Candidate gene analysis of organ pigmentation loci in the Solanaceae

Ten structural genes from the Capsicum (pepper) carotenoid biosynthetic pathway have been localized on a (Capsicum annuum x Capsicum chinense)F(2) genetic map anchored in Lycopersicon (tomato). The positions of these genes were compared with positions of the same genes in tomato when known, and with loci from pepper, potato, and tomato that affect carotenoid levels in different tissues. C2, one of three phenotypically defined loci determining pepper fruit color, cosegregated with phytoene synthase. The capsanthin-capsorubin synthase (Ccs) locus, shown previously to cosegregate with Y, another pepper fruit color locus, mapped to pepper chromosome 6. Other structural genes in pepper corresponded to loci affecting carotenoid production as follows: Ccs in pepper and the B locus for hyperaccumulation of beta-carotene in tomato fruit mapped to homeologous regions; the position of the lycopene beta-cyclase gene in pepper may correspond to the lutescent-2 mutation in tomato; and the lycopene epsilon-cyclase locus in pepper corresponded to the lycopene epsilon-cyclase locus/Del mutation for hyperaccumulation of delta-carotene in tomato fruit. Additional associations were seen between the structural genes and previously mapped loci controlling quantitative variation in pepper and tomato fruit color. These results demonstrate that comparative analyses using candidate genes may be used to link specific metabolic phenotypes and loci that affect these phenotypes in related species.     

Systemin activates synthesis of wound-inducible tomato leaf polyphenol oxidase via the octadecanoid defense signaling pathway.

Tomato plants overexpressing a prosystemin gene that encodes the precursor of a mobile wound signal called systemin have been shown previously to constitutively synthesize extraordinarily high levels of two defensive proteinase inhibitor proteins in leaves in the absence of wounding. We herein report that leaves of these transgenic plants possess enhanced levels of another defensive protein, polyphenol oxidase (PPO) at levels that are up to 70-fold higher than levels found in leaves of wild-type plants. Supplying young wild-type tomato plants with systemin through cut stems induced PPO activity in leaves, and wounding lower leaves of young tomato plants induced PPO activity in both wounded and unwounded leaves to levels equal to those induced by systemin. Exposing young tomato plants to methyl jasmonate vapor caused an increase in PPO activity equivalent to levels found in plants overexpressing the prosystemin gene. The data indicate that PPO and proteinase inhibitor genes are coactivated systemically by wounding via the octadecanoid signal transduction pathway and that systemin has a much broader role in signaling plant defensive genes than was previously known.     

Signals involved in wound-induced proteinase inhibitor II gene expression in tomato and potato plants

Chemical and physical signals have been reported to mediate wound-induced proteinase inhibitor II (Pin2) gene expression in tomato and potato plants. Among the chemical signals, phytohormones such as abscisic acid (ABA) and jasmonic acid (JA) and the peptide systemin represent the best characterized systems. Furthermore, electrical and hydraulic mechanisms have also been postulated as putative Pin2-inducing systemic signals. Most of the chemical agents are able to induce Pin2 gene expression without any mechanical wounding. Thus, ABA, JA, and systemin initiate Pin2 mRNA accumulation in the directly treated leaves and in the nontreated leaves (systemic) that are located distal to the treated ones. ABA-deficient tomato and potato plants do not respond to wounding by accumulation of Pin2 mRNA, therefore providing a suitable model system for analysis of the signal transduction pathway involved in wound-induced gene activation. It was demonstrated that the site of action of JA is located downstream to the site of action of ABA. Moreover, systemin represents one of the initial steps in the signal transduction pathway regulating the wound response. Recently, it was reported that heat treatment and mechanical injury generate electrical signals, which propagate throughout the plant. These signals are capable of inducing Pin2 gene expression in the nontreated leaves of wounded plants. Furthermore, electrical current application to tomato leaves leads to an accumulation of Pin2 mRNA in local and systemic tissues. Examination of photosynthetic parameters (assimilation and transpiration rate) on several types of stimuli suggests that heat-induced Pin2 gene expression is regulated by an alternative pathway from that mediating the electrical current and mechanical wound response.     

The Arabidopsis repressor of light signaling, COP1, is regulated by nuclear exclusion: mutational analysis by bioluminescence resonance energy transfer

Bioluminescence resonance energy transfer (BRET) between Renilla luciferase and yellow fluorescent protein has been adapted to serve as a real-time reporter on protein-protein interactions in live plant cells by using the Arabidopsis Constitutive photomorphogenesis 1 (COP1) protein as a model system. COP1 is a repressor of light signal transduction that functions as part of a nuclear E3 ubiquitin ligase. COP1 possesses a leucine-rich nuclear-exclusion signal that resides in a domain implicated in COP1 dimerization. BRET was applied in conjunction with site-directed mutagenesis to explore the respective contributions of the nuclear-exclusion and dimerization motifs to the regulation of COP1 activity in vivo. One specific mutant protein, COP1(L105A), showed increased nuclear accumulation but retained the ability to dimerize, as monitored by BRET, whereas other mutations inhibited both nuclear exclusion and COP1 dimerization. Mutant rescue and overexpression experiments indicated that nuclear exclusion of COP1 protein is a rate-limiting step in light signal transduction.     

Oligogalacturonides and chitosan activate plant defensive genes through the octadecanoid pathway

Jasmonic acid, synthesized from linolenic acid (the octadecanoid pathway), has been proposed to be part of a signal transduction pathway that mediates the induction of defensive genes in plants in response to oligouronide and polypeptide signals generated by insect and pathogen attacks. We report here that the induction of proteinase inhibitor accumulation in tomato leaves by plant-derived oligogalacturonides and fungal-derived chitosan oligosaccharides is severely reduced by two inhibitors (salicylic acid and diethyldi-thiocarbamic acid) of the octadecanoid pathway, supporting a role for the pathway in signaling by oligosaccharides. Jasmonic acid levels in leaves of tomato plants increased several fold within 2 hr after supplying the polypeptide systemin, oligogalacturonides, or chitosan to the plants through their cut stems, as expected if they utilize the octadecanoid pathway. The time course of jasmonic acid accumulation in tomato leaves in response to wounding was consistent with its proposed role in signaling proteinase inhibitor mRNA and protein synthesis. The cumulative evidence supports a model for the activation of defensive genes in plants in response to insect and pathogen attacks in which various elicitors generated at the attack sites activate the octadecanoid pathway via different recognition events to induce the expression of defensive genes in local and distal tissues of the plants.     

Ripening in the tomato Green-ripe mutant is inhibited by ectopic expression of a protein that disrupts ethylene signaling

To achieve full ripening, climacteric fruits, such as tomato require synthesis, perception and signal transduction of the plant hormone ethylene. The nonripening phenotype of the dominant Green-ripe (Gr) and Never-ripe 2 (Nr-2) mutants of tomato is the result of reduced ethylene responsiveness in fruit tissues. In addition, a subset of ethylene responses associated with floral senescence, abscission, and root elongation are also impacted in mutant plants, but to a lesser extent. Using positional cloning, we have identified an identical 334-bp deletion in a gene of unknown biochemical function at the Gr/Nr-2 locus. Consistent with a dominant gain of function mutation, this deletion causes ectopic expression of Gr/Nr-2, which in turn leads to ripening inhibition. A CaMV35::GR transgene recreates the Gr/Nr-2 mutant phenotype but does not lead to a global reduction in ethylene responsiveness, suggesting tissue-specific modulation of ethylene responses in tomato. Gr/Nr-2 encodes an evolutionary conserved protein of unknown biochemical function that we associate here with ethylene signaling. Because Gr/Nr-2 has no sequence homology with the previously described Nr (Never-ripe) ethylene receptor of tomato we now refer to this gene only as GR. Identification of GR expands the current repertoire of ethylene signaling components in plants and provides a tool for further elucidation of ethylene response mechanisms and for controlling ethylene signal specificity in crop plants.     

Derepression of the NC80 motif is critical for the photoactivation of Arabidopsis CRY2

Cryptochromes are blue light receptors that regulate photomorphogenesis in plants and the circadian clock in animals and plants. Arabidopsis cryptochrome 2 (CRY2) mediates blue light inhibition of hypocotyl elongation and photoperiodic control of floral initiation. CRY2 undergoes blue light-induced phosphorylation, which was hypothesized to be associated with CRY2 photoactivation. To further investigate how light activates CRY2, we analyzed the physiological activities and phosphorylation of various CRY2 fusion proteins in transgenic plants. Our results showed that an 80-residue motif, referred to as NC80, was sufficient to confer the physiological function of CRY2. The GUS-NC80 fusion protein expressed in transgenic plants is constitutively active but unphosphorylated, suggesting that the blue light-induced CRY2 phosphorylation causes a conformational change to derepress the NC80 motif. Consistent with this hypothesis, the CRY2 C-terminal tail was found to be required for the blue light-induced CRY2 phosphorylation but not for the CRY2 activity. We propose that the PHR domain and the C-terminal tail of the unphosphorylated CRY2 form a "closed" conformation to suppress the NC80 motif in the absence of light. In response to blue light, the C-terminal tail of CRY2 is phosphorylated and electrostatically repelled from the surface of the PHR domain to form an "open" conformation, resulting in derepression of the NC80 motif and signal transduction to trigger photomorphogenic responses.     

In vitro phosphorylation of plant plasma membrane proteins in response to the proteinase inhibitor inducing factor

A polygalacturonide purified from a tomato leaf pectic polysaccharide that induces the systemic synthesis of proteinase inhibitors in tomato plants enhances the phosphorylation of specific proteins in plasma membrane fractions isolated from tomato and potato leaves. In tomato plasma membranes, two proteins of 34 and 29 kDa show enhanced phosphorylation in response to the polyuronide. In potato plasma membranes, only a protein of 34 kDa exhibited enhanced phosphorylation due to the polyuronide. A noncarbohydrate class of proteinase inhibitor inducing factor, recently identified by workers in this laboratory, resulted in the in vitro hyperphosphorylation of a family of proteins of approximately 27 kDa. The phosphorylation of specific polypeptides in leaves in response to the same factors that induce the expression of proteinase inhibitor genes suggests that protein kinases may play an important role in the mechanism of signal transduction leading to defense gene expression.     

SPINDLY, a tetratricopeptide repeat protein involved in gibberellin signal transduction in Arabidopsis.

Gibberellins (GAs) are a major class of plant hormones that control many developmental processes, including seed development and germination, flower and fruit development, and flowering time. Genetic studies with Arabidopsis thaliana have identified two genes involved in GA perception or signal transduction. A semidominant mutation at the GIBBERELLIN INSENSITIVE (GAI) locus results in plants resembling GA-deficient mutants but exhibiting reduced sensitivity to GA. Recessive mutations at the SPINDLY (SPY) locus cause a phenotype that is consistent with constitutive activation of GA signal transduction. Here we show that a strong allele of spy is completely epistatic to gai, indicating that SPY acts downstream of GAI. We have cloned the SPY gene and shown that it encodes a new type of signal transduction protein, which contains a tetratricopeptide repeat region, likely serving as a protein interaction domain, and a novel C-terminal region. Mutations in both domains increase GA signal transduction. The presence of a similar gene in Caenorhabditis elegans suggests that SPY represents a class of signal transduction proteins that is present throughout the eukaryotes.     

A new class of regulatory genes underlying the cause of pear-shaped tomato fruit

A common, recurring theme in domesticated plants is the occurrence of pear-shaped fruit. A major quantitative trait locus (termed ovate) controlling the transition from round to pear-shaped fruit has been cloned from tomato. OVATE is expressed early in flower and fruit development and encodes a previously uncharacterized, hydrophilic protein with a putative bipartite nuclear localization signal, Von Willebrand factor type C domains, and an approximately equal 70-aa C-terminal domain conserved in tomato, Arabidopsis, and rice. A single mutation, leading to a premature stop codon, causes the transition of tomato fruit from round- to pear-shaped. Moreover, ectopic, transgenic expression of OVATE unevenly reduces the size of floral organs and leaflets, suggesting that OVATE represents a previously uncharacterized class of negative regulatory proteins important in plant development.     

Chlamydomonas mutants affected in the light-dependent step of sexual differentiation.

Sexual differentiation of Chlamydomonas reinhardtii is induced by the consecutive action of two extrinsic cues--nitrogen deprivation and blue light. The definition of a blue light-dependent step in gamete formation provided a basis for the isolation of mutants altered in the signal transduction pathway by which light controls sexual differentiation. In one mutant (lrg1), gamete formation has become light independent. In the other mutant (lrg2), perception or transduction of the light signal appears to be partially impaired. In both mutants, the expression of genes activated by light in the late phase of gamete formation is affected. Genetic analyses showed that genes LRG1 and LRG2 are linked. The recessive nature of the lrg1-1 mutation implies that the gene encodes a negative factor or a protein that controls the activity of a negative factor. In the case of lrg2-1, neither wild-type nor mutant allele was dominant. Rather, two copies of the lrg2-1 gene simulate a wild-type phenotype. The identification of genetic loci in the pathway for blue light-mediated differentiation provides a basis for the isolation of signal transduction genes in Chlamydomonas.     

Expression of an Arabidopsis cryptochrome gene in transgenic tobacco results in hypersensitivity to blue, UV-A, and green light.

The Arabidopsis HY4 gene, required for blue-light-induced inhibition of hypocotyl elongation, encodes a 75-kDa flavoprotein (CRY1) with characteristics of a blue-light photoreceptor. To investigate the mechanism by which this photoreceptor mediates blue-light responses in vivo, we have expressed the Arabidopsis HY4 gene in transgenic tobacco. The transgenic plants exhibited a short-hypocotyl phenotype under blue, UV-A, and green light, whereas they showed no difference from the wild-type plant under red/far-red light or in the dark. This phenotype was found to cosegregate with overexpression of the HY4 transgene and to be fluence dependent. We concluded that the short-hypocotyl phenotype of transgenic tobacco plants was due to hypersensitivity to blue, UV-A, and green light, resulting from over-expression of the photoreceptor. These observations are consistent with the broad action spectrum for responses mediated by this cryptochrome in Arabidopsis and indicate that the machinery for signal, transduction required by the CRY1 protein is conserved among different plant species. Furthermore, the level of these photoresponses is seen to be determined by the cellular concentration of this photoreceptor.