Hyperosmotic stress induces the rapid phosphorylation of a soybean phosphatidylinositol transfer protein homolog through activation of the protein kinases SPK1 and SPK2.

Although phosphatidylinositol transfer proteins (PITPs) are known to serve critical functions in regulating a varied array of signal transduction processes in animals and yeast, the discovery of a similar class of proteins in plants occurred only recently. Here, we report the participation of Ssh1p, a soybean PITP-like protein, in the early events of osmosensory signal transduction in plants, a function not attributed previously to animal or yeast PITPs. Exposure of plant tissues to hyperosmotic stress led to the rapid phosphorylation of Ssh1p, a modification that decreased its ability to associate with membranes. An osmotic stress-activated Ssh1p kinase activity was detected in several plant species by presenting recombinant Ssh1p as a substrate in in-gel kinase assays. Elements of a similar osmosensory signaling pathway also were conserved in yeast, an observation that facilitated the identification of soybean protein kinases SPK1 and SPK2 as stress-activated Ssh1p kinases. This study reveals the activation of SPK1 and/or SPK2 and the subsequent phosphorylation of Ssh1p as two early successive events in a hyperosmotic stress-induced signaling cascade in plants. Furthermore, Ssh1p is shown to enhance the activities of a plant phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase, an observation that suggests that the ultimate function of Ssh1p in cellular signaling is to alter the plant's capacity to synthesize phosphoinositides during periods of hyperosmotic stress.

Induction of lipid metabolic enzymes during the endoplasmic reticulum stress response in plants.

The endoplasmic reticulum (ER) stress response is a signal transduction pathway activated by the perturbation of normal ER metabolism. We used the maize (Zea mays) floury-2 (fl2) mutant and soybean (Glycine max) suspension cultures treated with tunicamycin (Tm) to investigate the ER stress response as it relates to phospholipid metabolism in plants. Four key phospholipid biosynthetic enzymes, including DG kinase and phosphatidylinositol (PI) 4-phosphate 5-kinase were up-regulated in the fl2 mutant, specifically in protein body fractions where the mutation has its greatest effect. The third up-regulated enzyme, choline-phosphate cytidylyltransferase, was regulated by fl2 gene dosage and developmental signals. Elevated accumulation of the fourth enzyme, PI 4-kinase, was observed in the fl2 endosperm and soybean cells treated with Tm. The activation of these phospholipid biosynthetic enzymes was accompanied by alterations in membrane lipid synthesis and accumulation. The fl2 mutant exhibited increased PI content in protein body membranes at 18 d after pollination and more than 3-fold higher triacylglycerol accumulation in the endosperm by 36 d after pollination. Incorporation of radiolabeled acetate into phospholipids in soybean culture cells increased by about 30% with Tm treatment. The coordinated regulation of ER stress related proteins and multiple components of phospholipid biosynthesis is consistent with signaling through a common pathway. We postulate that the plant ER stress response has an important role in general plant metabolism, and more specifically in integrating the synthesis of protein and lipid reserves to allow proper seed formation.

Phosphoinositide kinases and the synthesis of polyphosphoinositides in higher plant cells.

Phosphoinositides are a family of inositol-containing phospholipids which are present in all eukaryotic cells. Although in most cells these lipids, with the exception of phosphatidylinositol, constitute only a very minor proportion of total cellular lipids, they have received immense attention by researchers in the past 15-20 years. This is due to the discovery that these lipids, rather than just having structural functions, play key roles in a wide range of important cellular processes. Much less is known about the plant phosphoinositides than about their mammalian counterparts. However, it has been established that a functional phosphoinositide system exists in plant cells and it is becoming increasingly clear that inositol-containing lipids are likely to play many important roles throughout the life of a plant. It is not our intention to give an exhaustive overview of all aspects of the field, but rather we focus on the phosphoinositide kinases responsible for the synthesis of all phosphorylated forms of phosphatidylinositol. Also, we mention some of the aspects of current phosphoinositide research which, in our opinion, are most likely to provide a suitable starting point for further research into the role of phosphoinositides in plants.

Expression of a soybean cytochrome P450 monooxygenase cDNA in yeast and tobacco enhances the metabolism of phenylurea herbicides.

A strategy based on the random isolation and screening of soybean cDNAs encoding cytochrome P450 monooxygenases (P450s) was used in an attempt to identify P450 isozymes involved in herbicide metabolism. Nine full-length (or near-full-length) P450 cDNAs representing eight distinct P450 families were isolated by using PCR-based technologies. Five of the soybean P450 cDNAs were expressed successfully in yeast, and microsomal fractions generated from these strains were tested for their potential to catalyze the metabolism of 10 herbicides and 1 insecticide. In vitro enzyme assays showed that the gene product of one heterologously expressed P450 cDNA (CYP71A10) specifically catalyzed the metabolism of phenylurea herbicides, converting four herbicides of this class (fluometuron, linuron, chlortoluron, and diuron) into more polar compounds. Analyses of the metabolites suggest that the CYP71A10 encoded enzyme functions primarily as an N-demethylase with regard to fluometuron, linuron, and diuron, and as a ring-methyl hydroxylase when chlortoluron is the substrate. In vivo assays using excised leaves demonstrated that all four herbicides were more readily metabolized in CYP71A10-transformed tobacco compared with control plants. For linuron and chlortoluron, CYP71A10-mediated herbicide metabolism resulted in significantly enhanced tolerance to these compounds in the transgenic plants.

Novel developmentally regulated phosphoinositide binding proteins from soybean whose expression bypasses the requirement for an essential phosphatidylinositol transfer protein in yeast.

Phosphatidylinositol transfer proteins (PITPs) have been shown to play important roles in regulating a number of signal transduction pathways that couple to vesicle trafficking reactions, phosphoinositide-driven receptor-mediated signaling cascades, and development. While yeast and metazoan PITPs have been analyzed in some detail, plant PITPs remain entirely uncharacterized. We report the identification and characterization of two soybean proteins, Ssh1p and Ssh2p, whose structural genes were recovered on the basis of their abilities to rescue the viability of PITP-deficient Saccharomyces cerevisiae strains. We demonstrate that, while both Ssh1p and Ssh2p share approximately 25% primary sequence identity with yeast PITP, these proteins exhibit biochemical properties that diverge from those of the known PITPs. Ssh1p and Ssh2p represent high-affinity phosphoinositide binding proteins that are distinguished from each other both on the basis of their phospholipid binding specificities and by their substantially non-overlapping patterns of expression in the soybean plant. Finally, we show that Ssh1p is phosphorylated in response to various environmental stress conditions, including hyperosmotic stress. We suggest that Ssh1p may function as one component of a stress response pathway that serves to protect the adult plant from osmotic insult.

Characterization of soybean choline kinase cDNAs and their expression in yeast and Escherichia coli.

An expressed sequence tag from Arabidopsis that displayed sequence homology to mammalian and yeast choline kinases was used to isolate choline kinase-like cDNAs from soybean (Glycine max L.). Two distinct cDNAs, designated GmCK1 and GmCK2, were recovered that possessed full-length reading frames, each sharing approximately 32% identity at the predicted amino acid level with the rat choline kinase. A third unique choline kinase-like cDNA, GmCK3, was also identified but was not full length. Heterologous expression of GmCK1 in yeast (Saccharomyces cerevisiae) and GmCK2 in both yeast and Escherichia coli demonstrated that each encodes choline kinase activity. In addition to choline, other potential substrates for the choline kinase enzyme include ethanolamine, monomethylethanolamine (MME), and dimethylethanolamine (DME). Both soybean choline kinase isoforms demonstrated negligible ethanolamine kinase activity. Competitive inhibition assays, however, revealed very distinct differences in their responses to DME and MME. DME effectively inhibited only the GmCK2-encoded choline kinase activity. Although MME failed to effectively inhibit either reaction, an unexpected enhancement of choline kinase activity was observed specifically with the GmCK1-encoded enzyme. These results show that choline kinase is encoded by a small, multigene family in soybean comprising two or more distinct isoforms that exhibit both similarities and differences with regard to substrate specificity.

The AAPT1 gene of soybean complements a cholinephosphotransferase-deficient mutant of yeast.

Aminoalcoholphosphotransferases (AAPTases) utilize diacylglycerols and cytidine diphosphate (CDP)-aminoalcohols as substrates in the synthesis of the abundant membrane lipids phosphatidylcholine and phosphatidylethanolamine. A soybean cDNA encoding an AAPTase that demonstrates high levels of CDP-choline:sn-1,2-diacylglycerol cholinephosphotransferase activity was isolated by complementation of a yeast strain deficient in this function and was designated AAPT1. The deduced amino acid sequence of the soybean cDNA showed nearly equal similarity to each of the two characterized AAPTase sequences from yeast, cholinephosphotransferase and ethanolaminephosphotransferase (CDP-ethanolamine:sn-1,2-diacylglycerol ethanolaminephosphotransferase). Moreover, assays of soybean AAPT1-encoded enzyme activity in yeast microsomal membranes revealed that the addition of CDP-ethanolamine to the reaction inhibited incorporation of 14C-CDP-choline into phosphatidylcholine in a manner very similar to that observed using unlabeled CDP-choline. Although DNA gel blot analysis suggested that AAPT1-like sequences are represented in soybean as a small multigene family, the same AAPT1 isoform isolated from a young leaf cDNA library was also recovered from a developing seed cDNA library. Expression assays in yeast using soybean AAPT1 cDNAs that differed only in length suggested that sequences in the 5'leader of the transcript were responsible for the negative regulation of gene activity in this heterologous system. The inhibition of translation mediated by a short open reading frame located 124 bp upstream of the AAPT1 reading frame is one model proposed for the observed down-regulation of gene activity.

Pathogen, salicylic acid and developmental dependent expression of a beta-1,3-glucanase/GUS gene fusion in transgenic tobacco plants.

The 5' flanking region of a gene encoding an acidic beta-1,3-glucanase from Nicotiana tabacum was isolated and characterized. A chimeric gene composed of 1759 bp of the promoter sequence from the PR-2 gene was fused to the beta-glucuronidase (GUS) coding region and used to transform tobacco. Transcriptional activation of the PR-2 promoter was investigated in response to inoculation with tobacco mosaic virus (TMV), after treatment of leaves with salicylic acid (SA), and in specific tissues during the normal development of healthy plants. In TMV-inoculated transgenic plants, GUS activity was induced locally around necrotic viral lesions and systemically in uninoculated leaves. GUS activity was also induced by treatment of leaves with SA. The chimeric gene was expressed in floral organs of healthy plants and in newly germinated seedlings. Analyses of a series of 5' deletions of the glucanase promoter indicated that the cis-acting elements necessary for induction by all these signals are localized in the region between -321 bp and -607 bp upstream of the transcription start site.

Chimeric mitochondrial genes expressed in the C male-sterile cytoplasm of maize.

Aberrant recombinations involving the mitochondrial atp9, atp6 and coxII genes have created unique chimeric sequences in the C male-sterile cytoplasm (cms-C) of maize. An apparent consequence of the rearrangements is the interchanging of transcriptional and/or translational regulatory signals for these genes, and alterations in the reading frames encoding the atp6 and coxII genes in the C cytoplasm. Particularly unusual is the organization of the atp6 gene in cms-C mitochondria, designated atp6-C. The atp6-C sequence is a triple gene fusion product comprised of DNAs derived from atp9, atp6 and an open reading frame of unknown origin. Although there is no direct evidence indicating that these chimeric genes are responsible for the cytoplasmic male sterility (cms) trait, their novel arrangements and the strong correlation between these genes and the C type of male sterility suggest such a role.

A 13-kilodalton maize mitochondrial protein in E. coli confers sensitivity to Bipolaris maydis toxin.

The Texas male-sterile cytoplasm (cms-T) of maize carries the cytoplasmically inherited trait of male sterility. Mitochondria isolated from cms-T maize are specifically sensitive to a toxin (BmT-toxin) produced by the fungal pathogen Bipolaris maydis, race T, and the carbamate insecticide methomyl. A mitochondrial gene unique to cms-T maize, which produces a 13-kilodalton polypeptide associated with cytoplasmic male sterility, was expressed in Escherichia coli. After addition of BmT-toxin or methomyl, inhibition of whole cell respiration and swelling of spheroplasts were observed in Escherichia coli cultures producing the novel mitochondrial protein; these effects are similar to those observed with isolated cms-T mitochondria. The amino-terminal region of the 13-kilodalton polypeptide appears to be essential for proper interaction with the BmT-toxin and methomyl. These results implicate the 13-kilodalton polypeptide in conferring toxin sensitivity to mitochondria of cms-T maize.

A mitochondrial protein associated with cytoplasmic male sterility in the T cytoplasm of maize.

Mitochondrial DNA in the T male-sterile cytoplasm (cms-T) of maize contains an open reading frame (ORF 13) associated with the T type of sterility. Antibodies raised to a chemically synthesized oligopeptide corresponding to ORF 13 were used to establish the expression of a 13-kDa protein from this reading frame. The 13-kDa polypeptide is synthesized uniquely in cms-T maize and purifies with the membrane fraction of T mitochondria. We assign the symbol urf13-T to designate this mitochondrial gene. Presence of the nuclear restorer gene Rf1 in cms-T plants results in a decrease in abundance of 13-kDa protein and alteration in the transcripts of urf13-T.

Characterization of the gene urf13-T and an unidentified reading frame, ORF 25, in maize and tobacco mitochondria.

We have previously identified two large open reading frames, designated ORF13 and ORF25, in the Texas male-sterile cytoplasm (cms-T) of maize mitochondrial DNA (mtDNA). ORF13 is a single copy gene of chimeric origin that is uniquely transcribed and translated in the mitochondria of cms-T maize, where it produces a polypeptide of approximately 13,000 Mr. The ORF13 reading frame does not occur in the maize N, C or S cytoplasms or Nicotiana tabacum. ORF25 exists as a single copy and is transcribed in the four major maize cytoplasms (N, T, C and S) and N. tabacum. The predicted ORF25 polypeptide has a molecular weight of 24,374 in normal maize and 22,439 in tobacco. Several nucleotide and predicted amino acid changes have occurred in the ORF25 gene among the four maize cytoplasms and N. tabacum. Properties such as transcription and conservation of the sequence between two diverse species suggests that ORF25 encodes a functional plant mitochondrial gene. The ORF25 sequence of maize contains a chloroplast DNA insert homologous to a tRNA-Arg gene; this chloroplast DNA insert is absent in the tobacco ORF25 sequence. Comparison of the ORF25 and ORF13 sequences in restored and non-restored cms-T indicates no differences in their nucleotide sequences. Thus fertility restoration does not alter the primary sequences of ORF13 or ORF25.

Sequence of the 18S-5S ribosomal gene region and the cytochrome oxidase II gene from mtDNA of Zea diploperennis.

The coding and flanking sequences of the 18S-5S ribosomal RNA genes and the cytochrome oxidase subunit II gene of Zea diploperennis mitochondrial DNA have been determined and compared to the corresponding sequences of normal maize (Zea mays L.) Both length and substitution mutations are found in the coding region of the 18S rRNA gene, whereas only one substitution mutation is found in the coding region of cytochrome oxidase II. Sequence divergence between maize and Zea diploperennis is about one-tenth of that between wheat and maize. The rate of nucleotide divergence by base substitution is less for plant mitochrondrial genes than for comparable genes in animal mitochondria.

Novel recombinations in the maize mitochondrial genome produce a unique transcriptional unit in the Texas male-sterile cytoplasm.

We have characterized a 3547 bp DNA fragment from male-sterile (cms-T) maize mitochondria, designated TURF 2H3, selected because of its unique and abundant transcripts. Sequence analysis indicated that TURF 2H3 originated by recombinations among portions of the flanking and/or coding regions of the maize mitochondrial 26S ribosomal gene, the ATPase subunit 6 gene, and the chloroplast tRNA-Arg gene. TURF 2H3 contains two long open reading frames that could encode polypeptides of 12,961 Mr and 24,675 Mr. The larger open reading frame hybridizes to transcripts in all maize cytoplasms, the smaller to transcripts only in T cytoplasm. TURF 2H3 transcripts appear to be uniquely altered in cms-T plants restored to fertility by the nuclear restorer genes Rf1 and Rf2. A possible relationship between TURF 2H3, nuclear restorer genes, and the male sterility trait in T cytoplasm is suggested.

Nucleotide sequence of ATPase subunit 6 gene of maize mitochondria.

The ATPase subunit 6, located in the inner mitochondrial membrane, is encoded by mitochondrial genomes in animals and fungi. We have isolated and characterized a mitochondrial gene, designated atp 6, that encodes the subunit 6 polypeptide of Zea mays. Nucleotide and predicted amino acid sequence comparisons have revealed a homology of 44.6 and 33.2% with the yeast ATPase subunit 6 gene and polypeptide, respectively. The predicted protein in maize contains 291 amino acids with a molecular weight of 31,721. Hydropathy profiles generated for the maize and yeast polypeptides are very similar and contain large hydrophobic domains, characteristic of membrane bound proteins. RNA transfer blot analysis indicates that atp 6 is actively transcribed. Interestingly, 122 base pairs of nucleotide sequence interior to atp 6 have extensive homology with the 5' end of the cytochrome oxidase subunit II gene of maize mitochondria, suggesting recombination between the two genes.

Nucleotide sequence of F(0)-ATPase proteolipid (subunit 9) gene of maize mitochondria.

The F(0)-ATPase proteolipid, also referred to as subunit 9 or the dicyclohexylcarbodiimide-binding protein, is encoded by a mitochondrial gene in maize that we have designated atp 9. The clone containing atp 9 was selected for investigation from a mitochondrial DNA library because of its abundant transcript in total maize mitochondrial RNA preparations. Sequence analysis of the clone revealed an open reading frame that was readily identified by its nucleotide homology with the ATPase subunit 9 gene of yeast. As deduced from the nucleotide sequence, the maize ATPase subunit 9 protein contains 74 amino acids with a molecular weight of 7368. Substantial amino acid sequence homology is conserved among maize, yeast, bovine, and Neurospora mitochondrial ATPase subunit 9 proteins, regardless of whether the gene is nuclearly encoded (bovine and Neurospora) or mitochondrially encoded (yeast and maize). RNA transfer blot analysis indicated that the gene sequence is actively transcribed, producing an initial transcript that is large and extensively processed.