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PubMed1993

Chromoplast-Targeted Proteins in Tomato (Lycopersicon esculentum Mill.) Fruit.

The chloroplast to chromoplast transition during tomato (Lycopersicon esculentum Mill.) fruit ripening is characterized by a dramatic change in plastid structure and function. We have asked whether this process is mediated by an increase in the steady-state level of RNA for plastid targeted proteins. Assays for import of radiolabeled translation products into isolated pea (Pisum sativum L.) chloroplasts were used to monitor levels of chromoplast-targeted proteins at four stages of tomato fruit development. We have found striking increases during development in levels of translatable RNA for two such proteins. Additionally, the import of in vitro translation products was examined for seven individual cDNA clones known to encode RNA that increase during fruit ripening. Three of these clones produced in vitro translation products that were imported into pea chloroplasts. This implies that there is synthesis and import of new proteins during the transition from chloroplast to chromoplast and that the plastid conversion is an active developmental program rather than a simple decline in synthesis of the photosynthetic apparatus. Furthermore, our results demonstrate the utility of this method for identification of structural genes involved in plastid morphogenesis.

Journal Article↗

PubMed1993

C4 Photosynthetic Gene Expression in Light- and Dark-Grown Amaranth Cotyledons.

The patterns of expression for genes encoding several C4 photosynthetic enzymes were examined in light-grown and dark-grown (etiolated) cotyledons of amaranth (Amaranthus hypochondriacus), a dicotyledonous C4 plant. The large subunit and small subunit of ribulose-1,5-bisphosphate carboxylase (RuBPCase), phosphoenolpyruvate carboxylase (PEPCase), and pyruvate orthophosphate dikinase (PPdK) were all present in the cotyledons by d 2 after planting when the seedlings first emerged from the seed coat. Kranz anatomy was apparent in light-grown cotyledons throughout development, and the overall patterns of C4 gene expression were similar to those recently described for developing amaranth leaves (J.L. Wang, D.F. Klessig, J.O. Berry [1992] Plant Cell 4: 173-184). RuBPCase mRNA and proteins were present in both bundle sheath and mesophyll cells in a C3-like pattern during early development and became progressively more localized to bundle sheath cells in the C4-type pattern as the cotyledons expanded over 2 to 7 d. PEPCase and PPdK polypeptides were localized to mesophyll cells throughout development, even though PEPCase transcripts were detected in both bundle sheath and mesophyll cells. Kranz anatomy also developed in cotyledons grown in complete darkness. In 7-d-old dark-grown cotyledons, RuBPCase, PPdK, and PEPCase were all localized to the appropriate cell types, although at somewhat lower levels than in light-grown cotyledons. These findings demonstrate that the leaves and postembryonic cotyledons of amaranth undergo common developmental programs of C4 gene expression during maturation. Furthermore, light is not required for the cell-type-specific expression of genes encoding RuBPCase and other photosynthetic enzymes in this dicotyledonous C4 plant.

Journal Article↗

PubMed2004

Gene expression profiling of the tetrapyrrole metabolic pathway in Arabidopsis with a mini-array system.

Tetrapyrrole compounds, such as chlorophylls, hemes, and phycobilins, are synthesized in many enzymatic steps. For regulation of the tetrapyrrole metabolic pathway, it is generally considered that several specific isoforms catalyzing particular enzymatic steps control the flow of tetrapyrrole intermediates by differential regulation of gene expression depending on environmental and developmental factors. However, the coordination of such regulatory steps and orchestration of the overall tetrapyrrole metabolic pathway are still poorly understood. In this study, we developed an original mini-array system, which enables the expression profiling of each gene involved in tetrapyrrole biosynthesis simultaneously with high sensitivity. With this system, we performed a transcriptome analysis of Arabidopsis seedlings in terms of the onset of greening, endogenous rhythm, and developmental control. Data presented here clearly showed that based on their expression profiles at the onset of greening, genes involved in tetrapyrrole biosynthesis can be classified into four categories, in which genes are coordinately regulated to control the biosynthesis. Moreover, genes in the same group were similarly controlled in an endogenous rhythmic manner but also by a developmental program. The physiological significance of these gene clusters is discussed.

Arabidopsis↗

PubMed2005

Functional analysis of the pyrimidine de novo synthesis pathway in solanaceous species.

Pyrimidines are particularly important in dividing tissues as building blocks for nucleic acids, but they are equally important for many biochemical processes, including sucrose and cell wall polysaccharide metabolism. In recent years, the molecular organization of nucleotide biosynthesis in plants has been analyzed. Here, we present a functional analysis of the pyrimidine de novo synthesis pathway. Each step in the pathway was investigated using transgenic plants with reduced expression of the corresponding gene to identify controlling steps and gain insights into the phenotypic and metabolic consequences. Inhibition of expression of 80% based on steady-state mRNA level did not lead to visible phenotypes. Stepwise reduction of protein abundance of Asp transcarbamoylase or dihydro orotase resulted in a corresponding inhibition of growth. This was not accompanied by pleiotropic effects or by changes in the developmental program. A more detailed metabolite analysis revealed slightly different responses in roots and shoots of plants with decreased abundance of proteins involved in pyrimidine de novo synthesis. Whereas in leaves the nucleotide and amino acid levels were changed only in the very strong inhibited plants, the roots show a transient increase of these metabolites in intermediate plants followed by a decrease in the strong inhibited plants. Growth analysis revealed that elongation rates and number of organs per plant were reduced, without large changes in the average cell size. It is concluded that reduced pyrimidine de novo synthesis is compensated for by reduction in growth rates, and the remaining nucleotide pools are sufficient for running basic metabolic processes.

Aspartate Carbamoyltransferase↗

PubMed2006

Developmentally controlled farnesylation modulates AtNAP1;1 function in cell proliferation and cell expansion during Arabidopsis leaf development.

In multicellular organisms, organogenesis requires tight control and coordination of cell proliferation, cell expansion, and cell differentiation. We have identified Arabidopsis (Arabidopsis thaliana) nucleosome assembly protein 1 (AtNAP1;1) as a component of a regulatory mechanism that connects cell proliferation to cell growth and expansion during Arabidopsis leaf development. Molecular, biochemical, and kinetic studies of AtNAP1;1 gain- or loss-of-function mutants indicate that AtNAP1;1 promotes cell proliferation or cell expansion in a developmental context and as a function of the farnesylation status of the protein. AtNAP1;1 was farnesylated and localized to the nucleus during the cell proliferation phase of leaf development when it promotes cell division. Later in leaf development, nonfarnesylated AtNAP1;1 accumulates in the cytoplasm when it promotes cell expansion. Ectopic expression of nonfarnesylated AtNAP1;1, which localized to the cytoplasm, disrupts this developmental program by promoting unscheduled cell expansion during the proliferation phase.

Adenosine Triphosphatases↗

PubMed1995

Regulation of Photosynthesis during Leaf Development in RbcS Antisense DNA Mutants of Tobacco.

We have previously characterized RbcS antisense DNA mutants of tobacco that have drastic reductions in their content of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; S.R. Rodermel, M.S. Abbott, L. Bogorad [1988] Cell 55: 673-681). In this report we examine the impact of Rubisco loss on photosynthesis during tobacco (Nicotiana tabacum) leaf development. Photosynthetic capacities are depressed in the antisense leaves, but the patterns of change in photosynthetic rates during the development of these leaves are similar to those in wild-type plants: after attaining a maximum in young leaves, photosynthetic capacities undergo a prolonged senescence decline in older leaves. The alterations in photosynthetic capacities in both the wild type and mutant are closely correlated with changes in Rubisco activity and content. During wild-type leaf development, Rubisco accumulation is regulated by coordinate changes in RbcS and rbcL transcript accumulation, whereas in the antisense leaves, Rubisco content is a function of RbcS, but not rbcL, transcript abundance. This indicates that large subunit protein production is controlled posttranscriptionally in the mutants. The antisense leaves accumulate near-normal levels of chlorophyll and representative photosynthetic proteins throughout development, suggesting that photosynthetic gene expression is not feedback regulated by Rubisco abundance. Considered together, the data in this paper indicate that leaf developmental programs are generally insensitive to sharp reductions in Rubisco content and emphasize the metabolic plasticity of plant cells in achieving optimal photosynthetic rates.

Journal Article↗

PubMed1997

Differential Expression and Turnover of the Tomato Polyphenol Oxidase Gene Family during Vegetative and Reproductive Development.

Polyphenol oxidases (PPOs) are encoded by a highly conserved, seven-member gene family clustered within a 165-kb locus on chromosome 8 of tomato (Lycopersicon esculentum). Using gene-specific probes capable of differentiating between PPO A/C, PPO B, PPO D, and PPO E/F, we examined the spatial and temporal expression of this gene family during vegetative and reproductive development. RNA blots and in situ hybridization using these probes showed that although PPO expression is primarily confined to early stages of development, the steady-state mRNA levels of these genes are subject to complex patterns of spatial and temporal regulation in vegetative and reproductive organs. Young tomato leaves and flowers possess the most abundant PPO transcripts. PPO B is the most abundant in young leaves, whereas in the inflorescence PPO B and E/F transcripts are dominant. Differential expression of PPOs is also observed in various trichome types. PPO A/C are specifically expressed in type I and type IV trichomes. In contrast, PPO D is only expressed in type VI trichomes. Type I, IV, and VI trichomes possess PPO E/F transcripts. Immunolocalization verified the translational activity of PPOs identified by in situ hybridization and suggested cell-type-specific, developmentally programmed PPO turnover. In addition, immunolocalization demonstrated the accumulation of PPO in specific idioblast cells of stems, leaves, and fruits.

Journal Article↗

PubMed1997

Proteinase Activity during Tracheary Element Differentiation in Zinnia Mesophyll Cultures.

The zinnia (Zinnia elegans) mesophyll cell culture tracheary element (TE) system was used to study proteinases active during developmentally programmed cell death. Substrate-impregnated gels and single-cell assays revealed high levels of proteinase activity in differentiating TEs compared with undifferentiated cultured cells and expanding leaves. Three proteinases (145, 28, and 24 kD) were exclusive to differentiating TEs. A fourth proteinase (59 kD), although detected in extracts from all tissues examined, was most active in differentiating TEs. The 28- and 24-kD proteinases were inhibited by thiol proteinase inhibitors, leupeptin, and N-[N-(L-3-trans-carboxirane-2-carbonyl)-L-leucyl]-agmatine (E-64). The 145- and 59-kD proteinases were inhibited by the serine proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF). Extracts from the TE cultures contained sodium dodecyl sulfate-stimulated proteolytic activity not detected in control cultures. Sodium dodecyl sulfate-stimulated proteolysis was inhibited by leupeptin or E-64, but not by PMSF. Other tissues, sucrose-starved cells and cotyledons, that contain high levels of proteolytic activity did not contain TE-specific proteinases, but did contain higher levels of E-64-sensitive activities migrating as 36- to 31-kD enzymes and as a PMSF-sensitive 66-kD proteinase.

Journal Article↗

PubMed1997

The dark-adaptation response of the de-etiolated pea mutant lip1 is modulated by external signals and endogenous programs.

The lip1 mutant of pea (Pisum sativum L.) exhibits a de-etiolated phenotype. When grown in darkness, lip1 plants have several characteristics normally associated only with light-grown plants. Young wild-type (WT) seedlings accumulate high levels of transcripts from plastid-related genes (such as those encoding chlorophyll a/b-binding proteins, ferredoxin, and the small subunit of Rubisco) only in the light. In contrast, regardless of the light conditions under which the plants are grown, young mutant seedlings accumulate transcript levels equal to or greater than those seen in light-grown WT seedlings of the same age. Under some conditions, light-grown lip1 seedlings failed to respond to dark treatment. The largest response to darkness observed in the mutant occurred when older seedlings were first grown under low-light conditions before transfer to darkness. The mutant's inability to respond to darkness is not due to a gross disturbance in the circadian clock. We conclude that environmental signals (light) and endogenous programs (developmental and circadian) regulate gene expression in both WT and mutant plants. However, mutant seedlings exhibit a developmentally regulated and exaggerated response to light. In addition, the effect of the mutation may be greatest during a brief period early in development.

Adaptation, Physiological↗

PubMed1999

Abscisic acid induction of vacuolar H+-ATPase activity in mesembryanthemum crystallinum is developmentally regulated

Abscisic acid (ABA) has been implicated as a key component in water-deficit-induced responses, including those triggered by drought, NaCl, and low- temperature stress. In this study a role for ABA in mediating the NaCl-stress-induced increases in tonoplast H+-translocating ATPase (V-ATPase) and Na+/H+ antiport activity in Mesembryanthemum crystallinum, leading to vacuolar Na+ sequestration, were investigated. NaCl or ABA treatment of adult M. crystallinum plants induced V-ATPase H+ transport activity, and when applied in combination, an additive effect on V-ATPase stimulation was observed. In contrast, treatment of juvenile plants with ABA did not induce V-ATPase activity, whereas NaCl treatment resulted in a similar response to that observed in adult plants. Na+/H+ antiport activity was induced in both juvenile and adult plants by NaCl, but ABA had no effect at either developmental stage. Results indicate that ABA-induced changes in V-ATPase activity are dependent on the plant reaching its adult phase, whereas NaCl-induced increases in V-ATPase and Na+/H+ antiport activity are independent of plant age. This suggests that ABA-induced V-ATPase activity may be linked to the stress-induced, developmentally programmed switch from C3 metabolism to Crassulacean acid metabolism in adult plants, whereas, vacuolar Na+ sequestration, mediated by the V-ATPase and Na+/H+ antiport, is regulated through ABA-independent pathways.

Journal Article↗

PubMed2000

Differential screening indicates a dramatic change in mRNA profiles during grape berry ripening. Cloning and characterization of cDNAs encoding putative cell wall and stress response proteins.

We used differential screening to isolate ripening-associated cDNAs from a Shiraz grape (Vitis vinifera L.) berry cDNA library. A rapid increase in the mRNA levels of a number of cDNAs not present in unripe fruit occurred in grape berries at the onset of ripening. The putative translation products of some of these clones had homologs in other species that are involved in cell wall structure. These included four proline-rich proteins, a small protein that is similar to the non-catalytic, N-terminal domain of some pectin methylesterases, and two other glutamate-rich proteins. The remainder of the clones encoded putative stress response proteins. These included two thaumatin-like proteins, a metallothionein, a transcription factor, a cytochrome P450 enzyme, and proteins induced by water, sugar, and/or cold stress in other species. Many of the homologs of the grape cDNAs thought to be involved in cell wall structure or stress-related responses also accumulate in a developmental manner in other plants. This may indicate that the grape mRNAs accumulate in response to stresses such as the storage of high concentrations of sugars and rapid cell expansion, or they may accumulate as part of the ripening developmental program.

Amino Acid Sequence↗

PubMed2000

Gibberellins and seed development in maize. I. Evidence that gibberellin/abscisic acid balance governs germination versus maturation pathways.

Abscisic acid (ABA) is required for the regulation of seed maturation in maize (Zea mays L.). Mutants blocked in ABA synthesis (such as viviparous-5) do not mature to quiescent, desiccation-tolerant seeds, but germinate on the ear midway through kernel development. Because gibberellins (GA) and ABA act antagonistically in many aspects of plant development, we hypothesized that ABA antagonizes a positive GA signal for precocious germination in maize. In these experiments, we show that a GA deficiency early in seed development, induced genetically or via biosynthesis inhibitors, suppresses vivipary in ABA-deficient developing kernels. The resulting seeds have both desiccation tolerance and storage longevity. Temporal analysis of GA accumulation in wild-type kernels revealed the accumulation of bioactive GA(1) and GA(3) prior to the peak in ABA content. We speculate that these GAs stimulate a developmental program leading to vivipary in the absence of normal amounts of ABA, and that a reduction of GA content re-establishes an ABA/GA ratio appropriate for suppression of germination and induction of maturation. In contrast, the induction of a GA deficiency did not suppress vivipary in viviparous-1 mutant kernels, suggesting that VP1 acts downstream of both GA and ABA in programming seed development.

Abscisic Acid↗

PubMed1978

Carrier-mediated Uptake of Arginine and Urea by Volvox carteri f. nagariensis.

Volvox carteri f. nagariensis takes up arginine via a high affinity, highly specific carrier, whereas carriers for neutral and acidic amino acids cannot be detected (even in nitrogen-starved cultures). Exogenous arginine is accumulated against a steep concentration gradient and is incorporated into protein with high efficiency, but it is not catabolized to any significant extent and will not serve as a nitrogen source adequate to support growth. Urea is also taken up by a saturable carrier, but several lines of evidence indicate that the arginine and urea carriers are distinct and different. Preexposure to arginine suppresses arginine uptake while stimulating urea uptake. The K(i) values observed for reciprocal, competitive inhibition of uptake by arginine and urea are orders of magnitude different from the respective K(m) values for uptake. The two uptake systems show entirely different patterns of sensitivity to inhibition by structural analogs. Finally, the V(max) values for arginine and urea uptake fluctuate independently (but in a regular pattern) during the asexual life cycle. The fluctuations of urea uptake activity are of considerable magnitude and appear to be linked to key phases of the developmental program.

Journal Article↗

PubMed1985

Differential control of ethylene-induced gene expression and respiration in carrot roots.

Ethylene treatment of carrot roots elicits a developmental program encompassing an increase in respiration rate and changes in gene expression. Both phenomena are potentiated when ethylene is administered in O(2). Our previous studies showed that both respiration and a number of ethylene specific mRNAs increase together in response to ethylene through some 21 hours, whereas thereafter respiration continues to rise, while the level of induced mRNAs drops. Herein we ask whether an experimentally effected drop in the respiration rate within the first 21 hours caused by the withdrawal of ethylene, or substitution of air for O(2) in the continued presence of ethylene, is linked to a drop in the level of ethylene-induced mRNA. Quantitative estimation of two ethylene evoked mRNAs by dot blot hybridization with appropriate cDNA clones has shown that under the specified treatment the induced mRNA levels remain constant while the respiration rate drops, suggesting that gene expression, as reflected in induced mRNA levels, and respiration rate are separately regulated facets of the ethylene response.

Journal Article↗

PubMed1988

PEPCase Transcript Levels in Mesembryanthemum crystallinum Decline Rapidly upon Relief from Salt Stress.

Mesembryanthemum crystallinum plants respond to water stress by changing their pathway of carbon assimilation from C(3) to Crassulacean acid metabolism (CAM). Stressed plants are characterized by elevated levels of phosphoenolpyruvate carboxylase (PEPCase) mRNA, protein, and enzyme activity. We wanted to determine whether CAM is a reversible response to environmental conditions or a developmentally programmed adaptation that is irreversibly expressed once induced. Plants were osmotically stressed by irrigation with 500 millimolar NaCl for 12 days to elicit CAM. Salt was then thoroughly flushed from the soil and PEPCase protein and transcript levels were monitored. PEPCase mRNA levels dropped by 77% within 2.5 hours after salt removal. PEPCase activity and polypeptide levels declined more slowly, with a half-life of 2 to 3 days. These results show that PEPCase expression in M. crystallinum is a reversible response to stress that is regulated at the level of transcription or stability of the PEPCase mRNA.

Journal Article↗

PubMed1989

Immunological Detection and Quantitation of Tryptophan Decarboxylase in Developing Catharanthus roseus Seedlings.

l-Tryptophan decarboxylase (TDC) (EC 4.2.1.27) enzyme activity was induced in cell suspension cultures of Catharanthus roseus after treatment with a Pythium aphanidermatum elicitor preparation. The enzyme was extracted from lyophilized cells containing high levels of TDC and the protein was purified to homogeneity. The pure protein was used to produce highly specific polyclonal antibodies, and an enzyme-linked immunosorbent assay (ELISA) was developed to quantitate the level of TDC antigen during seedling development and in leaves of the mature plant. Western immunoblotting of proteins after SDS-PAGE with anti-TDC antibodies detected several immunoreactive proteins (40, 44, 54.8, 55, and 67 kilodaltons) which appeared at different stages during seedling development and in leaves of the mature plant. The major 54.8 and 55 kilodalton antigenic proteins in immunoblots appeared transiently between days 1 to 5 and 5 to 8 of seedling development, respectively. The 54.8 kilodalton protein was devoid of TDC enzyme activity, whereas the appearance of the 55 kilodalton protein coincided with the appearance of this decarboxylase activity. The minor immunoreactive proteins (40, 44, and 67 kilodaltons) appeared after day 5 of seedling development and in older leaves of the mature plant, and their relationship, if any, to TDC is presently unknown. Results suggest that the synthesis and degradation of TDC protein is highly regulated in Catharanthus roseus and that this regulation follows a preset developmental program.

Journal Article↗

PubMed1990

Developmental control of crassulacean Acid metabolism inducibility by salt stress in the common ice plant.

Ice plant (Mesembryanthemum crystallinum) is a facultative halophyte that responds to water stress in the form of drought or high salinity by switching from C(3) photosynthesis to Crassulacean acid metabolism (CAM), a physiological adaptation that increases water conservation. Although CAM is clearly environmentally controlled, and reversible upon removal of water stress, the competence to switch is developmentally determined. We have demonstrated this by measuring three parameters in the expression of a gene encoding a stress-specific isoform of a key enzyme of CAM, phosphoenolpyruvate carboxylase (PEPCase, Ppc1): (a) protein accumulation; (b) steady-state amounts of mRNA; and (3) transcriptional activity in isolated nuclei. Young plants (3 weeks of age) show little induction of PEPCase protein, mRNA, or transcription when stressed. In contrast, salt stress elicits a strong induction at all three levels of expression at 6 weeks of age. By 9 weeks of age, plants have already accumulated PEPCase protein and mRNA without being stressed. More importantly, transcriptional activation of Ppc1 by salt stress in 9-week-old plants is no longer observed despite an increase of both Ppc1 mRNA and protein. From these results we suggest that a developmental program exists that regulates PEPCase transcription and mRNA stability. This program appears to be synchronized with the climatic conditions in the plant's native environment.

Journal Article↗

PubMed1992

Accumulation of Group 3 Late Embryogenesis Abundant Proteins in Zea mays Embryos : Roles of Abscisic Acid and the Viviparous-1 Gene Product.

Several different types of proteins that are modulated by abscisic acid (ABA) accumulate in developing embryos of maize (Zea mays L.). Some of these proteins are specific to the developing seed, such as the storage globulin, GLB1, whereas others are involved in general responses to water deficit. Here we describe a maize protein family of this second type, a Group 3 late embryogenesis abundant (MLG3). Like other proteins of this class, MLG3 polypeptides are ABA-responsive. They are found in maturing seeds and in dehydrating plant tissues. Antigenically related proteins are found in other cereals. To distinguish the regulation of developmentally programmed ABA responses from those that are environmentally induced, we compared the ontological pattern and accumulation requirements of MLG3 polypeptides with those we previously described for GLB1. GLB1 accumulation begins early in the maturation phase and specifically requires high levels of ABA and the participation of the Viviparous-1 (Vp1) gene product. Vp1 is required for other ABA-modulated events in maize seed development as well. In experiments using vp1 mutants and mutants deficient in ABA synthesis (vp5 mutation), we show that MLG3 accumulation also is dependent upon ABA, but it shows striking differences from GLB1. MLG3 accumulates much later in embryogenesis, coincident with the onset of dehydration. In contrast to GLB1, MLG3 proteins can be induced by de novo ABA synthesis in response to culturing in high osmoticum. Unlike GLB1, MLG3 has no specific requirement for the Vp1 gene product.

Journal Article↗