Ralph A. Backhaus

Induction and control of chromoplast-specific carotenoid genes by oxidative stress

The differentiation of chloroplasts into chromoplasts involves a series of biochemical changes that culminate with the intense accumulation of long chain chromophore carotenoids such as lycopene, rhodoxanthin, astaxanthin, anhydroeschsoltzxanthin, capsanthin, and capsorubin. The signal pathways mediating these transformations are unknown. Chromoplast carotenoids are known to accumulate in green tissues experiencing stress conditions, and studies indicate that they provide efficient protection against oxidative stress. We tested the role of reactive oxygen species (ROS) as regulators of chromoplast carotenoid biosynthesis in vivo. The addition of ROS progenitors, such as menadione,tert-butylhydroperoxide, or paraquat and prooxidants such as diamide or buthionine sulfoximine to green pericarp discs of pepper fruits rapidly and dramatically induce the simultaneous expression of multiple carotenogenic gene mRNAS that give rise to capsanthin. Similarly, down-regulation of catalase by amitrole induces expression of carotenogenic gene mRNAs leading to the synthesis of capsanthin in excised green pericarp discs. ROS signals from plastids and mitochondria also contribute significantly to this process. Analysis of the capsanthin-capsorubin synthase promoter in combination with a β-glucuronidase reporter gene reveals strong activation in transformed pepper protoplasts challenged with the above ROS. Collectively these data demonstrate that ROS act as a novel class of second messengers that mediate intense carotenoid synthesis during chromoplast differentiation.

Rubber transferase activity in rubber particles of guayule

Abstract Rubber transferase (RuT) activity, measured as incorporation of radiolabelled isoprene from [ 14 C]isopentenyl pyrophosphate (IPP) into rubber, was assayed in suspensions of washed rubber particles (WRPs) purified from stembark tissue of Parthenium argentatum . Isolated WRPs had high RuT activity which was not diminished even after repeated washing, demonstrating the firm association of the enzyme with the particles. The activity of RuT was characterized with respect to substrate and WRP concentration. The rate of IPP-incorporation was dependent upon the concentration of two substrates, IPP and the allylic pyrophosphate starter molecule E , E -farnesyl pyrophosphate (FPP). The enzyme present in 6 × 10 10 WRPs per ml was saturated by 1 mM IPP and 20 μM FPP. Under saturating cosubstrate concentrations the apparent K m of RuT was ca 300 μM IPP and 3 μM FPP. Analysis of WRPs by SDS-PAGE revealed a simple protein profile characteristic of guayule rubber particles. A successful and facile assay for IPP-polymerization by isolated rubber particles is described.

In vitro culture as a potential method for the conservation of endangered plants possessing crassulacean acid metabolism

Rare and endangered plants possessing crassulacean acid metabolism (CAM), such as cacti, usually present limited reproductive capacities and very slow growth rates. The use of in vitro culture can overcome these difficulties. The massive in vitro production of new propagules which result in totally regenerated plants is described for two endangered cacti, Obregonia denegrii Fric. and Coryphantha minima Baird. A comparison of in vitro and ex vitro growth rates demonstrated that the in vitro environment notably accelerates cacti growth. Malic acid titratable acidity indicated that increase of the net carbon dioxide uptake is associated with active growth. This might be related to particular factors of the in vitro environment such as the high relative humidity inside the culture vessels, or growth regulators supplemented to the growth media. In vitro-derived cacti showed a proficient re-establishment capability which could be related to their succulence since water loss during transplantation did not represent a crucial hydric stress. Succulence and plasticity of the CAM metabolic pathway in plants like cactus, represent some possible advantageous for the application of in vitro propagation techniques in a number of endangered, succulent plants like members of the Cactaceae, Agavaceae, Orchidaceae, or Bromeliaceae families.

Alterations in growth and crassulacean acid metabolism (CAM) activity of in vitro cultured cactus

Unlike C-3 plants, cacti possess a crassulacean acid metabolism (CAM) physiology that can alter the pattern of carbon uptake and affect plant growth under artificial environmental conditions, especially in tissue culture. In vitro-derived plantlets of Coryphantha minima grew 7-fold larger than plants cultured under similar ex vitro conditions. Growth regulators incorporated into the culture media during shoot proliferation stage of micropropagation had a strong influence on this increased growth. Other important factors that contributed to increased growth under in vitro conditions were high relative humidity and sugar in the culture medium. An analysis of gas exchange and daily fluctuations of malic acid levels revealed an increase in net photosynthetic rate, in terms of carbon assimilation, by in vitro plants compared with that of ex vitro plants. This stimulated photosynthesis in the presence of an external carbon source was unexpected but apparently true for cacti exhibiting CAM physiology. Unlike CAM plants grown in ex vitro conditions, net CO2 uptake by in vitro-cultured cacti occurred continuously in the light as well as the dark. Once regenerated, cacti were transferred to ex vitro conditions where the normal CAM pathway resumed with a concomitant reduction in growth and CO2 uptake. These results showed that growth of cacti can be considerably accelerated by in vitro culture.

RUBBER FORMATION IN PLANTS—A MINI-REVIEW

The ability of plants to synthesize rubber is based on a single enzyme, rubber transferase, which causes the cis-polymerization of isoprene units, as isopentenyl pyrophosphate, into long, single stranded hydrocarbons. These are assembled and accumulated in the cell as rubber particles which range in size from 0.01 to 15 μm and are bounded by an osmiophilic film. Parthenium argentatimi (guayule) and Hevea brasiliensis represent two species which accumulate large quantities of high molecular weight rubber by different intercellular routes. Rubber synthesis takes place in parenchyma cells in guayule and in specialized latex vessels in Hevea. Biochemical questions concerning rubber synthesis are discussed.

Effects of atmospheric CO2 enrichment on the growth and development of Hymenocallis littoralis (Amaryllidaceae) and the concentrations of several antineoplastic and antiviral constituents of its bulbs

Two 2-yr crops of tropical spider lily (Hymenocallis littoralis) plants were grown in field soil in clear-plastic-wall open-top enclosures in the Sonoran Desert environment of central Arizona. Half of the plants were exposed to ambient air of 400 ppm atmospheric CO(2) concentration and half of them were exposed to air of 700 ppm CO(2). This 75% increase in the airs CO(2) content resulted in a 48% increase in aboveground plant biomass and a 56% increase in belowground (bulb) biomass. It also increased the concentrations of five bulb constituents that have been demonstrated to possess anticancer and antiviral activities. Mean percentage increases in these concentrations were 6% for a two-constituent (1:1) mixture of 7-deoxynarciclasine and 7-deoxy-trans-dihydronarciclasine, 8% for pancratistatin, 8% for trans-dihydronarciclasine, and 28% for narciclasine, for a mean active ingredient percentage concentration increase of 12%. Combined with the 56% increase in bulb biomass, these percentage concentration increases resulted in a mean active ingredient increase of 75% for the 75% increase in the airs CO(2) concentration used in our experiments.

PURIFICATION AND CHARACTERIZATION OF AN ABUNDANT RUBBER PARTICLE PROTEIN FROM GUAYULE

Abstract Guayule rubber particles, which have high rubber transferase (RuT) activity, contain several characteristic proteins. The most abundant of these was purified by preparative gel electrophoresis and characterized. Designated the rubber particle protein (RPP), it is present in all guayule lines studied to date. RPP has an apparent M r of 48 500 and an isoelectric point of pI 6.2 which corresponds to a pI of 6.17 predicted by amino acid analysis. Antibodies were raised successfully against RPP and used for immunological analysis. Concanavalin A binds to blotted RPP indicating that it is a glycoprotein. When concanavalin A is added to purified rubber particles, it results in their rapid agglutination. However, protease treatments of purified rubber particles indicate that RPP is positioned inside the particle. Thus, it appears that RPP is positioned within the rubber particle, but at the surface, and that its oligosaccharide moiety extends to the particle exterior.

Soil and environmental factors related to nodulation in Cowania and Purshia

SummaryNitrogen fixing trees and shrubs may be useful in revegetation efforts. The possibility that soil and environmental factors may influence a soils capability to produce nodulated seedlings was explored.Purshia tridentata andCowania mexicana var. Stansburiana seedlings were grown in greenhouse trials using ten soils from native sites for each of the two genera. Treatments included a control and a six mmole nitrogen amendment as NH4NO3 for both surface and subsurface samples. Nodulation was often sparse for seedlings grown in surface collected samples. Although nodulation was usually better in subsoil samples, even some subsoils produced few or no nodules. Nitrogen additions inhibit nodulation and although soil nitrogen may be inhibitory in some unamended surface soils it is probably not a general cause of sparse nodulation. Nodule masses showed the same trends as nodule number but varied less with treatment and depth of soil source. Seedlings compensated for sparse nodulation with an increase in mass per nodule. Incidence of nodulation was related to some soil and environmental factors. Multiple regression analysis explained a substantial portion of nodulation variability. Soils from lower elevations with less precipitation did not produce well nodulated seedlings even in well watered greenhouse trials. Micronutrient cations, potassium, and phosphorus are positively correlated with nodulation incidence. The two genera were generally similar in nodulation responses to soil and environmental factors.

In vitro propagation of Agave arizonica Gentry & Weber

Agave arizonica Gentry & Weber, an extremely rare and endangered species native to Arizona, was successfully propagated in vitro using modified Murashige and Skoog media. Adventitious shoots developed from callus which formed on bulbil explants grown in a medium supplemented with 1.4 μM 2,4-dichlorophenoxyacetic acid. These shoots proliferated by subculture in media supplemented with 44.4 μM 6-benzylaminopurine, and either 0.5 or 5.4 μM naphthaleneacetic acid. Rooting occurred on shoots transferred to a growth regulator free medium. Rooted plants transferred to potting soil could be established under greenhouse conditions following gradual acclimatization indoors.

Nucleotide Sequence of a cDNA for a P2 60S Acidic Ribosomal Protein from Parthenium argentatum

In screening a stem-bark cDNA library from guayule (Parthenium argentatum) for genes involved in isoprenoid biogenesis, we isolated a full-length gene for a P2 60S acidic ribosomal protein that shows sequence similarity to vertebrate (Rich and Steitz, 1987), invertebrate (Qian et al., 1987), and yeast (Beltrame and Bianchi, 1987, 1990; Remacha et al., 1988) 60S acidic ribosomal proteins (Table I). The guayule P2 sequence is a full-length cDNA; a partial cDNA has been described for Arabidopsis (C. Bardet, M. Axelos, D. Tremousaygue, M. Lebas, T. Lagravere, and B. Lescure, unpublished data; EMBL accession number Z17464) with 65.5% identity at the nucleic acid level and 54.5% identity and 74.5% similarity at the amino acid level. Our interest in this gene extends to its potential use in experimental studies of Parthenium, from which few genes are at present available. Being a ribosomal protein, it should display minimal variations in expression and thus serve as a suitable internal control for gene expression studies in this species. The P2 60S acidic ribosomal protein plays a crucial role in the elongation step of protein synthesis. The P2 proteins exist as heterodimers composed of a and d subunits. Comparison of the P2 Parthenium sequence with both the a (Beltrame and Bianchi, 1987) and p (Beltrame and Bianchi, 1990) subunits of fission yeast shows nearly the same identity, 52.7 versus 50.9%, respectively, making it difficult to classify the guayule protein as P2 a or P2 F. Comparison of the P2 Parthenium sequence to the human LA2 protein (Rich and Steitz, 1987) shows 59.0% identity at the nucleic acid level and 54.5% identity and 79.5% similarity on the amino acid level, with a similar level of identity for the Drosophila gene (Qian et al., 1987).