Salinity tolerance--mechanisms, models and the metabolic engineering of complex traits.
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Genes, Fungal↗
Biomedical subjects
H J Bohnert
Publications and source records attributed to H J Bohnert.
Plant stress adaptations--making metabolism move.
Persistently sub-optimal environmental conditions constitute stress. Perception and signaling lead to protein expression changes, the activation of new biochemical pathways, and repression of others which are characteristic of the unstressed state. Protective metabolic adaptations alter physiological reactions of the whole plant. Paramount among the mechanisms are oxygen radical scavenging, maintenance of ion uptake and water balance, and reactions altering carbon and nitrogen allocation, such that reducing power is defused. Elements of the stress signaling pathways and proteins that lead to stress protection have recently become known.
Adaptation, Physiological↗
Chimeric Arabidopsis thaliana ribulose-1,5-bisphosphate carboxylase/oxygenase containing a pea small subunit protein is compromised in carbamylation.
A cDNA of pea (Pisum sativum L.) RbcS 3A, encoding a small subunit protein (S) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), has been expressed in Arabidopsis thaliana under control of the cauliflower mosaic virus 35S promoter, and the transcript and mature S protein were detected. Specific antibodies revealed two protein spots for the four Arabidopsis S and one additional spot for pea S. Pea S in chimeric Rubisco amounted to 15 to 18% of all S, as judged by separation on two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels from partially purified enzyme preparations and quantitation of silver-stained protein spots. The chimeric enzyme had 11 +/- 1% fewer carbamylated sites and a 11 +/- 1% lower carboxylase activity than wild-type Arabidopsis Rubisco. Whereas pea S expression, preprotein transport, and processing and assembly resulted in a stable holoenzyme, the chimeric enzyme was reproducibly catalytically less efficient. We suggest that the presence of, on average, one foreign S per holoenzyme is responsible for the altered activity. In addition, higher-plant Rubisco, unlike the cyanobacterial enzyme, seems to have evolved species-specific interactions between S and the large subunit protein that are involved in carbamylation of the active site.
Amino Acid Sequence↗
Regulation of cell-specific inositol metabolism and transport in plant salinity tolerance.
myo-Inositol and its derivatives are commonly studied with respect to cell signaling and membrane biogenesis, but they also participate in responses to salinity in animals and plants. In this study, we focused on L-myo-inositol 1-phosphate synthase (INPS), which commits carbon to de novo synthesis, and myo-inositol O-methyltransferase (IMT), which uses myo-inositol for stress-induced accumulation of a methylinositol, D-ononitol. The Imt and Inps promoters are transcriptionally controlled. We determined that the transcription rates, transcript levels, and protein abundance are correlated. During normal growth, INPS is present in all cells, but IMT is repressed. After salinity stress, the amount of INPS was enhanced in leaves but repressed in roots. IMT was induced in all cell types. The absence of myo-inositol synthesis in roots is compensated by inositol/ononitol transport in the phloem. The mobilization of photosynthate through myo-inositol translocation links root metabolism to photosynthesis. Our model integrates the transcriptional control of a specialized metabolic pathway with physiological reactions in different tissues. The tissue-specific differential regulation of INPS, which leads to a gradient of myo-inositol synthesis, supports root growth and sodium uptake. By inducing expression of IMT and increasing myo-inositol synthesis, metabolic end products accumulate, facilitating sodium sequestration and protecting photosynthesis.
Acclimatization↗
Induction of a ribosome-inactivating protein upon environmental stress.
Transcripts of altered abundance in RNA from unstressed and 500 mm salt-shocked Mesembryanthemum crystallinum (common ice plant) were detected by reverse-transcription differential display (RT-DD). One transcript, Rip1, was of very low abundance in unstressed plants and was strongly induced by stress. RNA blot hybridizations showed strong induction and a diurnal rhythm of transcript abundance with a maximum each day around the middle of the light phase. Rip1 encodes a reading frame of 289 amino acids (molecular mass 32,652), RIP1, with homology to single-chain ribosome inactivating proteins (rRNA N-glycosidases). The deduced amino acid sequence is 31.7% identical to pokeweed antiviral protein RIP-C (overall similarity 66.5%) with highest identity in domains of documented functional importance. RT-DD also detected mRNA for pyruvate, orthophosphate dikinase (PPDK) which has already been shown to be stress-induced in the ice plant [16]. RIP1, expressed in Escherichia coli, showed rRNA N-glycosidase activity against ice plant and rabbit reticulocyte ribosomes. The induction of Rip1 coincides with the transition period during which global changes in translation lead to adaptation of the ice plant to salt stress.
Amino Acid Sequence↗
The expression of an aquaporin promoter from Mesembryanthemum crystallinum in tobacco.
The promoter region of the MipB gene encoding an aquaporin from Mesembryanthemum crystallinum was isolated and used in a transcriptional fusion to control uidA expression in tobacco. The sequence of the promoter was determined for 2 kb upstream of the translation initiation site. Three start sites were utilized with approximately equal frequency, located 176, 170, and 161 bases, respectively, upstream of the translation initiation site. As judged by analysis of GUS expression, promoter MipB retains its specificity in transgenic tobacco. In germinating seedlings, all cells showed GUS expression of different intensities with the strongest signals in root meristems. In older seedlings, GUS staining was observed in rapidly expanding cells--root and apical meristem, and lateral root primordia. In mature plants, strong GUS activity was located to glandular trichomes, subepidermal cells of the stem and petioles, to cells surrounding vascular tissues as well as in xylem parenchyma cells. In immature floral organs, GUS expression was strong in sepals, petals, stamen, and pistil. The intensity declined as they matured. In general, this promoter was active in rapidly expanding cells and cells with high water flux capacity, especially in the xylem parenchyma.
Aquaporins↗
Increased resistance to oxidative stress in transgenic plants by targeting mannitol biosynthesis to chloroplasts.
To investigate the potential role of a polyol, mannitol, in oxidative stress protection, a bacterial mannitol-1-phosphate dehydrogenase gene was targeted to chloroplasts by the addition of an amino-terminal transit peptide. Transgenic tobacco (Nicotiana tabacum) lines accumulate mannitol at concentrations ranging from 2.5 to 7 mumol/g fresh weight. Line BS1-31 accumulated approximately 100 mM mannitol in chloroplasts and was identical to the wild type in phenotype and photosynthetic performance. The presence of mannitol in chloroplasts resulted in an increased resistance to methyl viologen (MV)-induced oxidative stress, documented by the increased retention of chlorophyll in transgenic leaf tissue following MV treatment. In the presence of MV, isolated mesophyll cells of BS1-31 exhibited higher CO2 fixation than the wild type. When the hydroxyl radical probe dimethyl sulfoxide was introduced into cells, the initial formation rate of methane sulfinic acid was significantly lower in cells containing mannitol in the chloroplast compartment than in wild-type cells, indicating an increased hydroxyl radical-scavenging capacity in BS1-31 tobacco. We suggest that the chloroplast location of mannitol can supplement endogenous radical-scavenging mechanisms and reduce oxidative damage of cells by hydroxyl radicals.
Carbon Dioxide↗
Mutations in the small subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase increase the formation of the misfire product xylulose-1,5-bisphosphate.
The small subunit (S) increases the catalytic efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) by stabilizing the active sites generated by four large subunit (L) dimers. This stabilization appears to be due to an influence of S on the reaction intermediate 2,3-enediol, which is formed after the abstraction of a proton from the substrate ribulose-1,5-bisphosphate. We tested the functional significance of residues that are conserved among most species in the carboxy-terminal part of S and analyzed their influence on the kinetic parameters of Synechococcus holoenzymes. The replacements in S (F92S, Q99G, and P108L) resulted in catalytic activities ranging from 95 to 43% of wild type. The specificity factors for the three mutant enzymes were little affected (90-96% of wild type), but Km(CO2) values increased 0.5- to 2-fold. Mutant enzymes with replacements Q99G and P108L showed increased mis-protonation, relative to carboxylation, of the 2,3-enediol intermediate, forming 2 to 3 times more xylulose-1,5-bisphosphate per ribulose-1,5-bisphosphate utilized than wild-type or F92S enzymes. The results suggest that specific alterations of the L/S interfaces and of the hydrophobic core of S are transmitted to the active site by long-range interactions. S interactions with L may restrict the flexibility of active-site residues in L.
Amino Acid Sequence↗
Abundant accumulation of the calcium-binding molecular chaperone calreticulin in specific floral tissues of Arabidopsis thaliana.
Calreticulin (CRT) is a calcium-binding protein in the endoplasmic reticulum (ER) with an established role as a molecular chaper-one. An additional function in signal transduction, specifically in calcium distribution, is suggested but not proven. We have analyzed the expression pattern of Arabidopsis thaliana CRTs for a comparison with these proposed roles. Three CRT genes were expressed, with identities of the encoded proteins ranging from 54 to 86%. Protein motifs with established functions found in CRTs of other species were conserved. CRT was found in all of the cells in low amounts, whereas three distinct floral tissues showed abundant expression: secreting nectaries, ovules early in development, and a set of subepidermal cells near the abaxial surface of the anther. Localization in the developing endosperm, which is characterized by high protein synthesis rates, can be reconciled with a specific chaperone function. Equally, nectar production and secretion, a developmental stage marked by abundant ER, may require abundant CRT to accommodate the traffic of secretory proteins through the ER. Localization of CRT in the anthers, which are degenerating at the time of maximum expression of CRT, cannot easily be reconciled with a chaperone function but may indicate a role for CRT in anther maturation or dehiscence.
Arabidopsis↗
In vitro import of pre-ferredoxin-NADP+-oxidoreductase from Cyanophora paradoxa into cyanelles and into pea chloroplasts.
Using a novel cyanelle isolation procedure we showed that pre-ferredoxin-NADP+-oxidoreductase (pre-FNR) from C. paradoxa is translocated in vitro across the peptidoglycan-containing cyanelle envelope. Efficient import was also observed in a heterologous system with pea chloroplasts as the recipient organelles. These results support the conclusion derived from comparative analysis of plastid genome organization, that all plastids originate from a common semi-autonomous endosymbiotic ancestor.
Cell Fractionation↗
Coordinate transcriptional induction of myo-inositol metabolism during environmental stress.
The pathway from glucose 6-phosphate (G 6-P) to myoinositol 1-phosphate (Ins 1-P) and myo-inositol (Ins) is essential for the synthesis of various metabolites. In the halophyte Mesembryanthemum crystallinum (common ice plant), two enzymes, myo-inositol O-methyltransferase (IMT1) and ononitol epimerase (OEP1), extend this pathway and lead to the accumulation of methylated inositols, D-ononitol and D-pinitol, which serve as osmoprotectants. This paper describes transcripts for the enzyme, Inps1, encoding myo-inositol 1-phosphate synthase (INPS1), from the ice plant. Two Inps-like sequences are present in the genome. The deduced amino acid sequences of the cloned transcript are 49.5% and 87-90%, respectively, identical to those of yeast and other higher plant sequences. Inps1 RNA amounts are upregulated at least fivefold and amounts of free Ins accumulate approximately 10-fold during salinity stress. Inps1 induction is by transcription, similar to the induction of Imt1. In contrast, Arabidopsis thaliana does not show upregulation of Inps1 or increased amounts of Ins when salt-stressed. The lack of Inps1 induction in Arabidopsis exemplifies differences in glycophytic and halophytic regulation of gene expression at the point of entry into a pathway that leads to osmoprotection. The stress-induced coordinate upregulation of this pathway and its extension by novel enzymes in the ice plant also highlights biochemical differences.
Amino Acid Sequence↗
Ribonuclease-P RNA gene of the plastid chromosome from Cyanophora paradoxa.
The gene, rnpB, encoding the RNA portion of ribonuclease-P has been found in the cyanelle DNA of Cyanophora paradoxa. A secondary structure model for the cyanelle RNA fits into that for eubacterial Rapb-RNAs.
Base Sequence↗
Characterization of IMT1, myo-inositol O-methyltransferase, from Mesembryanthemum crystallinum.
A full-length transcript, Imt1, encoding myo-inositol O-methyltransferase (EC 2.1.1.X) from the halophyte Mesembryanthemum crystallinum was expressed in Escherichia coli. The enzyme, IMT1, uses S-adenosyl-L-methionine to methylate myo-inositol to form D-ononitol. IMT1 with a monomeric mass of 41,000 was isolated by ammonium sulfate fractionation, gel filtration and ion exchange chromatography to apparent purity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified recombinant enzyme was identical to that encoded by the cDNA sequence. The apparent Km for S-adenosylmethionine was 0.18 mM with a Vmax of 1550 pkat/mg protein. The Km for myo-inositol was 1.32 mM. The reaction became substrate-inhibited by concentrations of S-adenosylmethionine greater than 0.5 mM. Inositol methyltransferase was competitively inhibited 50% with 0.01 mM S-adenosyl-homocysteine, while 1 mM homocysteine, homoserine, or adenosine did not inhibit. The enzyme exhibited a pH optimum of 7.8 and a temperature optimum of 37 degrees C. Activity of the isolated inositol methyltransferase was stable when stored at 4 degrees C.
Amino Acid Sequence↗
Light-induced expression of ipt from Agrobacterium tumefaciens results in cytokinin accumulation and osmotic stress symptoms in transgenic tobacco.
Cytokinins are plant growth regulators that induce shoot formation, inhibit senescence and root growth. Experiments with hydroponically grown tobacco plants, however, indicated that exogenously applied cytokinin led to the accumulation of proline and osmotin. These responses were also associated with environmental stress reactions, such as salt stress, in many plant species. To test whether increased endogenous cytokinin accumulation led to NaCl stress symptoms, the gene ipt from Agrobacterium tumefaciens, encoding isopentenyl transferase, was transformed into Nicotiana tabacum cv. SR-1 under the control of the light-inducible rbcS-3A promoter from pea. In high light (300 mumol PPFD m-2 s-1), ipt mRNA was detected and zeatin/zeatin glucoside levels were 10-fold higher than in control plants or when transformants were grown in low light (30 mumol PPFD m-2 s-1). High light treatment was accompanied by increased levels of proline and osmotin when compared to low light grown transformed and untransformed control plants. Elevated in planta cytokinin levels induced responses also stimulated by salt stress, suggesting either common or overlapping signaling pathways are initiated independently by cytokinin and NaCl, setting in motion gene expression normally elicited by developmental processes such as flowering or environmental stress.
Adenine↗
Age-dependent induction of pyruvate, orthophosphate dikinase in Mesembryanthemum crystallinum L.
A full-length transcript for pyruvate, orthophosphate dikinase (PPDK; EC 2.7.9.1), has been characterized from Mesembryanthemum crystallinum. Under salt stress or with increasing age, this plant shows a transition from C3 to Crassulacean acid metabolism (CAM). The PPDK plays a central role in gluconeogenesis during the light phase of CAM. The transcript is 3165 bases in length with a single open reading frame of 2739 nucleotides specifying a protein of molecular mass 103098, including a transit peptide of mass 7902 for chloroplast import. The protein shares 44-77% sequence identity with PPDK from C4-plants and microorganisms. Known functional and regulatory amino acids are conserved. Southern-type hybridizations indicated one copy or very few closely related copies of the gene per haploid genome. We investigated the induction of PPDK at the mRNA and protein levels, using the well characterized induction of a CAM-form of phosphoenol pyruvate carboxylase (PEPCase) as internal standard. During wilting of excised leaves PEPCase mRNA amounts increased strongly within 8 h. Under these conditions amounts of PPDK mRNA remained constant. Re-hydrating leaves from previously stressed plants led to a decrease in PEPCase and PPDK mRNA amounts. During salt stress, no correlation between PEPCase and PPDK was observed. Analysis of plants of different ages indicated that, even in well-watered plants, PPDK-specific protein and mRNA increased when the plants reached a certain age. In old plants, salt stress failed to further increase PPDK mRNA or protein levels.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
A family of transcripts encoding water channel proteins: tissue-specific expression in the common ice plant.
Seawater-strength salt stress of the ice plant (Mesembryanthemum crystallinum) initially results in wilting, but full turgor is restored within approximately 2 days. We are interested in a mechanistic explanation for this behavior and, as a requisite for in-depth biochemical studies, have begun to analyze gene expression changes in roots coincident with the onset of stress. cDNAs that suggested changes in mRNA amount under stress were found; their deduced amino acid sequences share homologies with proteins of the Mip (major intrinsic protein) gene family and potentially encode aquaporins. One transcript, MipB, was found only in root RNA, whereas two other transcripts, MipA and MipC, were detected in roots and leaves. Transcript levels of MipB were of low abundance. All transcripts declined initially during salt stress but later recovered to at least prestress level. The most drastic decline was in MipA and MipC transcripts. MipA mRNA distribution in roots detected by in situ hybridization indicated that the transcript was present in all cells in the root tip. In the expansion zone of the root where vascular bundles differentiate, MipA transcript amounts were most abundant in the endodermis. In older roots, which had undergone secondary growth, MipA was highly expressed in cell layers surrounding individual xylem strands. MipA was also localized in leaf vascular tissue and, in lower amounts, in mesophyll cells. Transcripts for MipB seemed to be present exclusively in the tip of the root, in a zone before and possibly coincident with the development of a vascular system. MipA- and MipB-encoded proteins expressed in Xenopus oocytes led to increased water permeability. mRNA fluctuations of the most highly expressed MipA and MipC coincided with turgor changes in leaves under stress. As the leaves regained turgor, transcript levels of these water channel proteins increased.
Amino Acid Sequence↗
Environmental stress-mediated differential 3' end formation of chloroplast RNA-binding protein transcripts.
We report the characterization of transcripts from the halophyte, Mesembryanthemum crystallinum, encoding a protein with high homology to chloroplast RNA-binding proteins (cRBP). In this plant chloroplast-related functions are largely protected against salt stress. cRBP transcripts are derived from a single gene, Mc32crbp, although three size classes of polyadenylated mRNAs are detected. Transcription rate and steady state amounts of mRNA are developmentally regulated and light controlled with strong transcriptional activity as functional chloroplasts are established, and with lower maintenance activity thereafter. Upon salt stress, the rate of transcription decreases, although transcript levels increase. Accompanying stress, a change in the distribution of transcript size classes is observed as the longest transcript with an untranslated 3' end of 381 nucleotides increases relative to transcripts with shorter 3' ends. The long transcript is characterized by the presence of five sequence elements in the 3'-untranslated region that are present in cRBP mRNAs from a variety of plants, although not all elements are found in each mRNA. The results may indicate a mechanism by which mRNA levels of constitutively light-regulated genes may be modulated without enhanced transcription in response to environmental cues.
Adaptation, Physiological↗
Structure and function of the cyanelle genome.
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Biological Evolution↗