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J E Mullet

Publications and source records attributed to J E Mullet.

At least 37 records · Page 2Linked to original sources
PubMed1996

RNA-binding proteins of 37/38 kDa bind specifically to the barley chloroplast psbA 3'-end untranslated RNA.

The stability of the psbA mRNA increases during barley chloroplast development eventually reaching a half-life of over 40 h. Translation of psbA mRNA is also regulated in a complex way. Sequence-specific RNA binding proteins may modulate the translation or stability of the psbA mRNA during chloroplast development. UV cross-linking assays revealed that chloroplast proteins of 37 and 38 kDA bind specifically to the 3' end of psbA transcripts and not to the 5' end of psbA or rbcL transcripts. The two RNA-binding proteins were partially purified by ammonium sulfate precipitation followed by heparin agarose chromatography. Deletion and site-directed mutation analysis demonstrated that the 37/38RNPs bind in a 30 nucleotide region immediately downstream from the translation termination codon and upstream of sequences capable of forming a stem-loop structure in the 3' end of psbA transcripts. Single-base changes that diminish the binding of the 37RNP also reduce binding of the 38RNP suggesting that these proteins may bind as a heterodimer. The 37/38RNPs that bind within the 3' end of psbA transcripts could modulate transcription termination, translation or mRNA stability.

Base Sequence↗
PubMed1995

A chloroplast lipoxygenase is required for wound-induced jasmonic acid accumulation in Arabidopsis.

Plant lipoxygenases are thought to be involved in the biosynthesis of lipid-derived signaling molecules. The potential involvement of a specific Arabidopsis thaliana lipoxygenase isozyme, LOX2, in the biosynthesis of the plant growth regulators jasmonic acid (JA) and abscisic acid was investigated. Our characterization of LOX2 indicates that the protein is targeted to chloroplasts. The physiological role of this chloroplast lipoxygenase was analyzed in transgenic plants where cosuppression reduced LOX2 accumulation. The reduction in LOX2 levels caused no obvious changes in plant growth or in the accumulation of abscisic acid. However, the wound-induced accumulation of JA observed in control plants was absent in leaves of transgenic plants that lacked LOX2. Thus, LOX2 is required for the wound-induced synthesis of the plant growth regulator JA in leaves. We also examined the expression of a wound- and JA-inducible Arabidopsis gene, vsp, in transgenic and control plants. Leaves of transgenic plants lacking LOX2 accumulated less vsp mRNA than did control leaves in response to wounding. This result suggests that wound-induced JA (or some other LOX2-requiring component of the wound response pathway) is involved in the wound-induced regulation of this gene.

Abscisic Acid↗
PubMed1995

Jasmonic acid distribution and action in plants: regulation during development and response to biotic and abiotic stress.

Jasmonic acid (JA) is a naturally occurring growth regulator found in higher plants. Several physiological roles have been described for this compound (or a related compound, methyl jasmonate) during plant development and in response to biotic and abiotic stress. To accurately determine JA levels in plant tissue, we have synthesized JA containing 13C for use as an internal standard with an isotopic composition of [225]:[224] 0.98:0.02 compared with [225]:[224] 0.15:0.85 for natural material. GC analysis (flame ionization detection and MS) indicate that the internal standard is composed of 92% 2-(+/-)-[13C]JA and 8% 2-(+/-)-7-iso-[13C]JA. In soybean plants, JA levels were highest in young leaves, flowers, and fruit (highest in the pericarp). In soybean seeds and seedlings, JA levels were highest in the youngest organs including the hypocotyl hook, plumule, and 12-h axis. In soybean leaves that had been dehydrated to cause a 15% decrease in fresh weight, JA levels increased approximately 5-fold within 2 h and declined to approximately control levels by 4 h. In contrast, a lag time of 1-2 h occurred before abscisic acid accumulation reached a maximum. These results will be discussed in the context of multiple pathways for JA biosynthesis and the role of JA in plant development and responses to environmental signals.

Journal Article↗
PubMed1995

Developmental expression of a turgor-responsive gene that encodes an intrinsic membrane protein.

We previously reported that the pea (Pisum sativum) gene, Trg31, shows increased transcription and elevated mRNA levels in plant tissues which are dehydrated and lose turgor. The protein encoded by Trg31 is homologous to members of the MIP intrinsic membrane protein superfamily. Expression of Trg31 was characterized during pea seedling development and in transgenic tobacco using Trg31 promoter::Gus fusions. In pea, Trg31 mRNA abundance was highest in roots followed by flowers, stems and leaves. In roots, Trg31 mRNA levels were highest in non-elongating regions and low in root tips. In dark-grown seedlings, Trg31 mRNA levels were high in stems and illumination caused mRNA abundance in stems to decrease. Histochemical analysis of transgenic tobacco expressing Trg31 promoter::Gus constructs showed high GUS activity in root to shoot and hypocotyl to cotyledon junctions and cotyledons in germinating seedlings. High activity was also observed in the leaf marginal meristem and trichomes. In more mature seedlings, Trg31 promoter activity was observed in the non-elongating portion of the root and in stems especially in the vascular tissue. A gradient of expression was noted in leaf to stem junction zones with highest expression in the younger tissues. Very high expression was observed in stems of flowers and other floral tissues including the calyx, corolla, style, ovules, pods and pollen. This expression pattern suggests that the Trg31 gene product may facilitate transport from sources, through transmitting tissues to sinks.

Aquaporins↗
PubMed1995

A salt- and dehydration-inducible pea gene, Cyp15a, encodes a cell-wall protein with sequence similarity to cysteine proteases.

The pea (Pisum sativum) gene Cyp15a encodes a protein with sequence similarity to cysteine proteases. Expression of Cyp15a was investigated during pea seedling development and in response to environmental stress. Cyp15a shows increased transcription and elevated mRNA levels in plant tissues that are partially dehydrated or treated with 0.6 M mannitol. Cyp15a mRNA levels also increase in seedlings treated with 0.2-0.25 M NaCl or KCl. During development, Cyp15a mRNA levels increase within 6 to 12 h in cotyledons and axes during germination and continue to increase for at least 96 h. Illumination of dark-grown seedlings increased Cyp15a mRNA abundance in elongating and non-elongating stem tissues. GA and ABA, which modulate the abundance of many seed-localized cysteine proteases, did not significantly modulate Cyp15a mRNA levels in stems. The protein encoded by Cyp15a contains a typical amino-terminal secretory targeting domain. This domain is followed by a pro-sequence containing ca. 110 amino acids that is found in other cysteine proteases. Polyclonal antibodies, directed against CYP15a, recognized both the larger pro-form and the cleaved mature form of CYP15a on western blots. Immunolocalization assays indicated that both forms of the protein are located in cell walls of stem cortical cells.

Cell Wall↗
PubMed1995

Arabidopsis thaliana Atvsp is homologous to soybean VspA and VspB, genes encoding vegetative storage protein acid phosphatases, and is regulated similarly by methyl jasmonate, wounding, sugars, light and phosphate.

The soybean vegetative storage proteins, VSP alpha and VSP beta, are acid phosphatases that accumulate to very high levels in hypocotyls, young leaves and flowers and pods. The genes encoding the soybean VSP are activated by jasmonate, wounding, sugars and light and down regulated by phosphate and auxin. In this study, expression of an Arabidopsis thaliana gene (Atvsp) encoding a protein homologous to soybean Vsp alpha and Vsp beta, was examined and compared to expression of the soybean Vsp genes. Atvsp mRNA was present at high levels in flowers and buds and at low levels in roots, stems, leaves and siliques. Expression of Atvsp in leaves could be induced by wounding or by treatment of illuminated plants with methyl jasmonate and sucrose. Roots of plants with wounded leaves also accumulated Atvsp mRNA indicating that this gene can be regulated by a transmissible wound signal. Phosphate partially inhibited expression of Atvsp. Arabidopsis proteins of 29 and 30 kDa crossreacted with antibodies against soybean VSP. These proteins were very abundant in flowers and the proteins accumulated in leaves and roots of plants treated with methyl jasmonate. The level of these proteins in flowers was similar to the levels of soybean VSP in young soybean leaves. Overall, these data indicate that Arabidopsis Atvsp and soybean VspA/B genes are regulated similarly and that in both plants, the gene products can accumulate to high levels. This suggests that genes homologous to VspA/B may be of greater general significance than previously recognized.

Acetates↗
PubMed1995

Characterization and expression of rpoC2 in CMS and fertile lines of sorghum.

A 165 bp deletion in the middle of rpoC2, the plastid gene which encodes the RNA polymerase beta" subunit, was identified in the small-anthered types of CMS sorghum, Sorghum bicolor (L.). Moench, containing A1, A2, A5, and A6 cytoplasms. It was previously shown that the amino acid sequence deleted in these CMS lines is in a monocot-specific region that contains several protein motifs that are characteristic of several transcription factors. Using primers flanking the deletion in PCR analyses, various types of CMS lines, some of which are used in hybrid sorghum production, were classified into two groups. CMS lines containing A1, A2, A5, A6 cytoplasms display the deletion in rpoC2. These lines have small anthers in which pollen development is arrested at an early stage and in which usually only empty exines are found. CMS lines containing A3, A4, and 9E cytoplasms do not possess the deletion. These lines have large anthers in which pollen degenerates at a later stage. Run-on transcription assays using 15 chloroplast genes showed that chloroplast gene transcription rates are similar in CMS and fertile (maintainer and restorer) lines and F1 in seedling leaves. Analyses of RNA blots indicated that rbcL, rpoB and rpoC2 transcripts are accumulated mainly in the leaves and low in the inflorescence tissues and pollen. These data document plastid gene expression in leaves and non-photosynthetic tissues from CMS and fertile lines of sorghum.

Base Sequence↗
PubMed1995

Sink limitation induces the expression of multiple soybean vegetative lipoxygenase mRNAs while the endogenous jasmonic acid level remains low.

The response of individual members of the lipoxygenase multigene family in soybeans to sink deprivation was analyzed. RNase protection assays indicated that a novel vegetative lipoxygenase gene, vlxC, and three other vegetative lipoxygenase mRNAs accumulated in mature leaves in response to a variety of sink limitations. These data suggest that several members of the lipoxygenase multigene family are involved in assimilate partitioning. The possible involvement of jasmonic acid as a signaling molecule regulating assimilate partitioning into the vegetative storage proteins and lipoxygenases was directly assessed by determining the endogenous level of jasmonic acid in leaves from plants with their pods removed. There was no rise in the level of endogenous jasmonic acid coincident with the strong increase in both vlxC and vegetative storage protein VspB transcripts in response to sink limitation. Thus, expression of the vegetative lipoxygenases and vegetative storage proteins is not regulated by jasmonic acid in sink-limited leaves.

Amino Acid Sequence↗
PubMed1995

Identification of a sequence-specific DNA binding factor required for transcription of the barley chloroplast blue light-responsive psbD-psbC promoter.

The plastid gene psbD encodes the photosystem II reaction center chlorophyll protein D2. psbD is located in a complex operon that includes psbC, psbK, psbl, orf62, and trnG. The operon is transcribed from at least three different promoters. One of the psbD promoters is differentially activated when plants are exposed to blue light. In this study, the psbD blue light-responsive promoter was accurately transcribed in vitro in high-salt extracts of barley plastids. Transcription required supercoiled templates and was inhibited by tagetitoxin, an inhibitor of plastid transcription. Escherichia coli RNA polymerase did not recognize the psbD light-responsive promoter with the same specificity as plastid RNA polymerase. Deletion analyses demonstrated that sequences between -39 and -68, upstream of the transcription initiation site, were required for transcription of the psbD blue light-responsive promoter. This DNA region is highly conserved among plant species and contains multiple AAG sequences. Gel shift assays and DNase I footprinting experiments demonstrated that the AAG-rich DNA sequence interacts with a sequence-specific DNA binding factor termed AGF. Point mutations in the AAG cis element decreased binding of AGF and inhibited transcription from the psbD light-responsive promoter. We concluded that AGF is an essential factor required for transcription of the psbD light-responsive promoter.

Base Sequence↗
PubMed1994

Synthesis and turnover of photosystem II reaction center protein D1. Ribosome pausing increases during chloroplast development.

The chloroplast photosystem II reaction center protein D1 contains five membrane-spanning helices and binds chlorophyll, carotenoid, quinone, iron, and probably manganese. Turnover of pulse-labeled D1 in isolated plastids was found to involve cleavage between helix IV and helix V, which releases a 23-kDa N-terminal peptide and two C-terminal peptides of 10 and 8 kDa. Ribosomes pause at specific sites during translation of D1, which results in the accumulation of D1 translation intermediates. Pulse-labeling assays followed by polysome isolation and immunoprecipitation identified paused D1 translation intermediates of 9, 12.5, 15-18, 20, 21, 24, and 28-32 kDa. Ribosome pausing was not altered when dark-grown seedlings were illuminated for up to 1 h, even though this treatment stimulated accumulation of chlorophyll and D1. However, illumination of plants for 16-72 h resulted in increased ribosome pausing and the build-up of D1 translation intermediates. We hypothesize that ribosome pausing during synthesis of D1 improves the efficiency of chlorophyll binding of D1 nascent chains and enhances accumulation of D1 in mature chloroplasts, which have reduced rates of chlorophyll biosynthesis.

Amino Acid Sequence↗
PubMed1994

Ribosome-binding sites on chloroplast rbcL and psbA mRNAs and light-induced initiation of D1 translation.

Chloroplast ribosome-binding sites were identified on the plastid rbcL and psbA mRNAs using toeprint analysis. The rbcL translation initiation domain is highly conserved and contains a prokaryotic Shine-Dalgarno (SD) sequence (GGAGG) located 4 to 12 nucleotides upstream of the initiator AUG. Toeprint analysis of rbcL mRNA associated with plastid polysomes revealed strong toeprint signals 15 nucleotides downstream from the AUG indicating ribosome binding at the translation initiation site. Escherichia coli 30S ribosomes generated similar toeprint signals when mixed with rbcL mRNA in the presence of initiator tRNA. These results indicate that plastid SD sequences are functional in chloroplast translation initiation. The psbA initiator region lacks a SD sequence within 12 nucleotides of the initiator AUG. However, toeprint analysis of soluble and membrane polysome-associated psbA mRNA revealed ribosomes bound to the initiator region. E. coli 30S ribosomes did not associate with the psbA translation initiation region. E. coli and chloroplast ribosomes bind to an upstream region which contains a conserved SD-like sequence. Therefore, translation initiation on psbA mRNA may involve the transient binding of chloroplast ribosomes to this upstream SD-like sequence followed by scanning to localize the initiator AUG. Illumination 8-day-old dark-grown barley seedlings caused an increase in polysome-associated psbA mRNA and the abundance of initiation complexes bound to psbA mRNA. These results demonstrate that light modulates D1 translation initiation in plastids of older dark-grown barley seedlings.

Bacterial Proteins↗
PubMed1994

Vir-115 gene product is required to stabilize D1 translation intermediates in chloroplasts.

The nuclear gene mutant of barley, vir-115, shows a developmentally induced loss of D1 synthesis that results in inactivation of Photosystem II. Translation in plastids isolated from 1 h illuminated vir-115 seedlings is similar to wild type. In wild-type barley, illumination of plants for 16 to 72 h results in increased radiolabel incorporation into the D1 translation intermediates of 15-24 kDa. In contrast, these D1 translation intermediates were not observed in vir-115 plastids isolated from plants illuminated for 16-72 h. In addition, after 72 h of illumination, radiolabel incorporation into D1 was undetectable in vir-115 plastids. The level and distribution of psbA mRNA in membrane-associated polysomes was similar in wild-type and vir-115 mutant plastids isolated from plants illuminated for 16-72 h. Toeprint analysis showed similar levels of translation initiation complexes on psbA mRNA in vir-115 and wild-type plastids. These results indicate that translation initiation and elongation of D1 is not significantly altered in the mutant plastids. Ribosome pausing on psbA mRNA was observed in wild-type and vir-115 mutant plastids. Therefore, the absence of D1 translation intermediates in mutant plastids is not due to a lack of ribosome pausing on psbA mRNA. Based on these results, it is proposed that vir-115 lacks or contains a modified nuclear-encoded gene product which normally stabilizes the D1 translation intermediates. In wild-type plastids, ribosome pausing and stabilization of D1 translation intermediates is proposed to facilitate assembly of cofactors such as chlorophyll with D1 allowing continued D1 synthesis and accumulation in mature chloroplasts.

Bacterial Proteins↗
PubMed1994

Chlorophyll regulates accumulation of the plastid-encoded chlorophyll proteins P700 and D1 by increasing apoprotein stability.

Chlorophyll protein accumulation in barley (Hordeum vulgare L.) chloroplasts is controlled posttranscriptionally by light-induced formation of chlorophyll a. The abundance of translation initiation complexes associated with psbA, psaA, and rbcL mRNAs was measured using extension and inhibition analysis in plants grown in the dark for 4.5 d and then illuminated for up to 16 h. Light-induced accumulation of the chlorophyll proteins was not accompanied by changes in the abundance of translation initiation complexes, indicating that regulation of chlorophyll protein accumulation at this stage of development does not occur at the level of translation initiation. Translational runoff assays were performed in the presence of lincomycin, an inhibitor of translation initiation, to determine whether chlorophyll protein accumulation was regulated at the level of translation elongation. The extent of ribosome runoff of psaA and psbA mRNAs was similar in the presence or absence of chlorophyll, indicating that chlorophyll did not alter chlorophyll protein translation elongation. Polysome-associated D1 translation intermediates were radiolabeled in the presence or absence of chlorophyll, even though full-length D1 accumulated only in the presence of chlorophyll. Chlorophyll influenced the stability of D1 translation intermediates to a small extent and greatly increased D1 stability after release from ribosomes. Overall, these results demonstrate that light-induced chlorophyll biosynthesis triggers the accumulation of the chlorophyll proteins D1 and P700 in barley chloroplasts by enhancement of chlorophyll apoprotein stability.

Apoproteins↗
PubMed1994

Separate photosensory pathways co-regulate blue light/ultraviolet-A-activated psbD-psbC transcription and light-induced D2 and CP43 degradation in barley (Hordeum vulgare) chloroplasts.

We studied the effects of spectral quality and fluence on the expression of several chloroplast-encoded photosynthesis genes and on the stability of their protein products in barley (Hordeum vulgare). During light-dependent chloroplast maturation, mRNA levels for psbD-psbC and psbA were maintained at higher levels compared with mRNAs encoding proteins for other photosynthesis functions (atpB, rbcL). Maintenance of psbD-psbC mRNA levels was accounted for by differential activation of the psbD-psbC light-responsive promoter by high-irradiance blue light and, secondarily, ultraviolet A (UV-A) radiation. Promoter activation was fluence dependent and required continuous illumination for 2 h at threshold fluences of 1.3 (blue light), 7.5 (white light), or 10 (UV-A) mumol m-2 s-1. From immunoblot analysis experiments, we showed that the psbD-psbC gene products D2 and CP43 undergo light-mediated turnover similar to light-labile D1. Other photosynthesis proteins such as the beta subunit of ATP synthase and the large subunit of ribulose-1,5-bisphosphate carboxylase were relatively stable. In the absence of protein synthesis, D2 degradation paralleled the degradation of D1 (relative half-lives, 9.5-10 h). CP43 decay was about half of D2 and D1 decay. In contrast with activation of the light-responsive promoter, the fluence-dependent degradation of D1, D2, and CP43 required 50- to 100-fold higher fluences of photosynthetically active white, red, blue, or UV-A irradiation. We interpret the different fluence and wavelength requirements to indicate that separate photosensory systems regulate activation of psbD-psbC transcription and turnover of D1, D2, and CP43. We propose that a blue light/UV-A photosensory pathway activates the psbD-psbC light-responsive promoter, differentially maintaining the capacity of mature chloroplasts to synthesize D2 and CP43, which are damaged and turned over in illuminated plants.

Chloroplasts↗
PubMed1993

Direct evidence for selective modulation of psbA, rpoA, rbcL and 16S RNA stability during barley chloroplast development.

The turnover of RNAs encoded by seven different barley chloroplast genes was analyzed after treatment of barley shoots with tagetitoxin, a selective inhibitor of chloroplast transcription. Changes in RNA stability were examined during chloroplast development using basal and apical leaf sections of 4.5-day-old dark-grown seedlings and apical leaf sections of 4.0-day-old dark-grown seedlings which had been illuminated for 12 h. Of the RNAs examined, a 2.6 kb unspliced precursor of tRNA(lys) exhibited the shortest half-life, which was estimated to be 3 h. The 16S rRNA and psbA mRNA had the longest estimated half-lives, which were greater than 40 h. Among mRNAs, half-lives were estimated to range from 6 h for psaA mRNA, to over 40 h for psbA mRNA. Therefore, barley chloroplast mRNAs have long half-lives relative to bacterial mRNAs. The stability of atpB mRNA and the unspliced precursor of tRNA-lys was not altered during chloroplast development, while the stability of psaA mRNA decreased 2-fold. In contrast, the stability of the 16S rRNA and mRNAs for rpoA, psbA and rbcL increased during chloroplast development. The stability of 16S rRNA increased markedly during chloroplast development in the dark and this increase was maintained in illuminated seedlings. The stability of rbcL mRNA increased 2.5-fold during chloroplast development in the dark, and then decreased 2-fold in chloroplasts of light-grown plants. The initial increase in rpoA and psbA mRNA stability was also light-independent, with total increases in stability of at least 5-fold. In the case of rpoA, the stability of 2 of the 13 polycistronic rpoA transcripts that were detected in dark-grown plants was selectively increased during chloroplast development. In conclusion, the stability of some transcripts is selectively increased and further modulated during chloroplast development in barley. We propose that the selective stabilization of chloroplast mRNA, which occurred independent of light, is an indication that non-light regulated developmental signals are involved in barley chloroplast mRNA stability.

Base Sequence↗
PubMed1993

Characterization of an Arabidopsis lipoxygenase gene responsive to methyl jasmonate and wounding.

A cDNA corresponding to the gene AtLox2 was isolated from an Arabidopsis thaliana library using a lipoxygenase (LOX) probe from soybean. AtLox2 encodes a 102-kD protein, AtLOX2, which has 42 to 45% amino acid sequence identity with other plant LOX sequences. The AtLOX2 sequence is more than 30 amino acids longer at the amino terminus than other plant LOX sequences, and this extension has features reminiscent of chloroplast transit peptides, suggesting that AtLOX2 may be chloroplast localized. AtLox2 mRNA levels are high in leaves and inflorescences but very low in seeds, roots, and stems. AtLox2 mRNA accumulation is rapidly induced in leaves in response to methyl jasmonate. Leaves that have been wounded and adjacent leaves on the same plant also accumulate AtLox2 mRNA.

Acetates↗
PubMed1993

Identification of a methyl jasmonate-responsive domain in the soybean vspB promoter.

Soybean vspB encodes a highly expressed vegetative storage protein-acid phosphatase. In soybean, vspB expression is stimulated by methyl jasmonate (MeJA) and sugars. The vspB promoter was studied by transforming tobacco with fusions of 5' noncoding vspB DNA and the gene encoding beta-glucuronidase (GUS). Constructs containing 833 bp of vspB 5' DNA showed high expression of GUS in stems, leaf veins and trichomes, sepals, and pollen. Sucrose (0.2 M) and MeJA (10(-5) M) increased gene expression when applied to leaf tissue. Deletion of the region -787 to -520 with respect to the transcription initiation site rendered the vspB promoter noninducible by MeJA but still sucrose responsive. This result indicates that DNA elements capable of modulating vspB by MeJA can be separated from carbon response elements. Further 5' end deletion from -520 to -403 or 3' end deletion from -165 to -289 removed DNA sequences involved in carbon modulation of gene expression. A DNA domain that mediates the MeJA response was further localized to a 50-bp region between -535 and -585. This domain when fused to a cauliflower mosaic virus (CaMV) 35S truncated (-88) promoter makes the CaMV promoter responsive to MeJA. The MeJA-responsive domain contains a G-box motif (CACGTG) and a C-rich sequence. A similar 50-bp DNA region is present in the putative promoter of vspA. Related sequences are located in a wound- and MeJA-responsive domain of the proteinase inhibitor II gene and a UV-responsive promoter domain of chs, the gene encoding chalcone synthase that is also responsive to MeJA.

Acetates↗