Influences of Light Quality, Growth Regulators and Inhibitors of Protein Synthesis on Respiration of Protoplasts from Meristematic Cells in Barley Seedlings

The influences of light of different wavelengths and plant growthregulators on the respiration of protoplasts isolated from tissue0 to 5 mm above the basal intercalary meristem of barley (Hordeumvulgare L. cv. Patty) leaves were studied. Respiration was measuredusing oxygen electrodes and a Cartesian-diver technique. Red,far-red and blue light all stimulated respiration in the protoplastsbut not in mitochondria isolated from them. Gibberellic acid stimulated respiration in protoplasts but abscisicacid had the opposite effect. Physiological concentrations ofindole-3-acetic acid and kinetin had no influence in eitherdirection. Combinations of gibberellic acid with light of anywavelength always increased respiration. Red or far-red light treatments in the presence of abscisicacid decreased dark respiration and only blue light significantlyreversed the inhibitory effect of abscisic acid. Cycloheximidemarkedly increased dark respiratory activity; chloramphenicolwas without effect. These results indicate that mitochondrialactivity in the leaf basal intercalary meristem was partiallycontrolled through phytochrome and a blue light receptor, andby gibberellic and abscisic acids. Changes in cytosolic proteinsynthesis were important for the initiation of enhanced mitochondrialactivity in meristems. Hordeum vulgare L., barley, abscisic acid, Cartesian-diver microrespirometry, gibberellic acid, meristematic respiration, protoplasts     

Inhibition of Respiration in Protoplasts from Meristematic Tissues3

Owen, J. H., Hetherington, A. M. and Wellburn, A. R. 1986. Inhibitionof respiration in protoplasts from meristematic tissues by abscisicacid in the presence of calcium ions.—J. exp. Bot. 38:498–505. A study was made of the influences of abscisic acid (ABA) andcalcium ions on mitochondrial respiration in protoplasts fromcells close to the basal intercalary meristem of light-grownbarley (Hordeum vulgare L. cv. Patty) seedlings. This respirationwas inhibited by ABA only when calcium ions were present. Thecalcium channel agonist BAY K8644 caused a significant inhibitionof protoplast dark respiration, similar to that observed usingABA and calcium, presumably because it imitated the action ofABA by increasing calcium influx into protoplasts. These resultssuggest that ABA increases the permeability of the plasmamembraneto calcium and that calcium acts as a second messenger to regulatemitochondrial respiratory activity and thus the very early eventsassociated with plastid and meristematic cell development. Key words: Abscisic acid, calcium, meristematic respiration     

Chloroplast development in low light-grown barley seedlings

Segments of 7-d low light-grown barley laminae cut at 0.5 cm intervals up from the intercalary meristem were examined ultrastructurally and biochemically. The different regions upwards showed the succession of plastid development in light-grown tissues of eoplasts, amyloplasts, amoeboid, immature and mature plastids as described by Whatley (1977). Semi-crystalline bodies were detected in all of them. The eoplast-amyloplast regions are characterised by a greater proportion of mitochondria and high levels of ATP and 3-phosphoglyceric acid, together with low levels of inorganic phosphate conducive to the activation of ADP glucose pyrophosphorylase. The amoeboid and immature plastid regions have higher levels of inhibitory phosphate and starch breakdown may be responsible for the release of metabolites and energy for development. Segments containing amoeboid and immature plastids also have reduced levels of ATP (and 3-phosphoglyceric acid) as photosynthetic components are synthesised. Using ultrastructural assessments of areas of thylakoids, first -carotene and violaxanthin, followed by chlorophyll a and lutein and, lastly, chlorophyll b are concentrated in the developing lamellar systems of the immature and mature chloroplasts. The formation of additional membraneous material which spreads these pigment systems over a greater thylakoid area within the plastids is the final stage of plastid morphogenesis in low light-grown seedlings.Abbreviations Chl chlorophyll - 3-PGA 3 phosphoglyceric acid     

Plastid stromules are induced by stress treatments acting through abscisic acid

Stromules are highly dynamic stroma-filled tubules that extend from the surface of all plastid types in all multi-cellular plants examined to date. The stromule frequency (percentage of plastids with stromules) has generally been regarded as characteristic of the cell and tissue type. However, the present study shows that various stress treatments, including drought and salt stress, are able to induce stromule formation in the epidermal cells of tobacco hypocotyls and the root hairs of wheat seedlings. Application of abscisic acid (ABA) to tobacco and wheat seedlings induced stromule formation very effectively, and application of abamine, a specific inhibitor of ABA synthesis, prevented stromule induction by mannitol. Stromule induction by ABA was dependent on cytosolic protein synthesis, but not plastid protein synthesis. Stromules were more abundant in dark-grown seedlings than in light-grown seedlings, and the stromule frequency was increased by transfer of light-grown seedlings to the dark and decreased by illumination of dark-grown seedlings. Stromule formation was sensitive to red and far-red light, but not to blue light. Stromules were induced by treatment with ACC (1-aminocyclopropane-1-carboxylic acid), the first committed ethylene precursor, and by treatment with methyl jasmonate, but disappeared upon treatment of seedlings with salicylate. These observations indicate that abiotic, and most probably biotic, stresses are able to induce the formation of stromules in tobacco and wheat seedlings.     

Loss or retention of chloroplast DNA in maize seedlings is affected by both light and genotype

We examined the chloroplast DNA (cpDNA) from plastids obtained from wild type maize (Zea mays L.) seedlings grown under different light conditions and from photosynthetic mutants grown under white light. The cpDNA was evaluated by real-time quantitative PCR, quantitative DNA fluorescence, and blot-hybridization following pulsed-field gel electrophoresis. The amount of DNA per plastid in light-grown seedlings declines greatly from stalk to leaf blade during proplastid-to-chloroplast development, and this decline is due to cpDNA degradation. In contrast, during proplastid-to-etioplast development in the dark, the cpDNA levels increase from the stalk to the blade. Our results suggest that DNA replication continues in the etioplasts of the upper regions of the stalk and in the leaves. The cpDNA level decreases rapidly, however, after dark-grown seedlings are transferred to light and the etioplasts develop into photosynthetically active chloroplasts. Light, therefore, triggers the degradation of DNA in maize chloroplasts. The cpDNA is retained in the leaf blade of seedlings grown under red, but not blue light. We suggest that light signaling pathways are involved in mediating cpDNA levels, and that red light promotes replication and inhibits degradation and blue light promotes degradation. For five of nine photosynthetic mutants, cpDNA levels in expanded leaves are higher than in wild type, indicating that nuclear genotype can affect the loss or retention of cpDNA.     

The structure of chloroplast DNA molecules and the effects of light on the amount of chloroplast DNA during development in Medicago truncatula

We used pulsed-field gel electrophoresis and restriction fragment mapping to analyze the structure of Medicago truncatula chloroplast DNA (cpDNA). We find most cpDNA in genome-sized linear molecules, head-to-tail genomic concatemers, and complex branched forms with ends at defined sites rather than at random sites as expected from broken circles. Our data suggest that cpDNA replication is initiated predominantly on linear DNA molecules with one of five possible ends serving as putative origins of replication. We also used 4',6-diamidino-2-phenylindole staining of isolated plastids to determine the DNA content per plastid for seedlings grown in the dark for 3 d and then transferred to light before being returned to the dark. The cpDNA content in cotyledons increased after 3 h of light, decreased with 9 h of light, and decreased sharply with 24 h of light. In addition, we used real-time quantitative polymerase chain reaction to determine cpDNA levels of cotyledons in dark- and light-grown (low white, high white, blue, and red light) seedlings, as well as in cotyledons and leaves from plants grown in a greenhouse. In white, blue, and red light, cpDNA increased initially and then declined, but cpDNA declined further in white and blue light while remaining constant in red light. The initial decline in cpDNA occurred more rapidly with increased white light intensity, but the final DNA level was similar to that in less intense light. The patterns of increase and then decrease in cpDNA level during development were similar for cotyledons and leaves. We conclude that the absence in M. truncatula of the prominent inverted repeat cpDNA sequence found in most plant species does not lead to unusual properties with respect to the structure of plastid DNA molecules, cpDNA replication, or the loss of cpDNA during light-stimulated chloroplast development.     

Acetyl-CoA carboxylase during development of plastids in wild-type and mutant barley seedlings.

A soluble acetyl-CoA carboxylase in homogenates of leaves from wild-type barley seedlings was studied. Centrifuging the homogenate at 150,000 X g did not reduce the total activity, but raised the specific activity. During chloroplast development in light-grown seedlings or during light-dependent greening of leaves grown in the dark, both the total activity of the carboxylase per plant and the specific activity per mg of protein in homogenates of the seedlings increased rapidly. The soluble leaf acetyl-CoA carboxylase was studied in a number of barley mutants with lesions in chloroplast development. In a group of three mutants light elicited an increase in acetyl-CoA carboxylase activity as in the wild-type. In two mutants light caused a decrease in activity. Dark-grown leaves of mutant albina-f17 contained levels of soluble acetyl-CoA carboxylase reached only in the light by the wild-type, whereas light-grown albina-f17 seedlings lacked carboxylase activities. The possibility is discussed that leaf cells contain two forms of acetyl-CoA carboxylase, one soluble with unknown location and a dissociable form located in the chloroplast.     

Ethylene is not involved in the blue light-induced growth inhibition of red light-grown peas

Although the growth of intact plants is inhibited by irradiation with blue light, the growth rate of isolated stem segments is largely unaffected by blue light. We hypothesized that this loss of responsiveness was a result of ethylene production as part of the wounding response. However, we found no interaction between ethylene- and blue light-induced growth inhibition in dark- or red light-grown seedlings of pea (Pisum sativum L.). Inhibition of growth begins in dark-grown seedlings exposed to blue light within 3 min of the onset of blue light, as was known for red light-grown seedlings. By contrast, ethylene-induced inhibition of growth occurs only after a lag of 20 to 30 min or more (dark-grown seedlings) or 60 min (red light-grown seedlings). Also, the inhibition response of red light-grown seedlings is the same whether ethylene is present from the onset of continuous blue-light treatment or not. Finally the spatial distribution of inhibition following blue light was different from that following ethylene treatment.     

Accumulation of D1 polypeptide in tobacco plastids is regulated via the untranslated region of the psbA mRNA.

The plastid psbA mRNA is present in all tissues, while the encoded 32 kDa D1 protein of photosystem II accumulates tissue-specifically and in response to light. To study the regulation of D1 accumulation, a chimeric uidA gene encoding beta-glucuronidase (GUS) under control of the psbA 5'- and 3'-regulatory regions (224 and 393 bp, respectively), was integrated into the tobacco plastid genome. A high level of GUS accumulation in leaves and the lack of GUS in roots, with uidA mRNA present in both tissues, indicated tissue-specific accumulation of the chimeric gene product. Light-regulated accumulation of GUS in seedlings was shown. (i) Light-induced accumulation (100-fold) of GUS in etiolated cotyledons was accompanied by only a modest increase in mRNA levels. (ii) Inhibition of GUS synthesis was observed in cotyledons when light-grown seedlings were transferred to the dark, with no reduction in mRNA levels. Tissue-specific and light-regulated accumulation of GUS indicates that D1 accumulation is controlled via cis-acting regulatory elements in the untranslated region of the psbA mRNA. We propose that in tobacco, control of translation initiation is the primary mechanism regulating D1 protein accumulation.     

Cell death of barley aleurone protoplasts is mediated by reactive oxygen species

The barley aleurone layer is a terminally differentiated secretory tissue whose activity is hormonally controlled. The plant hormone gibberellic acid (GA) stimulates the secretion of hydrolytic enzymes and triggers the onset of programmed cell death (PCD). Abscisic acid (ABA) antagonizes the effects of GA and inhibits enzyme secretion and PCD. Reactive oxygen species (ROS) are key players in many types of PCD, and data presented here implicate ROS in hormonally regulated death of barley aleurone cells. Incubation of aleurone layers or protoplasts in H(2)O(2)-containing media results in death of GA-treated but not ABA-treated aleurone cells. Cells that are programmed to die are therefore less able to withstand ROS than cells that are programmed to remain alive. Illumination of barley aleurone protoplasts with blue or UV-A light results in a rapid increase in intracellular H(2)O(2) production. GA-treated protoplasts die rapidly in response to this increase in intracellular H(2)O(2) production, but ABA-treated protoplasts do not die. The rate of light-induced death could be slowed by antioxidants, and incubating protoplasts in the dark with the antioxidant butylated hydroxy toluene reduces the rate of hormonally induced death. Taken together, these data demonstrate that GA-treated aleurone protoplasts are less able than ABA-treated protoplasts to tolerate internally generated or exogenously applied H(2)O(2), and strongly suggest that ROS are components of the hormonally regulated cell death pathway in barley aleurone cells.     

Inhibitory Effect of Gabaculine on the Biosynthesis of 5-Aminolevulinate in the Tigrina d12 Mutant of Barley

Six-day-old, dark-grown, seedlings of barley homozygous forthe recessive mutation tigrina d¹² accumulated 5-aminolevulinicacid (ALA) and protochlorophyll (ide) in amounts exceeding thewild type level. Transferring the etiolated mutant to lightresulted in the destruction of pigments and the deteriorationof the ALA forming system. Such deleterious effects did notoccurusing light-grown mutant or etiolated and greened wildtype seedlings. Gabaculine (GAB) at 50 µM inhibited ALAsynthesis by about 85% when etiolated wild type seedlings wereexposed to light. In light-grown leaves of either wild typeor mutant strain, ALA production was also sharply (ca. 75%)inhibited by GAB. During dark incubation, however, the inhibitionof ALA accumulation did not exceed 50% in all types of tissues.The results give further evidence for the operation of the C₅pathway in such seedlings since GAB decreased the biosynthesisof ALA to the same extent in both tigrina d¹² mutant and wildtype of barley. (Received July 2, 1990; Accepted May 7, 1991)     

Phytochrome control of plastid mRNA in mustard (Sinapis alba L.)

The steady-state levels of plastid RNA sequences in dark-grown and light-grown mustard (Sinapis alba L.) seedlings have been compared. Total cellular RNAs were labeled in vitro with ³²P and hybridized to separated restriction fragments of plastid DNA. Cloned DNA fragments which encode the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxylase (dimerizing), EC 4.1.1.39] and a 35,000 plastid polypeptide were used as probes to assess the levels of these two plastid mRNAs. The 1.22-kilobase-pair mRNA for the 35,000 polypeptide is almost undetectable in dark-grown seedlings, but is a major plastid mRNA in light-grown seedlings. The hybridization analysis of RNA from seedlings which were irradiated with red and far-red light indicates that the level of this mRNA, but not of LS mRNA, is controlled by phytochrome.Abbreviations LS large subunit - RuBP ribulose-1,5-bisphosphate - ptDNA plastid DNA     

Plastid movement in statocytes of the arg1 (altered response to gravity) mutant

The ability of a plant to respond to gravity is crucial for growth and development throughout the life cycle. A key player in the cellular mechanisms of gravitropism is ARG1 (altered response to gravity), a DnaJ-like protein that associates with components of the vesicular trafficking pathway and carries a C-terminal domain with similarities to cytoskeleton-associated proteins. The arg1-2 mutant of Arabidopsis thaliana has reduced and delayed gravitropism in roots, shoots, and inflorescence stems when grown in the light or dark. We performed light microscopic studies of plastid movement in the gravity-perceiving statocytes (endodermal cells) of hypocotyls of arg1-2 and WT light-grown seedlings following reorientation to better characterize the role of ARG1 in gravitropism. Cryofixation/freeze substitution procedures were used because they provide a reliable indication of rapid cellular events within the statocytes. Our results suggest that ARG1 affects gravitropism by reducing plastid movement/sedimentation, a process known to be essential for early phases of signaling cascades in the statocytes.     

Endonucleolytic activation directs dark-induced chloroplast mRNA degradation

Plastid mRNA stability is tightly regulated by external signals such as light. We have investigated the biochemical mechanism responsible for the dark-induced decrease of relative half-lives for mRNAs encoding photosynthetic proteins. Protein fractions isolated from plastids of light-grown and dark-adapted plants correctly reproduced an RNA degradation pathway in the dark that is downregulated in the light. This dark-dependent pathway is initiated by endonucleolytic cleavages in the petD mRNA precursor substrate proximal to a region that can fold into a stem–loop structure. Polynucleotide phosphorylase (PNPase) polyadenylation activity was strongly increased in the protein fraction isolated from plastids in dark-adapted plants, but interestingly PNPase activity was not required for the initiation of dark-induced mRNA degradation. A protein factor present in the protein fraction from plastids of light-grown plants could inactivate the endonuclease activity and thereby stabilize the RNA substrate in the protein fraction from plastids of dark-adapted plants. The results show that plastid mRNA stability is effectively controlled by the regulation of a specific dark-induced RNA degradation pathway.     

Light Promotion of Hypocotyl Gravitropism of a Starch-Deficient Tobacco Mutant Correlates with Plastid Enlargement and Sedimentation

Dark-grown hypocotyls of a starch-deficient mutant (NS458) of tobacco (Nicotiana sylvestris) lack amyloplasts and plastid sedimentation, and have severely reduced gravitropism. However, gravitropism improved dramatically when NS458 seedlings were grown in the light. To determine the extent of this improvement and whether mutant hypocotyls contain sedimented amyloplasts, gravitropic sensitivity (induction time and intermittent stimulation) and plastid size and position in the endodermis were measured in seedlings grown for 8 d in the light. Light-grown NS458 hypocotyls were gravitropic but were less sensitive than the wild type (WT). Starch occupied 10% of the volume of NS458 plastids grown in both the light and the dark, whereas WT plastids were essentially filled with starch in both treatments. Light increased plastid size twice as much in the mutant as in the WT. Plastids in light-grown NS458 were sedimented, presumably because of their larger size and greater total starch content. The induction by light of plastid sedimentation in NS458 provides new evidence for the role of plastid mass and sedimentation in stem gravitropic sensing. Because the mutant is not as sensitive as the WT, NS458 plastids may not have sufficient mass to provide full gravitropic sensitivity.     

Cartesian-diver microrespirometry: a technique for the accurate measurement of respiratory flux in plant cells

Abstract. A Cartesian-diver microrespirometer system is described which can be used to measure respiratory fluxes of oxygen accurately for cells of higher plants in a liquid phase. This microrespirometry technique has been adapted from protozoological and microfaunal studies to plant physiology. The Cartesian-diver has considerable scope for investigation of oxygen flux in plant cells and has several advantages compared to the oxygen electrode in terms of sensitivity to changing oxygen levels in respiring material. Because the volumes of liquid are small in the Cartesian-divers, diffusional distances arc measured in micrometres and there is no need for stirring to overcome diffusional problems, thus minimizing the risk of mechanical damage to the experimental material. In addition, only very small quantities of experimental material are required for the Cartesian-diver which is invaluable where only limited amounts of tissue or numbers of cells can be obtained. Examples of respiratory oxygen consumption by protoplasts from intercalary meristematic regions of light-grown barley ( Hordeum vulgare L.c.v. Patty) seedlings, in response to abscisic and gibberellic acids, are presented. The advantages and disadvantages of Cartesian-diver microrespirometry compared to oxygen electrodes are also discussed.