2018 Volume 87 Issue 1 Pages 80-88
Effects of photoperiod and temperature on rhizome enlargement (dormancy induction) and accompanied dormancy depth were investigated in this study. Nine-day-old seedlings were transplanted from 26 July at 1 week intervals, and they were grown under a natural photoperiod for 5 weeks in an unheated greenhouse in Fukuoka Prefecture, Japan. Although subterranean stems elongated in the plants grown until 30 August or 6 September, enlarged rhizomes were formed in those grown until 13 September or 20 September. It was revealed from these results that the lotus recognizes a natural photoperiod after 6 September as a short day. When 9 treatments of day length combinations (LD0+SD8–LD8+SD0) were applied to the seedlings, the plants grown under short day after long day treatment of 0 (LD0+SD8), 1 (LD1+SD7), 2 (LD2+SD6), 3 (LD3+SD5), 4 (LD4+SD4), 5 (LD5+SD3), 6 (LD6+SD2), or 7 (LD7+SD1) weeks formed enlarged rhizomes from the fifth, fifth, sixth, seventh, eighth, tenth, twelfth, and fourteenth internodes, respectively. Although photoperiodic treatment in the first week was different between LD0+SD8 and LD1+SD7 treatments, subterranean stems began to enlarge from the same internode (fifth internode) in both treatments. This indicates that photoperiod treatments for the first week do not affect morphology of subterranean stems. Seven treatments of day length combinations (LD2+SD0+LD6–LD2+SD6+LD0) were applied to seedlings after long day treatment for 2 weeks. Enlarged subterranean stems were observed in the plants grown under short day for 6 weeks (LD2+SD6+LD0), but not in those under long day for 6 weeks (LD2+SD0+LD6). On the other hand, subterranean stems elongated again after rhizome enlargement under a subsequent long day following 1 (LD2+SD1+LD5), 2 (LD2+SD2+LD4), 3 (LD2+SD3+LD3), or 4 (LD2+SD4+LD2) weeks of short day. This clarified that morphogenesis in subterranean stems is completely dependent on photoperiod. Further, it is expected that such growth resumption may be attributed to a weak dormant state in the enlarged rhizome. The enlarged rhizomes were exposed to natural low temperatures to examine environmental factors for deepening dormancy. Rhizomes sprouted in all treatments irrespective of exposure to low temperatures when they were transferred to ideal conditions. Rapid growth in leaves and subterranean stems was particularly observed by exposure to low temperature. It was suggested that low temperature is an environmental factor for releasing dormancy, but not for deepening dormancy. It is proposed from these results that subterranean stem growth is completely dependent on photoperiod, and that enlarged rhizomes show weak dormancy.
Storage organs are classified into bulbs, tubers, rhizomes, and corms by modified organ and morphology among geophytes. The mechanisms of the storage organ formation have been poorly understood because there are few plants with strict regulation of storage organ formation by environmental factors, temperature and/or photoperiod. These few plants are restricted to only bulbous and tuberous plants such as Dutch iris (Iris hollandica) (Okubo and Uemoto, 1981), garlic (Allium sativum) (Aoba, 1966; Takagi, 1979), onion (Allium cepa) (Kato, 1964; Magruder and Allard, 1937), Allium x wakegi (Okubo et al., 1981; Yamasaki et al., 1999, 2003), potato (Solanum tuberosum) (Snyder and Ewing, 1989), Jerusalem artichoke (Helianthus tuberosus) (Hamner and Long, 1939), and Begonia evansiana (Esashi and Nagao, 1958).
The lotus plant (Nelumbo nucifera) forms enlarged rhizomes as storage organs. A few floating leaves appear from rhizomes as temperature rises in early spring (more than 10°C water temperature), and newly subterranean stems from them begin to elongate underground in a single direction. Subsequently, the main subterranean stem and axially subterranean stems have upright leaves in each node. Enlargement of a few subterranean stems formed by large girth subterranean stems and short subterranean stems occurs in late summer. It has been demonstrated in a previous study that each long and short day induces subterranean stem elongation and subterranean stem enlargement in lotus plant independently of temperature (Masuda et al., 2006). Furthermore, it has been reported that the photoperiodic response is regulated by the phytochrome in subterranean stem morphogenesis (Masuda et al., 2007). This is the first report on strict regulation of rhizome by environmental factors. A more detailed investigation can help improve our understanding of rhizome morphogenesis.
It is known that environmental factors during storage organ formation affect their development restrictively in some species. Begonia evansiana and the potato form storage organs as aerial tubers or tubers under short day like the lotus. The plants lost their induced state and began to sprout from tubers when they were transferred to a long day condition after relatively short cycles of short day (Esashi and Nagao, 1958; Snyder and Ewing, 1989). Sprout growth, however, was not observed from aerial tubers or tubers transferred to long day after the plants were grown under short day over 21 short days in the potato and 23 short days in B. evansiana. Thus, there was an effect of environmental factors on subsequent growth from storage organs when a shorter inductive period for storage organ formation was used. This may be perhaps attributed to depth of dormancy during storage organ formation.
The phenomenon of dormancy or developmental arrest is widespread in the plant kingdom. A definition that ‘dormancy is a temporary suspension of visible growth of any plant structure containing a meristem’ was proposed by Lang et al. (1987), and it seems to be well accepted. Okubo (2000) has also discussed this from growth cycle perspective that commencement of dormancy (dormancy induction) is ‘the change of the primordia that cease growing for a while or that initiate special organs instead of producing shoots’.
Examples of dormancy can be found in nearly all meristematic organs, including seeds, apical and lateral vegetative and floral buds, bulbs, corms, and tubers (Nooden and Weber, 1978; Saunders, 1978). In bulbous and tuberous plants such as garlic, onion, A. x wakegi, Dutch iris, potato, B. evansiana, and Jerusalem artichoke, the newly formed storage organs enter a deep dormant state, and they do not sprout immediately after harvesting (Esashi, 1969; Esashi and Leopold, 1969; Okubo and Uemoto, 1981; Wiltshire and Cobb, 1996; Yamasaki et al., 1999).
Environmental factors inducing storage organ formation deepened dormancy in Dutch iris (Okubo and Uemoto, 1981), potato (Wiltshire and Cobb, 1996), and A. x wakegi (Yamasaki et al., 1999) etc. In B. evansiana, dormancy was deepened by not only environmental factors for aerial tuber formation, but also other environmental factors (Esashi, 1969; Esashi and Leopold, 1969). Thus, depth of dormancy is determined by several environmental factors according to plant species.
We report here the effects of photoperiod and temperature on rhizome formation (dormancy induction) and accompanied dormancy depth in the lotus.
Open pollinated seeds of N. nucifera ‘Chugoku’ were used in all the experiments in this study. The seeds were prepared for germination by soaking in conc. H2SO4 for three hours and rinsing with distilled water. They were then soaked in distilled water for one day at 25°C. After removing softened seed coats, the seeds were incubated in distilled water at 25°C under continuous fluorescent light conditions (approximately 40 μmol·m−2·s−1 PPFD) until germination (about one week). The seedlings were transplanted into soil containing 30 g slow-release fertilizer (N:P:K = 16:5:10) per plastic container (45 × 32 × 23.5 cm). The containers were filled with water to a height of 5 cm above ground, and the water was replaced every week.
Effects of natural photoperiod on rhizome enlargementNine-day-old seedlings were transplanted on 26 July, 2 August, 9 August, and 16 August, 2010 at one week intervals. Five or more seedlings per treatment were grown for five weeks under a natural photoperiod in an unheated greenhouse. Namely, they were grown from 26 July to 30 August, from 2 August to 6 September, from 9 August to 13 September, and from 16 August to 20 September, respectively. The latitude in Fukuoka Prefecture in Japan is 33.35°N, and the natural photoperiod during the cultivation period from 26 July to 20 September gradually decreased. The photoperiods ranged from13 h 57 min to 12 h 56 min between 26 July and 30 August, from 13 h 47 min to 12 h 42 min between 2 August and 6 September, from 13 h 35 min to 12 h 28 min between 9 August and 13 September, and from 13 h 23 min to 12 h 14 min between 16 August and 20 September, respectively. The average ambient temperature in Fukuoka gradually increased from July (28°C) to August (30°C) in 2010, whereas it decreased from August (30°C) to September (26°C). After culture for 5 weeks, investigation of subterranean stem growth (maximum diameter and length in each internode in main subterranean stem) was carried out and the subterranean stem enlargement index (= maximum internode diameter / internode length) was calculated. Enlargement indices in subterranean stems from the first to fourth internodes were not measured in all the treatments because these internodes were always extremely short whenever the seeds were sown. The value of 0.2 in the enlargement index in subterranean stems was used to judge whether an internode of subterranean stems elongated or enlarged as reported previously (Masuda et al., 2006, 2007).
Effects of commencement of short day treatment on rhizome enlargementSeven-day-old seedlings were transplanted on 12 June, 2002, and two or more seedlings per treatment were grown for 8 weeks in an unheated greenhouse. Nine treatments were designed by combination with short day (SD) and long day (LD) conditions to investigate when photoperiod affected morphology in subterranean stems. A schematic diagram of photoperiodic treatments is shown in Figure 1A. They were exposed under a short day condition after a long day condition for 0, 1, 2, 3, 4, 5, 6, 7, or 8 weeks. Long day treatment was performed by exposing the plants to a natural photoperiod ranging from 13 h 49 min to 14 h 17 min. An 8 h photoperiod was achieved by exposing the plants to 8 h natural light and covering them from 17:00 to 9:00. The average ambient temperature in Fukuoka gradually increased from June (24°C) to August (28°C) through cultivation in 2002. After culture for 8 weeks, the enlargement index in subterranean stems was investigated in each internode of the main rhizomes by measuring the maximum internode diameter and internode length.
Schematic diagram of photoperiodic treatments. (A) Effects of commencement of short day treatment on rhizome enlargement (B) Effects of long day treatment after rhizome enlargement on subsequent subterranean stem growth. The gray and black rectangles represent short day (SD) and long day (LD) treatments, respectively.
After seeds were sown on 3 June, 2002, seedlings were transplanted on 10 June, 2002. Three or more seedlings per treatment were cultivated for 8 weeks in an unheated greenhouse in all treatments. After they were grown under a long day condition for 2 weeks, they were transferred to a short day condition for 0, 1, 2, 3, 4, 5, or 6 weeks as indicated in Figure 1B. Subsequently, they were returned to a long day condition for the remaining period. Photoperiod treatments were carried out by exposing plants to a natural photoperiod ranging from 13 h 17 min to 14 h 23 min for long day treatment or 8 h natural light by covering them from 17:00 to 9:00 for 8 h photoperiod. The average ambient temperature in Fukuoka gradually increased from June (24°C) to August (28°C) through cultivation in 2002. The maximum diameter and length of each internode in the main subterranean stem were measured 8 weeks after transplanting, and then the enlargement index in subterranean stem was calculated.
Effects of natural low temperature on depth of dormancy in enlarged rhizomesNine-day-old seedlings were transplanted into each container on 21 September, 2010, and they were grown under a natural photoperiod in an unheated greenhouse until 21 October, 2010, 21 November, 2010, 21 December, 2010, and 21 January, 2011. Subsequently, four or more plants were transferred to a 30°C phytotron glass room of the Biotron Institute, Kyushu University and cultivated for four weeks under a 14 h photoperiod by exposing them to 8 h natural light (9:00–17:00) with 6 h supplemental lights (FL20SSBRN/18; Toshiba Lighting and Technology Co., Tokyo, Japan) both 3 h (6:00–9:00 and 17:00–20:00) before and after 8 h natural light. Light intensity of approximately 40 μmol·m−2·s−1 PPFD was provided as supplemental lighting. Average ambient temperature gradually decreases from 21 September, 2010 to 21 January, 2011 in Fukuoka, and that on 21 September–21 October, 21 October–21 November, 21 November–21 December, and 21 November–21 January fluctuates from 24°C to 18°C, 18°C to 13°C, 13°C to 8°C, and 8°C to 6°C, respectively. The number of leaves were counted every day after transferring plants to a 30°C phytotron glass room. This indicates whether the new subterranean stem started to grow or not. After culture of 4 weeks, sprouting rate and number of internodes in main subterranean stem sprouted from rhizomes were investigated.
Enlargement indices in subterranean stems were lower than 0.06 in all internodes in plants grown until 30 August or 6 September under a natural photoperiod (Fig. 2). In both treatments, there were no plants showing subterranean stems with enlargement indices more than 0.2. When the plants were grown until 13 September, they showed slightly higher enlargement indices in the eighth (enlargement index; 0.1) and ninth (enlargement index; 0.1) internodes. Subterranean stems with high values (>0.2) enlargement indices were observed in approximately 40% of used plants (data not shown). On the other hand, enlargement indices were higher than 0.2 in the eighth and ninth internodes in plants grown until 20 September, and all plants formed rhizomes with values of more than 0.2 enlargement indices (data not shown). In addition, they did not develop subterranean stems in the tenth internode.
Enlargement index in each internode of the main subterranean stem in plants grown under a natural photoperiod. Nine-day-old seedlings were transplanted from 26 July at one week intervals, and they were grown until 30 August, 6 September, 13 September, or 20 September under a natural photoperiod in an unheated greenhouse. Values express mean ± SE (n = 5~7).
In this experiment, we investigated when the lotus could enlarge its subterranean stems. Enlargement indices in subterranean stems were lower than 0.2 in all internodes in the plants grown under long day (LD8+SD0), but they increased from the fifth internode in LD0+SD8 treatment (Fig. 3). LD1+SD7, LD2+SD6, LD3+SD5, LD4+SD4, LD5+SD3, LD6+SD2, and LD7+SD1 treatments brought about high value indices (>0.2) from the fifth, sixth, seventh, eighth, tenth twelfth, and fourteenth internodes, respectively. In both LD1+SD7 and LD0+SD8 treatments, they showed high enlargement indices from the fifth internode. Furthermore, a one week delay of short day treatment led to one internode with a low value in the plants within 4 weeks of transplanting (LD1+SD7, LD2+SD6, LD3+SD5, and LD4+SD4 treatments) before enlargement indices increased in subterranean stems. The plants exposed to long day treatment over 4 weeks (LD5+SD3, LD6+SD2, and LD7+SD1 treatments), however, formed two internodes with low values before an increase in the enlargement index with a one week delay of short day treatment.
Enlargement index in each internode of the main subterranean stem in plants grown under short day after long day culture during the respective period. Plants were grown for 8 weeks in an unheated greenhouse, and subjected to short day during the remaining period after 0 (LD0+SD8), 1 (LD1+SD7), 2 (LD2+SD6), 3 (LD3+SD5), 4 (LD4+SD4), 5 (LD5+SD3), 6 (LD6+SD2), 7 (LD7+SD1), and 8 (LD8+SD0) weeks of long day. Values express mean ± SE (n = 2~4).
When the seedlings were cultivated under long day for 6 weeks (LD2+SD0+LD6), enlargement indices were lower than 0.2 in all internodes (Fig. 4). Although enlargement indices were lower than 0.2 from the fifth to the sixth internodes in LD2+SD6+LD0 treatment, they gradually increased from the seventh internode and reached 1.14 in the twelfth internode. Enlargement indices were more than 0.2 from the sixth or seventh internode in LD2+SD1+LD5, LD2+SD2+LD4, LD2+SD3+LD3, LD2+SD4+LD2, and LD2+SD5+LD1 treatments. Subsequently, they showed lower values than 0.2 in enlargement indices from the eleventh or twelfth internode after formation of subterranean stems with high enlargement indices in LD2+SD1+LD5, LD2+SD2+LD4, LD2+SD3+LD3, and LD2+SD4+LD2 treatments. However, plants remained high in enlargement indices in the LD2+SD5+LD1 treatment.
Enlargement index in each internode of the main subterranean stem in plants grown under long day after rhizome formation. Seven-day-old seedlings were grown for 8 weeks in an unheated greenhouse. They were subjected to short day for 0 (LD2+SD0+LD6), 1 (LD2+SD1+LD5), 2 (LD2+SD2+LD4), 3 (LD2+SD3+LD3), 4 (LD2+SD4+LD2), 5 (LD2+SD5+LD1), or 6 (LD2+SD6+LD0) weeks after 2 weeks of long day, and then transferred to long day during the remaining period. Values express mean ± SE (n = 3~4).
In all treatments, leaves appeared from rhizomes between 7 and 12 days after transferring to a 30°C phytotron of glass room (Fig. 5). Rapid leaf proliferation was observed from rhizomes transferred on November, December, and January, and they developed 3 or 4 leaves after 4 weeks of planting. On the other hand, subsequent leaf appearance tended to be slow in the plants grown on October, and 2 leaves arose from rhizomes after 4 weeks of planting. All rhizomes sprouted in all treatments (Fig. 6). When rhizomes were planted on November, December, and January, they produced about 3 internodes over 4 weeks. Rhizomes planted on October, however, produced a lower number (1.5) of internodes.
Number of leaves from rhizomes exposed to natural low temperature. Nine-day-old seedlings were grown under a natural photoperiod in an unheated greenhouse in cultures from 21 September–21 October, 21 September–21 November, 21 September–21 December, and 21 September–21 January. They were then cultivated for 4 weeks under long day in a 30°C phytotron glass room. Values express mean ± SE (n = 4~6).
Number of internodes and sprouting rate from enlarged rhizomes. Nine-day-old seedlings were transplanted on 21 September, and grown under a natural photoperiod in an unheated greenhouse until 21 October, 21 November, 21 December, and 21 January. They were transferred to a 30°C phytotron glass room subsequently and grown for 4 weeks under long day. The percentage in brackets indicates sprouting rate from enlarged rhizomes. Values express mean ± SE (n = 4~6). The different letters indicate significant difference (P < 0.05) using Tukey’s HSD test.
Although subterranean stems elongated in the plants grown until 30 August or 6 September, enlargement in subterranean stem was observed in those grown until 13 September or 20 September. In addition, fewer subterranean stems were developed in culture until 20 September, indicating that they cease to elongate for rhizome enlargement. These results are compatible with the previous results that rhizomes were observed in plants grown from 6 August to 6 October under a natural photoperiod in an unheated greenhouse (Masuda et al., 2006). Additionally, it was shown that the lotus recognizes the natural photoperiod between 6 September and 20 September as short day. This indicates that the critical photoperiod for rhizome enlargement is between 12 h 14 min and 12 h 43 min in view of the photoperiod from 6 September and 20 September. The present results were in accordance with the previous results that the critical photoperiod in rhizome enlargement lay between 12 hours and 13 hours in the lotus (Masuda et al., 2007).
Subterranean stem morphology was not affected by long day treatment until the first week after transplanting because subterranean stems enlarged from the fifth internode in both LD1+SD7 and SD8 treatments. This may be attributed to the fact that extremely short internodes from the first to fourth are formed regardless of environmental conditions when seeds were sown in the lotus. Therefore, it seems that photoperiod does not affect the morphology of subterranean stems until subterranean stems from the first to fourth are formed within one week after transplanting.
Enlarged rhizomes were observed even if the plants were exposed to 7 short days (LD7+SD1 or LD2+SD1+LD5). In B. evansiana, intact plants required least 17 short days for initiation of aerial tubers (Esashi and Nagao, 1958). In the potato, tubers formed in a small number of plants in 7 short days, although tuber formation occurred in all plants that received 14 or more short days (Chapman, 1958). Furthermore, exposure of potato cutting plants to 10 short days induced tubers (Ewing and Wareing, 1978). Thus, it was found that sensitivity to short days in the lotus is similar to that in the potato.
Solanum tuberosum develops tubers in response to short day, and they require a photoperiod of 12 hours or less to tuberize (Snyder and Ewing, 1989). Tubers in potato plants exposed to only 14 short days developed stolons when they were placed back into non-inducing conditions. On the other hand, stolons from tubers were not observed in potato plants exposed to 21 short days even if they were returned to non-inducing conditions. Similar results were also obtained in B. evansiana. The buds of the aerial tubers formed under short day below 19 cycles began to sprout when transferred to subsequent long day conditions, whereas the aerial tubers did not sprout when they were transferred from 23 short days or more to long day (Esashi and Nagao, 1958). Thus, a restricted effect of environmental factors after storage organ formation on subsequent growth was observed in the potato and B. evansiana. In the lotus, rhizomes sprouted when transferred to a long photoperiod even if they were grown under short days for 28 days (4 weeks). Furthermore, long day treatment of more than 1 week is essential for subterranean stem elongation after rhizome formation. Thus, it was revealed that morphogenesis in subterranean stem elongation enlargement is completely dependent on photoperiod. These results may be attributed to the depth of dormancy in rhizomes because the extent of suppression in sprouting is lower. We hypothesized why rhizomes showed weak dormancy in the present results as follows (1) short day is an environmental factor for rhizome enlargement, but not for suppression in sprouting, and other factors are needed for suppression of sprouting (2) lotus is a plant in which the mechanism of sprouting suppression does not exist.
The lotus is exposed to low temperatures following short day under natural conditions when subterranean stems begin to enlarge. Therefore, it seems that low temperature may be an environmental factor for deepening dormancy in lotus rhizomes. It has been reported that other environmental factors affect depth of dormancy in B. evansiana (Esashi, 1969; Esashi and Leopold, 1969). Rhizomes, however, showed a 100% sprouting rate with expanding leaves when transferred to their favorite condition, a long photoperiod and high temperature irrespective of the length of exposure to natural low temperatures. This showed that low temperature is not environmental factor that deepens dormancy. In addition, it was observed that the plants transferred to their favorite condition in November, December, and January developed more expanding leaves and subterranean stems than those in October. This indicates that low temperature is an environmental factor for dormancy release because low temperature hastens rapid growth in leaf proliferation and subterranean stems. Furthermore, the above may indicate that the chilling requirement for dormancy release is satisfied between October and November. From these results, it was clarified that the lotus has weak dormancy in rhizomes, and they can sprout at any time when transferred to their favorite condition.
It is well known that low temperature affects enzymes involve in regulation of the sucrose/starch ratio in storage organs in many geophytes. Sato and Okubo (2006) reported that exposure of dormant bulbs to low temperature induced the breakdown of starch and accumulation of sucrose in shoot leaves in hyacinths and led to rapid growth of flower stalks. Similarly, effects of low temperature on conversion of starch to soluble sugars in storage organs have been investigated in geophytes such as the lily, tulip, and potato (Komiyama et al., 1997; Lambrechts et al., 1994; Matsuura-Endo et al., 2004; Miller and Langhans, 1990; Shin et al., 2002). Thus, carbohydrate metabolism plays a major role in normal growth from storage organs, and soluble sugars are essential for rapid plant growth. Therefore, the promotional effects of low temperature on growth in above-ground and underground parts may be due to increases in soluble sugar content.
Most bulbous and tuberous plants such as Dutch iris (I. hollandica), garlic (A. sativum), onion (A. cepa), A. x wakegi, potato (S. tuberosum), Jerusalem artichoke (H. tuberosus), and B. evansiana enter a state of deep dormancy during storage organ formation (Aoba, 1963; Esashi, 1969; Esashi and Leopold, 1969; Kays and Nottingham, 2008; Okubo and Uemoto, 1981; Suttle, 2004; Takagi, 1979; Yamasaki et al., 1999). Namely, it seems that two mechanisms controlling storage organ formation (dormancy induction) and deepening dormancy (suppression of sprouting) act at the same time. Such complex mechanisms have made it difficult to understand whether physiological changes take place for storage organ formation or for suppression of sprouting (Yamasaki et al., 1999). Using the lotus with weak rhizome dormancy, physiological changes during storage organ formation are directly linked to the mechanism controlling storage organ formation because they have no clear suppression of sprouting. Furthermore, the lotus has advantage in that we can observe physiological changes easily by transferring from long day to short day or vice versa. Considering these characteristics, the lotus may be a suitable plant to elucidate the mechanism controlling storage organ formation.
Based on these results, we demonstrated that in the lotus (1) photoperiod affects morphogenesis of subterranean stems one week after transplanting, (2) morphogenesis in subterranean stems is completely dependent on photoperiod, and (3) rhizome dormancy is weak because enlarged rhizomes sprout at any time under favorite conditions. We propose that the lotus is a suitable plant to elucidate the mechanism controlling storage organ formation because it has weak dormancy.
