M. Koornneef

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.

A Temporarily Red Light-Insensitive Mutant of Tomato Lacks a Light-Stable, B-Like Phytochrome

We have selected four recessive mutants in tomato (Lycopersicon esculentum Mill.) that, under continuous red light (R), have long hypocotyls and small cotyledons compared to wild type (WT), a phenotype typical of phytochrome B (phyB) mutants of other species. These mutants, which are allelic, are only insensitive to R during the first 2 days upon transition from darkness to R, and therefore we propose the gene symbol tri (temporarily red light insensitive). White light-grown mutant plants have a more elongated growth habit than that of the WT. An immunochemically and spectrophotometrically detectable phyB-like polypeptide detectable in the WT is absent or below detection limits in the tri1 mutant. In contrast to the absence of an elongation growth response to far-red light (FR) given at the end of the daily photoperiod (EODFR) in all phyB-deficient mutants so far characterized, the tri1 mutant responds to EODFR treatment. The tri1 mutant also shows a strong response to supplementary daytime far-red light. We propose that the phyB-like phytochrome deficient in the tri mutants plays a major role during de-etiolation and that other light-stable phytochromes can regulate the EODFR and shade-avoidance responses in tomato.

High pigment mutants of tomato exhibit high sensitivity for phytochrome action

Summary Anthocyanin synthesis and hypocotyl growth have been studied in tomato ( Lycopersicon esculentum Mill.) seedlings of high pigment ( hp ) mutants, aurea ( au ) mutants deficient in the labile type of phytochrome, an auhp double mutant and wild types. The phytochrome controlled [red light (RL)/far-red light reversible] anthocyanin synthesis occurring in a 24 h dark period after a 12 h pretreatment with RL or blue light (BL) is similar in the hp mutant, whereas in the case of the wild type, pretreatment with BL is more effective than RL. When grown under continuous broad-band UV-A, BL and RL for 5 d the au and auhp double mutants only accumulate low levels of anthocyanin compared to the wild type and the hp mutant. Under these conditions the hp mutant accumulates the highest levels of anthocyanin, but the relative effectiveness of RL and BL in the hp mutant and the wild type is reversed (BL being more effective than RL in wild type, whereas in the hp mutant RL is more effective than BL). These results suggest that the hp mutation enables maximal anthocyanin synthesis to be achieved without activation of the BL photoreceptor. When grown for 7 d in a regime of 14 h white light/10 h darkness the activation of anthocyanin synthesis is reduced in the au and auhp mutants compared to the wild type and the hp mutant. After the same treatment, the hypocotyls of hp -mutant seedlings of each cultivar are similar in length to those of their corresponding wild types and those of the au and auhp mutants are longer. However, after 5 d continuous low fluence rate (3 μ-tmol m- 2 s- 1 ) UV-A, BL and RL, the hypocotyl growth of the hp mutant is inhibited more than the wild type. The auhp double mutant shows a small but significantly higher anthocyanin accumulation than the au mutant, as well as increased inhibition of hypocotyl growth. It is proposed that the hp mutation increases the sensitivity to the labile phytochrome pool.

Photophysiology of a Tomato Mutant Deficient in Labile Phytochrome

Summary Photomorphogenetic responses have been studied in a tomato (Lycopersicon esculentum Mill.) aurea (au) mutant which has yellow-green leaves and elongated hypocotyls when grown in white light. Whereas etiolated tissue of the au mutant contains

Photomorphogenetic Responses of a Long Hypocotyl Mutant of Cucumis sativus L.

Summary Photomorphogenetic responses have been studied in a Cucumis sativus mutant (lh) which has long hypocotyls in white light (WL) compared to its wild type. Seeds and dark-grown etiolated seedlings of the lh mutant and wild type contain similar levels of spectrophotometrically detectable phytochrome. In continuous WL after 20 d there is a six-fold difference in hypocotyl length as a result of differences in cell elongation since the hypocotyls were estimated to be composed of similar numbers of cells. Although de-etiolation was retarded in the lh mutant in red light (RL), RL/far-red light reversibility of hypocotyl elongation and cotyledon expansion was shown. However, de-etiolated seedlings (seedlings pre-irradiated with 8 h WL) showed no inhibition of hypocotyl growth or promotion of cotyledon expansion in response to RL. Covering of one cotyledon of de-etiolated wildtype seedlings resulted in a marked curvature of the hypocotyl in RL given from above; the lh mutant did not exhibit this simulated phototropism. Little difference in responsiveness of wild type and lh mutant to UV-A and blue light (BL) was observed. It is provisionally concluded that this lh mutant lacks phytochrome function in light-grown plants perhaps due to the absence or lack of function of light stable phytochrome. The usefulness of the lh mutant for understanding the role of phytochrome and the BL-photoreceptor in controlling photomorphogenesis in light-grown plants is discussed.

Phytochrome control of anthocyanin biosynthesis in tomato seedlings : Analysis using photomorphogenic mutants

Anthocyanin biosynthesis has been studied in hypocotyls and whole seedlings of tomato (Lycoperskon esculentum Mill.) wild types (WTs) and photomorphogenic mutants. In white light (WL)/dark (D) cycles the fri1 mutant, deficient in phytochrome A (phyA), shows an enhancement of anthocyanin accumulation, whereas the tri1 mutant, deficient in phytochrome Bl (phyBl) has a WT level of anthocyanin. Under pulses of red light (R) or R followed by far‐red light (FR) given every 4 h, phyA is responsible for the non‐R/FR reversible response, whereas phyBl is partially responsible for the R/FR reversible response. From R and blue light (B) pretreatment studies, B is most effective in increasing phytochrome responsiveness, whereas under R itself it appears to be dependent on the presence of phyBl. Anthocyanin biosynthesis during a 24 h period of monochromatic irradiation at different flu‐ence rates of 4 day‐old D‐grown seedlings has been studied. At 660 nm the fluence rate‐response relationships for induction of anthocyanin in the WT are similar, yet complex, showing a low fluence rate response (LFRR) and a fluence rate‐dependent high irradiance response (HIR). The high‐pigment‐1 (hp‐1) mutant exhibits a strong amplification of both the LFRR and HIR. The fri1 mutant lacks the LFRR while retaining a normal HIR. In contrast, a transgenic tomato line overexpressing the oat PHYA3 gene shows a dramatic amplification of the LFRR. The tri1 mutant, retains the LFRR but lacks the HIR, whereas the fri1, tri1 double mutant lacks both components. Only an LFRR is seen at 729 nm in WT; however, an appreciable HIR is observed at 704 nm, which is retained in the tri1 mutant and is absent in the fri1 mutant, indicating the labile phyA pool regulates this response component.

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.

The mapping of phytochrome genes and photomorphogenic mutants of tomato.

The map positions of five previously described phytochrome genes have been determined in tomato (Lycopersicon esculentum Mill.) The position of the yg-2 gene on chromosome 12 has been confirmed and the classical map revised. The position of the phytochrome A (phy A)-deficient fri mutants has been refined by revising the classical map of chromosome 10. The position of the PhyA gene is indistinguishable from that of the fri locus. The putative phyB1-deficient tri mutants were mapped by classical and RFLP analysis to chromosome 1. The PhyB1 gene, as predicted, was located at the same position. Several mutants with the high pigment (hp) phenotype, which exaggerates phytochrome responses, have been reported. Allelism tests confirmed that the hp-2 mutant is not allelic to other previously described hp (proposed here to be called hp-1) mutants and a second stronger hp-2 allele (hp-2j) was identified. The hp-2 gene was mapped to the classical, as well as the RFLP, map of chromosome 1.

Physiological characterization of exaggerated photoresponse mutants of tomato.

Summary Four monogenic mutants in tomato (Lycopersicon esculentum Mill.), i.e. three recessive mutations, high-pigment-1 (hp-1), high-pigment-2 (hp-2), and atroviolacea (atv), and one dominant mutation, Intense pigmentation (Ip), were used in this study. These mutants all show exaggerated photoresponses during deetiolation and seedlings having shorter hypocotyls and higher anthocyanin levels. The hp-1 and hp-2 have higher chlorophyll levels in immature fruit, giving them a dark green colour. Spectrophotometrical and immunological analyses of phytochrome A and phytochrome B revealed no differences between the mutants and the wild types (WTs), suggesting that the mutants are not photoreceptor mutants. Both hp-1 and hp-2 accumulate high levels of anthocyanin in continuous blue (B) and red (R) broad-band light. In contrast, atv has a WT level of anthocyanin in B and an exaggerated response in R. The Ip mutant has the opposite response: a WT level of anthocyanin in R and an exaggerated response in B. In B and R pretreat-ment studies, all mutants show an enhanced R/far-red light (FR)-reversible response compared with WT, but the Ip mutant shows a preferentially enhanced response in B. The hp-1 mutant exhibits a strong amplification of both the low fluence rate response and high irradiance response components of anthocyanin biosynthesis in red light. The atv mutant shows the strongest amplification of the HIR component. The Ip mutant exhibits an exaggerated anthocyanin response in B. All four mutants exhibit a normal elongation response to supplementary FR during the daily photoperiod.