A. Van Tuinen

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.

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.

Photomorphogenic mutants of tomato

Photomorphogenesis of tomato is being studied with the aid of mutants which are either modified in their photoreceptor composition or in their signal transduction chain(s). Several mutants affecting the phytochrome family of photoreceptors, some of which appear deficient for specific genes encoding phytochrome apoproteins have been isolated. In addition, other mutants, including transgenic lines overexpressing phytochrome A, exhibit exaggerated photomorphogenesis during de-etiolation. Anthocyanin biosynthesis and plastid development are being used as model systems for the dissection of the complex interactions among photomorphogenic photoreceptors and to elucidate the nature of their transduction chains.

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.

Molecular analysis of tri-mutant alleles in tomato indicates the Tri locus is the gene encoding the apoprotein of phytochrome B1

Four monogenic recessive tomato (Lycopersicon esculentum Mill.) mutants at the temporarily red light-Insensitive (tri) locus (tri1, tri2in the genetic background breeding line GT; tri3, tri4in the genetic background cultivar Moneymaker) were studied. These mutants had slightly longer hypocotyls under white light than the wild type (WT). Western-blot analysis showed that the tri1mutant was deficient in a relatively lightstable phytochrome apoprotein (116 kDa) that was recognized in the WT by an antibody to tobacco phytochrome B; tri2had a 166-kDa band reduced in abundance; and tri2and tri4had bands reduced in molecular mass, approx. 105 and 95 kDa, respectively. These patterns were also found in light-grown plants. Northern-blot analysis for PHYB1 mRNA showed for tri2a transcript approx. 2 kb larger, for tri4, a transcript of WT size, but much reduced in abundance and for tri1and tri3transcripts equivalent in size and abundance to WT. In these mutants the transcripts of other members of the tomato phytochrome gene family (PHYA, PHYB2, PHYE, PHYF) were indistinguishable in size and abundance from WT. Thus, it appears that the tri locus specifically affects PHYB1 gene expression. Unlike phytochrome-B mutants in other plants, de-etiolated seedlings of the tri mutants exhibited normal responses to end-of-day far-red (EODFR) light and supplementary far-red light during the day. Since the holophytochromes of types B1 and B2 (phyB1 and phyB2) are closely related, it is proposed that there might be redundancy between them for these responses.

The Physiology of Photomorphogenetic Tomato Mutants

Photomorphogenesis of higher plants is a complex process resulting from the co-action of at least 3 different photoreceptors: phytochrome, a blue light (B)/UV-A photoreceptor (cryptochrome) and a UV-B photoreceptor (Mohr, 1986). The existence of multiple photoreceptor types, eg. type I (PI) or light-labile phytochrome and type II (PH) or light-stable phytochrome, adds to the complexity (Furuya, 1989; Tomizawa et al., 1990). The assignment of specific functions to the distinct molecular species of the photoreceptor is therefore being studied with the aid of photomorphogenetic mutants in which certain parts of the morphogenetic pathway are eliminated or altered. The relevance of the changed part in the mutant is directly indicated by its difference in response compared to its isogenic wild type (Kooraneef and Kendrick, 1986). Mutants can be found (isolated) from natural populations or varieties (cultivars) or more efficiently after mutagenic treatment: using e.g. chemicals, irrradiation; somaclonal variation; transposon insertion; transformation; introduction of antisense RNA.