RESEARCH PAPER |
Hsp90 canalizes developmental perturbation
Laboratory of Molecular Biology, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece
To whom correspondence should be addressed. E-mail: phat{at}aua.gr
Received 4 June 2007; Revised 23 July 2007 Accepted 24 July 2007
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Abstract |
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Stochastic processes are intrinsic phenomena that perturb developmental processes. However, the canalization process restricts the magnitude of perturbation and hence the magnitude of morphological variation during development. Heat-shock protein 90 (Hsp90) chaperones are a class of proteins stabilizing a network of ‘client’ proteins that are involved in diverse signal transduction pathways affecting development. Here it is reported that a reduction of Hsp90 gene dose creates canalization perturbations that affect many aspects of Arabidopsis development and results in a plethora of morphological alterations. Hence, Hsp90 restricts stochastic phenomena by minimizing perturbations, thereby canalizing development. It is also shown that morphogenesis is determined by three mutually inter-related parameters: genotype, environment, and time. Hsp90 is involved in the interaction of these three parameters which ultimately affect developmental processes. The amount of phenotypic variation upon the reduction of Hsp90 function could be perceived as adaptive and could have an impact on the evolutionary process.
Key words: Arabidopsis, genetic analysis, Hsp90, phenotypic variation, stochastic development
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
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Stochastic mechanisms during developmental processes of multicellular organisms result in polymorphisms (Gartner, 1990; Mc Adams and Arkin, 1997). This condition is distinct from genetic variation. In a given inbred population genetic variation tends towards equilibrium and hence phenotype uniformity (Powell, 1997). Subtle genetic variations do not usually substantiate phenotypic variants (Kitano, 2004). Stochastic development, being more flexible, allows a better adaptation to environmental changes during the life span of an organism and might generate another platform for evolution. However, radical stochastic mechanisms are restricted by a buffering system that canalizes canonical development (Waddington, 1942; Schmahausen, 1986).
Harsh environmental conditions can reveal new phenotypic polymorphisms presumably because of perturbation of this buffering capacity. These new phenotypic polymorphisms are heritable and genetically assimilated even though sometimes genetic polymorphisms are not present (Waddington, 1953, 1956; Sollars et al., 2003). The chaperone heat-shock protein 90 (Hsp90) can function as a buffering capacitor, thus operating as a periphery restricting individual polymorphisms. This phenomenon was apparent in evolutionarily distant organisms (Rutherford and Lindquist, 1998; Queitsch et al., 2002; Sollars et al., 2003).
When the function of Hsp90 was disturbed by mutation, pharmacological inhibition, or environmental stress in an obligatory outcrossing species such as Drosophila, a plethora of polymorphic phenotypes was uncovered for almost every part of the body structure or developmental pathways (Rutherford and Lindquist, 1998). Consequently, the chaperone Hsp90 system creates a network buffering the canalization during development and ultimately suppresses individual polymorphisms. Interestingly, when multiple polymorphisms were enriched by selection, these features were heritable even after Hsp90 functional restoration (Sollars et al., 2003).
In the inbreeding plant species Arabidopsis thaliana, the function of Hsp90 was challenged only by pharmacological inhibition while the phenotypic traits were measured and characterized during a restricted time period of the plant life, e.g. in seedlings (Queitsch et al., 2002). Reduced Hsp90 function generated an array of seedling morphological phenotypes apparently based on underlying genetic variation of different genetic backgrounds. This indicates that the Hsp90 chaperone system might influence morphogenetic responses to environmental cues and buffer normal development from destabilizing effects of stochastic processes (Queitsch et al., 2002; Sangster et al., 2004).
Hsp90 is a ubiquitous highly conserved molecular chaperone found in all organisms induced by heat stress. In all eukaryotes examined, Hsp90 is an abundant protein at normal temperatures. Under normal conditions, the dynamic interaction of Hsp90 with its client proteins is diverse, but nevertheless highly selective (Buchner, 1999; Young et al., 2001; Zhao et al., 2005). Recent work in yeast provided evidence that almost 10% of the genetic pool acts as client proteins to Hsp90, interacting physically, genetically, or chemically, thus creating a dynamic network in signal transduction pathways and cellular activities (Zhao et al., 2005). Typically, Hsp90 in an ATP-dependent mode retains these metastable proteins at a dynamic status quo before they are stabilized by conformational changes (Buchner, 1999; Mayer and Bukau, 1999; Young et al., 2001; Picard, 2002).
Heterozygocity is an important parameter in maintaining genetic variability and phenotypic robustness (Thoday, 1953). However, since heterozygocity per individual in Arabidopsis is extremely low (Abbott and Gomes, 1989; Kuittinen et al., 1997) and becomes even lower in self-fertilizing species, it is unclear whether Hsp90 action restricts variations resulting from heterozygocity at multiple loci or whether it predominantly restricts bona fide stochastic mechanisms. Here, using a genetic approach, it is demonstrate that Hsp90 restricts stochastic processes during developmental canalization rather than releasing cryptic genetic variations. Moreover, it is shown that Hsp90 plays a key role in the manifestation of phenotype plasticity and buffers development against environmental perturbations. The present data also indicate that the accumulation of stochastic phenomena over developmental time has a major influence on development and that Hsp90 canalizes development by counteracting these effects. The multiplicity of Hsp90 genes in Arabidopsis provides a fine tuning in the understanding of canalization and stochastic mechanisms during development that could lead to adaptation and evolution.
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Materials and methods |
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Plant material and normal or stress growth conditions
SALK collections seeds from the Arabidopsis Biological Resource Center (ABRC) and the Nottingham Arabidopsis Stock Center (NASC) were used: 007614 for AtHsp90-1 (At5g52640); 013240 for AtHsp90-2 (At5g56010); 038646 for AtHsp90-3 (At5g56030), and 084059 for AtHsp90-4 (At5g56000). Col seeds were laboratory stocks. Accessions were self-fertilized and their progeny (F1–F5) were scored. Surface-sterilized seeds were plated and left to grow under standard conditions, at 27 °C or 32 °C. Seeds and seedlings were heat stressed for appropriate periods at 27, 32, or 37 °C and left to grow under normal conditions or the indicated stress conditions. In order to provide objective evaluations, Col seeds were plated independently but left to grow under the same conditions and observed simultaneously with the mutant plants. Growth variations were eliminated by letting mutants and Col plants grow for longer, and phenotypes were observed at different time points. Digital images were taken at different time points of growth under different magnifications as indicated. Seeds were placed in 20% glycerol, dissected, and embryos were observed and photographed using Olympus scopes.
Molecular techniques
Genomic DNA was isolated using the standard cetyltrimethyl ammonium bromide (CTAB) method. In order to determine the T-DNA insertion point and the genes that were disrupted, pairs of primers specific for the insertion and for the Hsp90 genes were used. For the T-DNA insertion, the following primers were used: primer nosf 5'-GCATGACGTTATTTATGAGATGGG-3', primer nosR 5'-GACACCGCGCGCGATAATTTATCC-3'; and for the NPTII gene, primer npt1 5'-TCTGTTGTGCCCAGTCATAGCCGAATAG-3', primer npt3 5'-CATCTTGTTCAATCATGCGAAACGATCC-3', and primer npst 5'-TCAAGACCGACCTGTCCGGTGCCCTGAA-3'. For the Hsp90 genes, the following primers were used: for AtHsp90-1 primer 81revA 5'-CGTTGGCTGCAGCCATAAGAGCAATTTCTTCATCTC-3'; for AtHsp90-2 primer 5utr812 5'-TTTCCGATCAACGAGAATGG-3', primer 812ex3 5'-TCCTCATCCTTCTTCYCYTCCTCC-3', and primer 2intr812 5'-TCAGATCTGAACCTTGG-3'; for AtHsp90-3 primer Sma813a 5'-AGTACCCGGGCCGATCAACGAGAATGGCGGA-3' and primer Pst813a 5'-CTAGCTGCAGCGCAAACCATAGTCTTATAACACCGCT-3', and for AtHsp90-4 primer f814 5'-CTTTTCATTCATATCATTCCG-3' and primer h814 5'-TTTGTGCAATATTTAGTCG-3'.
RNA was isolated using the SDS/phenol method (Haralampidis et al. 2002). Total RNA was then treated with RNase-free DNase I (Invitrogen) and about 800 ng were used as template in first-strand cDNA synthesis using SuperscriptTM II RNase H– Reverse Transcriptase (Invitrogen), according to the manufacturer's protocol. Unless otherwise stated, the first-strand cDNA was primed by the poly(A) tail with the reverse transcription primer T17XHO (5'-GTCGACCTCGAGTTTTTTTTTTTTTTTTT-3'). Transcript analysis was performed using specific primers (for AtHsp90-1 forward 81FORA 5'-TTAATGGATCCAAGTTCGTTGCGATGGCGGATG-3' and reverse 81REVA 5'-CGTTGGCTGCAGCCATAAGAGCAATTTCTTCATCTC-3'; for AtHsp90-2 forward f812 5'-AGAGCTCTTCATTCACATC-3' and reverse h812 5'-TCTGTGTGATAATTTAGTCA-3'; for AtHsp90-3 forward Sma813a 5'-AGTACCCGGGCCGATCAACGAGAATGGCGGA-3' and reverse Pst813a 5'-CTAGCTGCAGCGCAAACCATAGTCTTATAACACCGCT-3'; and for AtHsp90-4 forward f814 5'-CTTTTCATTCATATCATTCCG-3' and reverse h814 5'-TTTGTGCAATATTTAGTCG-3') and reverse transcription–PCRs (RT-PCRs). We found that either the Hsp90 genes were not transcribed at all or the transcript length was decreased.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
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Hsp90 is required for pattern formation
The Arabidopsis cytosolic group comprises four Hsp90 members (Milioni and Hatzopoulos, 1997). To verify subtle or extreme phenotypes at any stage of development and to determine the effect of the Hsp90 system on developmental pathways or morphogenesis, >25 000 seeds, embryos, seedlings, and Arabidopsis plants were scored for at least four generations of all four hsp90 mutants.
Homozygous plants mutated at any of the four cytosolic members are not lethal, although a noticeable percentage (an average of 40% for the Athsp90-1 mutant and 15% for the other three Athsp90 mutants) resulted in non-germinated seeds. For Col plants, an average of 1.5% resulted in non-germinating seeds. Athsp90-1 mutant seeds showed the most pronounced phenotypes, being shrivelled and having various shapes, while a few seeds were almost dry, indicating the absence of embryo or endosperm tissue (Fig. 1). Embryos were dissected out from the abnormal non-germinating seeds of all four hsp90 mutants and showed defective root integrity and morphology, an unorganized central cylinder, deficiency in pattern formation and cell fate, and Siamese twin-like single cotyledon embryos (Fig. 1)
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The phenotypes of the Athsp90-1 mutant defective embryos were most prominent in the root system in which the central cylinder was either split, interrupted, or misplaced. All the scored Athsp90-2 mutant defective embryos had a pronounced and aberrant root system (e.g. a second primary root emerged, a split at the central cylinder). For the Athsp90-3 mutants, the aberrant phenotypes were obvious mainly in cotyledons. They showed an ectopic pattern formation of the phloem and xylem, single cotyledon embryos, a split root, and loss of cell fate. The Athsp90-4 mutant defective embryos were severely affected in cotyledon and root morphogenesis. In most cases the split in the root or cotyledon was more prominent, while in other cases Siamese twin-like single cotyledon embryos were scored. Disorganization or misplacement of cotyledon development was also scored (Fig. 1). In many cases hsp90 mutants showed a phenotypic appearance reminiscent of homeotic gene defects. There is, at least partly, functional overlap of the individual members of the Hsp90 cytosolic gene family; nevertheless, each mutant member had a certain class of phenotype. The mutant phenotypes observed here corroborate recent work showing that AtHsp90-1 is expressed mainly in embryo root tissues, while AtHsp90-3:GUS activity was intense in cotyledons (Haralampidis et al., 2002; Prasinos et al., 2005).
The germinated seeds from all four mutants produced mainly healthy seedlings, although a noticeable percentage (30% on average for all four mutants) exhibited a number of phenotypes ranging from weak to strong (Fig. 2). Aberrant mutant seedlings showed morphological defects affecting cotyledon number, morphology, shape, and development; shape and length of hypocotyls; appearance and growth of the first pair of leaves; and root presence or architecture. Few seedlings were etiolated or had an ethylene-like phenotype (Fig. 2 and data not shown). A spectrum of seedling phenocopies was also observed when AtHsp90 function was pharmacologically inhibited (Queitsch et al., 2002; Sangster et al., 2004).
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Even though there was an overlap in the spectrum of phenotypes for the four mutants, certain mutants had distinct phenotypes (Fig. 2). Root morphological abnormalities were found mostly in Athsp90-4 mutants, while leaf defects were more profound in Athsp90-1 mutants. The most severe phenotypes (e.g. lack of root), representing a small proportion, failed to prosper, while the majority eventually advanced to, more or less, wild type-like phenotypes acquiring a normal developmental process. Col seedlings showed a narrow range of subtle phenotypes. This phenomenon emphasizes that Hsp90 restricts developmental plasticity. The results also show that there is a degree of overlap in function among the members of the Hsp90 cytosolic gene family.
Perturbation of phenotypic morphs upon reduction of Hsp90 activity
Hsp90 has a strategic role in the developmental dynamics that govern early morphogenesis. To determine whether the process towards adult development was also affected, about 1000 seedlings from each hsp90 mutant line, irrespective of phenotypic appearance, were randomly collected, transferred to soil, and allowed to grow under standard conditions. A small proportion of the seedlings were unable to cope with this abrupt environmental change and died almost immediately.
Five distinct phenotypic classes (A–E) were observed when the AtHsp90-1 gene was mutated. Each class represented a group of plants showing a more or less similar phenotype (Fig. 3). Class A comprised seedlings growing for up to 10 d on plates having epinasty-like phenotypes with highly deformed leaves, and they eventually died. Class B comprised mature plants characterized by a bushy rosette while the inflorescence was either absent or grew up to 4 cm. In some cases the inflorescence appeared normal, the mutant plant had fewer inflorescences than the wild type, and bolting was delayed. Class C comprised plants having a small sized rosette with fewer leaves. Class D contained dwarf mature plants, while class E were wild-type-like plants (Fig. 3).
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Five distinct phenotypic classes were also observed when the AtHsp90-2 gene was mutated. Class A comprised small plants having a small sized rosette and the inflorescence was short. Class B contained bushy plants some with short or no inflorescence at all, and had saw-edged leaves. Class C comprised plants similar to class B. However, they were generally taller with many but short inflorescences implying the absence of apical dominance. Class D contained plants with altered morphology and a higher number of rosette leaves, while class E plantts had a wild-type-like phenotype (Fig. 3).
Athsp90-3 mutant plants showed four distinct classes. Class A contained plants with fewer rosette leaves and a delay in flowering. Class B was composed of plants with a bushy rosette appearance while the inflorescences crawled and did not grow from the centre of the rosette. Class C showed a defect in rosette architecture that grew at different levels, while class D comprised plants with an absence of apical dominance. Also, Athsp90-4 mutant plants could be divided into four distinct classes. Class A contained plants with delayed bolting and small sized rosettes. Class B plants showed absence of apical dominance. Class C plants showed defects in rosette architecture. Class D plants had a bushy rosette appearance while inflorescences crawled and did not grow from the centre of the rosette (Fig. 3). Col plants had uniform wild-type phenotypes when grown under the same growth conditions.
Within each mutant plant line peculiar phenotypes were recovered. These phenotypes were not grouped in any of the above classes and represented a rather low proportion of nevertheless distinctive phenotypic appearance (Fig. 3). Rosette leaves were highly deformed and developed at different levels (reminiscent of homeoitic mutants), developmental arrest was apparent, and seed production was minimal.
All these plants are inbred lines and therefore any heterozygocity that may emerge will depend solely on a single self-fertilizing cross. Hence heterozygocity should be minimal, indicating that any divergence from the norm depends exclusively on the hsp90 mutation. Although there were certain classes of different mutants showing a similar phenotype (e.g. class B or D of Athsp90-3 mutants to class D or B of Athsp90-4 mutants, respectively), other classes (such as class B of Athsp90-2 mutants, and class A or D of Athsp90-1 mutants) showed a rather distinctive phenotype (Fig. 3). The results showed that besides the overlapping function of the individual Hsp90 cytosolic members, there are specific aspects of developmental processes affected mainly by a single gene or a subgroup of genes.
Hsp90 determines developmental plasticity
The predisposition of the four different mutants to show different phenotypic classes illustrates the buffering capacity of the Hsp90 function in plant development. Similarly, the buffering capacity of Hsp90 was observed in Drosophila (Rutherford and Lindquist, 1998). It also suggests that the Hsp90 chaperones are engaged in stochastic mechanisms during development. To verify this concept and to examine whether any mutant phenotypic class is imprinted at any level of cellular activity or differentiation process, seeds were collected independently from each class of all four mutants after self-fertilization. About 100 seeds were grown to maturity. Surprisingly, each class of any mutant gave rise to phenotypes characteristic of all classes (Fig. 4). The percentage of each offspring class was almost distinctive and showed no clear pattern or type of direction towards a particular phenotype. Again peculiar unclassified phenotypes were recovered (data not shown). This phenomenon was repeated during the next generations. These results show that any mutant phenotypic class was partially penetrant and that the navigation of the chaperone network channels development through stochastic mechanisms.
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Hsp90 influences morphogenetic effects of environmental cues
In yeast and Drosophila if the Hsp90 activity is lowered from a threshold point, then it leads to abnormal development, more peculiar phenotypes, and finally to lethality, as in the case of homozygous mutants (Rutherford and Lindquist, 1998; Sollars et al., 2003). To test vigorously if this also holds for plants, the content of Hsp90 available for client proteins was diluted, thus increasing the demand for Hsp90. It is known that the highest expression of Arabidopsis Hsp90 genes occurs upon exposure at 37 °C for 3 h (Milioni and Hatzopoulos, 1997). All four mutants were grown at 22 °C for 10 d and then were subjected to short but severe heat stress (27 °C and/or 37 °C). Under these harsh environmental conditions, Col plants showed defects in development. However, extreme variegated phenotype appearances were observed in all mutants. Almost every individual plant of all hsp90 mutants had a distinct phenotype, and basic patterns of plant development were affected (Fig. 5). Therefore, many morphogenetic pathways were affected and the phenotype of each mutant plant was not strictly defined. However, mutant plants that were stressed at 37 °C and then transferred to 22 °C were more vigorous than the wild-type plants, as has been also observed in seedlings using geldanamycin (GDA; Queitsch et al., 2002; Sangster et al., 2004).
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Development is coordinated by time, environment, and genotype, and proceeds through stochastic mechanisms buffered by Hsp90
To investigate the interplay of environment and time in the stochastic processes and its dependence on the Hsp90 system, we artificially incorporated the parameter time during the course of development by placing all four Arabidopsis hsp90 mutants to grow under moderate (27 °C or 32 °C) stress conditions, for different lengths of time. If stochastic mechanisms contribute to multiple polymorphic traits, then time or environment might affect the proportion of each phenotypic class arbitrarily. Also, if Hsp90 restricts and buffers stochastic mechanisms, the distorted Hsp90 genes would increase phenotypic polymorphism.
The earlier or the greater the stress to which the Arabidopsis Col plants were subjected, the higher the frequencies of subtle altered phenotypes observed (
30%, compared with 2% at 22 °C). In the distorted hsp90 plants, the frequency of wild-type-like phenotypes decreased dramatically (by almost 15% for the Athsp90-1 mutant), while new and extreme phenotypes were detected (e.g. formation of multiple buds). The rest of the classes of Athsp90-1 mutants and all classes of the other three mutants showed a shift towards severe phenotypes when plants were grown at 27 °C (Fig. 6 and see Supplementary Fig. S1 available at JXB online). Mutant seeds or seedlings growing in a more severe environment at 32 °C showed even more extreme seedling phenotypes than plants grown at 27 °C. When mutant seeds were placed at 32 °C there was an abrupt reduction in the germination rate, ranging from 80% for the Athsp90-1 mutant to an average of 40% for the other three mutants. Again, the earlier the stress, the more extreme the phenotypes recovered. When 10-day-old Athsp90-1 or -2 mutant seedlings were transferred to 32 °C, they revealed a rather wide range of polymorphic phenotypes and grew up to the stage of the third pair of true leaves. Athsp90-3 and Athsp90-4 mutants survived longer, reverted to class A or C (dwarf), formed inflorescences, and eventually developed distorted and dried buds. Col plants showed fewer polymorphisms, formed inflorescences, and eventually dried.
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When 20-day-old Athsp90-1 mutant plants were placed at 32 °C, about 50% survived, showing the polymorphic classes B–E, while additional phenotypes were scored (e.g. large rosette leaves, chlorotic petioles, and dwarf phenotypes). Classes B–E of Athsp90-1 or D and E of Athsp90-2 formed chlorotic inflorescences, developed buds, and eventually died. Athsp90-3 or -4 mutant plants reverted to class A 20 d later. Novel phenotypes were also observed. New rosette leaves began to develop when Athsp90-3 mutants remained for 30 d at 32 °C. This phenomenon was less apparent for the Athsp90-4 mutant. Col plants were variegated at the rosette level, and showed a large petiole and green leaves with a small blade. No mutant or wild-type plant was able to set seeds at 32 °C, and most plants died. The diversity of the proportion of the phenotypic classes was not due mainly to reduced survival rates.
To investigate whether environmental changes influence multiple polymorphic traits and whether these are further affected by the Hsp90 buffering system, seeds from wild-type and hsp90 mutant plants set at 27 °C were collected in pools relative to the time that seeds, seedlings, or plants were placed at 27 °C and left to grow at 22 °C. The progeny of all hsp90 mutants showed defects in most aspects of development or body architecture, resulting in severe phenotypes. Col seedlings were also affected, though the variance or the percentage of peculiar phenotypes was less profound (see Supplementary Fig. S2 at JXB online). The longer the mutant parental plants remained at 27 °C the greater was the shift towards severe phenotypes and the higher the level of polymorphism of the offspring grown at 22 °C (Fig. 6; see Supplementary Fig. S1 and S2 at JXB online).
However, the germination rates of the Athsp90-1 offspring were surprisingly diverse. When parental seeds, 5-, 10-, or 20-day-old mutants were transferred to 27 °C, the germination rates were 88, 75, 66, or 35%, respectively. For the other mutants or Col, the germination rates of the offspring were stable at an average of 77% or 95%, respectively. Therefore, the earlier the parents were placed at 27 °C the better the adaptation of the offspring, i.e. the greater were the survival rates of the Athsp90-1 mutant offspring. These results also show that the functional activity of the members of the Hsp90 gene family is not completely overlapping, and particular aspects of development are undertaken by specific members. Adult mutant offspring plants also showed new and distinct traits that were similar to those observed in the previous generation, supporting previous results showing that the Hsp90 system is also involved in epigenetic phenomena (Sollars et al., 2003; Zhao et al., 2005).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
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The Arabidopsis Hsp90 gene family is divided into two groups (Milioni and Hatzopoulos, 1997; Krishna and Gloor, 2001). The organelle-type Hsp90 group comprises three members that play important roles in developmental pathways related to the organelles (Lin and Cheng, 1997; Cao et al., 2000, 2003). The cytosolic group comprises four members: AtHsp90-1, -2, -3, and -4. Even though organelles such as chloroplasts are vital components during the normal developmental process of a plant (Leech, 1984; Budziszewski et al., 2001; Tetlow et al., 2004), this study focused on the cytosolic members to avoid any interference of the organelle function in relation to the development and differentiation of a multicellular organism.
All four cytosolic proteins are very similar; however, three of them have the highest homology in amino acid sequence as well as in gene structure (Milioni and Hatzopoulos, 1997). Therefore, overlapping functions could be plausible. Since there are four Hsp90 genes, any single mutation in these members will not manifest a lethal phenotype as has been found in other organisms containing a single gene (Rutherford and Lindquist, 1998). If this overlap in expression and/or function was complete, a mutation in any given member of the cytosolic Hsp90 gene family would not necessarily be noticeable. However, mutations in an individual Hsp90 cytosolic member confer sensitivity to plant pathogens (Hubert et al., 2003; Takahashi et al., 2003).
A variety of seedling phenotypes were revealed when the Hsp90 protein function was decreased by pharmacological application (GDA) to progeny of Arabidopsis outcrosses (Queitsch et al., 2002; Sangster et al., 2004). Nevertheless, there are crucial limitations on the use of GDA. The drug is inactivated by light, therefore the effective selective pressure varies and consequently the phenocopies are displayed and scored only during a limited time period, e.g. at the seedling level.
The present results based on genetic analyses of nearly isogenic lines and observations at different developmental stages of Arabidopsis growth revealed that Hsp90 strongly affects developmental plasticity. Hsp90 mutations affect almost every part of the seed and, more intensively, every part of the embryo. Consequently, Hsp90 proteins are crucial determinants in embryo morphogenesis and pattern formation, as has also been described in Drosophila and zebra fish (Ding et al., 1993; Krone et al., 2003). Since the acquired phenotypes cover most norms of seedling growth, it is plausible that Hsp90 chaperone activity has a vital role in the progression of seedling development. Hsp90 also affects adult body formation and is essential in most developmental pathways. Therefore, the data presented herein showed that Hsp90 functions as a networking system in a wide range of developmental pathways affecting many aspects of morphogenesis at different stages. The nature or the degree to which the plasticity of morphs increases depends mainly on individual defective Hsp90 cytosolic gene. However, overlapping functions among the individual members are not excluded. Evidently, the Hsp90 system affects the likelihood that a particular trait might appear, thus resulting in polymorphism. Otherwise a single mutation or a nearly isogenic inbred line could fix a particular phenotype. Therefore, it is suggested that Hsp90 buffers the plasticity, while a decrease by mutation will provide an avenue through which populations can evolve different phenotypic states.
It is known that the function of the chaperone Hsp90 is to assist metastable ‘client’ proteins by conformational stabilization, therefore triggering their ability to be activated in a proper manner, transiently and spatially. The diverse class of ‘client’ proteins also encompasses regulators of the multicomplex network of development (Buchner, 1999; Young et al., 2001; Picard, 2002; Zhao et al., 2005). It is conceivable that a shortage of the Hsp90 system could provoke an interruption in the continuity of the pathway. Since Hsp90 cross-talks with many components of the regulatory pathway (Zhao et al., 2005), the lack of a particular member of the Hsp90 family could potentially interfere stochastically with a specific networking pathway. The random divergence of the adult phenotypes from the seedling phenotype appearance of all hsp90 mutants draws a direct line between stochastic mechanisms in development and Hsp90. It also indicates that the Hsp90 system actually restricts the magnitude of perturbations in development, a prerequisite in polymorphism of morphogenesis of multicellular organisms besides the genetic variation, and buffers the canalization of development.
Morphogenetic appearances of all four hsp90 mutants were developmentally stochastic and independently resulted in classes of phenotypic appearance. Each hsp90 mutant showed this classification of phenotypes reminiscent of each other. Nevertheless, discrete phenotypic categories in each mutant line indicated a fine tuning of stochastic mechanisms in specific aspects of development. The inhibition of Hsp90 should be modest in order to uncover these diverse and specific phenotypic classes, i.e. mutation in one of the four genes or allowing a threshold in the networking of chaperone activity as was observed in Drosophila (Rutherford and Lindquist, 1998). If reduction of Hsp90 activity passes a certain threshold, then it could interfere stochastically with a particular developmental process that is dependent on Hsp90 action. Therefore, each individual plant from any mutant might represent a distinct phenotypic class (as those that were not classified). However, the canalization process during development and most plausibly the intrinsic capacity of the Hsp90 system and a degree of overlap in function of the four genes restrict this phenomenon, and consequently these mutant morphs are clustered. Partial fixation of certain phenotypes was also observed. Thus, Hsp90 normally restricts the stochastic mechanisms that are potentially capable of producing changes in phenotypic traits within the dynamic network of signals and pathways of the developmental processes.
Misfolding of proteins which are also provoked by stress conditions induces Hsp90. When Hsp90 competence to sustain a functional pathway is exceeded by increased demand, mutation, and heat stress, then these pathways become highly discontinuous, resulting in a diverse range of developmental defects and phenotypes at any point during the course of development. The more severe the stress was, the broader the range of polymorphic traits observed. Heat stress on Col plants led to the emergence of polymorphic traits. The severity of phenotypes was directly related to the level of stress. Even more peculiar phenotypes were apparent when the Hsp90 system was additionally challenged by mutation, meaning that the erroneous switch could be almost anywhere or at almost any point in the developmental process, and multiple polymorphic morphs will emerge when Hsp90 is strongly challenged. This again indicates that the Hsp90 chaperone is networking developmental plasticity and therefore stochastic mechanisms. The phenotypes recovered through the hsp90 mutant lines did not emerge from general loss of vigour since they were more robust than Col plants under short but severe stress. Hence, environmental stress intensifies stochastic mechanisms in hsp90 mutant backgrounds as it reinforces the demand for Hsp90 activity in regulating gene networks.
The phenotypic classes of the individual hsp90 mutants were varied in relation to the time for which the stress was applied. There was not a clear direction towards a particular proportion of phenotypic class when the stress was applied for different time periods, and morphogenesis changed arbitrarily. The earlier the Hsp90 was challenged the greater the perturbation events during development, thus the broader the magnitude of polymorphic phenotypes.
Given that development and morphogenetic appearance are influenced by time and environment, and virtually by the genetic background, it was envisaged that development passes through a dynamic cone in which any point in space represents a distinct morphogenetic appearance at a particular time frame of development. If the phenotype at a specific time of development could be represented as a point in space, then the next position or phenotype appearance cannot be predicted precisely, apparently due to stochastic mechanisms in development. However, there is a given frame or restricted space for possible phenotypes to emerge. The present results show that this cone, made up from these points in space representing phenotypes at a particular developmental stage, is directly related to genome, environment, and time. The smaller the volume of the cone, the less the polymorphism. The width of the cone, representing the range of the morphs, is restricted by the Hsp90 chaperone system. The more or the earlier in development the Hsp90 was challenged, the wider the angle of the cone, the more the perturbation of stochastic events, and the higher the number of variants of morphs uncovered (Fig. 7). When the Hsp90 system was additionally challenged by elevated temperature at any given time point of development, more polymorphism and peculiar phenotypes were detected. Therefore, abrupt environmental alterations broaden the angle of the cone, and therefore diverse morphs appear (Fig. 7). This dynamic scheme symbolizes how the Hsp90 challenge allows more freedom in stochastic development. According to this model, it was envisaged that if the challenge of the Hsp90 system is complete, the angle of the cone might become wide enough to be represented eventually by a straight line, meaning lethality. Therefore, the canalization process is manifested by the Hsp90 system through the interaction with ‘client’ proteins via their activation in proper time and space, in order to exhibit proper developmental pathways and normal or canalized morphogenesis.
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‘Mild’ traits or noise are manifested in isogenic Col plants, though in an extremely low proportion, since the canalization process orchestrates canonical development. However, when Col plants are stressed, phenotypes having defects in development appear. Consequently, Hsp90 restricts polymorphic appearances by controlling stochastic mechanisms in development that otherwise occur when the functional status of a protein is continuously challenged in the dynamic state of cell differentiation through the rigorous interplay of genome, environment, and time.
Crucially, ‘phenocopying’ mechanisms under a suitable environment uncover polymorphisms, as in the case of Hsp90 challenge. These apparent polymorphisms could be perceived as adaptive forms and a rapid response of a population to sudden unforeseen environmental events without the intervening period of reduced fitness. Most polymorphisms could be deleterious and may be periodically eliminated from the population, as in the case of highly deformed embryos, seedlings, or plants of all hsp90 mutants due to selective abortions of residuals with high noise loads. Curiously, certain polymorphisms can uncover advantageous phenotypes. The enlargement of the angle of the cone of morphogenetic appearances (Fig. 7) allows more perturbations or adaptive peak shifts to occur in populations, without passing an adaptive valley. When the parents were subjected to mild stress, the offspring were less polymorphic and adaptive while the longer the stress, the more the polymorphism and adaptability in the next progeny. Consequently, there is a positive correlation between polymorphism and adaptability. Hence, from the evolutionary point of view, polymorphisms based on perturbations due to protein activation/inactivation in a developmental pathway could be crucial determinants for the adaptive peak. The more sensitive a system is, the more responsive to environmental changes, and hence it is evolvable. AtHsp90-1 appears to be the most responsive to abrupt environmental changes, while its expression level is rather low at 22 °C (Haralampidis et al., 2002). Evidently, these advantageous, but nevertheless highly polymorphic, phenotypes were recovered when the Athsp90-1 mutant was challenged by heat stress at early stages of development, permitting higher germination rates and therefore better adaptation in the next generation.
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Supplementary material |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
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The following supplementary material is available at JXB on line.
Fig. S1. Effect of time and environment on developmental canalization.
Fig. S2. Seedling polymorphisms from seeds set at 27 °C.
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Acknowledgements |
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The authors thank the Arabidopsis Biological Resource Center (ABRC) and Nottingham Arabidopsis Stock Center (NASC). We are grateful to G Banilas for his critical comments. This research was partly supported by a grant to P.H. from the GSRT, Greece (PENED 01/148) and Pythagoras I.
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* Present address: Mount Sinai School of Medicine, Graduate School of Biological Sciences, One Gustave L. Levy Place, Box 1022, New York, NY 10029-6574, USA
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