Abundance of mRNAs encoding HMG1/HMG2 class high-mobility-group DNA-binding proteins are differentially regulated in cotyledons of Pharbitis nil

The abundance of an mRNA encoding an HMG1/2 protein from Pharbitis nil (HMG1) has been previously shown to be regulated by light and an endogenous rhythm in cotyledons. A second Pharbitis nil HMG cDNA (HMG2) was characterized. The sequence of HMG2 was 82% and 86% identical to HMG1 at the nucleotide and amino acid level, respectively. As with HMG1, HMG2 mRNA was detected in all vegetative tissues and was most abundant in roots. However, unlike HMG1, HMG2 mRNA abundance did not increase upon transfer of cotyledons to darkness and did not exhibit regulation by an endogenous circadian rhythm when maintained in continuous darkness over a 68 h period. Similarly, while the abundance of HMG1 mRNA during a dark period that induced photoperiodically controlled flowering was dramatically affected by brief light exposure (night break), this treatment had no effect on HMG2 mRNA abundance. Collectively, these data are consistent with a role of HMG1 in contributing to the circadian-regulated and/or dark-regulated gene expression with constitutive expression of HMG2 playing a housekeeping role in the general regulation of gene expression in Pharbitis nil cotyledons.     

Gibberellins in the control of photoperiodic flower transition in Pharbitis nil

The role of gibberellins in the photoperiodic flower induction of short-day plant Pharbitis nil has been investigated. It has been found that the endogenous content of gibberellins in the cotyledons of P. nil is low before and after a 16-h-long inductive dark period. During the inductive night the content of gibberellins is high at the beginning of darkness and about the middle of the dark period. Exogenous GA₃ when applied to the cotyledons of non-induced plants does not replace the effect of the inductive night but it can stimulate the intensity of flowering in plants cultivated on suboptimal photoperiods. GA₃ could also reverse the inhibitory effect of end-of-day far-red light irradiation on P. nil flowering. 2-Chloroethyltri-methylammonium chloride (CCC) applied to the cotyledons during the inductive night also inhibited flowering. GA₃ could reverse the inhibitory effect of CCC. The obtained results strongly suggest that gibberellins are involved in the phytochrome controlled transition of P. nil to flowering. Their effect could be additive to that of photoperiodic induction.     

Light Rquirements for Photoperiodic Sensitivity in Cotyledons of Dark-grown Pharbitis nil

The light requirements for induction of flowering by a long dark period were investigated in dark-grown seedlings of Pharbitis nil Chois, cv. Violet. The cotyledons bcame photoperiodically sensitive to a 24 h dark period by two 1 min red irradiations (6.3 μmol m⁻² S⁻¹) separated by a 24 h dark period. The reversibility of the effect of brief red irradiations, and the effectiveness of low energies of red irradiation suggest the involvement of phytochrome in the induction of photoperiodic sensitivity. Partial de-etiolation occurred after these brief periods of red irradiation but the seedlings were not capable of net CO₂ uptakeeven 7 h after the start of the main light period that followed the critical dark period. A changing response to the duration of the priod of darkness given between the two short red irradiations showed the the correct phasing of an endogenous photoperiodic rhythm is needed for the attainment of photoperiodic snsitivity.     

Characterization of transcriptional oscillation of an Arabidopsis homolog of PnC401 related to photoperiodic induction of flowering in Pharbitis nil

AtC401 is an Arabidopsis homolog of PnC401 that is related to photoperiodic induction of flowering in Pharbitis nil. These genes show free-running rhythms. To study the free-running rhythm of AtC401, we fused a firefly luciferase reporter to the AtC401 promoter and transformed it into Arabidopsis plants. The observed bioluminescence oscillated under continuous light and continuous dark only with sucrose supplementation. The free-running period of bioluminescence was temperature-compensated between 22 degrees C and 30 degrees C. Light-pulse experiments under continuous darkness produced a phase-response curve typical of circadian rhythms. We conclude that rhythmic expression of AtC401 is controlled by a circadian oscillator.     

Phytochrome gene expression and phylogenetic analysis in the short-day plant Pharbitis nil (Convolvulaceae): Differential regulation by light and an endogenous clock

To investigate the role of distinct phytochrome pools in photoperiodic timekeeping, we characterized four phytochrome genes in the short-day plant Pharbitis nil. Each PHY gene had different photosensory properties and sensitivity to night break that inhibits flowering. During extended dark periods, PHYE, PHYB, and PHYC mRNA accumulation exhibited a circadian rhythmicity indicative of control by an endogenous clock. Phylogenetic analysis recovered four clades of angiosperm phytochrome genes, phyA, phyB, phyC, and phyE. All except the phyE clade included sequences from both monocots and eudicots. In addition, phyA is sister to phyC and phyE sister to phyB, with gymnosperm sequences sister to either the phyA-phyC clade or to the phyB-phyE clade. These results suggest that a single duplication occurred in an ancestral seed plant before the divergence of extant gymnosperms from angiosperms and that two subsequent duplications occurred in an ancestral angiosperm before the divergence of monocots from eudicots. Thus in P. nil, a multigene family with different patterns of mRNA abundance in light and darkness contributes to the total phytochrome pool: one pool is light labile (phyA), whereas the other is light stable (phyB and phyE). In addition, PHYC mRNA represents a third phytochrome pool with intermediate photosensory properties.     

Involvement of signal transduction pathway components in photoperiodic flower induction in Pharbitis nil

The possible participation of several major components of the signal transduction pathway in photoperiodic flower induction was examined in Pharbitis cotyledons. Exogenous applications of GTP-γ-S (1–10 μ M ) or of the phorbol ester, phorbol 12-myristate-13-acetate (PMA, 0.1–5.0 μ M ) to Pharbitis plants held under a marginal inductive period (11.5 h dark) significantly increased their flowering response. Membrane lipid fluidity, GTP-binding and protein kinase activity were increased following a single flowering-inducing dark period of 16 h; however, a light-break of 10 min that abolished flower induction failed to reverse the dark-induced increase in these processes. Photo-inductive dark conditions significantly increased the content of diacylglycerol (DAG) and phosphoinositides in the cotyledon membranes, together with the activities of their kinases, and a light break decreased them to control levels and below. In addition, a single spraying with GTP-γ-S or PMA at 1 μ M significantly increased both the lipid content and the kinase activities. These compounds also enhanced the kinase activities in vitro. It is concluded that DAG and phosphoinositide metabolism play a role in the linking of the photoperiodic induction of the phytochrome with the flowering response in Pharbitis nil .     

Abundance of an mRNA encoding a high mobility group DNA-binding protein is regulated by light and an endogenous rhythm

A cDNA clone encoding an HMG1 protein from Pharbitis nil was characterized with regard to its sequence, genomic organization and regulation in response to photoperiodic treatments that control floral induction. The HMG1 cDNA contains an open reading frame of 432 nucleotides encoding a 144 amino acid protein of approximately 16 kDa. The predicted polypeptide has the characteristic conserved motifs of the HMG1 and HMG2 class of proteins including an N-terminal basic region, one of two HMG-box domains, and a polyacidic carboxy terminus. Within the HMG-box region, Pharbitis HMG1 deduced amino acid sequence shares 47%, 67% and 69% identity with its animal, maize, and soybean counterparts, respectively. Southern blot hybridization analysis suggests that HMG1 is a member of a multigene family. Analysis of mRNA abundance indicates that the HMG1 gene is expressed to higher levels in dark-grown tissue, such as roots, and at lower levels in light-grown tissue, such as cotyledons and stems. Following the transition to darkness, the levels of HMG1 mRNA in cotyledons were initially stable, however, after a lag time of 8 h or more, HMG1 mRNA increased in abundance to a peak level at 20 h. A second peak in mRNA levels was observed about 24 h later, indicating that the expression of the HMG1 gene is regulated by an endogenous circadian rhythm. Abundance of the HMG1 mRNA during a dark period was dramatically affected by brief light exposure (night break), a treatment which inhibits floral induction. These data indicate that the expression of HMG1 is regulated by both an endogenous rhythm and the light/dark cycle and are consistent with a role for HMG1 in maintaining patterns of circadian-regulated gene expression activated upon the transition from light to darkness.     

Changes in cotyledon mRNA during floral induction of Pharbitis nil CV. Violet

Floral induction in seedlings of Pharbitis nil Choisy cv. Violet, with one cotyledon removed, was manipulated by applying various photoperiodic treatments to the remaining cotyledon. Populations of polyadenylated RNA from treated cotyledons were examined to identify messages specifically involved in floral induction. The RNA was translated in vitro using a wheat-germ system, and the resulting translation products were analysed by two-dimensional polyacrylamide gel electrophoresis. Substantial qualitative and quantitative differences were found between mRNA from cotyledons of seedlings kept in continuous light (non-induced) and of seedlings given a 16-h dark period (induced). In contrast, inhibition of flowering with a night-break resulted only in one detectable, quantitative difference in mRNA.Abbreviations CL continuous light - kDa kilodalton - NB 16 h darkness+10 min red-light break, 8 h into the dark period - poly(A)⁺ RNA polyadenylated RNA (isolated by binding to a cellulose oligodeoxythymidine affinity column) - SD short day (16 h dark) - SDP short-day plant - SDS sodium dodecyl sulfate     

长日照处理对牵牛开花抑制作用研究 Ⅱ.光周期处理与牵牛花芽分化及基因活动的关系

单个光周期暗期长度短于12h时,牵牛植株营养生长旺盛,开花受到抑制,并且出现了诱导光周期处理（ISD）子叶中没有的二种蛋白质或多肽（pI4.1,MW16.5kD;pI4.2,MW16．5kD）。连续光照处理（ICL）子叶内出现了短日照处理（ISD）子叶内没有的体外翻译蛋白质分子量为17．4kD的Poly（A~ ）mRNA。牵牛子叶内的这些变化可能与抑制牵牛花芽分化有一定的关系。     

Translational regulation of light-induced ribulose 1,5-bisphosphate carboxylase gene expression in amaranth.

The regulation of the genes encoding the large and small subunits of ribulose 1,5-bisphosphate carboxylase was examined in amaranth cotyledons in response to changes in illumination. When dark-grown cotyledons were transferred into light, synthesis of the large- and small-subunit polypeptides was initiated very rapidly, before any increase in the levels of their corresponding mRNAs. Similarly, when light-grown cotyledons were transferred to total darkness, synthesis of the large- and small-subunit proteins was rapidly depressed without changes in mRNA levels for either subunit. In vitro translation or in vivo pulse-chase experiments indicated that these apparent changes in protein synthesis were not due to alterations in the functionality of the mRNAs or to protein turnover, respectively. These results, in combination with our previous studies, suggest that the expression of ribulose 1,5-bisphosphate carboxylase genes can be adjusted rapidly at the translational level and over a longer period through changes in mRNA accumulation.     

Dark-induced and organ-specific expression of two asparagine synthetase genes in Pisum sativum.

Nucleotide sequence analysis of cDNAs for asparagine synthetase (AS) of Pisum sativum has uncovered two distinct AS mRNAs (AS1 and AS2) encoding polypeptides that are highly homologous to the human AS enzyme. The amino-terminal residues of both AS1 and AS2 polypeptides are identical to the glutamine-binding domain of the human AS enzyme, indicating that the full-length AS1 and AS2 cDNAs encode glutamine-dependent AS enzymes. Analysis of nuclear DNA shows that AS1 and AS2 are each encoded by single genes in P.sativum. Gene-specific Northern blot analysis reveals that dark treatment induces high-level accumulation of AS1 mRNA in leaves, while light treatment represses this effect as much as 30-fold. Moreover, the dark-induced accumulation of AS1 mRNA was shown to be a phytochrome-mediated response. Both AS1 and AS2 mRNAs also accumulate to high levels in cotyledons of germinating seedlings and in nitrogen-fixing root nodules. These patterns of AS gene expression correlate well with the physiological role of asparagine as a nitrogen transport amino acid during plant development.     

Ethylene and ABA interactions in the regulation of flower induction in Pharbitis nil

Hormones are included in the essential elements that control the induction of flowering. Ethylene is thought to be a strong inhibitor of flowering in short day plants (SDPs), whereas the involvement of abscisic acid (ABA) in the regulation of flowering of plants is not well understood. The dual role of ABA in the photoperiodic flower induction of the SDP Pharbitis nil and the interaction between ABA and ethylene were examined in the present experiments. Application of ABA on the cotyledons during the inductive 16-h-long night inhibited flowering. However, ABA application on the cotyledons or the shoot apices during the subinductive 12-h-long night resulted in slight stimulation of flowering. Application of ABA also resulted in enhanced ethylene production. Whereas nordihydroguaiaretic acid (NDGA) - an ABA biosynthesis inhibitor - applied on the cotyledons of 5-d-old seedlings during the inductive night inhibited both the formation of axillary and of terminal flower buds, application of 2-aminoethoxyvinylglycine (AVG) and 2,5-norbornadiene (NBD) - inhibitors of ethylene action - reversed the inhibitory effect of ABA on flowering. ABA levels in the cotyledons of seedlings exposed to a 16-h-long inductive night markedly increased. Such an effect was not observed when the inductive night was interrupted with a 15-min-long red light pulse or when seedlings were treated at the same time with gaseous ethylene during the dark period. Lower levels of ABA were observed in seedlings treated with NDGA during the inductive night. These results may suggest that ABA plays an important role in the photoperiodic induction of flowering in P. nil seedlings, and that the inhibitory effect of ethylene on P. nil flowering inhibition may depend on its influence on the ABA level. A reversal of the inhibitory effect of ethylene on flower induction through a simultaneous treatment of induced seedlings with both ethylene and ABA strongly supports this hypothesis.     

Induction of flowering by 5-azacytidine in some plant species: relationship between the stability of photoperiodically induced flowering and flower-inducing effect of DNA demethylation

The flower-inducing effect of 5-azacytidine, a DNA demethylating reagent, was examined in several plant species with a stable or unstable photoperiodically induced flowering state under non-inductive photoperiodic conditions. The long day plant Silene armeria , whose flowering state is stable and the short day plant Pharbitis nil , whose flowering state is unstable were induced to flower by 5-azacytidine under a non-inductive condition. Thus, the replacement of photoinduction by 5-azacytidine treatment is not specific to Perilla frutescens . On the other hand, 5-azacytidine did not induce flowering in Xanthium strumarium whose flowering state is stable and Lemna paucicostata whose flowering state is unstable. Thus, epigenetics caused by DNA demethylation may be involved in the regulation of photoperiodic flowering irrespective of the stability of the photoperiodically induced flowering state.     

Two <Emphasis Type="Italic">FLOWERING LOCUS T</Emphasis> (<Emphasis Type="Italic">FT</Emphasis>) homologs in <Emphasis Type="Italic">Chenopodium rubrum</Emphasis> differ in expression patterns

FLOWERING LOCUS T (FT) like genes are crucial regulators (both positive and negative) of flowering in angiosperms. We identified two FT homologs in Chenopodium rubrum, a short-day species used as a model plant for the studies of photoperiodic flower induction. We found that CrFTL1 gene was highly inducible by a 12-h dark period, which in turn induced flowering. On the other hand, photoperiodic treatments that did not induce flowering (short dark periods, or a permissive darkness interrupted by a night break) caused only a slight increase in CrFTL1 mRNA level. We demonstrated diurnal oscillation of CrFTL1 expression with peaks in the middle of a light period. The oscillation persisted under constant darkness. Unlike FT homologs in rice and Pharbitis, the CrFTL1 expression under constant darkness was very low. The CrFTL2 gene showed constitutive expression. We suggest that the CrFTL1 gene may play a role as a floral regulator, but the function of CrFTL2 remains unknown.     

Different circadian rhythms regulate photoperiodic flowering response and leaf movement in Pharbitis nil (L.) Choisy

The phase shifting effect of red light on both the leaf movement rhythm, and on the rhythm of responsiveness of photoperiodic flower induction towards short light breaks (10 min red light), has been studied in Pharbitis nil, strain Violet, and comparisons between the two rhythms have been made. The phase angle differences between the rhythms after a phase shift with 2 or 6 h of red light given at different times during a long dark period were not constant. The results indicate the involvement of two different clocks controlling leaf movement and photoperiodic flower induction.Abbreviations DD continuous darkness - l:D x:y light/dark cycles with x hours of light and y hours of darkness - PPR rhythm of photoperiodic responsiveness towards light break     

Gibberellins are not essential for photoperiodic flower induction of Pharbitis nil

The influence on photoperiodic flowering of (2-chloroethyl)trimethylmmonium chloride (CCC), an inhibitor of gibberellin (GA) biosynthesis, was studied in the short-day plant Pharbitis nil cv. Violet. The cotyledons contained high levels of endogenous bioactive gibberellins, whereas in the plumules and first leaves the levels were low or undetectable. The first leaf responded to a single'dark treatment by inducing flowering when it was 10 mm or wider. Similar seedlings, but without cotyledons, were used as the assay plants to study the effect of CCC on photoperiodic flowering. Treatment with CCC had no effect on flowering of seedlings without cotyledons, although stem elongation was inhibited. By contrast. CCC inhibited flowering of the intact seedlings with cotyledons. Gibberellic acid applied to the shoot apex or to the first leaf promoted flowering in the CCC-treated seedlings without cotyledons. The results indicate thai gibberellins are not essential for the flower induction process in leaves, but that they promote flower initiation and/or later processes in the shoot apices.     

Jasmonates Inhibit Flowering in Short-Day Plant Pharbitis nil

The role of jasmonates in the photoperiodic flower induction of short-day plant Pharbitis nil was investigated. The plants were grown in a special cycle: 72 h of darkness, 24 h of white light with lowered intensity, 24-h long inductive night, 14 days of continuous light. At 4 h of inductive night the cotyledons of non-induced plants contained about two times the amount of endogenous jasmonates (JA/JA-Me) compared to those induced. A 15-min long pulse of far red light (FR) applied at the end of a 24-h long white light phase inhibited flowering of P. nil. The concentration of jasmonates at 2 and 4 h of inductive night in the cotyledons of the plants treated with FR was similar. Red light (R) could reverse the effect of FR. R light applied after FR light decreased the content of jasmonates by about 50%. Methyl jasmonate (JA-Me) applied to cotyledons, shoot apices and cotyledon petioles of P. nil inhibited the formation of flower buds during the first half of a 24-h long inductive or 14-h long subinductive night. Application of JA-Me to the cotyledons was the most effective. None of the plants treated with JA-Me on the cotyledons in the middle of the inductive night formed terminal flower buds. The aspirin, ibuprofen and phenidone, jasmonates biosynthesis inhibitors partially reversed the effect of FR, stimulating the formation of axillary and terminal flower buds. Thus, the results obtained suggests that phytochrome system control both the photoperiodic flower induction and jasmonates metabolism. Jasmonates inhibit flowering in P. nil.