Abscisic Acid Refines the Synthesis of Chloroplast Proteins in Maize (Zea mays) in Response to Drought and Light

To better understand abscisic acid (ABA) regulation of the synthesis of chloroplast proteins in maize (Zea mays L.) in response to drought and light, we compared leaf proteome differences between maize ABA-deficient mutant vp5 and corresponding wild-type Vp5 green and etiolated seedlings exposed to drought stress. Proteins extracted from the leaves of Vp5 and vp5 seedlings were used for two-dimensional electrophoresis (2-DE) and subsequent matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). After Coomassie brilliant blue staining, approximately 450 protein spots were reproducibly detected on 2-DE gels. A total of 36 differentially expressed protein spots in response to drought and light were identified using MALDI-TOF MS and their subcellular localization was determined based on the annotation of reviewed accession in UniProt Knowledgebase and the software prediction. As a result, corresponding 13 proteins of the 24 differentially expressed protein spots were definitely localized in chloroplasts and their expression was in an ABA-dependent way, including 6 up-regulated by both drought and light, 5 up-regulated by drought but down-regulated by light, 5 up-regulated by light but down-regulated by drought; 5 proteins down-regulated by drought were mainly those involved in photosynthesis and ATP synthesis. Thus, the results in the present study supported the vital role of ABA in regulating the synthesis of drought- and/or light-induced proteins in maize chloroplasts and would facilitate the functional characterization of ABA-induced chloroplast proteins in C₄ plants.     

Proteomic identification of toxic volatile organic compound-responsive proteins in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The proteins that are responsive to toxic volatile organic compounds (VOC) such as formaldehyde and toluene were analyzed with proteome analysis using two-dimensional difference image gel electrophoresis (DIGE) technology. Twenty-one days after germination (DAG) seedlings of Arabidopsis thaliana were exposed either to the gaseous formaldehyde or toluene in an airtight box installed in a plant growth chamber maintained at 24°C under the long day condition with relatively low light condition. Comparative expression analysis revealed 14 and 22 protein spots as proteins displaying at least 1.5-fold differences in expression upon formaldehyde and toluene treatment, respectively, compared to those of untreated control. Most of the isolated spots were successfully identified by peptide analysis using LC-MS-MS. The VOC-responsive proteins contain ATP synthase CF1, ribulose-1,5-bisphosphate carboxylase/oxygenase, photosystem II light harvesting complex, and enolase, which are components of photosynthesis and carbohydrate metabolism. Despite the relatively low light intensity was applied, many identified VOC-induced proteins were previously known to be up-regulated upon high light stimulus. In addition, proteins involved in the toxin catabolic process and stress hormone-related proteins were identified as toluene-induced proteins. Although the exact function of most of the VOC-responsive proteins identified in these experiments had not been characterized, the protein expression analysis using DIGE was clearly demonstrated that plants are capable of responding actively to VOCs at translational level, and identified proteins may provide valuable tools to account for the effects of abiotic stress caused by air pollutants such as VOCs in plant.     

Regulation of phytochrome mRNA abundance in green oat leaves

Abstract. Avena sativa L. (oat) seedings were grown 4 d in continuous white light followed by 3 d in darkness. Probes derived from an oat phytochrome cDNA clone (pAP 3.2) were used in slot blot analyses to measure the abundance of phytochrome mRNA in the distinct etiolated and green portions of the leaves produced by these seedlings. Both the green and etiolated portions accumulated phytochrome mRNA to a level of about 85% of the etiolated seedling level. Subsequent experiments with similar seedlings showed that both the green and etiolated portions were capable of inducing a dramatic decline in phytochrome mRNA abundance in response to a saturating red light pulse. Despite the ability of green portions of oat leaves to accumulate phytochrome mRNA and to down-regulate phytochrome mRNA abundance in response to light, no substantial variation in phytochrome mRNA abundance was observed in green oat seedlings maintained on a 12-h day/12-h night cycle. In the same oat seedlings, the abundance of chlorophyll a/b binding protein mRNA fluctuated dramatically during the day/night cycle.     

Proteomic analysis of blue light-induced twining response in Cuscuta australis

The parasitic plant Cuscuta australis (dodder) invades a variety of species by entwining the stem and leaves of a host and developing haustoria. The twining response prior to haustoria formation is regarded as the first sign for dodders to parasitize host plants, and thus has been the focus of studies on the host-parasite interaction. However, the molecular mechanism is still poorly understood. In the present work, we have investigated the different effects of blue and white light on the twining response, and identified a set of proteins that were differentially expressed in dodder seedlings using a proteomic approach. Approximately 1,800 protein spots were detected on each 2-D gel, and 47 spots with increased or decreased protein levels were selected and analyzed with MALDI-TOF-MS. Peptide mass fingerprints (PMFs) obtained for these spots were used for protein identification through cross-species database searches. The results suggest that the blue light-induced twining response in dodder seedlings may be mediated by proteins involved in light signal transduction, cell wall degradation, cell structure, and metabolism.     

Effects of blue and red light on expression of nuclear genes encoding chloroplast glyceraldehyde-3-phosphate dehydrogenase of Arabidopsis thaliana.

We have characterized the effects of different light spectra on expression of the nuclear genes (GapA and GapB) encoding chloroplast glyceraldehyde-3-phosphate dehydrogenase in Arabidopsis thaliana. Steady-state mRNA levels for both genes in etiolated seedlings increased after a short exposure to red or blue light. However, these increases could not be reversed by immediate far-red light following the initial light treatment. In mature plants, a short light pulse, regardless of its spectrum, had no apparent effect on GapA or GapB mRNA levels in dark-adapted plants. In contrast, continuous exposure to red, blue, or white light resulted in increases of GapA and GapB mRNA levels, with blue and white light being far more efficient than red light. Similarly, continuous exposure of etiolated seedlings to red, blue, or white light also resulted in increased GapA and GapB mRNA levels. In addition, we show that illumination of red light-saturated Arabidopsis plants with continuous blue light results in further increases of GapA and GapB mRNA levels. Based on these results, we conclude that both blue light photoreceptor- and phytochrome-mediated pathways are involved in light regulation of GapA and GapB genes in Arabidopsis, with blue light acting as the dominant regulator.     

A Mung Bean Assay for Malformin-induced Growth Stimulation

A bioassay employing green or etiolated cuttings of Phaseolus aureus Roxb. was developed for determining malformin-induced growth stimulation in light. Growth enhancement of green cuttings was more rapid and relatively greater than that of etiolated cuttings. Cuttings from green seedlings responded less as seedlings aged; those from etiolated seedlings responded more. Malformin also stimulated the growth of green or etiolated seedlings in light. Most growth enhancement induced by malformin occurred in the upper 1 cm of the stem. Using green cuttings, malformin stimulated stem elongation relatively more when cotyledons, leaves, or especially apical buds were removed. Although malformin failed to stimulate elongation of 2-cm stem sections “floated” on solutions in Petri dishes, it stimulated elongation of sections when they were upright. High concentrations of indoleacetic acid inhibited growth enhancement by malformin. When gibberellin and malformin were combined, growth enhancement was nearly additive.     

Phototropism and geotropism in hypocotyls of cress (Lepidium sativum L.)

Abstract. The phototropic and the geotropic sensitivities of cress hypocotyls differed in etiolated and in green seedlings. In etiolated seedlings, phototropism was poorly developed and geotropism seemed to be the dominant orientation mechanism. In green seedlings, although geotropic sensitivity was slightly greater, phototropic responses were markedly enhanced, such that photo-signals could over-ride, or even reverse, geocurvature. The 'de-etiolation' light pretreatments required to bring about these changes in the photo-and geo-systems were different. The slight enhancement of geocurvature could be achieved by short-term exposure to red light and was reversed by far-red irradiation. The marked stimulation of photocurvature required extensive irradiation pretreatments.     

UV-B response of green and etiolated barley seedlings

7-d-old etiolated and green barley seedlings (Hordeum vulgare L. cv. Alfa) were irradiated with UV-B for 30 min and then kept for 24 h in light or darkness. Chlorophyll (Chl) synthesis was inhibited by about 30 % as a result of UV-B irradiation, but there were no significant changes in photochemical activity measured by variable to maximum fluorescence ratio (F_v/F_m), quantum yield (Φ_PS2) and oxygen evolution rate. Electron transport of etiolated seedlings was similar to that of green ones, nevertheless, the Chl content was more then 2-fold lower. Ribulose-1,5-bisphosphate carboxylase/oxygenase large and small subunits were diminished as a result of UV-B irradiation in etiolated and green plants, especially in those kept in the darkness. Catalase activity decreased and total superoxide dismutase activity increased in green and etiolated plants following UV-B treatment. When benzidine was used as a substrate, an isoform located between guaiacol peroxidases 2 and 3 (guaiacol peroxidase X) appeared, which was specific for UV-B treatment. As a result of irradiation, the contents of UV-B absorbing and UV-B induced compounds increased in green seedlings but not in etiolated seedlings.     

Lil3 assembles as chlorophyll-binding protein complex during deetiolation

Dark-grown angiosperm seedlings are etiolated and devoid of chlorophyll. Deetiolation is triggered by light leading to chlorophyll dependent accumulation of the photosynthetic machinery. The transfer of chlorophyll to the chlorophyll-binding proteins is still unclear. We demonstrate here that upon illumination of dark-grown barley seedlings, two new pigment-binding protein complexes are de novo accumulated. Pigments bound to both complexes are identified as chlorophyll a and protochlorophyll a. By auto-fluorescence tracking and mass spectrometry, we show that exclusively Lil3 is the pigment-binding complex subunit in both complexes.     

Two-dimensional gel electrophoresis of proteins as a tool in wheat genetics

In this minireview are reported several genetic investigations undertaken on wheat with the use of two-dimensional gel electrophoresis of total proteins extracted mainly from etiolated seedlings or from green leaves. Differences between developmental stages or organs of one genotype and nuclear and cytoplasmic genetic variations between genotypes are revealed by this method. We have also localized on the chromosomes structural genes coding for the proteins revealed and assigned their subcellular location to many polypeptides. We obtained new information concerning the regulation of protein amounts as well as the phylogenetic and homeology relationships between the A, B and D genomes.