Richard E. Kendrick

Photomorphogenic Responses of Long Hypocotyl Mutants of Tomato

Summary Tomato mutants at the au, yg-2, and yg-6 loci have yellow-green leaves, elongated hypocotyls and reduced anthocyanin content when grown in white light. Mutant auw, in contrast to its wild type, shows little or no effect of light on seed germination, anthocyanin synthesis, and hypocotyl elongation. In addition the chlorophyll content is reduced, the chlorophyll a/b ratio is increased and the stacking of the thylakoids in the chloroplasts is greatly reduced_ Spectrophotometrically determined phytochrome is absent or strongly reduced in its seeds, dark-grown hypocotyls, light-grown leaves, and roots. These results suggest that the phenotype of these mutants is correlated with a reduced phytochrome content.

PHOTOMORPHOGENETIC MUTANTS OF HIGHER PLANTS

List of abbreviationsPhotomorphogenesis of higher plants is a complex process resulting from the co-action of at least three different photoreceptors: phytochrome (P), blue light (BL)/UV-A photoreceptor (cryptochrome) and UV-B photoreceptor (Mohr, 1986). The possible existence of multiple photoreceptor types [e.g. light-labile [type I] and light-stable [type 111 P) adds to the complexity (Vierstra and Quail, 1986; Pratt and Cordonnier, 1987; Nagatani et al . , 1987)l. Since both these types have very similar absorption spectra it is impossible to say which is responsible for a particular response. In addition, there appear to be multiple working mechanisms of some photoreceptors, e.g. very low fluence response (VLFR), low fluence response (LFR) and high irradiance response (HIR) of P (Kronenberg and Kendrick, 1986). Genotypes (often as induced mutants) in which certain parts of the morphogenetic pathway are eliminated, provide the tools for further physiological analysis. Such genotypes will exhibit a photomorphogenesis different and often simpler than their wild type. The relevance of the deleted part in the mutant is directly indicated by its difference in response compared to its isogenic wild type. Mutants with other defects, such as chlorophyll (Chl) or carotenoid deficiency, can also be very useful in photomorphogenetic research. The available literature is reviewed and the potential of a genetic approach to photomorphogenesis is outlined.

Photocontrol of seed germination

It has been known for a long time that light influences the germination of seeds of many species. In the early 20th century it was found that germination of some species was inhibited by light, while in others germination was promoted by light. Flint and McAlister (1937) were the first to recognize the promotive effect of red light (R) and the inhibitory effect of far-red light (FR) on the germination of lettuce seeds (Fig. 1). They prepared their seed samples by giving them an initial promotive irradiation, estimated to bring them to 50% germination. Subsequently they irradiated for 24 h and noted promotive and inhibitory deviations from 50% germination. In fact Flint and McAlister were the first to make a preliminary action spectrum of the light mediated seed germination. However they were not aware of the great importance of the R/FR reversibility of the pigment system and their papers were neglected until the 1940’s when H. A. Borthwick, S. B. Hendricks, M. W. Parker and H. W. Siegelman studied the influence of light on flowering, at the United States Department of Agriculture, Beltsville (Butler 1982). They recognized the great importance of the R/FR reversible pigment in plant growth and development and they also introduced the name phytochrome (Part 1).

Physiological characterization of a high-pigment mutant of tomato.

Abstract— A high‐pigment (hp) mutant, which shows exaggerated phytochrome responses and three other genotypes of Lycopersicon esculenrum Mill. cv. Ailsa Craig: the aurea (au) mutant deficient in the bulk light‐labile phytochrome (PI) pool, the au, hp double mutant, and their isogenic wild type, were used in this study. Measurements of phytochrome destruction in red light (R) revealed that the exaggerated responses of the hp mutant are not caused by a higher absolute phytochrome level or a reduced rate of phytochrome destruction. Fluence‐response relationships for anthocyanin synthesis after a blue‐light pretreatment were studied to test if the hp mutant conveys hypersensitivity to the far‐red light (FR)‐absorbing form of phytochrome (Pfr), i.e. the threshold of Pfr required to initiate the response is lower. The response range for the hp mutant and wild type was identical, although the former exhibited a 6‐fold larger response. Moreover, the kinetics of anthocyanin accumulation in continuous R were similar in the wild‐type and hp‐mutant seedlings, despite the latter accumulating 9‐fold more anthocyanin. Since the properties of phytochrome are the same, the hp mutation appears to affect the state of responsiveness amplification, i.e. the same amount of Pfr leads to a higher response in the hp mutant. We therefore propose that the hp mutation is associated with an amplification step in the phytochrome transduction chain. Escape experiments showed that the anthocyanin synthesis after different light pretreatments terminated with a R pulse was still 50% FR reversible after 4–6 h darkness, indicating that the Pfr pool regulating this response must be relatively stable. However, fluence‐rate response relationships for anthocyanin synthesis and hypocotyl growth induced by a 24‐h irradiation with 451, 539, 649, 693, 704 and 729 nm light showed no or a severely reduced response in the au and au, hp mutants, suggesting the importance of PI in these responses. We therefore propose that the capacity for anthocyanin synthesis (state of responsiveness amplification) could be established by PI, while the anthocyanin synthesis is actually photoregulated via a stable Pfr pool. The Hp gene product is proposed to be an inhibitor of the state of responsiveness amplification for responses controlled by this relatively stable Pfr species.

Spotlight on phytochrome nomenclature.

For many years after its discovery over four decades ago, the regulatory photoreceptor phytochrome was widely considered, implicitly or otherwise, to be a single molecular species (Sage, 1992). However, steadily accumulating evidence from physiological, spectrophotometric, biochemical, and immunochemical studies made it increasingly difficult to reconcile the diversity of phytochrome-mediated responses with the action of asingle photoreceptor species (Smith and Whitelam, 1990; Quail, 1991). There is now direct molecular evidence that the phytochrome polypeptide is encoded by multiple, divergent genes, at least in higher plants (Quail, 1994). Moreover, studies with photomorphogenic mutants and transgenic plants overexpressing different phytochromes have established that individual family members perform discrete photosensory functions in interpreting and processing information from the light environment (Somers et al., 1991; McCormac et al., 1992, 1993; Smith, 1992; Dehesh et al., 1993; Nagatani et al., 1993; Parks and Quail, 1993; Reed et al., 1993, 1994; Whitelam et al., 1993). In Arabidopsis, the phytochrome polypeptide is encoded by a family of five genes (Sharrock and Quail, 1989; Clack et al., 1994). These genes were initially designated phyA, phyB, phyC, phyD, and phyf (note lower case), based on the bacteria1 system of gene nomenclature (Sharrock and Quail, 1989; Quail, 1991, 1994). However, as increasing numbers of publications on the different phytochrome family members have appeared from multiple laboratories, a variety of nomenclature systems and symbols have been used to describe not only the genes themselves, but also the gene products and photochemical forms of the individual family members. We believe that it is timely, therefore, to implement a more standardized terminology to describe the phytochrome system. In addition, the recent demonstration that a series of photomorphogenic mutants of Arabidopsis, isolated independently in

Acetylcholine in plants : presence, metabolism and mechanism of action

Acetylcholine (ACh) has been detected in representatives of many taxonomic groups throughout the plant kingdom. The site of its synthesis in plants is probably young leaves. In some plant species choline acetyltransferase (ChAT) activity has been found. This enzyme showing properties similar to animal ChAT, probably participates in ACh synthesis from its precursors, choline and acetyl-Coenzyme A. Acetylcholinesterase (AChE) activity has also been found in many plant tissues. This enzyme decomposes ACh and exhibits properties similar to animal AChE. The presence of both ChAT and AChE in plant tissues suggests that ACh undergoes similar metabolism in plants as it does in animals. Exogenous ACh affects phytochrome-controlled plant growth and development. Mimicking red light (R), ACh stimulates adhesion of root tips to a glass surface and influences leaf movement and membrane permeability to ions. It also affects seed germination and plant growth. Moreover, ACh can modify some enzyme activity and the course of some metabolic processes in plants. Acetylcholine in the presence of calcium ions (Ca2+), like R stimulates swelling of protoplast isolated from etiolated wheat leaves. It is proposed that the primary mechanism of action of ACh in plant cells is via the regulation of membrane permeability to protons (H+), potassium ions (K+), sodium ions (Na+) and Ca2+.ZusammenfassungAcetylcholin (ACh) wurde in Vertretern vieler taxonomischer Gruppen des Pflanzreiches gefunden. Es wird wahrscheinlich inden jungen Blättern synthetisiert. In einigen Pflanzen hat man daneben Cholin-Acetyltransferase (ChAT)-Activität nachweisen können; dieses Enzym ziegt ähnliche Eigenschaften wie tierische ChAT und ist offenbar an der ACh-Synthese aus sienen Vorstufen Cholin und Acetyl-Coenzym A beteiligt. Acetylcholineesterase (AChE)-Activität wurde ebenfalls in vielen Pflanzengeweben gefunden; dieses Enzym spaltet ACh und ziegt ähnliche Eigenschaften wie tierische AChE. Die Anwesenheit von ChAT und AChE in pflanzlichem Gewebe läßt vermuten, daß ACh in Pflanzen einem ähnlichen Metabolismus unterliegt wie im tierischen System.Ähnlich wie Rotlicht stimuliert ACh die Anheftung von Wurzelspitzen an Glasoberflächen und beeinflußt Blattbewegung und Membranpermeabilität für Ionen; darüber hinaus beeinflußt es Samenkeimung und pflanzliches Wachstum. Des weiteren kann ACh Enzym-Aktivitäten modifizieren und dadurch den Ablauf einiher metabolischer Prozesse in Pflanzen. Schließlich stimuliert ACh in Gegenwart von Calcium-Ionen (Ca2+), ähnlich wie Rotlicht, das Schwellen von Protoplasten etiolierter Weizenblätter. Es wird vermutet, daß die Primärwirkung von ACh in Pflanzenzellen durch Regulation der Membranpermeabilität für Protonen (H+), Kaliumionen (K+), Natriumionen (Na+) und Ca2+ erfolgt.

New lv mutants of pea are deficient in phytochrome B

The lv-1 mutant of pea (Pisum sativum L.) is deficient in responses regulated by phytochrome B (phyB) in other species but has normal levels of spectrally active phyB. We have characterized three further lv mutants (lv-2, lv-3, and lv-4), which are all elongated under red (R) and white light but are indistinguishable from wild type under far-red light. The phyB apoprotein present in the lv-1 mutant was undetectable in all three new lv mutants. The identification of allelic mutants with and without phyB apoprotein suggests that Lv may be a structural gene for a B-type phytochrome. Furthermore, it indicates that the lv-1 mutation results specifically in the loss of normal biological activity of this phytochrome. Red-light-pulse and fluence-rate-response experiments suggest that lv plants are deficient in the low-fluence response (LFR) but retain a normal very-low-fluence-rate-dependent response for leaflet expansion and inhibition of stem elongation. Comparison of lv alleles of differing severity indicates that the LFR for stem elongation can be mediated by a lower level of phyB than the LFR for leaflet expansion. The retention of a strong response to continuous low-fluence-rate R in all four lv mutants suggests that there may be an additional phytochrome controlling responses to R in pea. The kinetics of phytochrome destruction and reaccumulation in the lv mutant indicate that phyB may be involved in the light regulation of phyA levels.

PHOTOCONTROL OF ANTHOCYANIN SYNTHESIS IN TOMATO SEEDLINGS: A GENETIC APPROACH*

The photocontrol of anthocyanin synthesis in dark‐grown seedlings of tomato (Lycopersicon esculentum Mill.) has been studied in an aurea (au) mutant which is deficient in the labile type of phytochrome, a high pigment (hp) mutant which has the wild‐type level of phytochrome and the double mutant au/hp, as well as the wild type. The hp mutant demonstrates phytochrome control of anthocyanin synthesis in response to a single red light (RL) pulse, whereas there is no measurable response in the wild type and au mutant. After pretreatment with 12 h blue light (BL) the phytochrome regulation of anthocyanin synthesis is 10‐fold higher in the hp mutant than in the wild type, whilst no anthocyanin is detectable in the au mutant, thus suggesting that it is the labile pool of phytochrome which regulates anthocyanin synthesis. The au/hp double mutant exhibits a small (3% of that in the hp mutant) RL/far‐red light (FR)‐reversible regulation of anthocyanin synthesis following a BL pretreatment. It is proposed that the hp mutant is hypersensitive to the FR‐absorbing form of phytochrome (Pfr) and that this (hypersensitivity) establishes response to the low level of Pfl. (below detection limits in phytochrome assays) in the au/hp double mutant.

Interaction of light and temperature on the germination of Rumex obtusifolius L.

Seeds (nutlets) of Rumex obtusifolius L. fail to germinate in darkness at 25° C, but are stimulated by short exposure to red light (R) the effectiveness of which can be negated by a subsequent short exposure to far red light (F) indicating phytochrome control. Short periods of elevated temperature treatment (e.g. 5 min at 35° C) can induce complete germination in darkness. Although short F cannot revert the effect of 35° C treatment, cycling the phytochrome pool by exposure to short R before short F results in reversion of at least 50% of the population. Prolonged or intermittent F can also revert the germination induced by 35° C treatment. The effect of elevated temperature treatment is interpreted on the basis of two possible models; (i) that it increases the sensitivity of the seeds to a low level of pre-existing active form of phytochrome (Pfr) (ii) that it induces the appearance of Pfr in the dark. In both cases it is envisaged that elevated temperature treatment and Pfr control germination at a common point in the series of reactions that lead to germination.

The Phytochrome-Deficient pcd1 Mutant of Pea Is Unable to Convert Heme to Biliverdin IX[alpha].

We isolated a new pea mutant that was selected on the basis of pale color and elongated internodes in a screen under white light. The mutant was designated pcd1 for phytochrome chromophore deficient. Light-grown pcd1 plants have yellow-green foliage with a reduced chlorophyll (Chl) content and an abnormally high Chl a/Chl b ratio. Etiolated pcd1 seedlings are developmentally insensitive to far-red light, show a reduced response to red light, and have no spectrophotometrically detectable phytochrome. The phytochrome A apoprotein is present at the wild-type level in etiolated pcd1 seedlings but is not depleted by red light treatment. Crude phytochrome preparations from etiolated pcd1 tissue also lack spectral activity but can be assembled with phycocyanobilin, an analog of the endogenous phytochrome chromophore phytochromobilin, to yield a difference spectrum characteristic of an apophytochrome-phycocyanobilin adduct. These results indicate that the pcd1-conferred phenotype results from a deficiency in phytochrome chromophore synthesis. Furthermore, etioplast preparations from pcd1 seedlings can metabolize biliverdin (BV) IX[alpha] but not heme to phytochromobilin, indicating that pcd1 plants are severely impaired in their ability to convert heme to BV IX[alpha]. This provides clear evidence that the conversion of heme to BV IX[alpha] is an enzymatic process in higher plants and that it is required for synthesis of the phytochrome chromophore and hence for normal photomorphogenesis.