Elimination of chrysanthemum stunt viroid from an infected chrysanthemum cultivar by shoot regeneration from a leaf primordium-free shoot apical meristem dome attached to a root tip

In this research we eliminated chrysanthemum stunt viroid (CSVd) from a highly infected chrysanthemum cultivar using a newly established method. Piato is one of the most difficult cultivars in which to obtain CSVd-free plants by conventional methods. Leaf primordium-free shoot apical meristems (LP-free SAMs) of Piato plants were dissected and attached to CSVd-free chrysanthemum or cabbage root tips. As shown by nested-PCR, CSVd was not detected in some shoots regenerated on both types of root tip. The production rates of CSVd-free plants using chrysanthemum and cabbage root tips were 14% and 3%, respectively. Regeneration of plants from LP-free SAMs of chrysanthemum plants by attaching these SAMs to root tips is an efficient method of generating CSVd-free chrysanthemum plants.Communicated by K.K. Kamo     

Analysis of Flowering Time Control in Arabidopsis by Comparison of Double and Triple Mutants

Three genetic pathways promote flowering of Arabidopsis under long photoperiods. These pathways are represented by the genes CO, FCA, and GA1, which act in the long-day, autonomous, and gibberellin pathways, respectively. To test whether these are the only pathways that promote flowering under long photoperiods, the co-2 fca-1 ga1-3 triple mutant was constructed. These plants never flowered under long- or short-day conditions, indicating that the three pathways impaired by these mutations are absolutely required for flowering under these conditions. The triple mutant background represents a "vegetative ground state" enabling the roles of single pathways to be described in the corresponding double mutants. The phenotypes of plants carrying all eight combinations of wild-type and mutant alleles at the three loci were compared under long- and short-day conditions. This analysis demonstrated that under long photoperiods the long-day pathway promoted flowering most effectively, whereas under short photoperiods the gibberellin pathway had the strongest effect. The autonomous pathway had a weak effect when acting alone under either photoperiod but appeared to play an important role in facilitating the promotion of flowering by the other two pathways. The vegetative phenotype of the triple mutant could be overcome by vernalization, suggesting that a fourth pathway promoted flowering under these conditions. These observations are discussed in light of current models describing the regulation of flowering time in Arabidopsis.     

Flowering of strict photoperiodic <Emphasis Type="Italic">Nicotiana</Emphasis> varieties in non-inductive conditions by transgenic approaches

The genus Nicotiana contains species and varieties that respond differently to photoperiod for flowering time control as day-neutral, short-day and long-day plants. In classical photoperiodism studies, these varieties have been widely used to analyse the physiological nature for floral induction by day length. Since key regulators for flowering time control by day length have been identified in Arabidopsis thaliana by molecular genetic studies, it was intriguing to analyse how closely related plants in the Nicotiana genus with opposite photoperiodic requirements respond to certain flowering time regulators. SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and FRUITFULL (FUL) are two MADS box genes that are involved in the regulation of flowering time in Arabidopsis. SOC1 is a central flowering time pathway integrator, whereas the exact role of FUL for floral induction has not been established yet. The putative Nicotiana orthologs of SOC1 and FUL, NtSOC1 and NtFUL, were studied in day-neutral tobacco Nicotiana tabacum cv Hicks, in short-day tobacco N. tabacum cv Hicks Maryland Mammoth (MM) and long-day N. sylvestris plants. Both genes were similarly expressed under short- and long-day conditions in day-neutral and short-day tobaccos, but showed a different expression pattern in N. sylvestris. Overexpression of NtSOC1 and NtFUL caused flowering either in strict short-day (NtSOC1) or long-day (NtFUL) Nicotiana varieties under non-inductive photoperiods, indicating that these genes might be limiting for floral induction under non-inductive conditions in different Nicotiana varieties.     

EAF1 regulates vegetative-phase change and flowering time in Arabidopsis.

We have identified a new locus that regulates vegetative phase change and flowering time in Arabidopsis. An early-flowering mutant, eaf1 (early flowering 1) was isolated and characterized. eaf1 plants flowered earlier than the wild type under either short-day or long-day conditions, and showed a reduction in the juvenile and adult vegetative phases. When grown under short-day conditions, eaf1 plants were slightly pale green and had elongated petioles, phenotypes that are observed in mutants altered in either phytochrome or the gibberellin (GA) response. eaf1 seed showed increased resistance to the GA biosynthesis inhibitor paclobutrazol, suggesting that GA metabolism and/or response had been altered. Comparison of eaf1 to other early-flowering mutants revealed that eaf1 shifts to the adult phase early and flowers early, similarly to the phyB (phytochrome B) and spy (spindly) mutants. eaf1 maps to chromosome 2, but defines a locus distinct from phyB, clf (curly leaf), and elf3 (early-flowering 3). These results demonstrate that eaf1 defines a new locus involved in an autonomous pathway and may affect GA regulation of flowering.     

Rescue of shoot apical meristems of chrysanthemum by culturing on root tips

A new method to regenerate plants from leaf primordia-free shoot apical meristem domes (LP-free SAMs) was developed by establishing the meristem dome on the cut surface of root tips. Ten days after culture, the viable rate of LP-free SAMs of chrysanthemum Piato attached to chrysanthemum root tips was >40%. Shoot regeneration was not observed from LP-free SAMs without the root tips. When LP-free SAMs of chrysanthemum were transferred to root tips of either petunia, cabbage, or carnation, the highest shoot regeneration rate was observed with cabbage root tips. Microscopic observation documented that the LP-free SAM temporarily adhered to the cut surface of the root tip of cabbage.Communicated by K.K. Kamo     

CIRCANNUAL GROWTH RHYTHM AND PHOTOPERIODIC SORUS INDUCTION IN THE KELP LAMINARIA SETCHELLII (PHAEOPHYTA)1

Growth and reproduction of laboratory-grown sporophytes of Laminaria setchellii Silva were investigated in a tank system with controlled conditions of daylength, temperature, and nutrients (N and P). A circannual growth rhythm of the frond was detected under constant laboratory conditions. In continuous long-day and night-break conditions the period τ of the free-running rhythm varied between 11.3 and 17.3 months; in short-day conditions the frond grew indefinitely. The growth rhythm of individual plants could be synchronized by a simulated annual cycle of day-length with a period of T = 12 months. The four seasons of the year were simultaneously simulated by phase shifting the annual cycle of daylength by 3, 6, or 9 months in three out of four tanks. The annual growth cycle followed these phase shifts, and initiation of the new blade always started just after the winter daylength minimum. The formation of sori was induced by a genuine photoperiodic short-day reaction in 1- to 2-year-old plants. Sori became, visible 9–14 weeks after transfer of individual plants from long-day to short-day conditions, whereas plants cultured in continuous long-day or night-break conditions remained sterile. Sporophytes with or without blades were able to continue growth or produce new blades in continuous darkness.     

Flowering Induced by Nitrogen Deficiency in Lemna paucicostata 151

Lemna paucicostata 151 is a weakly responsive short-day plantwhen grown in non-aseptic ten-fold diluted E medium supplementedwith 1 µM 6-benzyladenine, but it flowered even underlong-day conditions (continuous light) when grown in this mediumfor more than 14 days. On the 14th day of culture, the levelof endogenous nitrogen in the plants decreased to about 60%of that in the plants inoculated at the start of the culture.The flowering obtained under long-day conditions was suppressedby raising the concentration of nitrogen in the medium, whileit was induced more rapidly by lowering the concentration ofnitrogen in the medium. Benzyladenine did not cause floweringby itself, but it was required for the flowering that was inducedby a reduction in the level of nitrogen in the medium. Thus,the flowering observed under long-day conditions is due to nitrogendeficiency in the plants. Two inhibitors of proteases, bestatin and elastatinal, clearlyinhibited the flowering induced by nitrogen deficiency. It appears,therefore, that the induction or activation of some protease(s)is involved in the flowering that is induced by nitrogen deficiency. (Received March 19, 1991; Accepted August 16, 1991)     

Modulation of flowering responses in different Nicotiana varieties

We have identified and characterized a FLOWERING PROMOTING FACTOR 1(FPF1) gene from tobacco (NtFPF1). Over-expression of NtFPF1 leads to early flowering in the day-neutral tobacco Nicotiana tabacum cv. Hicks, and under inductive photoperiods also in the short-day Nicotiana tabacum cv. Hicks Maryland Mammoth (MM) tobacco and the long-day plant Nicotiana sylvestris. N. sylvestris wild-type plants remained in the rosette stage and never flowered under non-inductive short-days, whereas 35S::NtFPF1 transgenic plants bolted but did not flower. However, if treated with gibberellins, transgenic N. sylvestrisplants flowered much faster under non-inductive short days than corresponding wild type plants, indicating an additive effect of gibberellins and the NtFPF1 protein in flowering time control. The day-neutral wild type cv. Hicks and the short-day cv. Hicks MM plants exhibit an initial rosette stage, both under short- and long-days. In the transgenic lines, this rosette stage was completely abolished. Wild-type plants of cv. Hicks MM never flowered under long days; however, all transgenic lines over-expressing NtFPF1 flowered under this otherwise non-inductive photoperiod.     

HYPERSENSITIVE TO RED AND BLUE 1 and its C-terminal regulatory function control FLOWERING LOCUS T expression

The red and far-red light-absorbing phytochromes and UV-A/blue light-absorbing cryptochromes regulate seedling de-etiolation and flowering responses. The signaling steps that mediate the photoreceptor regulation on key flowering genes remain largely unknown. We report that a previously identified photomorphogenic mutant, hypersensitive to red and blue 1 (hrb1), flowered late and showed attenuated expression of FLOWERING LOCUS T (FT) over both long days and short days. Transgenic plants that overexpress the full-length HRB1, or its C-terminal half, flowered early and accumulated more FT messages under short-day conditions. The transgenic plants also displayed hyposensitive de-etiolation phenotypes, and the expression of these phenotypes requires the action of PIF4. The double mutant of hrb1/cry2 showed a flowering phenotype and an FT expression pattern similar to hrb1 under long-day conditions, suggesting that HRB1 may function downstream of cry2 under long-day conditions. In contrast, hrb1/phyB-9 showed a flowering phenotype and an FT expression pattern similar to phyB-9 over both long days and short days, indicating a modulatory role of HRB1 in the flowering pathway mediated by phyB. Overexpression of HRB1 did not affect the expression of the central clock oscillators, TOC1 and CCA1. HRB1 therefore represents a signaling step that regulates FT expression downstream of red and blue light perception.     

Salicylic acid is involved in the regulation of starvation stress-induced flowering in Lemna paucicostata

The short-day plant, Lemna paucicostata (synonym Lemna aequinoctialis), was induced to flower when cultured in tap water without any additional nutrition under non-inductive long-day conditions. Flowering occurred in all three of the tested strains, and strain 6746 was the most sensitive to the starvation stress conditions. For each strain, the stress-induced flowering response was weaker than that induced by short-day treatment, and the stress-induced flowering of strain 6746 was completely inhibited by aminooxyacetic acid and l-2-aminooxy-3-phenylpropionic acid, which are inhibitors of phenylalanine ammonia-lyase. Significantly higher amounts of endogenous salicylic acid (SA) were detected in the fronds that flowered under the poor-nutrition conditions than in the vegetative fronds cultured under nutrition conditions, and exogenously applied SA promoted the flowering response. The results indicate that endogenous SA plays a role in the regulation of stress-induced flowering.     

GENETIC AND HORMONAL REGULATION OF GROWTH,FLOWERING, AND SEX EXPRESSION IN PLANTS

The interaction of the genetic and hormonal regulation of growth, flowering, and sex expression in plants is discussed. The genetic control of these processes is characterized, and data on their hormonal regulation are supplied. The interaction of genetic and hormonal regulation is considered with reference to tall-growing and genetic dwarf forms of the pea and wheat plants. It is shown that in the dwarf forms of the pea plant and in many other varieties, growth stimulation in response to treatment with the phytohormone gibberellic acid is clearly manifested and the expression of genetic dwarfism is eliminated, whereas in dwarf wheats it is expressed only slightly, if at all. At the same time both tall-growing and dwarf forms of both pea and wheat show a clearly defined growth retardation response to treatment with the growth inhibitor, abscisic acid, which causes the expression of physiological dwarfism. The short- and long-day characteristics of the photoperiodic response of plants are described as genetically controlled features, and data are given on the induction of flowering of a long-day variety coneflower grown under short-day conditions with the aid of gibberellins extracted from leaves of long-day vegetative plants of short-day Mammoth tobacco. Data are also supplied on the induction of flowering of a short-day variety, red-leaved goosefoot, grown under continuous light with the aid of metabolites extracted from leaves of the same Mammoth tobacco plants flowering under short-day conditions. This demonstrates the possibility of hormonal regulation of the genetically controlled long-day and short-day characteristics in photoperiodically sensitive plants. Genetic and hormonal regulation of sex expression in two dioecious plants, hemp and spinach, is discussed. It is shown that sex expression in these plants is regulated by gibberellins which are synthesized in leaves and cause male sex expression and by cytokinins which are synthesized in the roots and cause female sex expression. These data indicate that sex expression in dioecious plants is the result of interaction between the genetic apparatus and phytohormones.     

Phytotron experiments in Pisum

Summary The flowering behavior of 59 Pisum mutants and 228 recombinants was studied in the phytotron in four different photoperiods (continuous light, long-day 18/6 h, short-day 12/12 h, extreme short-day 6/18 h). There was no or little difference in the response of the genotypes to long-day and permanent light, whereas great differences were observed between long- and short-day 12/12 h and between the two short-day trials. About half the genotypes tested were unable to survive or to flower in extreme short-day. Some recombinants, however, had an almost normal development under these unfavorable conditions. Gene fis controls the photoperiodic reaction of the plants: they are unable to flower in short-day. Gene fds negatively influences gene efr for earliness: it causes a strong delay of flowering of efr recombinants in long-day and suppresses the formation of functionable flowers in short-day. Most of the genotypes tested showed a specific reaction to the four photoperiods different from that of the mother variety and the other genotypes. The practical aim of our phytotron experiments is the preselection of Pisum genotypes which might be suited for cultivation in countries with short-day climate.This paper is dedicated to Professor Karl-Ernst Wohlfarth-Bottermann on his 65th birthday     

The promotive effect of applying mixtures of (S)-(+)-abscisic acid and gibberellic acid on flowering in long-day plants

Application of mixtures of natural type abscisic acid,(S)-(+)-abscisic acid (SABA), and gibberellic acid (GA₃)promoted floral-bud initiation and flowering in the long-day plants, spinach,pansy, primrose and petunia, even under short-day conditions. The effectiveconcentrations for spray application of SABA/GA₃ were restrictedwithin the limits of 10–1 respectively. Applications ofSABAand/or GA₃ as high as 50 induced no flowering.Flowering in short-day plants, dahlia, morning-glory and Christmas-cactus wasnot promoted by the mixtures.     

Overexpression of <Emphasis Type="Italic">AP1-</Emphasis>like genes from <Emphasis Type="Italic">Asteraceae</Emphasis> induces early-flowering in transgenic <Emphasis Type="Italic">Chrysanthemum</Emphasis> plants

Chrysanthemum is one of the most important commercial cut flowers in the world. Early-flowering cultivars are required to produce quality chrysanthemum flowers with a lower cost of production. To shorten the vegetative growth phase of chrysanthemum, three AP1-like genes from Asteraceae were constitutively overexpressed in 80 independent transgenic chrysanthemum lines. All lines were characterized by PCR and RT-PCR and demonstrated that overexpression of compositae AP1-homologs in transgenic chrysanthemum under long-day conditions had no effect on plant development compared to non-transgenic controls. Conversely, under short-day conditions, transgenic plants commenced bud initiation 2 wk earlier than non-transgenic chrysanthemum plants. Subsequently, transgenic chrysanthemum flowers showed color earlier and resulted in full opening of inflorescences 3 wk prior to non-transgenic control plants. These results open new possibilities for genetic improvement and breeding of chrysanthemum cultivars.     

Induction and promotion of flowering by heat-treated catecholamines in Lemna paucicostata

Alkali- and heat-treated norepinephrine, a catecholamine, induced flowering of short-day (SD) plant Lemna paucicostata 151 even under long-day (LD) conditions. Flowering induced with a lower concentration of heat-treated norepinephrine was promoted under SD conditions but inhibited by a night break. The related compounds L-dopa and dopamine also promoted flowering under SD conditions when they were heat-treated.     

METABOLISM AND FLOWERING

Two earlier papers have established (1) that the late-flowering chrysanthemum, a typical "short-day" species, flowered in autumn because of a disability in nocturnal translocation which limited the amount of carbohydrate available at the growing-point during the short nights of summer (Grainger, 1938), and (2) that many plants which flower in spring and early summer actually begin the formation of their flower initials in the autumn and winter of the previous year (Grainger, 1939). Such plants might conceivably be "short-day" kinds, since the actual transformation from vegetative to floral organization of the growing-point was accomplished when the days were short. It therefore appears necessary to investigate their relative diurnal carbohydrate metabolism, in order to discover whether it has any causal relationship with the time of actual flower initiation, as was demonstrated for the late-flowering chrysanthemum. The present paper describes the results of such an investigation, and also of certain contributory studies as to the converse effect of flowering upon metabolism, the relative amount of energy used in flowering, and the possible inadequacy of nutrition in very floriferous plants.     

Photoperiodic induction of flowering in green and photobleachedChenopodium rubrum L. ecotype 184 - a short- day plant

Chenopodium rubrum L. ecotype 184 is a qualitative short-day plant with critical length of the night of eight hours that must be exceeded in order to flower: Five days after sowing, the plants were exposed to a various number of inductive cycles (14/10 h of däy/night cycle) to test the optimal photoperiodic conditions for flowering. In our experimental conditions the plants flowered with high percentage after more than four received inductive cycles, but there was no flowering below that. The plants grown on the herbicide Norflurazon (photobleached plants) showed different photoperiodic characteristics. There was negligible flowering of photobleached plants in the same experimental conditions as for the green ones.     

Interactions among leaf photosynthetic rates,flowering and pod set in soybeans

The seasonal maximum in photosynthetic CO₂ exchange rate (CER) and the cessation of leaf expansion in soybeans (Glycine max (L.) Merr) accompany fruiting under normal agricultural conditions. To investigate whether these phenomena were obligatively tied together, we caused early flowering of long-season varieties by imposing artificial short-day treatments. Comparisons of CER and leaf area between vegetative (long-day treatment) and fruiting (short-day treatment) plants of long-season cultivar confirmed the relationship of these phenomena. The same comparisons made between a long-season and a short-season cultivar, both at the same daylength, also confirmed the relationship.     

Induction and Promotion of Flowering by Heat-Treated Catecholamines in Lemna paucicostata

Alkali- and heat-treated norepinephrine, a catecholamine, induced flowering of short-day (SD) plant Lemna paucicostata 151 even under long-day (LD) conditions. Flowering induced with a lower concentration of heat-treated norepinephrine was promoted under SD conditions but inhibited by a night break. The related compounds L-dopa and dopamine also promoted flowering under SD conditions when they were heat-treated.