~OMMENT
Mutants shed light on plant development EDUARDO R. BEJARANO AND CONRAD LICHTENSTEIN DEPARTMENTOF BIOCHEMISTRY, IMPERIALCOLLEGE,LONDON S'~r7 2AZ, UK.
That light provides the source of energy, via photosynthesis, for ma~aufacturing organic molecules is obvious; but light is also an important environmental signal for some of the more dramatic decisions in plant development. These are collectively called photomorphogenesis. Such responses include seed germination, leaf formation, stem growth, flowering and fruit ripening, some of which also involve photoperiodic responses (i.e. to day length) and phototropism (growth towards light). Chloroplast formation, synthesis of components of the photosynthetic apparatus and anthocyanin accumulation are also affected by light (reviewed in Ref. 1). The web of responses is complex, bt, t begins with detection of light by photoreceptors. In higher plants, physiological experiments suggest that at least three photoreceptors exist: phytochrome (a farred/red light receptor), a blue/ UV-A light receptor and a UV-B light receptor. Only phytochrome has heen identified at the molecular and biochemical level 23. It consists of a protein attached to a tetrapyrmle chromophore that can exist in two photochemicaily different forms: the Pr form can absorb red light: the Pf~ form can absorb farred light. Red light converts P~ to Pf~; far-red light converts Pf~ to Pr" Pf~ is the physiologically active form. It is now known that a gene family encodes a set of slightly different phytochrome apoproteins. Subtle differences in the properties of these phytochromes, and different patterns of expression, may account for the diversity of phytochrome responses. The mechanisms by which light signals the cascade of events leading to regulation of gene expression are unknown. Recently, genetic approaches have been taken towards unmasking this signal transduction pathway. Curiously, the classical genetic approaches taken, though enhanced by gene cloning
technology, would have been possible long ago. The reason for their belated application to this problem is cultural; it is the recent worldwide adoption of Arabidopsis tbaliana as the plant model system ~. This small weed is a perfectly respectable flowering plant showing the typical photomorphogenic responses of dicotyledenous angiosperms, as summarized in Table 1. In the dark, the hypocotyl of seedlings elongates. In the light, hypocotyl elongation is inhibited, the cotyledons ()pen and enlarge, and develop chloroplasts and differentiated cell types. Leaves develop and anthocyanin synthesis commences. Plants with defects in these responses can be generated by mutagenesis and selfed to recover homozygous recessive progeny. One approach taken was to screen for mutant seedlings that failed to show white light-induced suppression of hypocotyl elongation ~.~'. These so-called h.r mutants fall into six complementation groups, indicating that at least six genetic loci exist, t0'1-6. In adult plants the i~.rl, hy2 and 10'6 mutants display pleiotropic effects to varying extents, such as increased apical dominance, paler green colour, and reduced leaf size.
TAmE1. Phenotypes for photomorphogenic responses of wild-type plants and constitutive Ught-induced mutants Wild tTpe Dark Light Chloroplast biogenesis Light-specific gene expression Seedling development Hypocotyl inhibition Cotyledon expansion Leaf formation Anthocyanin synthesis Germination Dark adaptation a
cop l
det l
det2
Dark
Dark
Dark
-
+ +
+ +
+ +
+
-
+ + + + + +
+ + + + -
+ + + + + +
+ + + + -
aDark adaptation is defined at the molecular level as the changes in the mRNA levels of some genes when a plant, grown in light, is transferred to the dark. The other characteristics were analysed in seedlings.
T1G JANI'ARY 1992 VOL 8 NO. 1 e 1092 Elsc~ier ."idt.ncc Publinht'r', lid (1"KP
These mutants lack fully functional (photoreversible) phytochrome but do express the apoprotein ~,.'. The other mutants have wild-type phytochrome, suggesting that a later step in control of hypocotyl growth is defective; interestingly, their pleiotropic effects are also less severe. Expression of anthocyanins in hi'l, hy2 and hy6 is normal, indicating that their expression is either not regulated by phytochrome or that other photoreceptors can compensate. Recently, Liscum and Hangarter s refined this approach by isolating mutants, designated bht (for blue light uninhibited), that have long hypocotyls when grown under intense blue light. But, as in wildtype plants, hypocotyl elcmgation is still inhibited by far-red light. Liscum and Hangarter defined three genetic loci responsible for this effect and showed that progeny of bhtxhl' crosses are wild type. In blu mutants, cotyledon expansion is also reduced btnt chlorophyll content is nortnal: mc)reover, there is little effect on mature pl:mts. Their data corroborate physiological evidence for an independent blue light signal transduction p:tthway: however, it is not known whether the genetic lesions affect
U
[~OMMENT
photoreceptor function or a later step. In a converse approach, Arabidopsis mutants were identified that showed constitutively active lightinduced responses in the dark. Three genetic loci - detl and det2 (de-etiolated) 9.1° and cop1 (constitutive photomorphogenesis) ~1 were isolated by screening for seedlings that grow in the dark more or less as wild-type seedlings grow in the light. The phenotypes of these mutants are summarized in Table 1. The recessive nature of all the mutations isolated in these three loci implies that a loss of function leads to a constitutive photomorphogenic response; thus these gene products must (directly or indirectly) repress the lightinduced developmental programme in the dark. But at what stage do these gene products act? Analysis of d e t b y double mutants shows that detl and det2 mutations are epistatic to several different by mutations, indicating that the DET1 and DET2 gene products act downstream of phytochrome activation. Also. phytochrome has been shown to be functional in both detl and det2 mutants. Other regulatory networks initiating from different receptors may also interact with the DE77 and DE72 gene products: both detl and det2 seedlings constitt, tively express anthocyanins in the dark - a pathway thought not to be regulated by phytochrome. Apart from the similar phenotypes of the dark-grown seedlings of the three mutant classes, the mutants also show some interesting differences. For example, both detl and cop1 dark-grown seedlings develop chloroplasts, but det2 cannot. Seed germination requires light (controlled by phytochrome). Mutants in copl, like wild-type plants, cannot germinate efficiently in the dark, but all detl mutants germinated in the dark. The phenotypes of adult plants are also interesting. The transcription of nuclearencoded photosynthesis genes is reduced in wild-type light-grown plants when they are transferred to the dark. While detl mutants fully retain this property, det2 and copl mutants do not. Tissue-specific expression in the light is also affected in detl mutants, for ex-
ample, roots produce chloroplasts and turn green, whereas wild-type roots remain white t2. Both detl and det2 mutants are smaller than wildtype plants, but detl plants are paler and det2 mutants are darker green. Furthermore, det2 plants enjoy a prolonged juvenile phase, producing more leaves, have reduced leaf senescence, and take longer to flower than wild type; their flowers are smaller and male fertility is reduced. The marked differences in the detl and det2 phenotypes and the additive effects in detl det2 double mutants suggest that DET1 and DET2 gene products must act in separate branches of a common signal transduction pathway or even in independent pathways. Very recently, Karlin-Neumann et al. 13 have developed a novel strategy to select Arabidopsis mutants that fail to respond to light. In this strategy, a positive selection for such mutants is made possible by fusion of light-inducible promoter(s) to a conditionally lethal gene, tins2 of Agrobacterium tumefaciens. This gene encodes an indole-3-acetamide hydrolase, which converts indole 3acetamide to indole 3-acetic acid, a member of the auxin class of phytohormones. A. tumefaciens is used to transfer this and other phytohormone genes to the genome of infected plant cells, By subverting endogenous plant hormone biochemistry, Agrobacterium infection causes rapid proliferation of infected tissue, resulting in a 'tumour'. Expression of tins2 in transgenic plants is lethal when synthetic auxin amides, added to the growth medium, are hydrolysed, since auxins are toxic at high concentrations. Transgenic seedlings containing light-inducible gene fusions to tins2 grow normally on media containing auxin amides as long as they are kept in the dark. Upon illumination, their growth is inhibited. In addition to allowing the
recovery of mutations early in the signal transduction pathway (like those discussed above), this strategy should also uncover trans-acting gene products interacting with cisacting DNA sequences required for expression of specifically selected light-inducible promoters. To complement this approach - to map these cis-acting sequences - other groups have initiated deletion analyses of light-inducible promoters fused to a reporter gene in transgenic plants TM. These molecular genetic approaches should provide valuable additional tools. It is premature to construct a model for the signal transduction pathway. But a path into the jungle has been cleared, and an exciting future in the study of this area lies ahead.
References 1 Kendrick, R.E. and Kronenberg, G.H.M. (1986) Photomorphogenesis in Plants, Junk Publishers 2 Colbert, J.T. (1988) Plant Cell Environ. 11, 305-318 3 Sharrock, R. and Quail, P. (1989) Genes Dev. 3, 1745-1757 4 Meyerowitz, E.M. (1989) Cell 56, 263-269 5 Koornneef, M., Rolff, E. and Spruit, C. (1980)Z. Pflanzenphysiol. 100S, 147-160 6 Chory,J. et al. (1989) Plant Cell 1, 867-880 7 Parks, B. et ai. (1989) Plant Mol. Biol. 12, 425--437 8 Liscum, E. and Hangarter, R.P. ( 1991) Plant Cell3, 685--694 9 Chory,J. etal. (1989) Ceil58, 991-999 10 Chory, J., Nagpal, P. and Peto, C. (1991) Plant Cell 3, 445--459 11 Deng, X., Caspar, T. and Quail, P.H. (1991) Genes Dev. 5,
1172-1182 12 Chory, J. and Peto, C. (1990) Proc. Naa Acad. Sci. USA 87, 8776--8780 13 Karlin-Neumann, G.A., Brusslan, J.A. and Tobin, E.M. (1991) Plant Cell3, 573-582 14 Gilmartin, P., Sarokin,J., Memelink, J. and Chua, N-H. (1990) Plant Cell 2, 369-378
Genome Mapping and Sequencing A special issue from Trends in Biotechnology The Jan/Feb issue of TIBTECH is a special issue on the innovative technologies and strategies being developed to tackle large-scale genome analysis. The organization and financial aspects of the national and international projects are also discussed.
TlG JANUARY 1992 VOL. 8 NO. 1
m
Mutants shed light on plant development EDUARDO R. BEJARANO AND CONRAD LICHTENSTEIN DEPARTMENTOF BIOCHEMISTRY, IMPERIALCOLLEGE,LONDON S'~r7 2AZ, UK.
That light provides the source of energy, via photosynthesis, for ma~aufacturing organic molecules is obvious; but light is also an important environmental signal for some of the more dramatic decisions in plant development. These are collectively called photomorphogenesis. Such responses include seed germination, leaf formation, stem growth, flowering and fruit ripening, some of which also involve photoperiodic responses (i.e. to day length) and phototropism (growth towards light). Chloroplast formation, synthesis of components of the photosynthetic apparatus and anthocyanin accumulation are also affected by light (reviewed in Ref. 1). The web of responses is complex, bt, t begins with detection of light by photoreceptors. In higher plants, physiological experiments suggest that at least three photoreceptors exist: phytochrome (a farred/red light receptor), a blue/ UV-A light receptor and a UV-B light receptor. Only phytochrome has heen identified at the molecular and biochemical level 23. It consists of a protein attached to a tetrapyrmle chromophore that can exist in two photochemicaily different forms: the Pr form can absorb red light: the Pf~ form can absorb farred light. Red light converts P~ to Pf~; far-red light converts Pf~ to Pr" Pf~ is the physiologically active form. It is now known that a gene family encodes a set of slightly different phytochrome apoproteins. Subtle differences in the properties of these phytochromes, and different patterns of expression, may account for the diversity of phytochrome responses. The mechanisms by which light signals the cascade of events leading to regulation of gene expression are unknown. Recently, genetic approaches have been taken towards unmasking this signal transduction pathway. Curiously, the classical genetic approaches taken, though enhanced by gene cloning
technology, would have been possible long ago. The reason for their belated application to this problem is cultural; it is the recent worldwide adoption of Arabidopsis tbaliana as the plant model system ~. This small weed is a perfectly respectable flowering plant showing the typical photomorphogenic responses of dicotyledenous angiosperms, as summarized in Table 1. In the dark, the hypocotyl of seedlings elongates. In the light, hypocotyl elongation is inhibited, the cotyledons ()pen and enlarge, and develop chloroplasts and differentiated cell types. Leaves develop and anthocyanin synthesis commences. Plants with defects in these responses can be generated by mutagenesis and selfed to recover homozygous recessive progeny. One approach taken was to screen for mutant seedlings that failed to show white light-induced suppression of hypocotyl elongation ~.~'. These so-called h.r mutants fall into six complementation groups, indicating that at least six genetic loci exist, t0'1-6. In adult plants the i~.rl, hy2 and 10'6 mutants display pleiotropic effects to varying extents, such as increased apical dominance, paler green colour, and reduced leaf size.
TAmE1. Phenotypes for photomorphogenic responses of wild-type plants and constitutive Ught-induced mutants Wild tTpe Dark Light Chloroplast biogenesis Light-specific gene expression Seedling development Hypocotyl inhibition Cotyledon expansion Leaf formation Anthocyanin synthesis Germination Dark adaptation a
cop l
det l
det2
Dark
Dark
Dark
-
+ +
+ +
+ +
+
-
+ + + + + +
+ + + + -
+ + + + + +
+ + + + -
aDark adaptation is defined at the molecular level as the changes in the mRNA levels of some genes when a plant, grown in light, is transferred to the dark. The other characteristics were analysed in seedlings.
T1G JANI'ARY 1992 VOL 8 NO. 1 e 1092 Elsc~ier ."idt.ncc Publinht'r', lid (1"KP
These mutants lack fully functional (photoreversible) phytochrome but do express the apoprotein ~,.'. The other mutants have wild-type phytochrome, suggesting that a later step in control of hypocotyl growth is defective; interestingly, their pleiotropic effects are also less severe. Expression of anthocyanins in hi'l, hy2 and hy6 is normal, indicating that their expression is either not regulated by phytochrome or that other photoreceptors can compensate. Recently, Liscum and Hangarter s refined this approach by isolating mutants, designated bht (for blue light uninhibited), that have long hypocotyls when grown under intense blue light. But, as in wildtype plants, hypocotyl elcmgation is still inhibited by far-red light. Liscum and Hangarter defined three genetic loci responsible for this effect and showed that progeny of bhtxhl' crosses are wild type. In blu mutants, cotyledon expansion is also reduced btnt chlorophyll content is nortnal: mc)reover, there is little effect on mature pl:mts. Their data corroborate physiological evidence for an independent blue light signal transduction p:tthway: however, it is not known whether the genetic lesions affect
U
[~OMMENT
photoreceptor function or a later step. In a converse approach, Arabidopsis mutants were identified that showed constitutively active lightinduced responses in the dark. Three genetic loci - detl and det2 (de-etiolated) 9.1° and cop1 (constitutive photomorphogenesis) ~1 were isolated by screening for seedlings that grow in the dark more or less as wild-type seedlings grow in the light. The phenotypes of these mutants are summarized in Table 1. The recessive nature of all the mutations isolated in these three loci implies that a loss of function leads to a constitutive photomorphogenic response; thus these gene products must (directly or indirectly) repress the lightinduced developmental programme in the dark. But at what stage do these gene products act? Analysis of d e t b y double mutants shows that detl and det2 mutations are epistatic to several different by mutations, indicating that the DET1 and DET2 gene products act downstream of phytochrome activation. Also. phytochrome has been shown to be functional in both detl and det2 mutants. Other regulatory networks initiating from different receptors may also interact with the DE77 and DE72 gene products: both detl and det2 seedlings constitt, tively express anthocyanins in the dark - a pathway thought not to be regulated by phytochrome. Apart from the similar phenotypes of the dark-grown seedlings of the three mutant classes, the mutants also show some interesting differences. For example, both detl and cop1 dark-grown seedlings develop chloroplasts, but det2 cannot. Seed germination requires light (controlled by phytochrome). Mutants in copl, like wild-type plants, cannot germinate efficiently in the dark, but all detl mutants germinated in the dark. The phenotypes of adult plants are also interesting. The transcription of nuclearencoded photosynthesis genes is reduced in wild-type light-grown plants when they are transferred to the dark. While detl mutants fully retain this property, det2 and copl mutants do not. Tissue-specific expression in the light is also affected in detl mutants, for ex-
ample, roots produce chloroplasts and turn green, whereas wild-type roots remain white t2. Both detl and det2 mutants are smaller than wildtype plants, but detl plants are paler and det2 mutants are darker green. Furthermore, det2 plants enjoy a prolonged juvenile phase, producing more leaves, have reduced leaf senescence, and take longer to flower than wild type; their flowers are smaller and male fertility is reduced. The marked differences in the detl and det2 phenotypes and the additive effects in detl det2 double mutants suggest that DET1 and DET2 gene products must act in separate branches of a common signal transduction pathway or even in independent pathways. Very recently, Karlin-Neumann et al. 13 have developed a novel strategy to select Arabidopsis mutants that fail to respond to light. In this strategy, a positive selection for such mutants is made possible by fusion of light-inducible promoter(s) to a conditionally lethal gene, tins2 of Agrobacterium tumefaciens. This gene encodes an indole-3-acetamide hydrolase, which converts indole 3acetamide to indole 3-acetic acid, a member of the auxin class of phytohormones. A. tumefaciens is used to transfer this and other phytohormone genes to the genome of infected plant cells, By subverting endogenous plant hormone biochemistry, Agrobacterium infection causes rapid proliferation of infected tissue, resulting in a 'tumour'. Expression of tins2 in transgenic plants is lethal when synthetic auxin amides, added to the growth medium, are hydrolysed, since auxins are toxic at high concentrations. Transgenic seedlings containing light-inducible gene fusions to tins2 grow normally on media containing auxin amides as long as they are kept in the dark. Upon illumination, their growth is inhibited. In addition to allowing the
recovery of mutations early in the signal transduction pathway (like those discussed above), this strategy should also uncover trans-acting gene products interacting with cisacting DNA sequences required for expression of specifically selected light-inducible promoters. To complement this approach - to map these cis-acting sequences - other groups have initiated deletion analyses of light-inducible promoters fused to a reporter gene in transgenic plants TM. These molecular genetic approaches should provide valuable additional tools. It is premature to construct a model for the signal transduction pathway. But a path into the jungle has been cleared, and an exciting future in the study of this area lies ahead.
References 1 Kendrick, R.E. and Kronenberg, G.H.M. (1986) Photomorphogenesis in Plants, Junk Publishers 2 Colbert, J.T. (1988) Plant Cell Environ. 11, 305-318 3 Sharrock, R. and Quail, P. (1989) Genes Dev. 3, 1745-1757 4 Meyerowitz, E.M. (1989) Cell 56, 263-269 5 Koornneef, M., Rolff, E. and Spruit, C. (1980)Z. Pflanzenphysiol. 100S, 147-160 6 Chory,J. et al. (1989) Plant Cell 1, 867-880 7 Parks, B. et ai. (1989) Plant Mol. Biol. 12, 425--437 8 Liscum, E. and Hangarter, R.P. ( 1991) Plant Cell3, 685--694 9 Chory,J. etal. (1989) Ceil58, 991-999 10 Chory, J., Nagpal, P. and Peto, C. (1991) Plant Cell 3, 445--459 11 Deng, X., Caspar, T. and Quail, P.H. (1991) Genes Dev. 5,
1172-1182 12 Chory, J. and Peto, C. (1990) Proc. Naa Acad. Sci. USA 87, 8776--8780 13 Karlin-Neumann, G.A., Brusslan, J.A. and Tobin, E.M. (1991) Plant Cell3, 573-582 14 Gilmartin, P., Sarokin,J., Memelink, J. and Chua, N-H. (1990) Plant Cell 2, 369-378
Genome Mapping and Sequencing A special issue from Trends in Biotechnology The Jan/Feb issue of TIBTECH is a special issue on the innovative technologies and strategies being developed to tackle large-scale genome analysis. The organization and financial aspects of the national and international projects are also discussed.
TlG JANUARY 1992 VOL. 8 NO. 1
m
