Photoperiodic Regulation of Tuber Enlargement in Water Yam

Abstract

In tuberous crops, tuber enlargement is one of the most important target traits for yield formation. It has long been known that tuber growth in yams is enhanced by short-day (SD) conditions, but the mechanism of tuber enlargement remains unknown. Here, we analyzed the photoperiodic regulation of tuber enlargement in water yam (Dioscorea alata L.). The photoperiod experiments in seedlings showed that tuber enlargement is initiated under SD conditions (≤10 h daylength) within 20 days of treatment. DaFT2, a FLOWERING LOCUS T (FT)-like gene, was upregulated in SD and downregulated in long-day (LD) conditions in tubers, suggesting that DaFT2 promotes tuber enlargement. DaFT1, the other FT-like gene, was significantly upregulated only in the leaves under LD, and its expression pattern was opposite to that of DaFT2 in the tubers. A night-break experiment showed that tuber growth was inhibited by red light in the dark period. These results suggest that the tuber enlargement of water yam is completely dependent on the photoperiod and that it involves an FT gene-mediated mechanism in response to the SD condition by red light sensing.

1. Introduction

Yams, Dioscorea spp., are major tuber crops and are widespread as important food sources in Africa, Asia, and Oceania [1]. In particular, yam is a major food and cash crop in West Africa, where about 95% of the global yam production occurs, and the value of agricultural production for the crop is by far the highest among all the staple crops [2]. Furthermore, yams have long been known as emergency crops and can grow vigorously under poor nutritional conditions [3,4]. Some species contain functional substances, which may increase their demand as medicinal and health foods [5,6]. Thus, yams have the potential to become an important crop due to their stable food production and useful components. However, differences in the daylength response of the tuber enlargement among species limit where yams can be grown.

Water yam (Dioscorea alata L.) is the most widely distributed of the cultivated Dioscorea species in tropical and temperate regions and is an important food and processing crop [7,8]. However, it is difficult to intercrop it with other crops because of its long growth duration (7–10 months in Kyushu, Japan). In addition, being tropical, it is sensitive to low temperatures and suffers from chilling injury at temperatures below 10 °C [9]. Early tuber enlargement would shorten the cultivation period and thus avoid the low temperatures in temperate areas and the late-onset drought in some parts of the tropics, reducing the cost of production (e.g., the number of times the farm needs weeding); therefore, it would be possible to expand the cropping area in temperate regions and establish perennial cultivation in the tropics and subtropics.

The edible part of yam, called a rhizophore, is the intermediate between the stem and the root [7]. Yams form rhizophores, commonly called tubers, both above and below ground; mainly, it is the underground part that is harvested. Therefore, to improve production, control of the underground tuber enlargement is important. Yam is a short-day (SD) plant, and the short daylength accelerates the tuber growth [10]. The initiation of tuber enlargement varies among species and cultivars owing to different photosensitivities [11,12]. In water yam, the tuber forms at the stem base (the seed tuber–stem junction) [7] in the early growth stage regardless of daylength [13,14]. However, tuber growth stagnates under long-day (LD) conditions, and the tuber begins to enlarge under SD conditions [15,16]. This SD response varies among cultivars and tuber growth stages [11]. However, the mechanisms of tuber enlargement are not clear, and the main factors regulating it are unknown.

The formation and enlargement of edible organs are crucial for the yield of crops and may be related to photoperiod sensitivity, but the mechanisms have long been unknown. In the 2010s, it became clear that a homolog of the floral inducer FLOWERING LOCUS T (FT) regulates not only flowering but also the formation of the storage organs in potato (Solanum tuberosum L.) [17] and onion (Allium cepa L.) [18]. FT is now recognized as a signaling molecule or long-distance molecule [19]. Many studies have been carried out to elucidate the molecular mechanisms of tuberization in potato, a major tuber crop (reviewed in [20]). Tuberization is induced by a homolog of FT called StSP6A [17]. As SD promotes tuber growth in both potato and water yam, we considered that the FT-like genes may also be involved in the tuber enlargement in water yam.

The whole genomes of white guinea yam (Dioscorea rotundata) [21] and water yam [22] (https://www.ncbi.nlm.nih.gov/nuccore/CZHE00000000.2 (accessed on 7 June 2022)) have been released by the International Institute of Tropical Agriculture. This information is valuable for the analysis of yam traits at the molecular level. However, there are no reports analyzing the control of tuber enlargement by using this information.

Here, to clarify the mechanism of tuber enlargement in water yam, we evaluated the effects of daylength and light quality on tuber enlargement and analyzed the expression patterns of the FT-like genes.

2. Materials and Methods

2.1. Plant Material and Growth Conditions

This study was conducted in 2019 in the Biotron Institute, Kyushu University, Fukuoka, Japan. Dioscorea alata L. ‘Yamatomakousha’ (JP175488), from the National Institute of Agrobiological Sciences Genebank, was used in all the experiments.

Seed setts from mature tubers grown in an experimental field at Kyushu University in 2018 were cut into pieces of ~1.5 g, treated with fungicide (benomyl), and incubated in a plastic bag with vermiculite at 30 °C in the dark for 1 week. The moisture content of the vermiculite was then adjusted to 50% (w/w) to induce sprouting. After 3 weeks, sprouted setts were selected and transplanted into plastic containers (35 cm × 50 cm, 15 cm high) containing vermiculite in bottomless nursery trays (35 setts per container). No fertilizer was applied. The seedlings were grown in a growth chamber under fluorescent light with a photon flux density of 60 µmol m⁻² s⁻¹ at 30 °C and 60% relative humidity, with sufficient water.

2.2. Photoperiod Treatments and Measurement of Tuber Traits

2.2.1. Experiment 1: Effects of Different Daylength Conditions on Tuber Enlargement

At least 16 seedlings were grown continuously in a container under four photoperiods: 8 h light/16 h dark (8 h), 10 h light/14 h dark (10 h), 12 h light/12 h dark (12 h), and 14 h light/10 h dark (14 h) at 30 °C and 60% relative humidity. When the primary leaf of a plant expanded vertically, that date was defined as the beginning of photoperiod treatment. At 40 DAT, the tuber fresh weight was weighed, and maximum tuber width was determined by electronic calipers.

2.2.2. Experiment 2: Changes in Tuber Growth and FT-like Gene Expression under SD and LD

The seedlings were grown in containers under SD (10 h light/14 h dark) or LD (14 h light/10 h dark) conditions as in Experiment 1. They were sampled at 10, 20, 30, and 40 DAT. The tuber width and FW were measured, and then, the leaf and tuber samples were frozen in liquid nitrogen and stored at −80 °C.

2.2.3. Experiment 3: Effects of Night-Break Treatments on Tuber Enlargement

The seedlings were grown at 30 °C and 60% relative humidity under a 14 h photoperiod (LD) until the primary leaf expanded vertically and then under a 10 h photoperiod (control), with a 2 h night-break treatment with white light (ISLM-150 × 150-WW, CCS Inc., Kyoto, Japan), red light (ISLM-150 × 150-RR, CCS Inc., Kyoto, Japan), or far-red light (ISLM-150 × 150-FF, CCS Inc., Kyoto, Japan) in the middle of the dark period. Tuber width and FW were measured at 40 DAT.

2.3. RNA Extraction and Quantitative Real-Time PCR

The frozen primary leaves and new tubers were ground in liquid nitrogen with a mortar and pestle. Total RNA was extracted by the CTAB method for Dioscorea spp. [23]. cDNA was synthesized the from total RNA (1 μg) by using ReverTra ACE qPCR RT Master Mix with a gDNA Remover kit (Toyobo, Osaka, Japan). Quantitative real-time PCR was performed in a CFX Connect Optics Module real-time PCR system (Bio-Rad, Hercules, CA, USA) with SYBR Green SYBR Green fluorescent dye (Toyobo) according to the manufacturer’s instructions.

The primer sequences are listed in Supplementary Table S1. Thermal cycling consisted of 94 °C for 2 min; 40 cycles of 94 °C for 20 s, primer-specific temperatures for 30 s, and 72 °C for 30 s, with a final 72 °C for 5 min. The melting curve was recorded from 50 to 95 °C. The amount of each gene transcript was normalized against that of the housekeeping gene (DaActin), chosen on the basis of the melting and standard curves.

3. Results and Discussion

3.1. Photoperiod Response of Tuber Enlargement

We evaluated the tuber traits in yam seedlings in a 2-month experiment to investigate their relationship with the photoperiod (Figure 1). Tuber width and fresh weight (FW) at 40 days after the onset of treatment (DAT) were significantly higher in the plants grown under 8 and 10 h photoperiods than in those under 12 and 14 h photoperiods, which resulted in the formation of enlarged tubers. In contrast, growing the plants under 12 and 14 h photoperiods, resulted in the formation of only small tubers (~0.5 cm width, ~0.1 g FW). The tuber length at 40 DAT showed the same pattern as the tuber width (data not shown). These observations indicate that a photoperiod of ≤10 h promotes the enlargement of formed tubers. The tuber width and FW at 40 DAT were significantly higher in the plants grown under 8 and 10 h photoperiods than in those under 12 and 14 h photoperiods, thus confirming that tuber enlargement is promoted by SD and that the critical photoperiod for this cultivar is a daylength of between 10 and 12 h. Vaillant et al. [16] have demonstrated using in vitro plantlets that a 12 h short-day photoperiod promotes the enlargement of water yam tubers, whereas a 16 h long-day photoperiod has the opposite effect.

To confirm when tuber enlargement begins, we then investigated the changes in tuber growth with time, under SD (10 h light/14 h dark) and LD (14 h light/10 h dark) conditions (Figure 2). Under LD, the tubers that had formed stopped growing, whereas under SD the tuber width and FW increased remarkably after 20 DAT and were significantly higher in SD than in LD from 30 DAT. Thus, tuber enlargement was initiated. At 40 DAT, tuber width in SD was 1.9×, and FW was 5.3× that in LD. Therefore, tuber enlargement in water yam seedlings is completely dependent on the photoperiod, and the cultivar has a photoperiodic response program that starts tuber enlargement about 20 days after sensing short daylength.

3.2. Changes in FT-like Gene Expression under SD and LD

The initiation of potato tuberization is promoted by the FT homolog StSP6A protein [17]. We performed homology analysis by searching its amino acid sequence in the water yam genome (Dioscorea alata v1.1, Phytozome 13). As a result, we detected the top two genes with the highest homology to the StSP6A amino acid sequence, and we named those two genes as DaFT1 and DaFT2 (Table S2).

In leaves, the DaFT1 was expressed at trace levels throughout the SD treatment period, but it increased in LD and was significantly higher in LD at 30 and 40 DAT (Figure 3A). In the tubers, DaFT1 was expressed at trace levels in SD and LD (Figure 3C). The DaFT2 expression in the leaves increased independently of daylength (Figure 3B), although it tended to be somewhat suppressed by LD after 10 DAT. The expression of DaFT2 in the tubers tended to increase after SD treatment, but it decreased after LD treatment (Figure 3D). The DaFT2 expression was significantly higher at 20 to 40 DAT in SD than in LD. These results indicate that the expression of DaFT1 in leaves and DaFT2 in tubers is a photoperiodic response. The expression of DaFT1 was increased in leaves only under LD. This gene expression pattern is consistent with that of FT-like StSP5G, which suppresses potato tuberization under LD [24]. On the other hand, the FT homolog StSP6A is upregulated in tubers under SD and promotes tuberization in potato [17]. Indeed, DaFT2 was highly expressed in tubers under SD. These two FT genes may positively or negatively act in the photoperiodic response of tuber enlargement in water yam.

3.3. Effects of Night-Break Treatments on Tuber Enlargement

To clarify whether light quality is involved in tuber growth, we evaluated the effect of night-break treatments (2 h irradiation with LED lights in the middle of the dark period) by red, far-red, or white lights on tuber enlargement in seedlings grown under SD (10 h light/14 h dark). The tuber width and FW at 40 DAT were significantly lower in the night-break treatment with the white light and red light than in the control (Figure 4). However, they were almost the same in the plants treated with far-red light and the control. These results show that interruption by white or red light during the dark period inhibits tuber enlargement. The red light wavelengths are included in the white light wavelength region. Therefore, it is suggested that red light is an effective light quality for measuring the photoperiod or perceiving the night break in the initiation of tuber enlargement in water yam.

In several tuberous crops, it has been proposed that SD responsive tuberization (potato and begonia [25]) or rhizome enlargement (lotus [26]) via red light sensing involves the phytochrome. The phytochrome may also be involved in the photoperiodic regulation of tuber enlargement in water yam.

4. Conclusions

In water yam, the tuber enlargement is an important agronomic trait for the yielding ability. In this study, we revealed that in water yam the tuber enlargement is initiated under SD conditions (≤10 h daylength) within 20 days of treatment, and it is completely dependent on the photoperiod. We found the candidate genes that may be involved in tuber enlargement: DaFT1 as a repressor under LD and DaFT2 as a promoter under SD. The photoperiodic regulation of tuber enlargement may involve a DaFT gene-mediated mechanism in response to SD by red light sensing. In addition, the pattern of increased DaFT1 expression in leaves under LD was opposite to the pattern of decreased DaFT2 expression in tubers under LD. Because FT is a mobile protein [17,19], the DaFT1 protein synthesized in leaves may be translocated to tubers and suppress the DaFT2 expression. To demonstrate the effects of DaFT1 and DaFT2 regulation on tuber growth, it is necessary to develop transgenic lines (overexpression or GFP-DaFTs) for each gene and to confirm the tuber traits and the mobility of the proteins.