An rbcL mRNA-binding protein is associated with C3 to C4 evolution and light-induced production of Rubisco in Flaveria.

Nuclear-encoded RLSB protein binds chloroplastic rbcL mRNA encoding the Rubisco large subunit. RLSB is highly conserved across all groups of land plants and is associated with positive post-transcriptional regulation of rbcL expression. In C3 leaves, RLSB and Rubisco occur in all chlorenchyma cell chloroplasts, while in C4 leaves these accumulate only within bundle sheath (BS) chloroplasts. RLSB's role in rbcL expression makes modification of its localization a likely prerequisite for the evolutionary restriction of Rubisco to BS cells. Taking advantage of evolutionarily conserved RLSB orthologs in several C3, C3-C4, C4-like, and C4 photosynthetic types within the genus Flaveria, we show that low level RLSB sequence divergence and modification to BS specificity coincided with ontogeny of Rubisco specificity and Kranz anatomy during C3 to C4 evolution. In both C3 and C4 species, Rubisco production reflected RLSB production in all cell types, tissues, and conditions examined. Co-localization occurred only in photosynthetic tissues, and both proteins were co-ordinately induced by light at post-transcriptional levels. RLSB is currently the only mRNA-binding protein to be associated with rbcL gene regulation in any plant, with variations in sequence and acquisition of cell type specificity reflecting the progression of C4 evolution within the genus Flaveria.

Evolution of RLSB, a nuclear-encoded S1 domain RNA binding protein associated with post-transcriptional regulation of plastid-encoded rbcL mRNA in vascular plants.

BACKGROUND: RLSB, an S-1 domain RNA binding protein of Arabidopsis, selectively binds rbcL mRNA and co-localizes with Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) within chloroplasts of C3 and C4 plants. Previous studies using both Arabidopsis (C3) and maize (C4) suggest RLSB homologs are post-transcriptional regulators of plastid-encoded rbcL mRNA. While RLSB accumulates in all Arabidopsis leaf chlorenchyma cells, in C4 leaves RLSB-like proteins accumulate only within Rubisco-containing bundle sheath chloroplasts of Kranz-type species, and only within central compartment chloroplasts in the single cell C4 plant Bienertia. Our recent evidence implicates this mRNA binding protein as a primary determinant of rbcL expression, cellular localization/compartmentalization, and photosynthetic function in all multicellular green plants. This study addresses the hypothesis that RLSB is a highly conserved Rubisco regulatory factor that occurs in the chloroplasts all higher plants. RESULTS: Phylogenetic analysis has identified RLSB orthologs and paralogs in all major plant groups, from ancient liverworts to recent angiosperms. RLSB homologs were also identified in algae of the division Charophyta, a lineage closely related to land plants. RLSB-like sequences were not identified in any other algae, suggesting that it may be specific to the evolutionary line leading to land plants. The RLSB family occurs in single copy across most angiosperms, although a few species with two copies were identified, seemingly randomly distributed throughout the various taxa, although perhaps correlating in some cases with known ancient whole genome duplications. Monocots of the order Poales (Poaceae and Cyperaceae) were found to contain two copies, designated here as RLSB-a and RLSB-b, with only RLSB-a implicated in the regulation of rbcL across the maize developmental gradient. Analysis of microsynteny in angiosperms revealed high levels of conservation across eudicot species and for both paralogs in grasses, highlighting the possible importance of maintaining this gene and its surrounding genomic regions. CONCLUSIONS: Findings presented here indicate that the RLSB family originated as a unique gene in land plant evolution, perhaps in the common ancestor of charophytes and higher plants. Purifying selection has maintained this as a highly conserved single- or two-copy gene across most extant species, with several conserved gene duplications. Together with previous findings, this study suggests that RLSB has been sustained as an important regulatory protein throughout the course of land plant evolution. While only RLSB-a has been directly implicated in rbcL regulation in maize, RLSB-b could have an overlapping function in the co-regulation of rbcL, or may have diverged as a regulator of one or more other plastid-encoded mRNAs. This analysis confirms that RLSB is an important and unique photosynthetic regulatory protein that has been continuously expressed in land plants as they emerged and diversified from their ancient common ancestor.

The unique structural and biochemical development of single cell C4 photosynthesis along longitudinal leaf gradients in Bienertia sinuspersici and Suaeda aralocaspica (Chenopodiaceae).

Temporal and spatial patterns of photosynthetic enzyme expression and structural maturation of chlorenchyma cells along longitudinal developmental gradients were characterized in young leaves of two single cell C4 species, Bienertia sinuspersici and Suaeda aralocaspica Both species partition photosynthetic functions between distinct intracellular domains. In the C4-C domain, C4 acids are formed in the C4 cycle during capture of atmospheric CO2 by phosphoenolpyruvate carboxylase. In the C4-D domain, CO2 released in the C4 cycle via mitochondrial NAD-malic enzyme is refixed by Rubisco. Despite striking differences in origin and intracellular positioning of domains, these species show strong convergence in C4 developmental patterns. Both progress through a gradual developmental transition towards full C4 photosynthesis, with an associated increase in levels of photosynthetic enzymes. Analysis of longitudinal sections showed undeveloped domains at the leaf base, with Rubisco rbcL mRNA and protein contained within all chloroplasts. The two domains were first distinguishable in chlorenchyma cells at the leaf mid-regions, but still contained structurally similar chloroplasts with equivalent amounts of rbcL mRNA and protein; while mitochondria had become confined to just one domain (proto-C4-D). The C4 state was fully formed towards the leaf tips, Rubisco transcripts and protein were compartmentalized specifically to structurally distinct chloroplasts in the C4-D domains indicating selective regulation of Rubisco expression may occur by control of transcription or stability of rbcL mRNA. Determination of CO2 compensation points showed young leaves were not functionally C4, consistent with cytological observations of the developmental progression from C3 default to intermediate to C4 photosynthesis.

Expression of Malus xiaojinensis IRT1 (MxIRT1) protein in transgenic yeast cells leads to degradation through autophagy in the presence of excessive iron.

Iron is essential for plants, but highly toxic when present in excess. Consequently, iron uptake by root transporters must be finely tuned to avoid excess uptake from soil under iron excess. The iron-regulated transporter of Malus xiaojinensis (MxIRT1), induced in roots under iron deficiency, is a highly effective iron(II) transporter. Here, we investigated how the presence of excessive iron leads to MxIRT1 degradation in yeast expressing this plant iron transporter protein. To determine the relationship between iron abundance and MxIRT1 degradation, relative levels of autophagy-related gene-8 (ATG8) mRNA and the active ATG8-phosphatidylethanolamine-conjugated (PE) protein were measured in wild-type yeast and the autophagic mutant strains atg1∆, atg5∆, atg7∆, ypt7∆ and tor1∆ under normal and excessive iron conditions. The data showed that the exposure of MxIRT1-eGFP-transformed wild-type and tor1∆ strains to excessive iron led to significantly increased levels of ATG8 transcript and ATG8-PE protein, which resulted in enhanced MxIRT1 degradation. Co-localization of mCherry-ATG8 and MxIRT1-eGFP provided evidence that these proteins interact during autophagy in yeast. While inhibition of autophagic initiation, autophagosome formation and vacuole fusion all decreased MxIRT1 degradation. PMSF inhibition of autophagy prevented degradation, leading to the accumulation of MxIRT1-containing vesicles in the vacuoles. MxIRT1-vesicles were sorted into autophagosomes for iron-induced degradation in yeast, whereas the endogenous iron(II) transporter Fet4 was degraded in an autophagy-independent manner. Moreover, immunoprecipitation showed that multimono-ubiquitins provided MxIRT1 with the ubiquitination signal. Together, three factors, iron excess, autophagy and mono-ubiquitination, affect the functional activity and stability of exogenous MxIRT1 in yeast, thereby preventing iron uptake via this root transporter.

Exploring mechanisms linked to differentiation and function of dimorphic chloroplasts in the single cell C4 species Bienertia sinuspersici.

BACKGROUND: In the model single-cell C4 plant Bienertia sinuspersici, chloroplast- and nuclear-encoded photosynthetic enzymes, characteristically confined to either bundle sheath or mesophyll cells in Kranz-type C4 leaves, all occur together within individual leaf chlorenchyma cells. Intracellular separation of dimorphic chloroplasts and key enzymes within central and peripheral compartments allow for C4 carbon fixation analogous to NAD-malic enzyme (NAD-ME) Kranz type species. Several methods were used to investigate dimorphic chloroplast differentiation in B. sinuspersici. RESULTS: Confocal analysis revealed that Rubisco-containing chloroplasts in the central compartment chloroplasts (CCC) contained more photosystem II proteins than the peripheral compartment chloroplasts (PCC) which contain pyruvate,Pi dikinase (PPDK), a pattern analogous to the cell type-specific chloroplasts of many Kranz type NAD-ME species. Transient expression analysis using GFP fusion constructs containing various lengths of a B. sinuspersici Rubisco small subunit (RbcS) gene and the transit peptide of PPDK revealed that their import was not specific to either chloroplast type. Immunolocalization showed the rbcL-specific mRNA binding protein RLSB to be selectively localized to the CCC in B. sinuspersici, and to Rubisco-containing BS chloroplasts in the closely related Kranz species Suaeda taxifolia. Comparative fluorescence analyses were made using redox-sensitive and insensitive GFP forms, as well comparative staining using the peroxidase indicator 3,3-diaminobenzidine (DAB), which demonstrated differences in stromal redox potential, with the CCC having a more negative potential than the PCC. CONCLUSIONS: Both CCC RLSB localization and the differential chloroplast redox state are suggested to have a role in post-transcriptional rbcL expression.

An uncleaved signal peptide directs the Malus xiaojinensis iron transporter protein Mx IRT1 into the ER for the PM secretory pathway.

Malus xiaojinensis iron-regulated transporter 1 (Mx IRT1) is a highly effective inducible iron transporter in the iron efficient plant Malus xiaojinensis. As a multi-pass integral plasma membrane (PM) protein, Mx IRT1 is predicted to consist of eight transmembrane domains, with a putative N-terminal signal peptide (SP) of 1-29 amino acids. To explore the role of the putative SP, constructs expressing Mx IRT1 (with an intact SP) and Mx DsIRT1 (with a deleted SP) were prepared for expression in Arabidopsis and in yeast. Mx IRT1 could rescue the iron-deficiency phenotype of an Arabidopsis irt1 mutant, and complement the iron-limited growth defect of the yeast mutant DEY 1453 (fet3fet4). Furthermore, fluorescence analysis indicated that a chimeric Mx IRT1-eGFP (enhanced Green Fluorescent Protein) construct was translocated into the ER (Endoplasmic reticulum) for the PM sorting pathway. In contrast, the SP-deleted Mx DsIRT1 could not rescue either of the mutant phenotypes, nor direct transport of the GFP signal into the ER. Interestingly, immunoblot analysis indicated that the SP was not cleaved from the mature protein following transport into the ER. Taken together, data presented here provides strong evidence that an uncleaved SP determines ER-targeting of Mx IRT1 during the initial sorting stage, thereby enabling the subsequent transport and integration of this protein into the PM for its crucial role in iron uptake.

A novel RNA binding protein affects rbcL gene expression and is specific to bundle sheath chloroplasts in C4 plants.

BACKGROUND: Plants that utilize the highly efficient C4 pathway of photosynthesis typically possess kranz-type leaf anatomy that consists of two morphologically and functionally distinct photosynthetic cell types, the bundle sheath (BS) and mesophyll (M) cells. These two cell types differentially express many genes that are required for C4 capability and function. In mature C4 leaves, the plastidic rbcL gene, encoding the large subunit of the primary CO2 fixation enzyme Rubisco, is expressed specifically within BS cells. Numerous studies have demonstrated that BS-specific rbcL gene expression is regulated predominantly at post-transcriptional levels, through the control of translation and mRNA stability. The identification of regulatory factors associated with C4 patterns of rbcL gene expression has been an elusive goal for many years. RESULTS: RLSB, encoded by the nuclear RLSB gene, is an S1-domain RNA binding protein purified from C4 chloroplasts based on its specific binding to plastid-encoded rbcL mRNA in vitro. Co-localized with LSU to chloroplasts, RLSB is highly conserved across many plant species. Most significantly, RLSB localizes specifically to leaf bundle sheath (BS) cells in C4 plants. Comparative analysis using maize (C4) and Arabidopsis (C3) reveals its tight association with rbcL gene expression in both plants. Reduced RLSB expression (through insertion mutation or RNA silencing, respectively) led to reductions in rbcL mRNA accumulation and LSU production. Additional developmental effects, such as virescent/yellow leaves, were likely associated with decreased photosynthetic function and disruption of associated signaling networks. CONCLUSIONS: Reductions in RLSB expression, due to insertion mutation or gene silencing, are strictly correlated with reductions in rbcL gene expression in both maize and Arabidopsis. In both plants, accumulation of rbcL mRNA as well as synthesis of LSU protein were affected. These findings suggest that specific accumulation and binding of the RLSB binding protein to rbcL mRNA within BS chloroplasts may be one determinant leading to the characteristic cell type-specific localization of Rubisco in C4 plants. Evolutionary modification of RLSB expression, from a C3 "default" state to BS cell-specificity, could represent one mechanism by which rbcL expression has become restricted to only one cell type in C4 plants.

Photosynthetic gene expression in higher plants.

Within the chloroplasts of higher plants and algae, photosynthesis converts light into biological energy, fueling the assimilation of atmospheric carbon dioxide into biologically useful molecules. Two major steps, photosynthetic electron transport and the Calvin-Benson cycle, require many gene products encoded from chloroplast as well as nuclear genomes. The expression of genes in both cellular compartments is highly dynamic and influenced by a diverse range of factors. Light is the primary environmental determinant of photosynthetic gene expression. Working through photoreceptors such as phytochrome, light regulates photosynthetic genes at transcriptional and posttranscriptional levels. Other processes that affect photosynthetic gene expression include photosynthetic activity, development, and biotic and abiotic stress. Anterograde (from nucleus to chloroplast) and retrograde (from chloroplast to nucleus) signaling insures the highly coordinated expression of the many photosynthetic genes between these different compartments. Anterograde signaling incorporates nuclear-encoded transcriptional and posttranscriptional regulators, such as sigma factors and RNA-binding proteins, respectively. Retrograde signaling utilizes photosynthetic processes such as photosynthetic electron transport and redox signaling to influence the expression of photosynthetic genes in the nucleus. The basic C3 photosynthetic pathway serves as the default form used by most of the plant species on earth. High temperature and water stress associated with arid environments have led to the development of specialized C4 and CAM photosynthesis, which evolved as modifications of the basic default expression program. The goal of this article is to explain and summarize the many gene expression and regulatory processes that work together to support photosynthetic function in plants.

Development of structural and biochemical characteristics of C(4) photosynthesis in two types of Kranz anatomy in genus Suaeda (family Chenopodiaceae).

Genus Suaeda (family Chenopodiaceae, subfamily Suaedoideae) has two structural types of Kranz anatomy consisting of a single compound Kranz unit enclosing vascular tissue. One, represented by Suaeda taxifolia, has mesophyll (M) and bundle sheath (BS) cells distributed around the leaf periphery. The second, represented by Suaeda eltonica, has M and BS surrounding vascular bundles in the central plane. In both, structural and biochemical development of C(4) occurs basipetally, as observed by analysis of the maturation gradient on longitudinal leaf sections. This progression in development was also observed in mid-sections of young, intermediate, and mature leaves in both species, with three clear stages: (i) monomorphic chloroplasts in the two cell types in younger tissue with immunolocalization and in situ hybridization showing ribulose bisphosphate carboxylase oxygenase (Rubisco) preferentially localized in BS chloroplasts, and increasing in parallel with the establishment of Kranz anatomy; (ii) vacuolization and selective organelle positioning in BS cells, with occurrence of phosphoenolpyruvate carboxylase (PEPC) and immunolocalization showing that it is preferentially in M cells; (iii) establishment of chloroplast dimorphism and mitochondrial differentiation in mature tissue and full expression of C(4) biochemistry including pyruvate, Pi dikinase (PPDK) and NAD-malic enzyme (NAD-ME). Accumulation of rbcL mRNA preceded its peptide expression, occurring prior to organelle positioning and differentiation. During development there was sequential expression and increase in levels of Rubisco and PEPC followed by NAD-ME and PPDK, and an increase in the (13)C/(12)C isotope composition of leaves to values characteristic of C(4) photosynthesis. The findings indicate that these two forms of NAD-ME type C(4) photosynthesis evolved in parallel within the subfamily with similar ontogenetic programmes.

Toxicity and reductions in intracellular calcium levels following uptake of a tetracycline antibiotic in Arabidopsis.

Plant responses to natural stresses have been the focus of numerous studies; however less is known about plant responses to artificial (i.e., man-made) stress. Chlortetracycline (CTC) is widely used in agriculture and becomes an environmental contaminant when introduced into soil from manure used as fertilizer. We show here that in the model plant Arabidopsis (Arabidopsis thaliana), root uptake of CTC leads to toxicity, with growth reductions and other effects. Analysis of protein accumulation and in vivo synthesis revealed numerous changes in soluble and membrane-associated proteins in leaves and roots. Many representative proteins associated with different cellular processes and compartments showed little or no change in response to CTC. However, differences in accumulation and synthesis of NAD-malic enzyme in leaves versus roots suggest potential CTC-associated effects on metabolic respiration may vary in different tissues. Fluorescence resonance energy transfer (FRET) analysis indicated reduced levels of intracellular calcium are associated with CTC uptake and toxicity. These findings support a model in which CTC uptake through roots leads to reductions in levels of intracellular calcium due to chelation. In turn, changes in overall patterns and levels of protein synthesis and accumulation due to reduced calcium ultimately lead to growth reductions and other toxicity effects.

Development of a rapid biolistic assay to determine changes in relative levels of intracellular calcium in leaves following tetracycline uptake by pinto bean plants.

Tetracycline antibiotics, such as chlortetracycline (CTC) and tetracycline (TC), are introduced into agricultural lands through the application of manure as fertilizer. These compounds are phytotoxic to certain crop plants, including pinto beans (Phaseolus vulgaris), the species used for this investigation. While the mechanism of this toxicity is not yet understood, CTC is known to be a calcium chelator. We describe here a novel method to show that CTC is taken up by pinto bean plants and chelates calcium in leaves. Cameleon fusion proteins can provide qualitative and quantitative imaging of intracellular calcium levels, but current methodology requires stable transformation. Many plant species, including pinto beans, are not yet transformable using standard Agrobacterium-based protocols. To determine the role of calcium chelation in this plant, a rapid, biolistic method was developed to transiently express the cameleon protein. This method can easily be adapted to other plant systems. Our findings provide evidence that chelation of intracellular calcium by CTC is related to phytotoxic effects caused by this antibiotic in pinto beans. Root uptake of CTC and TC by pinto beans and their translocation to leaves were further verified by fluorescence spectroscopy and liquid chromatography/mass spectrometry, confirming results of the biolistic method that showed calcium chelation by tetracyclines in leaves.

Rubisco gene expression in C4 plants.

In leaves of most C(4) plants, ribulose 1,5 bisphosphate carboxylase (Rubisco) accumulates only in bundle sheath (bs) cells that surround the vascular centres, and not in mesophyll (mp) cells. It has been shown previously that in the C(4) dicots amaranth and Flaveria bidentis, post-transcriptional control of mRNA translation and stability mediate the C(4) expression patterns of genes encoding the large and small Rubisco subunits (chloroplast rbcL and nuclear RbcS, respectively). Translational control appears to regulate bs cell-specific Rubisco gene expression during early dicot leaf development, while control of mRNA stability appears to mediate bs-specific accumulation of RbcS and rbcL transcripts in mature leaves. Post-transcriptional control is also involved in the regulation of Rubisco gene expression by light, and in response to photosynthetic activity. Transgenic and transient expression studies in F. bidentis provide direct evidence for post-transcriptional control of bs cell-specific RbcS expression, which is mediated by the 5' and 3' untranslated regions (UTRs) of the mRNA. Comparisons of Rubisco gene expression in these dicots and in the monocot maize indicates possible commonalities in the regulation of RbcS and rbcL genes in these divergent C(4) species. Now that the role of post-transcriptional regulation in C(4) gene expression has been established, it is likely that future studies of mRNA-protein interactions will address long-standing questions about the establishment and maintenance of cell type-specificity in these plants. Some of these regulatory mechanisms may have ancestral origins in C(3) species, through modification of pre-existing factors, or by the acquisition of novel C(4) processes.

CRISPR Gene Editing in Yeast: An Experimental Protocol for an Upper-Division Undergraduate Laboratory Course.

Clustered regularly interspaced short palindromic repeats (CRISPR) are a revolutionary tool based on a bacterial acquired immune response system. CRISPR has gained widespread use for gene editing in a variety of organisms and is an increasingly valuable tool for basic genetic research, with far-reaching implications for medicine, agriculture, and industry. This lab is based on the premise that upper division undergraduate students enrolled in a Life Sciences curriculum must become familiar with cutting edge advances in biotechnology that have significant impact on society. Toward this goal, we developed a new hands-on laboratory exercise incorporating the use of CRISPR-Cas9 and homology directed repair (HDR) to edit two well-characterized genes in the budding yeast, Saccharomyces cerevisiae. The two genes edited in this exercise, Adenine2 (ADE2) and Sterile12 (STE12) affect metabolic and developmental processes, respectively. Editing the premature stop codons in these genes results in clearly identifiable phenotypes that can be assessed by students in a standard laboratory course setting. Making use of this basic eukaryotic model organism facilitates a laboratory exercise that is inexpensive, simple to organize, set up, and present to students. This exercise enables undergraduate students to initiate and follow-up on all stages of the CRISPR gene editing process, from identification of guide RNAs, amplification of an appropriate HDR fragment, and analysis of mutant phenotypes. The organization of this protocol also allows for easy modification, providing additional options for editing any expressed genes within the yeast genome to produce new mutations, or recovery of existing mutants to wild type. © 2018 International Union of Biochemistry and Molecular Biology, 46(6):592-601, 2018.

Regulation of Rubisco gene expression in C4 plants.

Ribulose-1,5-bisphosphate-carboxylase/oxygenase (Rubisco) incorporates inorganic carbon into an organic form, making this chloroplastic enzyme one of the most essential factors for all life on earth. Despite its central role in photosynthesis, research into regulation of the chloroplast rbcL and nuclear RbcS genes that encode this enzyme has lagged behind other plant gene systems. A major characteristic of kranz-type C4 plants is the accumulation of Rubisco only within chloroplasts of internalized bundle sheath cells that surround the leaf vascular centers. In plants that utilize the less common single cell C4 system, Rubisco accumulates only within one type of dimorphic chloroplasts localized to a specific region of leaf chlorenchyma cells. Understanding regulatory processes that restrict Rubisco gene expression to only one cell type or chloroplast type is a major focus of C4 research. Regulatory steps may include transcriptional, post-transcriptional, and post-translational processes.

OsSEC24, a functional SEC24-like protein in rice, improves tolerance to iron deficiency and high pH by enhancing H(+) secretion mediated by PM-H(+)-ATPase.

Iron is abundant in the soil, but its low solubility in neutral or alkaline soils limits its uptake. Plants can rely on rhizosphere acidification to increase iron solubility. OsSEC27p was previously found to be a highly up-regulated gene in iron-deficient rice roots. Here, pH-dependent complementation assays using yeast mutants sec24Δ/SEC24 and sec27Δ/SEC27 showed that OsSEC27 could functionally complement SEC24 but not SEC27 in yeast; thus, it was renamed as OsSEC24. We found that OsSEC24-transgenic tobacco plants increased the length and number of roots under iron deficiency at pH 8.0. To explore how OsSEC24 confers tolerance to iron deficiency, we utilized transgenic tobacco, rice and rice protoplasts. H(+) flux measurements using Non-invasive Micro-test Technology (NMT) indicated that the transgenic OsSEC24 tobacco and rice enhanced H(+) efflux under iron deficiency. Conversely, the application of plasma membrane PM-H(+)-ATPase inhibitor vanadate elucidated that H(+) secretion increased by OsSEC24 was mediated by PM-H(+)-ATPase. OsPMA2 was used as a representative of iron deficiency-responsive PM-H(+)-ATPases in rice root via RT-PCR analysis. In transgenic rice protoplasts OsPMA2 was packaged into OsSEC24 vesicles after export from the ER through confocal-microscopy observation. Together, OsSEC24 vesicles, along with PM-H(+)-ATPases stimulate roots formation under iron deficiency by enhancing rhizosphere acidification.

Chlortetracycline detoxification in maize via induction of glutathione S-transferases after antibiotic exposure.

Soil contamination with nonmetabolized antibiotics is an emerging environmental concern, especially on agricultural croplands that receive animal manure as fertilizer. In this study, phytotoxicity of chlortetracycline (CTC) antibiotics on pinto beans (Phaseolus vulgaris) and maize (Zea mays) was investigated under controlled conditions. When grown in CTC-treated soil, a significant increase in the activities of the plant stress proteins glutathione S-transferases (GST) and peroxidases (POX) were observed in maize plants, but not in pinto beans. In vitro conjugation reactions demonstrated that the induced GST in maize catalyzed the conjugation of glutathione (GSH) with CTC, producing stable conjugates that were structurally characterized using liquid chromatography/mass spectrometry. The antibiotic-induced GST produced CTC-glutathione conjugate at relative concentrations 2-fold higher than that produced by constitutively expressed GST extracted from untreated maize. On the other hand, GST extracted from pinto beans (both treated and untreated) did not efficiently catalyze glutathione conjugation with CTC. These results suggest that maize is able to detoxify chlortetracycline via the glutathione pathway, whereas pinto beans cannot. This may explain the observed stunted growth of pinto beans after antibiotic treatment. This study demonstrates the importance of plant uptake in determining the fate of antibiotics in soil and their potential phytotoxicity to susceptible plants.

Determination of enzyme kinetics and glutathione conjugates of chlortetracycline and chloroacetanilides using liquid chromatography-mass spectrometry.

Glutathione S-transferases (GSTs) isolated from chlortetracycline (CTC)-treated maize catalyzed the conjugation of glutathione (GSH) with CTC, producing stable conjugates that were structurally characterized using liquid chromatography-ion-trap mass spectrometry (LC-IT-MS). Enzyme-mediated dechlorination of CTC resulted during GSH conjugation as revealed by the mass spectra of the CTC-GSH conjugate, which was characterized by the loss of the chlorine isotopic signature, and shorter chromatographic retention time relative to the chlorinated parent compound. Several fragmentation patterns in the mass spectrum of the CTC-GSH conjugate can be used to verify the identity of the enzyme reaction products. The expected molecular ion [M + H](+) of the CTC-GSH conjugate (m/z 751) with chlorine removal was not observed in the positive electrospray ionization. Instead, a base peak of m/z 677, corresponding to the loss of glycine (MW = 75 Da), was observed. When m/z 677 was subjected to further fragmentation, characteristic peaks corresponding to the loss of glutamic acid (m/z = 129) and water (m/z 18) were observed in the MS/MS spectrum. The catalytic activity of the CTC-induced GST towards dechlorination of chloroacetanilide herbicides (alachlor, metolachlor and propachlor), which are known to be detoxified in plants via the glutathione pathway, was also evaluated in vitro. Glutathione conjugates of chloroacetanilides also showed the losses of m/z 129 and m/z 18 that are characteristic of GSH conjugates when characterized by LC-IT-MS. Interestingly, the sensitivity of LC-IT-MS made it possible, for the first time, to detect chloroacetanilides that are conjugated with two GSH molecules, in addition to the known single GSH conjugates. This research demonstrates a more sensitive and specific method of measuring enzyme reaction products using LC-IT-MS.

A cucumber mosaic virus mutant lacking the 2b counter-defence protein gene provides protection against wild-type strains.

Several plant virus mutants, in which genes encoding silencing suppressor proteins have been deleted, are known to induce systemic or localized RNA silencing against themselves and other RNA molecules containing homologous sequences. Thus, it is thought that many cases of cross-protection, in which infection with a mild or asymptomatic virus mutant protects plants against challenge infection with closely related virulent viruses, can be explained by RNA silencing. We found that a cucumber mosaic virus (CMV) mutant of the subgroup IA strain Fny (Fny-CMVDelta2b), which cannot express the 2b silencing suppressor protein, cross-protects tobacco (Nicotiana tabacum) and Nicotiana benthamiana plants against disease induction by wild-type Fny-CMV. However, protection is most effective only if inoculation with Fny-CMVDelta2b and challenge inoculation with wild-type CMV occurs on the same leaf. Unexpectedly, Fny-CMVDelta2b also protected plants against infection with TC-CMV, a subgroup II strain that is not closely related to Fny-CMV. Additionally, in situ hybridization revealed that Fny-CMVDelta2b and Fny-CMV can co-exist in the same tissues but these tissues contain zones of Fny-CMVDelta2b-infected host cells from which Fny-CMV appears to be excluded. Taken together, it appears unlikely that cross-protection by Fny-CMVDelta2b occurs by induction of systemic RNA silencing against itself and homologous RNA sequences in wild-type CMV. It is more likely that protection occurs through either induction of very highly localized RNA silencing, or by competition between strains for host cells or resources.

Untranslated regions of FbRbcS1 mRNA mediate bundle sheath cell-specific gene expression in leaves of a C4 plant.

C4 photosynthesis typically requires two specialized leaf cell types, bundle sheath (bs) and mesophyll (mp), which provide the foundation for this highly efficient carbon assimilation pathway. In leaves of Flaveria bidentis, a dicotyledonous C4 plant, ribulose 1,5-bisphosphate carboxylase (rubisco) accumulates only in bs cells surrounding the vascular centers and not in mp cells. This is in contrast to the more common C3 plants, which accumulate rubisco in all photosynthetic cells. Many previous studies have focused on transcriptional control of C4 cell type-specificity; however, post-transcriptional regulation has also been implicated in the bs-specific expression of genes encoding the rubisco subunits. In this current study, a biolistic leaf transformation assay has provided direct evidence that the 5'- and 3'-untranslated regions (UTRs) of F. bidentis FbRbcS1 mRNA (from a nuclear gene encoding the rubisco small subunit), in themselves, confer strong bs cell-specific expression to gfpA reporter gene transcripts when transcribed from a constitutive CaMV promoter. In transformed leaf regions, strong bs cell-specific GFP expression was accompanied by corresponding bs cell-specific accumulation of the constitutively transcribed FbRbcS1 5'-UTR-gfpA-3'-UTR mRNAs. Control constructs lacking any RbcS mRNA sequences were expressed in all leaf cell types. These findings demonstrate that characteristic cell type-specific FbRbcS1 expression patterns in C4 leaves can be established entirely by sequences contained within the transcribed UTRs of FbRbcS1 mRNAs. We conclude that selective transcript stabilization (in bs cells) or degradation (in mp cells) plays a key role in determining bs cell-specific localization of the rubisco enzyme.

Inducible expression, enzymatic activity, and origin of higher plant homologues of bacterial RelA/SpoT stress proteins in Nicotiana tabacum.

All living cells possess adaptive responses to environmental stress that are essential to ensuring cell survival. For motile organisms, this can culminate in avoidance or attractile behavior, but for sessile organisms such as plants, stress adaptation is a process of success or failure within the confines of a given environment. Nearly all bacterial species possess a highly evolved system for stress adaptation, known as the "stringent response." This ancient and ubiquitous regulatory response is mediated by production of a second messenger of general stress, the nucleotide guanosine-3',5'-(bis)pyrophosphate (ppGpp), which mediates reprogramming of the global transcriptional output of the cell. Accumulation of ppGpp is stress-induced through the enzymatic activation of the well known bacterial ppGpp synthetases, RelA and SpoT. We have recently discovered homologues of bacterial relA/spoT genes in the model plant Nicotiana tabacum. We hypothesize that these homologues (designated RSH genes for RelA/SpoT homologues) serve a stress-adaptive function in plants analogous with their function in bacteria. In support of this hypothesis, we find 1) inducibility of tobacco RSH gene expression following treatment with jasmonic acid; 2) bona fide ppGpp synthesis activity of purified recombinant Nt-RSH2 protein, and 3) a wide spread distribution of RSH gene expression in the plant kingdom. Phylogenetic analyses identifies a distinct phylogenetic branch for the plant RSH proteins with two subgroups and supports ancient symbiosis and nuclear gene transfer as a possible origin for these stress response genes in plants. In addition, we find that Nt-RSH2 protein co-purifies with chloroplasts in subcellular fractionation experiments. Taken together, our findings implicate a direct mode of action of these ppGpp synthetases with regard to plant physiology, namely regulation of chloroplast gene expression in response to plant defense signals.