalate secretion has been shown to be quorum sensing dependent and to support pathogenicity and cell viability. In light of the critical roles of oxalate in Burkholderia as well as other organisms, it is surprising that our understanding of how this simple dicarboxylate is biosynthesized remains incomplete. Here we report the expression, purification, and partial characterization of the first intact bacterial oxalate biosynthetic enzyme, Obc1, from B. mallei. An N-terminal His-tagged Bmobc1 was cloned into pDUET, expressed in E. coli BLR (DE3), and the recombinant enzyme purified by affinity chromatography. Oxalate biosynthetic enzyme assays coupled with HPLC analysis revealed that BmObc1 catalyzed the biosynthesis of oxalate, acetoacetate, and free CoA from oxaloacetate and a short chain acyl-CoA following Michaelis-Menten kinetics. Optimal enzyme activity was measured at pH 8.0 and a temperature around 44°C. Kinetic analysis conducted under conditions of saturating acetyl-CoA and varying oxaloacetate concentrations resulted in a calculated Km value for oxaloacetate of 94.3± 9.2 μM (mean ± SE). Under conditions of saturating oxaloacetate concentration and varying acyl-CoA (acetyl- or propionyl-CoA) concentrations kinetic analysis generated a calculated Km value of 26.8 ± 2.3 μM (mean ± SE) for acetyl-CoA and 104.4 ± 12.7 μM for propionyl-CoA. The significantly lower Km for acetyl-CoA suggests that it is strongly favored as a substrate over propionyl-CoA.

CRISPR/Cas9 has evolved and transformed the field of biology at an unprecedented pace. From the initial purpose of introducing a site specific mutation within a genome of choice, this technology has morphed into enabling a wide array of molecular applications, including site-specific transgene insertion and multiplexing for the simultaneous induction of multiple cleavage events. Efficiency, specificity, and flexibility are key attributes that have solidified CRISPR/Cas9 as the genome-editing tool of choice by scientists from all areas of biology. Within the field of plant biology, several CRISPR/Cas9 technologies, developed in other biological systems, have been successfully implemented to probe plant gene function and to modify specific crop traits. It is anticipated that this trend will persist and lead to the development of new applications and modifications of the CRISPR technology, adding to an ever-expanding collection of genome-editing tools. We envision that these tools will bestow plant researchers with new utilities to alter genome complexity, engineer site-specific integration events, control gene expression, generate transgene-free edited crops, and prevent or cure plant viral disease. The successful implementation of such utilities will represent a new frontier in plant biotechnology.

Considering the widespread occurrence of oxalate in nature and its broad impact on a host of organisms, it is surprising that so little is known about the turnover of this important acid. In plants, oxalate oxidase is the most well studied enzyme capable of degrading oxalate, but not all plants possess this activity. Recently, an Acyl Activating Enzyme 3 (AAE3), encoding an oxalyl-CoA synthetase, was identified in Arabidopsis. AAE3 has been proposed to catalyze the first step in an alternative pathway of oxalate degradation. Whether this enzyme and proposed pathway is important to other plants is unknown. Here, we identify the Medicago truncatula AAE3 (MtAAE3) and show that it encodes an oxalyl-CoA synthetase activity exhibiting high activity against oxalate with a Km = 81 ± 9 μM and Vmax = 19 ± 0.9 μmoles min-1mg protein-1. GFP-MtAAE3 localization suggested that this enzyme functions within the cytosol of the cell. Mtaae3 knock-down line showed a reduction in its ability to degrade oxalate into CO2. This reduction in the capacity to degrade oxalate resulted in the accumulation of druse crystals of calcium oxalate in the Mtaae3 knock-down line and an increased susceptibility to oxalate-secreting phytopathogens such as Sclerotinia sclerotiorum. Taken together, these results suggest that AAE3 dependent turnover of oxalate is important to different plants and functions in the regulation of tissue calcium oxalate crystal accumulation and in defense against oxalate-secreting phytopathogens.

Crystals of calcium oxalate have been observed among members from most taxonomic groups of photosynthetic organisms ranging from the smallest algae to the largest trees. The biological roles for calcium oxalate crystal formation in plant growth and development include high-capacity calcium regulation, protection against herbivory, and tolerance to heavy metals. Using a variety of experimental approaches researchers have begun to unravel the complex mechanisms controlling formation of this biomineral. Given the important roles for calcium oxalate formation in plant survival and the antinutrient and pathological impact on human health through its presence in plant foods, researchers are avidly seeking a more comprehensive understanding of how these crystals form. Such an understanding will be useful in efforts to design strategies aimed at improving the nutritional quality and production of plant foods.

Chilling stress is a production constraint of tomato, a tropical origin, chilling-sensitive horticultural crop. The development of chilling tolerant tomato thus has significant potential to impact tomato production. Glutaredoxins (GRXs) are ubiquitous oxidoreductases, which utilize the reducing power of glutathione to reduce disulfide bonds of substrate proteins and maintain cellular redox homeostasis. Here, we report that tomato expressing Arabidopsis GRX gene AtGRXS17 conferred tolerance to chilling stress without adverse effects on growth and development. AtGRXS17-expressing tomato plants displayed lower ion leakage, higher maximal photochemical efficiency of photosystem II (Fv/Fm) and increased accumulation of soluble sugar compared with wild-type plants after the chilling stress challenge. Furthermore, chilling tolerance was correlated with increased antioxidant enzyme activities and reduced H2O2 accumulation. At the same time, temporal expression patterns of the endogenous C-repeat/DRE-binding factor 1 (SlCBF1) and CBF mediated-cold regulated genes were not altered in AtGRXS17-expressing plants when compared with wild-type plants, and proline concentrations remained unchanged relative to wild-type plants under chilling stress. Green fluorescent protein -AtGRXS17 fusion proteins, which were initially localized in the cytoplasm, migrated into the nucleus during chilling stress, reflecting a possible role of AtGRXS17 in nuclear signaling of chilling stress responses. Together, our findings demonstrate that genetically engineered tomato plants expressing AtGRXS17 can enhance chilling tolerance and suggest a genetic engineering strategy to improve chilling tolerance without yield penalty across different crop species.

Although it is known that oxalic acid provides a selective advantage to the secreting microbe our understanding of how this acid is biosynthesized remains incomplete. This study reports the identification, cloning, and partial characterization of the oxalic acid biosynthetic enzyme from the animal bacterial pathogen, Burkholderia mallei. The discovered gene was named oxalate biosynthetic component (obc)1. Complementation of Burkholderia oxalate defective (Bod)1, a Burkholderia glumae mutant that lacks expression of a functional oxalic acid biosynthetic operon, revealed that the obc1 was able to rescue the no oxalate mutant phenotype. This single gene rescue is in contrast to the situation found in B. glumae which required the expression of two genes, obcA and obcB, to achieve complementation. Enzyme assays showed that even though the two Burkholderia species differed in the number of genes required to encode a functional enzyme, both catalyzed the same acyl-CoA dependent biosynthetic reaction. In addition, mutagenesis studies suggested a similar domain structure of the assembled oxalate biosynthetic enzymes whether encoded by one or two genes.

Plant structural traits often act as defenses against herbivorous insects, causing them to avoid feeding on a given plant or tissue. Mineral crystals of calcium oxalate in Medicago truncatula Gaertn. (Fabaceae) leaves have previously been shown to be effective deterrents of lepidopteran insect feeding. They are also inhibitors of conversion of plant material into insect body mass during or after consumption. Growth of beet armyworm, Spodoptera exigua Hubner (Lepidoptera: Noctuidae), larvae was correspondingly greater on calcium oxalate-defective (cod) mutants of M. truncatula with lower levels of crystal accumulation. Data presented here show that insects feeding on M. truncatula leaves with calcium oxalate crystals experience greater negative effects on growth and mandible wear than those feeding on artificial diet amended with smaller amorphous crystals from commercial preparations. Commercial calcium oxalate can be added to insect artificial diet at levels up to 7.5-fold higher than levels found in wild-type M. truncatula leaves with minimal effect on insect growth or lepidopteran mandibles. These data suggest that negative impacts of calcium oxalate in the diet of larvae are due to physical factors, and not toxicity of the compound, as high levels of the commercial crystals are readily tolerated. In contrast to the dramatic physical effects that M. truncatula-derived crystals have on insect mandibles, we could detect no damage to insect peritrophic gut membranes due to consumption of these crystals. Taken together, the data indicate that the size and shape of prismatic M. truncatula oxalate crystals are important factors in determining effects on insect growth. If manipulation of calcium oxalate is to be used in developing improved insect resistance in plants, then our findings suggest that controlling not only the overall amount, but also the size and shape of crystals, could be valuable traits in selecting desirable plant lines.

Bioavailable calcium affects bone formation and calcification. Here we investigate how a single gene mutation altering calcium partitioning in the model forage crop Medicago truncatula affects calcium bioavailability. Previously, the cod5 M. truncatula mutant was identified which contains identical calcium concentrations to wild-type, but contains no oxalate crystals. In this study, equal number of male and female mice were randomly grouped and then fed one of four 45Ca-containing diets: M. truncatula extrinsically or intrinsically labeled, and cod5 extrinsically or intrinsically labeled. Absorption of the tracer was determined in the legs one day after consumption. The absorption was similar in the M. truncatula and cod5 extrinsically labeled diets; however, in the intrinsically labeled diets, calcium absorption was 22.87% (P < 0.001) higher in mice fed cod5. Our study presents the first genetic evidence demonstrating the nutritional impact of removing oxalate crystals from foods.

It is known that oxalate, present in edible plants, can bind calcium in a crystalline form that reduces the availability of the bound calcium for nutritional absorption by humans. It is unknown, however, the degree to which the calcium oxalate content of a plant can be genetically altered and how much such alterations can impact the nutritional availability of the calcium present in plant foods. The recent identification of near isogenic Medicago truncatula mutants that contain a varying range in calcium oxalate content allows us to begin to address this gap in our knowledge. Here we assess, using an in vitro dialysis system that simulates the processes of digestion and absorption, the availability of calcium present in the leaves of M. truncatula. The results showed that calcium availability generally correlates inversely with the amount of calcium sequestered in the oxalate crystal. The plants with more calcium oxalate were found to have reduced calcium availability while the plant with less calcium oxalate was found to have enhanced calcium availability, compared to controls. Overall, this study supports genetically manipulating the form of calcium in edible plants as a viable strategy to improve the nutritional quality.

The mechanisms controlling oxalate biosynthesis and calcium oxalate formation in plants remain largely unknown. As an initial step toward gaining insight into these regulatory mechanisms we initiated a mutant screen to identify plants that over-accumulate crystals of calcium oxalate. Four new mutants were identified, from an ethyl methanesulfonate (EMS)-mutagenized Medicago truncatula (cv. Jemalong genotype A17) population, that over-accumulated calcium oxalate crystals. The increased calcium oxalate content of these new mutants, as with the previously isolated mutant cod4, resulted from an increase in druse crystals accumulated within the mesophyll cells of leaves. Complementation and segregation analysis revealed that each mutant was affected at a different locus. This was confirmed through the genetic mapping of each mutation to different linkage groups. Together, these findings emphasize the complexity of factors that can contribute to oxalate biosynthesis and crystal formation in these plants. In addition, each mutant showed a common decrease in ascorbic acid content providing genetic support for ascorbic acid as a precursor in the oxalate biosynthetic pathway for druse crystal formation. Further support was obtained by the ability of an exogenous supply of ascorbate to induce druse crystal formation while other tested organic acids did not induce crystal production.

The CRISPR/Cas9 genome engineering system has ignited and swept through the scientific community like wildfire. Owing largely to its efficiency, specificity, and flexibility, the CRISPR/Cas9 system has quickly become the preferred genome-editing tool of plant scientists. In plants, much of the early CRISPR/Cas9 work has been limited to proof of concept and functional studies in model systems. These studies, along with those in other fields of biology, have led to the development of several utilities of CRISPR/Cas9 beyond single gene editing. Such utilities include multiplexing for inducing multiple cleavage events, controlling gene expression, and site specific transgene insertion. With much of the conceptual CRISPR/Cas9 work nearly complete, plant researchers are beginning to apply this gene editing technology for crop trait improvement. Before rational strategies can be designed to implement this technology to engineer a wide array of crops there is a need to expand the availability of crop-specific vectors, genome resources, and transformation protocols. We anticipate that these challenges will be met along with the continued evolution of the CRISPR/Cas9 system particularly in the areas of manipulation of large genomic regions, transgene-free genetic modification, development of breeding resources, discovery of gene function, and improvements upon CRISPR/Cas9 components. The CRISPR/Cas9 editing system appears poised to transform crop trait improvement.

The establishment of new approaches to control chewing insects has been sought not only for direct use in reducing crop loss but also in managing resistance to the pesticides already in use. Engineered formation of calcium oxalate crystals is a potential strategy that could be developed to fulfill both these needs. As a step toward this development, this study investigates the effects of transforming a non-calcium oxalate crystal accumulating plant, Arabidopsis thaliana, into a crystal accumulating plant. Calcium oxalate crystal accumulating A. thaliana lines were generated by ectopic expression of a single bacterial gene encoding an oxalic acid biosynthetic enzyme. Biochemical and cellular studies suggested that the engineered A. thaliana lines formed crystals of calcium oxalate in a manner similar to naturally occurring crystal accumulating plants. The amount of calcium oxalate accumulated in leaves also reached levels similar to those measured in the leaves of Medicago truncatula in which the crystals are known to play a defensive role. Visual inspection of the different engineered lines, however, suggested a phenotypic consequence on plant growth and development with higher calcium oxalate concentrations. The restoration of a near wild-type plant phenotype through an enzymatic reduction of tissue oxalate supported this observation. Overall, this study is a first to provide initial insight into the potential consequences of engineering calcium oxalate crystal formation in non-crystal accumulating plants.

Calcium oxalate is the most abundant insoluble mineral found in plants and its crystals have been reported in more than 200 plant families. In the barrel medic Medicago truncatula Gaertn., these crystals accumulate predominantly in a sheath surrounding secondary veins of leaves. Mutants of M. truncatula with decreased levels of calcium oxalate crystals were used to assess the defensive role of this mineral against insects. Caterpillar larvae of the beet armyworm Spodoptera exigua Hübner show a clear feeding preference for tissue from calcium oxalate-defective (cod) mutant lines cod5 and cod6 in choice test comparisons with wild-type M. truncatula. Compared to their performance on mutant lines, larvae feeding on wild-type plants with abundant calcium oxalate crystals suffer significantly reduced growth and increased mortality. Induction of wound-responsive genes appears to be normal in cod5 and cod6, indicating that these lines are not deficient in induced insect defenses. Electron micrographs of insect mouthparts indicate that the prismatic crystals in M. truncatula leaves act as physical abrasives during feeding. Food utilization measurements show that, after consumption, calcium oxalate also interferes with the conversion of plant material into insect biomass during digestion. In contrast to their detrimental effects on a chewing insect, calcium oxalate crystals do not negatively affect the performance of the pea aphid Acyrthosiphon pisum Harris, a sap-feeding insect with piercing-sucking mouthparts. The results confirm a long-held hypothesis for the defensive function of these crystals and point to the potential value of genes controlling crystal formation and localization in crop plants.

To regulate bulk Ca 2+ levels, many plants evolved specialized Ca 2+ oxalate accumulating cells called crystal idioblasts. The crystal idioblast appears to have modified some of its cellular components to accommodate the large fluxes of Ca 2+ that occur during calcium oxalate deposition. In this study we examine the modified endoplasmic reticulum (ER) and its involvement in Ca 2+ sequestration. Using a variety of microscopy techniques, we observe that the crystal idioblast of Pistia stratiotes possesses an elaborate network of ER with unique swollen regions. Light microscopy studies, using chlortetracycline and Calcium Green 2, revealed that idioblast ER contains an abundance of calcium. Transmission electron microscopy studies, using the potassium pyroantimonate technique, showed that the Ca 2+ accumulated primarily in the swollen regions. We hypothesize that these swollen areas function as specialized Ca 2+ storage sub-compartments. Whether these sub-compartments are required for proper ER functions or whether they are part of the Ca 2+ transport system involved in calcium oxalate formation remains to be determined.

Calreticulin (CRT) has the highest calcium-binding capacity of the endoplasmic reticulum (ER) proteins found thus far in plants. In this study, we isolated cDNAs encoding CRT from Pistia stratiotes L. (Araceae) and analyzed the temporal and spatial patterns of CRT expression in leaves. Northern analysis showed young leaves had the highest CRT transcript levels, which decreased as the leaves matured. To investigate differences in the expression and cellular localization of CRT, in situ hybridization and immunolocalization were performed. We observed an enrichment of both CRT transcript and protein in a unique cell type called the crystal idioblast. Crystal idioblasts are specialized cells that act as calcium sinks in tissues such as leaves. Sub-cellular localization showed that the CRT protein partitioned into the ER of the crystal idioblasts. At higher resolution, using transmission electron microscopy (TEM), it appeared that the CRT protein was more abundant in “dilated” regions of the ER. Although the specific role(s) CRT plays in the idioblast remains to be determined, we hypothesize that this high-capacity calcium-binding protein functions in the regulation of intracellular idioblast calcium levels.

Calcium oxalate crystal formation has been well documented in plants. The pathway(s) and regulatory mechanism(s) of crystal formation and function, however, remain largely unknown. As a step toward expanding our understanding of crystal formation and function, we characterize the oxalate over-accumulating mutant calcium oxalate defective 4 ( cod4 ). Measurements of oxalate content in the different cod4 tissues showed elevated levels in leaves, stems and pods, compared to the corresponding control tissues. Root oxalate content was similar between cod4 and controls. Microscopic examination of the cod4 tissues revealed that all of the tissues with elevated oxalate content accumulated druse crystals in addition to the prismatic crystals that are present in wild type. With the exception of pods, however, the calcium content did not increase in proportion to the increase in oxalate. Thus, more of the tissue calcium was partitioned into the crystalline form in the leaves and stems from cod4 than controls. Shoot biomass and chlorophyll measurements showed that the cod4 mutation also resulted in an overall reduction in plant growth and chlorophyll content.

Plants invest a considerable amount of resources and energy into the formation of calcium oxalate crystals. A number of roles for crystal formation in plant growth and development have been assigned based on the prevalence of crystals, their spatial distribution, and the variety of crystal shapes. As a step toward determining whether crystal formation plays a critical role in plant growth and development, we characterized the growth, oxalate content, and mineral content of the c alcium o xalate d efective mutant, cod5. Examination of control plants, using light microscopy, revealed the accumulation of prismatic crystals along the vascular strands in all the different plant tissues with the exception of roots, in which no crystals were observed. In contrast, no prismatic crystals were detected in any of the different tissues of the cod5 mutant. Crystals of calcium oxalate were observed in the pods of cod5, but they were of a different morphology. Measurements of the oxalate content in the different tissues confirmed the cod5 crystal phenotype by exhibiting low oxalate levels compared to those of controls. The cod5 pods did contain measurable oxalate levels, but at levels several times lower than controls. Although compromised in its ability to accumulate crystals of calcium oxalate, cod5 exhibited growth, which was similar to that of controls. Moreover, cod5 and controls contained similar amounts of calcium, sodium, and potassium. Our findings suggest that calcium oxalate crystal formation is not essential for plant growth or development in the case of Medicago truncatula.

In order to identify the genes expressed during calcium oxalate crystal formation, we devised a sequential subtractive protocol that allows for efficient isolation of rare differentially expressed transcripts. This new protocol combines the beneficial elements of suppression subtractive hybridization (SSH), subtractive hybridization (SH), and differential screening into a single method. The protocol was designed to increase retention of rare transcripts, efficiently remove unwanted transcripts, have the capacity to analyze numerous transcripts simultaneously, and reduce the number of false positives. The potential for this method was demonstrated through the identification of several transcripts that were differentially expressed in two c alcium o xalate d efective (cod) Medicago truncatula mutants: cod 4 (which has abundant crystal accumulation) and cod 5 (which lacks crystal accumulation). Transcripts were isolated that showed higher steady-state levels in cod 4 than cod 5. The observed differences in transcript levels appeared to occur via two different mechanisms. In most cases, the higher transcript levels were a result of an increase in the amount of transcript present in cod 4. In one instance, however, the higher steady-state transcript level resulted from a decrease in the amount of transcript present in cod 5 rather than an increase in cod 4. The protocol described is well suited for the identification of rare transcripts from complex transcript pools

The gram-negative bacterium, Burkeholderia glumae , is a pathogen of rice. As a step toward identifying pathogenicity determinants, a transposon mutagenized B. glumae library was generated. A transposition frequency of 2.1×10 6 cfu/μg of DNA was achieved using a recently developed Tn 5 -derived transposon system. Southern-blot analysis of several randomly selected colonies indicated that the transpositions were single, random events. Two genetic screens were conducted to assess the mutagenic frequency of the generated library. Screening of 1000 colonies for auxotrophs resulted in the identification of 46 mutants. The 46 mutants were subdivided into five classes based on their ability to grow on supplemented medias. Exposure of 1000 mutagenized colonies to UV light, as part of a second screen, identified two fluorescent mutants. The transposon library will be a useful tool in the investigation of metabolic pathways in B. glumae and allow a direct route to the affected gene. Current emphasis is directed toward identifying the factors required for plant infection.

Plants accumulate crystals of calcium oxalate in a variety of shapes, sizes, amounts, and spatial locations. How and why many plants form crystals of calcium oxalate remain largely unknown. To gain insight into the regulatory mechanisms of crystal formation and function, we have initiated a mutant screen to identify the genetic determinants. Leaves from a chemically mutagenized Medicago truncatula population were visually screened for alterations in calcium oxalate crystal formation. Seven different classes of calcium oxalate defective mutants were identified that exhibited alterations in crystal nucleation, morphology, distribution and/or amount. Genetic analysis suggested that crystal formation is a complex process involving more than seven loci. Phenotypic analysis of a mutant that lacks crystals, cod 5, did not reveal any difference in plant growth and development compared with controls. This finding brings into question the hypothesized roles of calcium oxalate formation in supporting tissue structure and in regulating excess tissue calcium.

Free-floating aquatic plants are useful in studies of plant biochemistry. Their value lies in the fact that they are naturally adapted to growth on liquid media which can be precisely manipulated. Critical biochemical studies require axenic plants free of contaminating organisms which contribute unwanted chemical products or bind/metabolize added compounds. Axenic plants of Lemna , Spirodela , and Wolffiella species have been available for some time. Access to axenic cultures of the free-floating aquatic plant, Pistia stratiotes L., Araceae, (water lettuce) would provide a model more representative of higher and terrestrial plants (leaves, stems, stolons, multiple roots). Our choice of P. stratiotes focuses on its value as a model to study oxalic acid formation and calcium regulation as related to calcium oxalate production. To this end, we have developed axenic Pistia cultures and compared non-axenic plants grown in greenhouse conditions to the axenic plants in terms of organic acid content and morphology/anatomy. Axenic Pistia were comparable to greenhouse-grown plants, the only difference being the smaller size of axenic plants. This study demonstrates the suitability of axenic Pistia for biochemical research and also indicates that such plants will be very useful in studies of bioremediation and other processes in aquatic systems.

Although oxalic acid is common in nature our understanding of the mechanism(s) regulating its turnover remains incomplete. In this study we identify Saccharomyces cerevisiae acyl-activating enzyme 3 (ScAAE3) as an enzyme capable of catalyzing the conversion of oxalate to oxalyl-CoA. Based on our findings we propose that ScAAE3 catalyzes the first step in a novel pathway of oxalate degradation to protect the cell against the harmful effects of oxalate derived from an endogenous process or an environmental source.

Background: How the distribution and sequestered form of plant macro/micro-nutrients influence their bioavailability, and ultimately impact human health, are poorly understood. The legume Medicago truncatula has a portion of its tissue calcium (Ca) sequestered in the form of the Ca oxalate (CaOx) crystal which reduces its nutritional value in terms of Ca bioavailability. The calcium oxalate deficient 5 (cod5) mutant has a total Ca content similar to wild-type (WT) plants, but sequesters less of its tissue Ca in the form of the CaOx crystal. Previous short-term mice feeding studies suggest that this difference is responsible for the improved Ca bioavailability of the cod5 plants compared to WT plants. Objectives: To perform long term feeding studies with Vitamin D Receptor Knockout (VDR-KO) mice and the nutritionally improved cod5 line to assess the impact of increased Ca bioavailability on VDR-KO Ca deficiency phenotypes. Methods: To assess the ability of diets containing cod5 plant material as the sole Ca source to rescue the Ca deficiency phenotypes of the VDR-KO mice we conducted both short term and long term experiments. Specifically, Ca absorption and utilization were measured short term (24-hour) in the hind limb bones and duodenum tissue of VDR-KO mice that were fed either an intrinsically 45Ca labeled cod5 or WT Medicago diet. Long term (20-day) bodyweight gain and change in Bone Mineral Density (BMD) were also measured over a 20 day period in VDR-KO mice fed either a cod5 or WT Medicago diet. Results: In the 24-hour feeding study, 45Ca incorporation was found to be 46.3% (male) or 53.9% (female) higher in hind limb bones (P