James L. Weller

Manipulation of the Blue Light Photoreceptor Cryptochrome 2 in Tomato Affects Vegetative Development, Flowering Time, and Fruit Antioxidant Content

Cryptochromes are blue light photoreceptors found in plants, bacteria, and animals. In Arabidopsis, cryptochrome 2 (cry2) is involved primarily in the control of flowering time and in photomorphogenesis under low-fluence light. No data on the function of cry2 are available in plants, apart from Arabidopsis (Arabidopsis thaliana). Expression of the tomato (Solanum lycopersicum) CRY2 gene was altered through a combination of transgenic overexpression and virus-induced gene silencing. Tomato CRY2 overexpressors show phenotypes similar to but distinct from their Arabidopsis counterparts (hypocotyl and internode shortening under both low- and high-fluence blue light), but also several novel ones, including a high-pigment phenotype, resulting in overproduction of anthocyanins and chlorophyll in leaves and of flavonoids and lycopene in fruits. The accumulation of lycopene in fruits is accompanied by the decreased expression of lycopene β-cyclase genes. CRY2 overexpression causes an unexpected delay in flowering, observed under both short- and long-day conditions, and an increased outgrowth of axillary branches. Virus-induced gene silencing of CRY2 results in a reversion of leaf anthocyanin accumulation, of internode shortening, and of late flowering in CRY2-overexpressing plants, whereas in wild-type plants it causes a minor internode elongation.

Conservation of Arabidopsis Flowering Genes in Model Legumes

The model plants Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) have provided a wealth of information about genes and genetic pathways controlling the flowering process, but little is known about the corresponding pathways in legumes. The garden pea (Pisum sativum) has been used for several decades as a model system for physiological genetics of flowering, but the lack of molecular information about pea flowering genes has prevented direct comparison with other systems. To address this problem, we have searched expressed sequence tag and genome sequence databases to identify flowering-gene-related sequences from Medicago truncatula, soybean (Glycine max), and Lotus japonicus, and isolated corresponding sequences from pea by degenerate-primer polymerase chain reaction and library screening. We found that the majority of Arabidopsis flowering genes are represented in pea and in legume sequence databases, although several gene families, including the MADS-box, CONSTANS, and FLOWERING LOCUS T/TERMINAL FLOWER1 families, appear to have undergone differential expansion, and several important Arabidopsis genes, including FRIGIDA and members of the FLOWERING LOCUS C clade, are conspicuously absent. In several cases, pea and Medicago orthologs are shown to map to conserved map positions, emphasizing the closely syntenic relationship between these two species. These results demonstrate the potential benefit of parallel model systems for an understanding of flowering phenology in crop and model legume species.

Pea Mutants with Reduced Sensitivity to Far-Red Light Define an Important Role for Phytochrome A in Day-Length Detection

In garden pea (Pisum sativum L.), a long-day plant, long photoperiods promote flowering by reducing the synthesis or transport of a graft-transmissible inhibitor of flowering. Previous physiological studies have indicated that this promotive effect is predominantly achieved through a response that requires long exposures to light and for which far-red (FR) light is the most effective. These characteristics implicate the action of phytochrome A (phyA). To investigate this matter further, we screened ethylmethane sulfonate-mutagenized pea seedlings for FR-unresponsive, potentially phyA-deficient mutants. Two allelic, recessive mutants were isolated and were designated fun1 for FR unresponsive. The fun1–1 mutant is specifically deficient in the PHYA apoprotein and has a seedling phenotype indistinguishable from wild type when grown under white light. However, fun1–1 plants grown to maturity under long photoperiods show a highly pleiotropic phenotype, with short internodes, thickened stems, delayed flowering and senescence, longer peduncles, and higher seed yield. This phenotype results in large part from an inability of fun1–1 to detect day extensions. These results establish a crucial role for phyA in the control of flowering in pea, and show that phyA mediates responses to both red and FR light. Furthermore, grafting and epistasis studies with fun1 and dne, a mutant deficient in the floral inhibitor, show that the roles of phyA in seedling deetiolation and in day-length detection are genetically separable and that the phyA-mediated promotion of flowering results from a reduction in the synthesis or transport of the floral inhibitor.

Pea LATE BLOOMER1 is a GIGANTEA ortholog with roles in photoperiodic flowering, deetiolation, and transcriptional regulation of circadian clock gene homologs

Genes controlling the transition to flowering have been studied in several species, including Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), but have not yet received much attention in legumes. Here, we describe a new allelic series of late-flowering, photoperiod-insensitive mutants in the pea (Pisum sativum) LATE BLOOMER1 (LATE1) gene and show that LATE1 is an ortholog of Arabidopsis GIGANTEA. Mutants display defects in phytochrome B-dependent deetiolation under red light and in the diurnal regulation of pea homologs of several Arabidopsis circadian clock genes, including TIMING OF CAB1, EARLY FLOWERING4, and CIRCADIAN CLOCK ASSOCIATED1/LATE ELONGATED HYPOCOTYL. LATE1 itself shows strongly rhythmic expression with a small but distinct acute peak following dark-to-light transfer. Mutations in LATE1 prevent the induction of a FLOWERING LOCUS T (FT) homolog FTL in long days but cause only minor alteration to the rhythmic expression pattern of the only known group Ia CONSTANS homolog COLa. The late-flowering phenotype of late1 mutants can be completely rescued by grafting to the wild type, but this rescue is not associated with a significant increase in FTL transcript level in shoot apices. Genetic interactions of late1 with the photoperiod-insensitive, early-flowering sterile nodes (sn) mutant and impairment of the LATE1 diurnal expression rhythm in sn plants suggest that SN may also affect the circadian clock. These results show that several functions of Arabidopsis GIGANTEA are conserved in its pea ortholog and demonstrate that genetic pathways for photoperiodic flowering are likely to be conserved between these two species. They also suggest that in addition to its role in the floral transition, LATE1 also acts throughout reproductive development.

The genetic control of flowering in pea

The transition from vegetative to reproductive growth is a key event during plant development. In many species, the timing of this change depends on the length and spectral quality of the photoperiod. Extensive research supports the notion that the site of light perception in the leaf and the site of flower initiation in the apex are linked by the action of mobile, hormone-like substances. Physiological characterization of flowering mutants in the garden pea has identified two distinct mobile regulators of flowering produced in the leafy shoot — a floral stimulus and an inhibitor. The stimulus is specific to the flowering process and is thought to be constitutively produced. In contrast, the inhibitor affects both reproductive and vegetative growth and is strongly regulated. Photoperiod sensitivity is mainly conferred by a phytochrome A-mediated reduction in inhibitor level.

Light-Induced Nuclear Translocation of Endogenous Pea Phytochrome A Visualized by Immunocytochemical Procedures

Although the physiological functions of phytochrome A (PhyA) are now known, the distribution of endogenous PhyA has not been examined. We have visualized endogenous PhyA apoprotein (PHYA) by immunolabeling cryosections of pea tissue, using PHYA-deficient mutants as negative controls. By this method, we examined the distribution of PHYA in different tissues and changes in its intracellular distribution in response to light. In apical hook cells of etiolated seedlings, PHYA immunolabeling was distributed diffusely in the cytosol. Exposure to continuous far-red (cFR) light caused a redistribution of the immunolabeling to the nucleus, first detectable after 1.5 hr and greatest at 4.5 hr. During this time, the amounts of spectrally active phytochrome and PHYA did not decline substantially. Exposure to continuous red (cR) light or to a brief pulse of red light also resulted in redistribution of immunolabeling to the nucleus, but this occurred much more rapidly and with a different pattern of intranuclear distribution than it did in response to cFR light. Exposures to cR light resulted in loss of immunolabeling, which was associated with PHYA degradation. These results indicate that the light-induced intracellular location of PHYA is wavelength dependent and imply that this is important for PhyA activity.

The Pea GIGAS Gene Is a FLOWERING LOCUS T Homolog Necessary for Graft-Transmissible Specification of Flowering but Not for Responsiveness to Photoperiod

The pea flowering gene GIGAS regulates a mobile flowering signal and is essential for flowering under long days but not for the ability to respond to photoperiod. This study characterizes the FLOWERING LOCUS T (FT) gene family in pea, identifies one gene (FTa1) as GIGAS, and associates another gene (FTb2) with a second mobile signal and a broader role in photoperiod responsiveness. Garden pea (Pisum sativum) was prominent in early studies investigating the genetic control of flowering and the role of mobile flowering signals. In view of recent evidence that genes in the FLOWERING LOCUS T (FT) family play an important role in generating mobile flowering signals, we isolated the FT gene family in pea and examined the regulation and function of its members. Comparison with Medicago truncatula and soybean (Glycine max) provides evidence of three ancient subclades (FTa, FTb, and FTc) likely to be common to most crop and model legumes. Pea FT genes show distinctly different expression patterns with respect to developmental timing, tissue specificity, and response to photoperiod and differ in their activity in transgenic Arabidopsis thaliana, suggesting they may have different functions. We show that the pea FTa1 gene corresponds to the GIGAS locus, which is essential for flowering under long-day conditions and promotes flowering under short-day conditions but is not required for photoperiod responsiveness. Grafting, expression, and double mutant analyses show that GIGAS/FTa1 regulates a mobile flowering stimulus but also provide clear evidence for a second mobile flowering stimulus that is correlated with expression of FTb2 in leaf tissue. These results suggest that induction of flowering by photoperiod in pea results from interactions among several members of a diversified FT family.

Tendril-less regulates tendril formation in pea leaves

Tendrils are contact-sensitive, filamentous organs that permit climbing plants to tether to their taller neighbors. Tendrilled legume species are grown as field crops, where the tendrils contribute to the physical support of the crop prior to harvest. The homeotic tendril-less (tl) mutation in garden pea (Pisum sativum), identified almost a century ago, transforms tendrils into leaflets. In this study, we used a systematic marker screen of fast neutron–generated tl deletion mutants to identify Tl as a Class I homeodomain leucine zipper (HDZIP) transcription factor. We confirmed the tendril-less phenotype as loss of function by targeting induced local lesions in genomes (TILLING) in garden pea and by analysis of the tendril-less phenotype of the t mutant in sweet pea (Lathyrus odoratus). The conversion of tendrils into leaflets in both mutants demonstrates that the pea tendril is a modified leaflet, inhibited from completing laminar development by Tl. We provide evidence to show that lamina inhibition requires Unifoliata/LEAFY-mediated Tl expression in organs emerging in the distal region of the leaf primordium. Phylogenetic analyses show that Tl is an unusual Class I HDZIP protein and that tendrils evolved either once or twice in Papilionoid legumes. We suggest that tendrils arose in the Fabeae clade of Papilionoid legumes through acquisition of the Tl gene.

A Study of Gibberellin Homeostasis and Cryptochrome-Mediated Blue Light Inhibition of Hypocotyl Elongation

Cryptochromes mediate blue light-dependent photomorphogenic responses, such as inhibition of hypocotyl elongation. To investigate the underlying mechanism, we analyzed a genetic suppressor, scc7-D (suppressors of cry1cry2), which suppressed the long-hypocotyl phenotype of the cry1cry2 (cryptochrome1/cryptochrome2) mutant in a light-dependent but wavelength-independent manner. scc7-D is a gain-of-expression allele of the GA2ox8 gene encoding a gibberellin (GA)-inactivating enzyme, GA 2-oxidase. Although scc7-D is hypersensitive to light, transgenic seedlings expressing GA2ox at a level higher than scc7-D showed a constitutive photomorphogenic phenotype, confirming a general role of GA2ox and GA in the suppression of hypocotyl elongation. Prompted by this result, we investigated blue light regulation of mRNA expression of the GA metabolic and catabolic genes. We demonstrated that cryptochromes are required for the blue light regulation of GA2ox1, GA20ox1, and GA3ox1 expression in transient induction, continuous illumination, and photoperiodic conditions. The kinetics of cryptochrome induction of GA2ox1 expression and cryptochrome suppression of GA20ox1 or GA3ox1 expression correlate with the cryptochrome-dependent transient reduction of GA4 in etiolated wild-type seedlings exposed to blue light. Therefore we propose that in deetiolating seedlings, cryptochromes mediate blue light regulation of GA catabolic/metabolic genes, which affect GA levels and hypocotyl elongation. Surprisingly, no significant change in the GA4 content was detected in the whole shoot samples of the wild-type or cry1cry2 seedlings grown in the dark or continuous blue light, suggesting that cryptochromes may also regulate GA responsiveness and/or trigger cell- or tissue-specific changes of the level of bioactive GAs.

Gibberellins and phytochrome regulation of stem elongation in pea

In garden pea (Pisum sativum L.) neither etiolation nor the phytochrome B (phyB)-response mutation lv substantially alters the level of the major active endogenous gibberellin, GA1 in the apical portion of young seedlings. The phyB-controlled responses to continuous red light and end-of-day far-red light are retained even in a GA-overproducing mutant (sln). Comparison of the effects of the lv mutation and GA1 application on seedling development shows important differences in rate of node development, cell extension and division, and leaf development. These results suggest that in pea the control of stem elongation by light in general and phyB in particular is not mediated by changes in GA1 content. Instead, the increased elongation of dark-grown and lv plants appears to result from increased responsiveness of the plant to its endogenous levels of GA1. Three GA1-deficient mutants, na, ls and le have been used to investigate these changes in responsiveness, and study of these and the double mutants na lv, ls lv and le lv has demonstrated that the relative magnitude of the change in responsiveness is dependent on GA1 level. The difference in pleiotropic effects of GA1 application and the lv mutation suggest that light and GA1 interact late in their respective transduction pathways. A model for the relationship between light, GA1 level and elongation in pea is presented and discussed.