Gymnosperm Ginkgo Research Articles

Genome-wide identification of the EIN3/EIL gene family in Ginkgo biloba and functional study of a GbEIL in the ethylene signaling pathway

ETHYLENE-INSENSITIVE3 (EIN3) or EIN3-Like (EIL) proteins, play critical roles in integrating ethylene signaling and physiological regulation in plants by modulating the expression of various downstream genes, such as ethylene-response factors (ERFs). However, little is known about the characteristics of EIN3/EILs in the gymnosperm Ginkgo biloba. In the present study, a genome-wide comparative analysis of Ginkgo EIN3/EIL gene family was performed with those from an array of species, including bryophytes (Physcomitrella patens), gymnosperms (Cycas panzhihuaensis), and angiosperms (Arabidopsis thaliana, Gossypium raimondii, Gossypium hirsutum, Oryza sativa, and Brachypodium distachyon). Within the constructed phylogenetic tree for the 53 EIN3/EILs identified, 5 GbEILs from G. biloba, 2 PpEILs from P. patens, and 3 CpEILs from C. panzhihuaensis were assigned to one cluster, suggesting that their derivation occurred after the split of their ancestors and angiosperms. Although considerable divergence accumulated in amino acid sequences along with the evolutionary process, the specific EIN3_DNA-binding domains were evolutionarily conserved among the 53 EIN3/EILs. Collinearity analysis indicated that whole-genome or segmental duplication and subsequent purifying selection might have prompted the generation and evolution of EIN3/EIL multigene families. Based on the expression patterns of five GbEILs at the four developmental stages of Ginkgo ovules, one GbEIL gene (Gb_03292) was further investigated for its role in mediating ethylene signaling. The functional activity of Gb_03292 was closely related to ethylene signaling, as it complemented the triple response via ectopic expression in ein3eil1 double mutant Arabidopsis. Additionally, GbEIL likely modulates the expression of a Ginkgo ERF (Gb_15517) by directly binding to its promoter. These results demonstrated that the GbEIL gene could have participated in mediating ethylene signal transduction during ovule development in G. biloba. The present study also provides insights into the conservation of ethylene signaling across the gymnosperm G. biloba and angiosperm species.

CfAPX, a cytosolic ascorbate peroxidase gene from Cryptomeria fortunei, confers tolerance to abiotic stress in transgenic Arabidopsis

Plants subjected to biotic or abiotic stresses produce a large amount of reactive oxygen species (ROS). If ROS cannot be cleared in time, they cause a series of harmful reactions in plants. Ascorbate peroxidase (APX) is a key enzyme that removes ROS from plant cells and plays a vital role in plant stress resistance. However, to date, no studies on APX homologs in Cryptomeria fortunei have been reported. In this study, we isolated complementary DNA (cDNA) encoding APXfrom C. fortunei needles, which is referred to as CfAPX, by rapid amplification of cDNA ends (RACE). The full-length CfAPX sequence was 1226 bp in length and included a 750-bp open reading frame (ORF) encoding a protein of 249 amino acids. Phylogenetic analysis showed that APXs of different plant species have been highly evolutionarily conserved. CfAPX was shown to belong to the cytoplasmic subgroup and was more closely related to GbAPX of the gymnosperm Ginkgo biloba. CfAPX showed no transcriptional activity in yeast cells but was highly expressed in cones. To better handle abiotic stresses, compared with wild-type (WT) Arabidopsis thaliana, 35S::CfAPX transgenic Arabidopsis strongly expressed CfAPX, presented increased antioxidant enzyme activities, ascorbic acid (AsA) contents, chlorophyll levels and fluorescence parameter and reduced malondialdehyde (MDA) and hydrogen peroxide (H2O2) contents. In addition, CfAPX expression in C. fortunei was mostly upregulated under stress. In summary, CfAPX confers abiotic stress responses to plants, which provides a scientific basis for subsequent breeding for increased stress resistance in C. fortunei.

Genome-wide identification and classification of MIKC-type MADS-box genes in Streptophyte lineages and expression analyses to reveal their role in seed germination of orchid

BackgroundMADS-box genes play crucial roles in plant floral organ formation and plant reproductive development. However, there is still no information on genome-wide identification and classification of MADS-box genes in some representative plant species. A comprehensive investigation of MIKC-type genes in the orchid Dendrobium officinale is still lacking.ResultsHere we conducted a genome-wide analysis of MADS-box proteins from 29 species. In total, 1689 MADS-box proteins were identified. Two types of MADS-box genes, termed type I and II, were found in land plants, but not in liverwort. The SQUA, DEF/GLO, AG and SEP subfamilies existed in all the tested flowering plants, while SQUA was absent in the gymnosperm Ginkgo biloba, and no genes of the four subfamilies were found in a charophyte, liverwort, mosses, or lycophyte. This strongly corroborates the notion that clades of floral organ identity genes led to the evolution of flower development in flowering plants. Nine subfamilies of MIKCC genes were present in two orchids, D. officinale and Phalaenopsis equestris, while the TM8, FLC, AGL15 and AGL12 subfamilies may be lost. In addition, the four clades of floral organ identity genes in both orchids displayed a conservative and divergent expression pattern. Only three MIKC-type genes were induced by cold stress in D. officinale while 15 MIKC-type genes showed different levels of expression during seed germination.ConclusionsMIKC-type genes were identified from streptophyte lineages, revealing new insights into their evolution and development relationships. Our results show a novel role of MIKC-type genes in seed germination and provide a useful clue for future research on seed germination in orchids.

Lacandonia granules are present in the cell nucleus of Welwitschia mirabilis

Background: Lacandonia granules are extranucleolar ribonucleoprotein (RNPs) particles, 32 nanometers in diameter that were first described in the nucleus of Lacandonia schismatica . Cytochemical and immunocytochemical studies suggest that these particles are equivalent to perichromatin and Balbiani ring granules described in mammals and salivary glands cells of the insect Chironomus tentans, respectively. Lacandonia granules are also present in the related Triuris brevystilis , and they were later described in the gymnosperm Ginkgo biloba . These findings suggest that Lacandonia granules have a wider distribution in the plant kingdom. Species study: The plant Welwitschia mirabilis, a gymnosperm of the order Gnetales.Hyphotesis: Lacandonia granules are present in the cell nucleus of W. mirabilis . Methods: Plants were cultivated in a germination chamber and samples of leaves were processed for transmission electron microscopy. Thin sections were stained with the EDTA technique preferential for ribonucleoproteins and osmium amine specific for DNA and observed with a microscopy.Results: Light, electronic and atomic force microscopy revealed that cell nuclei of W. mirabilis display a reticulated arrangement of chromatin. Moreover, granules of 32.17 ± 1.7 nm in diameter were observed among strands of reticulated chromatin.Conclusions: Our results indicate that Lacandonia granules are present in the nuclei of the gnetal W. mirabilis.

The gymnosperm ortholog of the angiosperm central cell‐specification gene CKI1 provides an essential clue to endosperm origin

A defining feature of angiosperms is double fertilization involving the female gametophyte central cell and formation of a nutrient-storing tissue called endosperm. The route for the evolutionary origin of endosperm from a gymnosperm ancestor, particularly the molecular steps involved, has remained elusive. Recently, the histidine kinase gene Cytokinin-Independent 1 (CKI1), an activator of cytokinin signaling, was described as a key to specification of the endosperm precursor central cell in Arabidopsis. Here, we have investigated the function and expression of a putative ortholog of CKI1 in the gymnosperm Ginkgo biloba. We demonstrate that Ginkgo CKI1 can partially rescue an Arabidopsis cki1 mutant and promote weak activation of the cytokinin signaling pathway in the Arabidopsis embryo sac, but does not confer central cell specification. Ginkgo CKI1 is expressed in both male and female gametophytes of Ginkgo. In the latter, it is expressed in the ventral canal cell, which is sister to the egg cell in the archegonium. As in Arabidopsis, Ginkgo CKI1 is not expressed in the egg cell. The similarities in expression patterns of CKI1 in Ginkgo and Arabidopsis female gametophytes suggest that extant gymnosperms possess an essential component of the molecular machinery required for angiosperm endosperm development, and provide new insights into endosperm origin from a gymnospermous ancestor.

Isolation and functional characterization of a circadian-regulated CONSTANS homolog (GbCO) from Ginkgo biloba.

This is the first report to clone and functionally characterize a flowering time gene GbCO in perennial gymnosperm Ginkgo biloba. GbCO complements the co mutant of Arabidopsis, restoring normal early flowering. CONSTANS (CO) is a central regulator of photoperiod pathway, which channels inputs from light, day length, and circadian clock to promote the floral transition. In order to understand the role of CO in gymnosperm Ginkgo biloba, which has a long juvenile phase (15-20years), a CO homolog (GbCO) was isolated and characterized from G. biloba. GbCO encodes a 1741-bp gene with a predicted protein of 400 amino acids with two zinc finger domains (B-box I and B-box II) and a CCT domain. Phylogenic analysis classified GbCO into the group 1a clade of CO families in accordance with the grouping scheme for Arabidopsis CO (AtCO). Southern blot analysis indicated that GbCO belongs to a multigene family in G. biloba. Real-time PCR analysis showed that GbCO was expressed in aerial parts of Ginkgo, with the highest transcript level of GbCO being observed in shoot apexes. GbCO transcript level exhibited a strong diurnal rhythm under flowering-inductive long days and peaked during early morning, suggesting that GbCO is tightly coupled to the floral inductive long-day signal. In addition, an increasing trend of GbCO transcript level was observed both in shoot tips and leaves as the shoot growth under long-day condition, whereas GbCO transcript level decreased in both tissues under short-day condition prior to growth cessation of shoot in G. biloba. GbCO complemented the Arabidopsis co-2 mutant, restoring normal early flowering. All the evidence being taken together, our findings suggested that GbCO served as a potential inducer of flowering in G. biloba.

Morintides: cargo-free chitin-binding peptides from Moringa oleifera

BackgroundHevein-like peptides are a family of cysteine-rich and chitin-binding peptides consisting of 29–45 amino acids. Their chitin-binding property is essential for plant defense against fungi. Based on the number of cysteine residues in their sequences, they are divided into three sub-families: 6C-, 8C- and 10C-hevein-like peptides. All three subfamilies contain a three-domain precursor comprising a signal peptide, a mature hevein-like peptide and a C-terminal domain comprising a hinge region with protein cargo in 8C- and 10C-hevein-like peptides.ResultsHere we report the isolation and characterization of two novel 8C-hevein-like peptides, designated morintides (mO1 and mO2), from the drumstick tree Moringa oleifera, a drought-resistant tree belonging to the Moringaceae family. Proteomic analysis revealed that morintides comprise 44 amino acid residues and are rich in cysteine, glycine and hydrophilic amino acid residues such as asparagine and glutamine. Morintides are resistant to thermal and enzymatic degradation, able to bind to chitin and inhibit the growth of phyto-pathogenic fungi. Transcriptomic analysis showed that they contain a three-domain precursor comprising an endoplasmic reticulum (ER) signal sequence, a mature peptide domain and a C-terminal domain. A striking feature distinguishing morintides from other 8C-hevein-like peptides is a short and protein-cargo-free C-terminal domain. Previously, a similar protein-cargo-free C-terminal domain has been observed only in ginkgotides, the 8C-hevein-like peptides from a gymnosperm Ginkgo biloba. Thus, morintides, with a cargo-free C-terminal domain, are a stand-alone class of 8C-hevein-like peptides from angiosperms.ConclusionsOur results expand the existing library of hevein-like peptides and shed light on molecular diversity within the hevein-like peptide family. Our work also sheds light on the anti-fungal activity and stability of 8C-hevein-like peptides.

Global comparative analysis of expressed genes in ovules and leaves of Ginkgo biloba L.

Ovules are an essential reproductive structure in all seed plants. However, limited information is available on the genetics and genomics of ovules in gymnosperms. Here, we used Illumina sequencing to derive comprehensive gene expression profiles for ovules and leaves in the extant basal gymnosperm Ginkgo biloba. A total of 100,124 transcripts, including 77,898 unigenes, were obtained by de novo assembly from leaf cDNA samples. These transcripts were functionally annotated by comparing their sequences to public protein databases as references. For the comparative analysis of gene expression levels between ovules and leaves, digital gene expression tag profiling was used. An analysis of the expressed genes revealed a high proportion of functional genes commonly expressed in both ovules and leaves. Functional gene expression analyses and microscopic observations implied that ovules and leaves share the photosynthetic structures of chloroplasts and stomata, indicating that they are homologous structures. Interestingly, many homologous genes associated with floral development were expressed in ovules, indicating that gymnosperm ovules share some gene regulatory mechanisms with angiosperm floral organs. The genes that showed highly differential expression levels between leaves and ovules were involved in flavonoid biosynthesis, cell division, hormone transport, transcriptional regulation, and starch and sucrose metabolism, and they indicated higher cell growth and division activity in the ovule. Thus, these results provide valuable gene expression information, which will contribute to an enhanced understanding of the diverse biological mechanisms in the ovules and leaves of G. biloba and provide important molecular insights into ovule evolution in early seed plants.

Characterization of an AGAMOUS gene expressed throughout development of the fleshy fruit-like structure produced by Ginkgo biloba around its seeds.

BackgroundThe involvement of MADS-box genes of the AGAMOUS lineage in the formation of both flowers and fruits has been studied in detail in Angiosperms. AGAMOUS genes are expressed also in the reproductive structures of Gymnosperms, yet the demonstration of their role has been problematic because Gymnosperms are woody plants difficult to manipulate for physiological and genetic studies. Recently, it was shown that in the gymnosperm Ginkgo biloba an AGAMOUS gene was expressed throughout development and ripening of the fleshy fruit-like structures produced by this species around its seeds. Such fleshy structures are evolutionarily very important because they favor the dispersal of seeds through endozoochory. In this work a characterization of the Ginkgo gene was carried out by over-expressing it in tomato.ResultsIn tomato plants ectopically expressing the Ginkgo AGAMOUS gene a macroscopic anomaly was observed only in the flower sepals. While the wild type sepals had a leaf-like appearance, the transgenic ones appeared connately adjoined at their proximal extremity and, concomitant with the development and ripening of the fruit, they became thicker and acquired a yellowish-orange color, thus indicating that they had undergone a homeotic transformation into carpel-like structures. Molecular analyses of several genes associated with either the control of ripening or the ripening syndrome in tomato fruits confirmed that the transgenic sepals behaved like ectopic fruits that could undergo some ripening, although the red color typical of the ripe tomato fruit was never achieved.ConclusionsThe ectopic expression of the Ginkgo AGAMOUS gene in tomato caused the homeotic transformation of the transgenic sepals into carpel-like structures, and this showed that the gymnosperm gene has a genuine C function. In parallel with the ripening of fruits the related transgenic sepals became fleshy fruit-like structures that also underwent some ripening and such a result indicates that this C function gene might be involved, together with other gens, also in the development of the Ginkgo fruit-like structures. It seems thus strengthened the hypothesis that AGAMOUS MADS-box genes were recruited already in Gymnosperms for the development of the fleshy fruit habit which is evolutionarily so important for the dispersal of seeds.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0418-x) contains supplementary material, which is available to authorized users.

Molecular Cloning and Characterization of Three Genes Encoding Dihydroflavonol-4-Reductase from Ginkgo biloba in Anthocyanin Biosynthetic Pathway

Dihydroflavonol-4-reductase (DFR, EC1.1.1.219) catalyzes a key step late in the biosynthesis of anthocyanins, condensed tannins (proanthocyanidins), and other flavonoids important to plant survival and human nutrition. Three DFR cDNA clones (designated GbDFRs) were isolated from the gymnosperm Ginkgo biloba. The deduced GbDFR proteins showed high identities to other plant DFRs, which form three distinct DFR families. Southern blot analysis showed that the three GbDFRs each belong to a different DFR family. Phylogenetic tree analysis revealed that the GbDFRs share the same ancestor as other DFRs. The expression of the three recombinant GbDFRs in Escherichia coli showed that their actual protein sizes were in agreement with predictions from the cDNA sequences. The recombinant proteins were purified and their activity was analyzed; both GbDFR1 and GbDFR3 could catalyze dihydroquercetin conversion to leucocyanidin, while GbDFR2 catalyzed dihydrokaempferol conversion to leucopelargonidin. qRT-PCR showed that the GbDFRs were expressed in a tissue-specific manner, and transcript accumulation for the three genes was highest in young leaves and stamens. These transcription patterns were in good agreement with the pattern of anthocyanin accumulation in G.biloba. The expression profiles suggested that GbDFR1 and GbDFR2 are mainly involved in responses to plant hormones, environmental stress and damage. During the annual growth cycle, the GbDFRs were significantly correlated with anthocyanin accumulation in leaves. A fitted linear curve showed the best model for relating GbDFR2 and GbDFR3 with anthocyanin accumulation in leaves. GbDFR1 appears to be involved in environmental stress response, while GbDFR3 likely has primary functions in the synthesis of anthocyanins. These data revealed unexpected properties and differences in three DFR proteins from a single species.

Distinct expression patterns of two Ginkgo biloba 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase/isopentenyl diphospahte synthase (HDR/IDS) promoters in Arabidopsis model

1-Hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) or isopentenyl diphosphate synthase (IDS) is an enzyme at the final step of the MEP pathway. The multi-copy nature of IDS gene in a gymnosperm Ginkgo biloba is known. To evaluate the function of each isogene, the roles of the promoters were examined in Arabidopsis model. Among the promoters of GbIDS series, about 1.3kb of GbIDS1pro and 1.5kb of GbIDS2pro were cloned and fused with GUS. The GbIDS1pro::GUS was introduced into Arabidopsis to show GUS expression in most organs except for roots, petals, and stamina, whereas the GbIDS2pro::GUS was expressed only in the young leaves, internodes where the flower and shoot branched, and notably in primary root junction. This pattern of GUS expression correlated with high transcript level of GbIDS2 compared to that of GbIDS1 in Ginkgo roots. Methyl jasmonate (MeJA) treatment resulted in down-regulated GbIDS1pro activity in Arabidopsis leaves and upregulated GbIDS2pro activity in roots. The same pattern of gene regulation in roots was also seen upon treatments of gibberellins, abscisic acid, and indole butyric acid.

Early evolutionary colocalization of the nuclear ribosomal 5S and 45S gene families in seed plants: evidence from the living fossil gymnosperm Ginkgo biloba

In seed plants, the colocalization of the 5S loci within the intergenic spacer (IGS) of the nuclear 45S tandem units is restricted to the phylogenetically derived Asteraceae family. However, fluorescent in situ hybridization (FISH) colocalization of both multigene families has also been observed in other unrelated seed plant lineages. Previous work has identified colocalization of 45S and 5S loci in Ginkgo biloba using FISH, but these observations have not been confirmed recently by sequencing a 1.8 kb IGS. In this work, we report the presence of the 45S-5S linkage in G. biloba, suggesting that in seed plants the molecular events leading to the restructuring of the ribosomal loci are much older than estimated previously. We obtained a 6.0 kb IGS fragment showing structural features of functional sequences, and a single copy of the 5S gene was inserted in the same direction of transcription as the ribosomal RNA genes. We also obtained a 1.8 kb IGS that was a truncate variant of the 6.0 kb IGS lacking the 5S gene. Several lines of evidence strongly suggest that the 1.8 kb variants are pseudogenes that are present exclusively on the satellite chromosomes bearing the 45S-5S genes. The presence of ribosomal IGS pseudogenes best reconciles contradictory results concerning the presence or absence of the 45S-5S linkage in Ginkgo. Our finding that both ribosomal gene families have been unified to a single 45S-5S unit in Ginkgo indicates that an accurate reassessment of the organization of rDNA genes in basal seed plants is necessary.

Differences in the scaling of area and mass of Ginkgo biloba (Ginkgoaceae) leaves and their relevance to the study of specific leaf area

The manner in which the area of the leaf lamina (A) scales with respect to the dry mass of the lamina (M) is an important functional trait that is correlated with whole-plant growth rates and habitat preferences across diverse species. However, the extent to which the scaling between these two variables differs among leaves collected from different types of shoots within the canopy of a tree is poorly understood. Should they exist, significant differences in the A vs. M scaling relationship within canopies would raise a number of important questions, in particular what constitutes an adequate sampling procedure to determine the whole-canopy A vs. M relationship. To address this issue, we used a large data set representing 13 biologically distinct categories of leaves sampled from mega- and microsporangiate trees of the dioecious gymnosperm Ginkgo biloba. Analyses of the data for these 13 categories of leaves identify seven statistically significantly different modes of A vs. M scaling that result in significant differences in how specific leaf area (SLA) changes as M varies within the canopies of Ginkgo. These results indicate that the protocols used to sample leaves for the analysis of foliar functional traits such as specific leaf area need to acknowledge and cope with the effects of leaf and shoot polymorphisms on the quantification of functional traits (and on the construction and testing of hypotheses about these traits).

Plants

Plants

Differences in photosynthetic activity, chlorophyll and carotenoid levels, and in chlorophyll fluorescence parameters in green sun and shade leaves of Ginkgo and Fagus

The differences in pigment levels and photosynthetic activity of green sun and shade leaves of ginkgo (Ginkgo biloba L.) and beech (Fagus sylvatica L.) are described. Sun leaves of both tree species possessed higher levels in chlorophylls (Chl) and carotenoids on a leaf area basis, higher values for the ratio Chl a/b and lower values for the ratio Chl/carotenoids (a+b)/(x+c) in comparison to shade leaves. The higher photosynthetic rates P(N) of sun leaves (ginkgo 5.4+/-0.9 and beech 8.5+/-2.1 micromol m(-2)s(-1)) were also reflected by higher values for the Chl fluorescence decrease ratios R(F)(d) 690 and R(F)(d) 735. In contrast, the shade leaves had lower P(N) rates (ginkgo 2.4+/-0.3 and beech 1.8+/-1.2 micromol m(-2)s(-1)). In both tree species the stomatal conductance G(s) was significantly higher in sun (range: 70-19 1 mmol m(-2)s(-1)) as compared to shade leaves (range: 5-55 mmol m(-2)s(-1)). In fact, at saturating light conditions there existed a close correlation between G(s) values and P(N) rates. Differences between sun and shade leaves also existed in several other Chl fluorescence ratios (F(v)/F(m), F(v)/F(o), and the stress adaptation index Ap). The results clearly demonstrate that the fan-shaped gymnosperm ginkgo leaves show the same high and low irradiance adaptation response as the angiosperm beech leaves.

Novel Plastocyanin Containing Phenylalanine-83 from the Gymnosperm Ginkgo biloba

Plastocyanin was purified from the gymnosperm Ginkgo biloba L., and its complete amino acid sequence was determined. The protein was shown to contain Phe-83 instead of Tyr-83 conserved in other land plant plastocyanins. This is the first report of the characterization and complete amino acid sequence of a gymnosperm plastocyanin.

Specificity patterns indicate that auxin exporters and receptors are the same proteins.

A study of transport and action of synthetic auxin analogues can help to identify transporters and receptors of this plant hormone. Both aspects--transportability and action on growth--were tested with 2-naphthoxyacetic acid (2-NOA) and compared across several plant species. 2-NOA stimulates elongation effectively at low concentrations in petioles of the gymnosperm Ginkgo biloba L., in hypocotyls or internodes of the dicot legumes, mung bean (Vigna mungo L.) and pea (Pisum sativum L.), in cotyledons of onion (Allium cepa L.) and in leaf bases of chive (Allium schoenoprasum L.), the latter two of the monocot order Asparagales. In contrast, elongation of coleoptile segments of maize (Zea mays L.) is poorly responsive to 2-NOA. Significant auxin-like transport of 2-NOA was observed in segments of mung bean hypocotyls, pea internodes, and chive leaf bases, but not in segments of the grass coleoptiles. Thus, for the two assays, elongation and polar transportability, the same difference in ligand specificity was observed between the grass and all other species assayed. This finding supports the hypothesis that a common protein mediates auxin efflux as well as auxin action on elongation.

Ginkgo biloba retains functions of both type I and type II flowering plant phytochrome.

While the photoreceptor systems of flowering plants have been well studied, the origins of these gene families and their functions are only partially understood. To begin to resolve the evolutionary origins of angiosperm photoreceptor function, we have studied the photomorphogenic responses of the early diverging gymnosperm Ginkgo biloba. Here, we describe the effects of continuous white light, red light, far-red light, and blue light on stem length, chlorophyll accumulation, Lhcb mRNA accumulation, and plastid development. Differences in the efficacy of these light regimes on de-etiolation in Ginkgo suggest separate but complementary roles for red and blue light-sensing systems. Additionally, the unique manner in which developmental regulation occurs in Ginkgo reveals a far-red high irradiance response different from both angiosperm and other gymnosperm species. We conclude from these data that Ginkgo contains a functional complement to both flowering plant type I and type II phytochromes, as well as independent blue light-sensing system(s). The implications of these findings are discussed with respect to the evolution of higher plant photoreceptors.

Circadian Rhythms of Leaf and Stomatal Movements in Gymnosperm Species

It is generally accepted that various physiological, morphological and gene expression phenomena are under the control of a circadian clock, and that this time keeping mechanism is universally present. Although such endogenously regulated phenomena have first been documented in plants more than 250 years ago and much work has been accumulated particularly in the past 70 years, it was not obvious from the literature whether such time keeping mechanisms exist in gymnosperms. Two prominent parameters were investigated in several gymnosperm species which have been demonstrated to be under the control of a circadian clock in many plants: (i) leaf movement and (ii) stomata movement. In young plants of Pinus sylvestris, Picea abies, Taxus baccata, Araucaria angustifolia, Araucaria heterophylla and Ginkgo biloba leaf oscillations could be recorded for about 5 days. However, compared to angiosperm plants, the amplitude was small. The period length under free running conditions (constant temperature and continuous light) was characteristic for the species. Stomatal movement was observed in Ginkgo biloba leaves by electron microscopy. Stomata were open at noon and closed at midnight under normal day/night conditions (LD) as well as under constant light conditions (LL), indicating that stomatal aperture is under circadian control in the gymnosperm Ginkgo biloba. Online recordings of stomata conductance however, exhibited diurnal but not circadian oscillations of net CO2-exchange in G. biloba leaves. Our results show that a circadian clock controls leaf and stomatal movements in gymnosperm species indicating that endogenous time keeping mechanisms are present.

Origins of phytochrome-modulated Lhcb mRNA expression in seed plants.

The levels of Lhcb mRNA in higher plants are regulated by phytochrome, cryptochrome, and an endogenous circadian oscillator. To determine whether similar regulatory mechanisms operate in the ancient gymnosperm Ginkgo biloba, we measured Lhcb mRNA levels in seedlings in response to different light conditions. Removal of a diurnally oscillating light stimulus caused dampening of maximal Lhcb mRNA accumulation levels, with little change in periodicity. Although low fluence pulses of both red and blue light given to etiolated seedlings caused maximal accumulation of Lhcb mRNAs characteristic of the phasic/circadian response seen in flowering plants, the additional initial acute response seen in flowering plants was absent. The induction of Lhcb gene expression in both cases was at least partially reversible by far-red light, and appeared biphasic over a range of red fluences. Together, these data indicate that Lhcb genes in G. biloba appear to be regulated in a manner similar to that of flowering plants, whereas signaling and attenuation of mRNA levels through the photoreceptor systems and circadian clock show features distinct from those characterized to date. The implications for these findings are discussed in light of the evolution of circadian clock input signaling.