Abstract
Many plant species can be induced to flower by responding to stress factors. The short-day plants Pharbitis nil and Perilla frutescens var. crispa flower under long days in response to the stress of poor nutrition or low-intensity light. Grafting experiments using two varieties of P. nil revealed that a transmissible flowering stimulus is involved in stress-induced flowering. The P. nil and P. frutescens plants that were induced to flower by stress reached anthesis, fruited and produced seeds. These seeds germinated, and the progeny of the stressed plants developed normally. Phenylalanine ammonialyase inhibitors inhibited this stress-induced flowering, and the inhibition was overcome by salicylic acid (SA), suggesting that there is an involvement of SA in stress-induced flowering. PnFT2, a P. nil ortholog of the flowering gene FLOWERING LOCUS T (FT) of Arabidopsis thaliana, was expressed when the P. nil plants were induced to flower under poor-nutrition stress conditions, but expression of PnFT1, another ortholog of FT, was not induced, suggesting that PnFT2 is involved in stress-induced flowering.
Key words: flowering, stress, phenylalanine ammonia-lyase, salicylic acid, FLOWERING LOCUS T, Pharbitis nil, Perilla frutescens
Flowering in many plant species is regulated by environmental factors, such as night-length in photoperiodic flowering and temperature in vernalization. On the other hand, a short-day (SD) plant such as Pharbitis nil (synonym Ipomoea nil) can be induced to flower under long days (LD) when grown under poor-nutrition, low-temperature or high-intensity light conditions.1–9 The flowering induced by these conditions is accompanied by an increase in phenylalanine ammonia-lyase (PAL) activity.10 Taken together, these facts suggest that the flowering induced by these conditions might be regulated by a common mechanism. Poor nutrition, low temperature and high-intensity light can be regarded as stress factors, and PAL activity increases under these stress conditions.11 Accordingly, we assumed that such LD flowering in P. nil might be induced by stress. Non-photoperiodic flowering has also been sporadically reported in several plant species other than P. nil, and a review of these studies suggested that most of the factors responsible for flowering could be regarded as stress. Some examples of these factors are summarized in Table 1. Thus, the evidence for stress-mediated flowering is accumulating. Based on this, we call this flowering ‘stress-induced flowering’.12–14
Table 1.
Some cases of stress-induced flowering
| Stress factor | Species | Flowering response | Reference |
| high-intensity light | Pharbitis nil | induction | 5 |
| low-intensity light | Lemna paucicostata | induction | 29 |
| Perilla frutescens var. crispa | induction | 14 | |
| ultraviolet C | Arabidopsis thaliana | induction | 23 |
| drought | Douglas-fir | induction | 30 |
| tropical pasture Legumes | induction | 31 | |
| lemon | induction | 32–35 | |
| Ipomoea batatas | promotion | 36 | |
| poor nutrition | Pharbitis nil | induction | 3, 4, 13 |
| Macroptilium atropurpureum | promotion | 37 | |
| Cyclamen persicum | promotion | 38 | |
| Ipomoea batatas | promotion | 36 | |
| Arabidopsis thaliana | induction | 39 | |
| poor nitrogen | Lemna paucicostata | induction | 40 |
| poor oxygen | Pharbitis nil | induction | 41 |
| low temperature | Pharbitis nil | induction | 9, 12 |
| high conc. GA4/7 | Douglas-fir | promotion | 42 |
| girdling | Douglas-fir | induction | 43 |
| root pruning | Citrus sp. | induction | 44 |
| Pharbitis nil | induction | 45 | |
| mechanical stimulation | Ananas comosus | induction | 46 |
| suppression of root elongation | Pharbitis nil | induction | 7 |
Stress-Induced Flowering of P. nil and Perilla frutescens
P. nil can flower under LD conditions when grown in tap water (poor-nutrition stress), at 12 to 15°C (low-temperature stress) or under 15,000 to 20,000 lux light (high-intensity light stress). The responses to different stress factors differ depending on the cultivars.7 For example, cv. Tendan was not induced to flower even when nutritionally stressed by growing in tap water, although vegetative growth was significantly inhibited,13 indicating that the plants were indeed stressed.12 Tendan did not flower by high-intensity light stress either. Cv. Kidachi was not induced to flower by poor nutrition or high-intensity light. On the other hand, the white-flowered mutant of cv. Violet responded sensitively to low-temperature stress.15 P. nil seedlings respond to these stress factors when the cotyledons expand as well as to an SD treatment during photoperiodic flowering. The flowering was delayed when the cotyledons were removed, indicating that the cotyledons are necessary for stress-induced flowering, similar to photoperiodic flowering.2,6
Recently, we found that an SD plant, Perilla frutescens var. crispa, was induced to flower under LD conditions when grown under low-intensity light (30 µmol m−2 s−1).14 Because vegetative growth was suppressed when flowering was induced, this is another example of stress-induced flowering. The flowering response was stronger in the red-leafed form than in the green-leafed form. We treated the red-leafed form with stress factors other than low-intensity light. The plants were grown in tap water or a diluted mineral nutrient solution (poor-nutrition stress), at 5 to 15°C (low-temperature stress), with 50 to 400 mM NaCl (salt stress), or with poor watering (water stress). None of these factors induced flowering, although they retarded vegetative growth. Thus, not all types of stress can induce flowering. The red-leafed P. frutescens, which were exposed to low-intensity light when their cotyledons had just expanded, were induced to flower by the 3-week treatment, and 100% flowering occurred after the 4-week treatment. The plants could respond to low-intensity light immediately after the cotyledons expanded. The flowering response decreased with an increase in plant age, and flowering was not induced when the low-intensity light treatment started 2 weeks after the cotyledons expanded or at any later time.
Transmissible Flowering Stimulus Produced by Stress
Stress-induced flowering of P. nil is inhibited by the PAL inhibitor aminooxyacetic acid (AOA),3,5 and therefore it is hypothesized that some compounds in the metabolic pathway regulated by PAL act as flowering stimuli.10,12 However, a transmissible flowering stimulus like florigen, which is involved in photoperiodic flowering, has not been reported in stress-induced flowering. To investigate this possibility, we performed grafting experiments to detect the transmission of stress-induced flowering stimuli in P. nil.13
Violet and Tendan were grafted in several combinations, and the grafted plants were grown in tap water under LD conditions. The Violet scions grafted onto the Violet rootstocks flowered. The flowering might have been caused by the influence of the rootstocks because all of the leaves were removed from the scions. This suggests that a transmissible flowering stimulus is involved in stress-induced flowering. We predicted that Tendan would not produce such a flowering stimulus because Tendan did not flower in response to the poor-nutrition stress conditions. However, the defoliated Violet scions grafted onto the Tendan rootstocks with cotyledons were induced to flower. Conversely, the Tendan scions grafted onto the Violet rootstocks were not induced to flower. These results indicate that Tendan produces a transmissible flowering stimulus but does not respond to it.
Production of Progeny by Plants that Flowered Under Stress Conditions
Plants can modify their development to adapt to stress conditions. Stressed plants might flower as an emergency response to produce the next generation. In this way, plants can preserve its species, even in an unfavorable environment. In order for this to be a biologically advantageous response, plants induced to flower by stresses must produce fertile seeds and the progeny must develop normally.
P. nil Violet was grown in a 1/10-strength nutrient solution or tap water throughout its life. The plants that were induced to flower by poor-nutrition stress conditions reached anthesis, fruited and produced seeds.13 All of these seeds germinated, and the progeny of the stressed plants developed normally. The progeny responded to SD treatment and formed floral buds. Furthermore, a normal second-generation from the stress progeny was produced. Red-leafed P. frutescens plants were grown under LD conditions with low-intensity light from the stage in which the cotyledons expanded. The plants induced to flower under these conditions reached anthesis and formed seeds.14 The seeds germinated, grew normally, and were induced to flower in response to SD treatments. These results indicate that the stressed plants do not need to await the arrival of a season when photoperiodic conditions are suitable for flowering, and such precocious flowering might assist in species preservation. Therefore, stress-induced flowering might have a biological benefit, and it should be considered to be as important as photoperiodic flowering and vernalization.
Involvement of Salicylic Acid (SA) in Stress-Induced Flowering
As mentioned above, some compound(s) in the metabolic pathway regulated by PAL might act as flowering stimuli in P. nil. Phenylpropanoids such as chlorogenic acid were a prominent candidate for this in earlier studies.3–5,8,9 However, exogenously applied chlorogenic acid failed to induce flowering.3,5,12 In addition to chlorogenic acid, several compounds including SA and anthocyanin are derived from t-cinnamic acid of which conversion from phenylalanine is catalyzed by PAL.11 A factor in the metabolic pathways derived from t-cinnamic acid might be involved in stress-induced flowering. We induced flowering of P. nil by low-temperature or poor-nutrition stress, inhibited the flowering by AOA, and co-applied several metabolic intermediates in the pathways. Among the intermediates, t-cinnamic, benzoic acids and SA were shown to negate the inhibitory effect of AOA while p-coumaric and caffeic acids did not.12,13 These results suggest that SA is involved in the stress-induced flowering of P. nil. Stress promotes the metabolism of t-cinnamic acid to SA via benzoic acid.16,17 SA plays several physiological roles in plant development.18 The treatment of P. nil with benzoic acid, SA or benzoic acid derivatives prior to a low-temperature treatment enhances the flower-inducing effect of low temperature.2,6,19 In addition, several derivatives of benzoic acid and SA induce flowering in P. nil,19–21 and benzoic acid enhances the flowering of cultured plumules excised from photoinduced P. nil seedlings.21 SA induces flowering in many species belonging to the Lemnaceae,22 and it has been implicated in the stress-induced flowering of A. thaliana.23,24 These data support the conclusions described above. However, SA alone did not induce flowering of P. nil under non-stress conditions.13 Stress conditions might induce not only SA biosynthesis but also other essential factors to induce flowering.
It was previously observed that the leaves of red-leafed P. frutescens were deep green when induced to flower under low-intensity light.14 The greening of the leaves was due to a decrease in anthocyanin content. There was a negative correlation between the anthocyanin content and the percent of flowering. PAL is a key enzyme in anthocyanin synthesis, and we can therefore assume that low-intensity light induces flowering through suppression of PAL activity. However, this conflicts with previous knowledge. Stress generally increases PAL activity and promotes anthocyanin biosynthesis,11,25,26 and PAL activity actually increases in the stress-induced flowering of P. nil, as mentioned above. Therefore, we examined the effect of the PAL inhibitor on the low-intensity light-induced flowering in P. frutescens. AOA and another PAL inhibitor, L-2-aminooxy-3-phenylpropionic acid (AOPP), did not induce flowering when applied under non-inductive normal-intensity light and inhibited flowering when applied under inductive low-intensity light.14 These results suggest that the same mechanism is involved in the flowering that is induced by low-intensity light in P. frutescens and the flowering that is induced by several stress factors in P. nil. The fact that PAL inhibitors inhibited stress-induced flowering suggests that the stress increased PAL activity. However, in P. frutescens, the fact that the anthocyanin content decreased under low-intensity light suggests that stress limited the activity of PAL. These contradictory results must be explained.
Involvement of PnFT Genes in Stress-Induced Flowering of P. nil
A molecular approach has not been previously applied to study stress-induced flowering. Therefore, we searched for genes involved in the stress-induced flowering of P. nil. Flowering of A. thaliana is induced not only by LD conditions but also by vernalization, autonomous cues and gibberellins, and these factors operate through a common pathway integrated by FLOWERING LOCUS T (FT).27 This suggests that FT could also be involved in the flowering induced by stress factors. Two orthologs of FT, PnFT1 and PnFT2, have been identified in P. nil, and these genes are expressed under inductive SD conditions to promote flowering.28 Therefore, we examined the expression of PnFT genes in response to poor-nutrient stress conditions.
P. nil Violet was induced to flower by growth in tap water; the cotyledons and true leaves of these plants were collected, and the expression of PnFT1 and PnFT2 was examined by RT-PCR.13 The expression of PnFT2 was induced in the plants grown under the poor-nutrition conditions for two weeks or longer. On the other hand, PnFT1 was not expressed regardless of the nutritional conditions. These results suggest that PnFT2, but not PnFT1, is involved in the stress-induced flowering of P. nil. SA might induce the expression of PnFT2, or the product of PnFT2 might induce the expression of genes involved in the biosynthesis of, response to, or signal transduction of SA. It is interesting to note that PnFT2 is involved in both photoperiodic flowering and stress-induced flowering, while PnFT1 is involved only in photoperiodic flowering. The two PnFT genes might have different roles in the regulation of flowering depending on the inductive cue. It is also possible that the essential gene for flowering is PnFT2 and that PnFT1 expression is induced only by SD treatment and redundantly enhances the activity of PnFT2.
Footnotes
Previously published online: www.landesbioscience.com/journals/psb/article/11826
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