Flooding stress: acclimations and genetic diversity.

Flooding is an environmental stress for many natural and man-made ecosystems worldwide. Genetic diversity in the plant response to flooding includes alterations in architecture, metabolism, and elongation growth associated with a low O(2) escape strategy and an antithetical quiescence scheme that allows endurance of prolonged submergence. Flooding is frequently accompanied with a reduction of cellular O(2) content that is particularly severe when photosynthesis is limited or absent. This necessitates the production of ATP and regeneration of NAD(+) through anaerobic respiration. The examination of gene regulation and function in model systems provides insight into low-O(2)-sensing mechanisms and metabolic adjustments associated with controlled use of carbohydrate and ATP. At the developmental level, plants can escape the low-O(2) stress caused by flooding through multifaceted alterations in cellular and organ structure that promote access to and diffusion of O(2). These processes are driven by phytohormones, including ethylene, gibberellin, and abscisic acid. This exploration of natural variation in strategies that improve O(2) and carbohydrate status during flooding provides valuable resources for the improvement of crop endurance of an environmental adversity that is enhanced by global warming.

Natural variation of submergence tolerance among Arabidopsis thaliana accessions.

• The exploitation of natural variation in Arabidopsis thaliana (Arabidopsis) provides a huge potential for the identification of the molecular mechanisms underlying this variation as a result of the availability of a vast array of genetic and genomic resources for this species. Eighty-six Arabidopsis accessions were screened for natural variation in flooding tolerance. This forms the first step towards the identification and characterization of the role of candidate genes contributing to flooding tolerance. • Arabidopsis accessions at the 10-leaf stage were subjected to complete submergence in the dark. Survival curves were plotted to estimate median lethal times as a measure of tolerance. Flooding-associated survival parameters, such as root and shoot oxygen content, initial carbohydrate content and petiole elongation under water, were also measured. • There was a significant variation in submergence tolerance among Arabidopsis accessions. However, the order of tolerance did not correlate with root and shoot oxygen content or initial amounts of shoot starch and total soluble sugars. A negative correlation was observed between submergence tolerance and underwater petiole elongation. • Arabidopsis accessions show considerable variation in the ability to tolerate complete submergence, making it a good species in which to identify and characterize genes and to study mechanisms that contribute to survival under water.

RNase Activities Are Reduced Concomitantly with Conservation of Total Cellular RNA and Ribosomes in O2-Deprived Seedling Roots of Maize.

The effect of O2 deprivation on the activities of RNases and levels of total cellular RNA and ribosomes in seedling roots of maize (Zea mays L.) was investigated. Sodium dodecyl sulfate-polyacrylamide gels containing RNA were used to distinguish RNase isoenzymes by apparent molecular mass. Since O2 deprivation causes a decrease in cytosolic pH from approximately pH 7.4 to 6.4 and an elevation in cytosolic Ca2+, RNase levels were examined in the physiological range of cytosolic pH and in the presence of Ca2+, Mg2+, Zn2+, ethylenediaminetetracetate, or ethyleneglycol-bis([beta]-aminoethyl ether)-N,N[prime]-tetraacetic acid. The activity of a number of RNases present in aerobic roots was reduced in response to O2 deprivation. Several RNases with a pH optimum of 6.4 were rapidly down-regulated by O2 deprivation. Spectrophotometric assay of extracts revealed that RNase activity was higher at pH 6.4 than at 7.2, and ethylenediaminetetracetate-insensitive RNase activity decreased in response to O2 deprivation. The decrease in RNase activity was correlated with no loss of total cellular RNA or ribosomes, despite a 4-fold decrease in run-on transcription of rRNA in isolated nuclei. Regulation of RNase activity may facilitate the conservation of nontranslating ribosomes and poorly translated mRNAs during O2 deprivation.

Ozone-Induced Alterations in the Accumulation of Newly Synthesized Proteins in Leaves of Maize.

We examined the response of leaves of 3-week-old maize (Zea mays L.) to short-term (5 h) fumigation with O3-enriched air (0, 0.12, 0.24, or 0.36 [mu]L/L). Older leaves and leaf tissue developed more severe visible damage at higher external O3 concentrations. To investigate the immediate effect of O3 exposure on the accumulation of newly synthesized leaf proteins, leaves were labeled with [35S]methionine after 2 h and fumigated for an additional 3 h. O3-induced alterations of leaf proteins were observed in a concentration-dependent manner. There was a significant decrease in [35S]methionine incorporation into protein at the highest O3 concentration. Developmental differences in accumulation of de novo-synthesized leaf proteins were observed when the leaf tip, middle, and basal sections were labeled under 0 [mu]L/L O3, and additional changes were apparent upon exposure to increasing O3 concentrations. Changes in leaf protein synthesis were observed in the absence of visible leaf injury. Subcellular fractionation revealed O3-induced alterations in soluble and membrane-associated proteins. A number of thylakoid membrane-associated proteins showed specific increases in response to O3 fumigation. In contrast, the synthesis of a 32-kD polypeptide associated with thylakoid membranes was reduced in response to O3 fumigation in parallel with reduced incorporation of [35S]methionine into protein. Immunoprecipitation identified this polypeptide as the D1 protein of photosystem II. A reduction in the accumulation of newly synthesized D1 could have consequences for the efficiency of photosynthesis and other cellular processes.

Microgenomics: genome-scale, cell-specific monitoring of multiple gene regulation tiers.

The expression of nuclear protein-coding genes is controlled by dynamic mechanisms ranging from DNA methylation, chromatin modification, and gene transcription to mRNA maturation, turnover, and translation and the posttranslational control of protein function. A genome-scale assessment of the spatiotemporal regulation of gene expression is essential for a comprehensive understanding of gene regulatory networks. However, there are major obstacles to the precise evaluation of gene regulation in multicellular plant organs; these include the monitoring of regulatory processes at levels other than steady-state transcript abundance, resolution of gene regulation in individual cells or cell types, and effective assessment of transient gene activity manifested during development or in response to external cues. This review surveys the advantages and applications of microgenomics technologies that enable panoramic quantitation of cell-type-specific expression in plants, focusing on the importance of querying gene activity at multiple steps in the continuum, from histone modification to selective translation.

Flooding tolerance: O2 sensing and survival strategies.

The investigation of flooding survival strategies in model, crop and wild plant species has yielded insights into molecular, physiological and developmental mechanisms of soil flooding (waterlogging) and submergence survival. The antithetical flooding escape and quiescence strategies of deepwater and submergence tolerant rice (Oryza sativa), respectively, are regulated by members of a clade of ethylene responsive factor transcriptional activators. This knowledge paved the way for the discovery that these proteins are targets of a highly conserved O2-sensing protein turnover mechanism in Arabidopsis thaliana. Further examples of genes that regulate transcription, root and shoot metabolism or development during floods have emerged. With the rapid advancement of genomic technologies, the mining of natural genetic variation in flooding tolerant wild species may ultimately benefit crop production.

Getting the message across: cytoplasmic ribonucleoprotein complexes.

mRNA-ribonucleoprotein (mRNP) complexes mediate post-transcriptional control mechanisms in the cell nucleus and cytoplasm. Transcriptional control is paramount to gene expression but is followed by regulated nuclear pre-mRNA maturation and quality control processes that culminate in the export of a functional transcript to the cytoplasm. Once in the cytosol, mRNPs determine the activity of individual mRNAs through regulation of localization, translation, sequestration and turnover. Here, we review how quantitative assessment of mRNAs in distinct cytoplasmic mRNPs, such as polyribosomes (polysomes), has provided new perspectives on post-transcriptional regulation from the global to gene-specific level. In addition, we explore recent genetic and biochemical studies of cytoplasmic mRNPs that have begun to expose RNA-binding proteins in an integrated network that fine-tunes gene expression.

Selective translation of cytoplasmic mRNAs in plants.

Translation of mRNA is emerging as an important mode of gene regulation in plants. It is frequently controlled at initiation and appears to be regulated by competition for limiting translational components, different requirements for specific factors and cis-acting mRNA elements. Recent studies indicate that interactions between the 5' and 3' ends of the message enhance translation, perhaps by facilitating recruitment of initiation factors or enhancing ribosome recycling. Normal development and environmental stimuli modulate the phosphorylation of components of the mRNA 5'-cap-binding complex, ribosomes and mRNA-binding proteins. These modifications might be responsible for changes in the hierarchy of mRNAs that are in competition for translation.

Synonymous codon usage in Zea mays L. nuclear genes is varied by levels of C and G-ending codons.

A multivariate statistical method called correspondence analysis was used to examine the codon usage of one-hundred-and-one nuclear genes of maize (Zea mays L.). Forty percent of the variation in codon usage was due to bias toward G or C-ending versus A or U-ending codons. Differences in levels of G-ending codons showed the weakest correlation with the major codon usage bias. The bias toward C or U versus A or G in the silent third nucleotide position of synonymous codons accounted for approximately 10% of the variation in codon usage. The G+C content of the silent third nucleotide position of coding regions was not strongly correlated with G+C content of introns. Codon usage was strongly biased toward codons ending in G or C for a number of highly expressed genes including most light-regulated chloroplast proteins, ABA-induced proteins, histones, and anthocyanin biosynthetic enzymes. Codon usage of genes encoding storage proteins and regulatory proteins, such as transposases, kinases, phosphatases and transcription factors, was more random than that of genes encoding cytosolic enzymes with similar bias toward G or C-ending codons. Codon usage in maize may reflect both regional bias on nucleotide composition and selection on the silent third nucleotide position.

Purification and characterization of cytosolic 6-phosphogluconate dehydrogenase isozymes from maize.

Cytosolic isozymes of 6-phosphogluconate dehydrogenase were purified from roots of maize (Zea mays L.). The final preparation contained two 55-kD proteins. Affinity-purified dehydrogenases from a maize line that is null for both cytosolic 6-phosphogluconate dehydrogenase isozymes (Pgd1-null, Pgd2-null) lacked the 55-kD proteins. The substrate kinetics of the purified enzyme were determined.

Both 5' and 3' sequences of maize adh1 mRNA are required for enhanced translation under low-oxygen conditions.

Alcohol dehydrogenase-1 (ADH1) synthesis in O2-deprived roots of maize (Zea mays L.) results from induced transcription and selective translation of ADH1 mRNA. The effect of ADH1 mRNA sequences on message stability and translation was studied in protoplasts of the maize cell line P3377.5' capped and 3' polyadenylated mRNA constructs containing the firefly gene (luc) for luciferase (LUC) or the Escherichia coli gene (uidA) for beta-glucuronidase (GUS) coding region were synthesized in vitro and electroporated into protoplasts that were cultured at 40 or 5% O2. A LUC mRNA with a 17-nucleotide polylinker 5' untranslated region (UTR) was expressed 10-fold higher under aerobic conditions than under hypoxic conditions. Expression of five chimeric ADH1-GUS mRNAs was measured relative to this LUC mRNA. An mRNA containing the 5'-UTR and the first 18 codons of adh1 in a translational fusion with the GUS coding region and followed by the 3'-UTR of adh1 was expressed 57-fold higher at 5% O2. Progressive deletion of adh1 5'-UTR and coding sequences reduced expression of the GUS-mRNA at 5% O2, but had little impact on expression of 40% O2. Enhancement of expression in hypoxic protoplasts conferred by the adh1 5'-UTR and the first 26 codons decreased more than 3-fold when the adh1 3'-UTR was removed. In addition, the adh1 3'-UTR slightly inhibited expression in aerobic protoplasts. The physical half-lives of the GUS and LUC mRNAs were similar under both anaerobic and hypoxic conditions, indicating that expression levels were largely independent of mRNA stability. Thus, both adh1 5' and 3' mRNA sequences are required for enhanced translation in protoplasts under O2 deprivation.

Hypoxic stress-induced changes in ribosomes of maize seedling roots.

The hypoxic stress response of Zea mays L. seedling roots involves regulation of gene expression at transcriptional and posttranscriptional levels. We investigated the effect of hypoxia on the translational machinery of seedling roots. The levels of monoribosomes and ribosomal subunits increased dramatically within 1 hour of stress. Prolonged hypoxia resulted in continued accumulation of nontranslating ribosomes, as well as increased levels of small polyribosomes. The return of seedlings to normal aerobic conditions resulted in recovery of normal polyribosome levels. Comparison of ribosomal proteins from control and hypoxic roots revealed differences in quantity and electrophoretic mobility. In vivo labeling of roots with [(35)S]methionine revealed variations in newly synthesized ribosomal proteins. In vivo labeling of roots with [(32)P]orthophosphate revealed a major reduction in the phosphorylation of a 31 kilodalton ribosomal protein in hypoxic stressed roots. In vitro phosphorylation of ribosomal proteins by endogenous kinases was used to probe for differences in ribosome structure and composition. The patterns of in vitro kinased phosphoproteins of ribosomes from control and hypoxic roots were not identical. Variation in phosphoproteins of polyribosomes from control and hypoxic roots, as well as among polyribosomes from hypoxic roots were observed. These results indicate that modification of the translational machinery occurs in response to hypoxic stress.

Regulated heterogeneity in 12-kDa P-protein phosphorylation and composition of ribosomes in maize (Zea mays L.).

Maize (Zea mays L.) possesses four distinct approximately 12-kDa P-proteins (P1, P2a, P2b, P3) that form the tip of a lateral stalk on the 60 S ribosomal subunit. RNA blot analyses suggested that the expression of these proteins was developmentally regulated. Western blot analysis of ribosomal proteins isolated from various organs, kernel tissues during seed development, and root tips deprived of oxygen (anoxia) revealed significant heterogeneity in the levels of these proteins. P1 and P3 were detected in ribosomes of all samples at similar levels relative to ribosomal protein S6, whereas P2a and P2b levels showed considerable developmental regulation. Both forms of P2 were present in ribosomes of some organs, whereas only one form was detected in other organs. Considerable tissue-specific variation was observed in levels of monomeric and multimeric forms of P2a. P2b was not detected in root tips, accumulated late in seed embryo and endosperm development, and was detected in soluble ribosomes but not in membrane-associated ribosomes that copurified with zein protein bodies of the kernel endosperm. The phosphorylation of the 12-kDa P-proteins was also developmentally and environmentally regulated. The potential role of P2 heterogeneity in P-protein composition in the regulation of translation is discussed.

Expression and distribution of cytosolic 6-phosphogluconate dehydrogenase isozymes in maize.

Maize (Zea mays L.) cytosolic 6-phosphogluconate dehydrogenase isozymes (EC 1.1.1.44; 6-PGD) are encoded by unlinked loci Pgd1 and Pgd2. Two families from a Robertson's Mutator line were isolated which have no detectable expression of Pgd2. These Pgd2-null mutants and a Pgd1-null line were used to generate plants homozygous for null alleles at both cytosolic 6-PGD loci. The specific activity of 6-PGD in the double-null mutant was between 20 and 30% of wild-type levels in root extracts. The double-null mutant was reproductively viable in a moderate environment, suggesting that wild-type levels of cytosolic 6-PGD activity are not essential for growth. Isozyme dimer ratios in roots, leaves, and scutellum were binomial and reflected the wild-type gene copy number. 6-PGD isozymes showed tissue- and cell type-specific expression.

Evolutionary analyses of the 12-kDa acidic ribosomal P-proteins reveal a distinct protein of higher plant ribosomes.

The P-protein complex of eukaryotic ribosomes forms a lateral stalk structure in the active site of the large ribosomal subunit and is thought to assist in the elongation phase of translation by stimulating GTPase activity of elongation factor-2 and removal of deacylated tRNA. The complex in animals, fungi, and protozoans is composed of the acidic phosphoproteins P0 (35 kDa), P1 (11-12 kDa), and P2 (11-12 kDa). Previously we demonstrated by protein purification and microsequencing that ribosomes of maize (Zea mays L.) contain P0, one type of P1, two types of P2, and a distinct P1/P2 type protein designated P3. Here we implemented distance matrices, maximum parsimony, and neighbor-joining analyses to assess the evolutionary relationships between the 12 kDa P-proteins of maize and representative eukaryotic species. The analyses identify P3, found to date only in mono- and dicotyledonous plants, as an evolutionarily distinct P-protein. Plants possess three distinct groups of 12 kDa P-proteins (P1, P2, and P3), whereas animals, fungi, and protozoans possess only two distinct groups (P1 and P2). These findings demonstrate that the P-protein complex has evolved into a highly divergent complex with respect to protein composition despite its critical position within the active site of the ribosome.

Oxygen deprivation stimulates Ca2+-mediated phosphorylation of mRNA cap-binding protein eIF4E in maize roots.

Flooding of maize seedlings causes O2 deprivation that leads to a global reduction in protein synthesis and selective translation of cytoplasmic mRNAs. Since selective translation in animal cells can involve the cap-binding protein eIF4E, we characterized the distinct mRNA cap-binding proteins eIF4E and eIFiso4E of maize. These proteins have 45% deduced amino acid sequence identity and are highly conserved at residues of eIF4E that function in intermolecular interactions in animals. Maize eIF4E is a phosphoprotein. O2 deprivation resulted in a decrease in the isoelectric point of eIF4E, consistent with additional phosphorylation. Modification of eIF4E was mimicked by treatment with caffeine under aerobic conditions and blocked by treatment with ruthenium red under O2 deprivation, implicating Ca2+ as a second messenger in eIF4E modification. In contrast, no isoelectric variants of eIFiso4E were detected. The possible role of cytosolic Ca2+ and pH in regulation of mRNA cap-binding protein activity under O2 deprivation is discussed.

Molecular characterization of the mitochondrial citrate synthase gene of an acidless pummelo (Citrus maxima).

Pummelo (Citrus maxima [Burm.] Merrill) cDNAs encoding mitochondrial citrate synthase (mCS) were cloned by reverse transcription of juice-sac poly(A)+ mRNA, followed by Taq Polymerase-mediated amplification. The nucleotide sequence of the citrus gene (cit1) is 77% conserved relative to plant mRNAs for mCS. The encoded polypeptide includes a mitochondrial targeting signal at its amino terminus; all 20 putative active-site residues of the citrus enzyme are conserved. Southern hybridization showed that citrus cit1 is a single-copy gene. A polymorphism associated with cit1 did not cosegregate with fruit acidity indicating that acitric, the gene causing the acidless phenotype of pummelo 2240, is not an allele of cit1 locus. Quantitative detection of cit1 mRNA showed that transcript levels are not developmentally regulated in juice sacs; no differences were observed between high- and low-acid genotypes.

Size distributions of circular molecules in plant mitochondrial DNAs.

Some physicochemical properties of the mitochondrial DNAs (mtDNA) from plants of flax, broad bean and mung bean, and from tissue culture cells of jimson weed, soybean, petunia and tobacco were determined. Circular molecules were observed in electron microscope preparations of each mtDNA. In soybean, petunia, broad bean and mung bean mtDNAs, the circular molecules had a continuous distribution of lengths (ranges between 1 to 36 kb, and 1 to 126 kb), heavily skewed toward smaller molecules. Eighty-six percent of the flax circular molecules were from 27 to 54 kb in size, and 78% of the jimson weed circular molecules were from 4 to 15 kb. Replicative forms of 1.2-1.6 kb circular molecules were observed in electron microscope preparations of broad bean mtDNA.

Nuclear-mitochondrial interactions in cytoplasmic male-sterile sorghum.

Variation in mitochondrial genome organization and expression between male fertile and sterile nuclear-cytoplasmic combinations of sorghum has been examined. Cytoplasmic genotypes were classified into eleven groups on the basis of restriction endonuclease digestion of mitochondrial DNA (mtDNA) and five groups on the basis of mitochondrial translation products. These cytoplasms were further characterized by hybridization of specific gene probes to Southern blots of EcoRI digested mtDNA, and identification of the fragment location of four mitochondrial genes. Variation was observed in the genomic location and copy number of the F1 ATPase α-subunit gene, as well as the genomic location and gene product of the cytochrome c oxidase subunit I gene. The effect of nuclear genotype on mitochondrial genome organization, expression and the presence of two linear plasmid-like mtDNA molecules was examined. Our results indicate that nuclear-mitochondrial interactions are required for regulation of mitochondrial gene expression. When a cytoplasm is transferred from its natural to a foreign nuclear background some changes in the products of in organello mitochondrial protein synthesis occur. In a number of cytoplasmic genotypes these changes correlate with the expression of cytoplasmic male sterile phenotype, suggesting a possible molecular basis for this mutation.

Acidic phosphoprotein complex of the 60S ribosomal subunit of maize seedling roots. Components and changes in response to flooding.

We determined that ribosomes of seedling roots of maize (Zea mays L.) contain the acidic phosphoproteins (P-proteins) known to form a flexible lateral stalk structure of the 60S subunit of eukaryotic ribosomes. The P-protein stalk, composed of P0, P1, and P2, interacts with elongation factors, mRNA, and tRNA during translation. Acidic proteins of 13 to 15.5 kD were released as a complex from ribosomes with 0.4 M NH4Cl/50% ethanol. Protein and cDNA sequence analysis confirmed that maize ribosomes contain one type of P1, two types of P2, and a fourth and novel P1/P2-type protein. This novel P-protein, designated P3, has the conserved C terminus of P1 and P2. P1, P2, and P3 are similar in deduced mass (11.4-12.2 kD) and isoelectric point (4.1-4.3). A 35.5- to 36-kD acidic protein was released at low levels from ribosomes with 1.0 M NH4Cl/50% ethanol and identified as P0. Labeling of roots with [32P]inorganic phosphate confirmed the in vivo phosphorylation of the P-proteins. Flooding caused dynamic changes in the P-protein complex, which affected the potential of ribosome-associated kinases and casein kinase II to phosphorylate the P-proteins. We discuss possible alterations of the ribosomal P-protein complex and consider that these changes may be involved in the selective translation of mRNA in flooded roots.