イネにおける株開張性と茎の負の重力屈性との関係

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Acccepted:January 29, 2017

Corresponding author: Tsuneo Kato (tkato@waka.kindai.ac.jp)

Relationship between the Spreading-Stub Phenotype of Rice and Negative Gravitropism of Stems

at Tillering Stage

Tsuneo Kato, Akira Horibata

Faculty of Biology-Oriented Science and Technology, Kindai University （930 Nishimitani, Kinokawa, Wakayama, 649-6493 Japan）

Summary: The spreading-stub phenotype in rice could be a novel plant architecture in rice breeding. As a

factor underlying this phenotype, weakened negative gravitropism of the stems caused by a shortage of starch granules in pulvinus cells has been proposed. This study evaluated one erect-type (non-spreading) cultivar and two spreading-type cultivars, which had the already reported to have nucleotide substitutions in the fourth intron of TAC1, for their negative gravitropism at the tillering stage. The degree of negative gravitropism was quantiﬁ ed from the responses to slanting treatment, in which plants in pots were slanted at 45° from the perpendicular, and also their recovery after plants were returned to the upright position. Starch content of the leaf sheath bases was also determined. Results showed that the two spreading-type cultivars clearly rose up less, and expressed weaker negative gravitropism than the erect-type cultivar in response to the two treatments during the tillering stage. The shortage of starch in the stem bases, including pulvinus cells, was also implied in these spreading-type cultivars. The present results, therefore, support the mechanism of spreading-stub expression in rice.

Key words: plant type, pulvinus cell, slanting treatment, starch granule, tilting angle

Introduction

In rice (Oryza sativa L.), the spreading of stems from the hill base, a spreading stub, is a well-known characteristics particularly in indica-type cultivars, although this is rare in japonica-type cultivars. This phenotype is controlled by a single dominant allele at a locus on the long arm of chromosome 9. This allele was originally identiﬁ ed as Spk (t) (Yamamoto et al. 1997, Miyata et al. 2005), and was ﬁ nely mapped and cloned as the TILLER ANGLE CONTROLL 1 (OsTAC1, TAC1, Os09g0529300) locus (Yu et al. 2007, Komori et al. 2009). At this locus, a single nucleotide polymorphism (SNP) at a splicing site at the end of the fourth intron, which is located downstream of the stop codon, is attributed to the spreading-stub phenotype: alleles containing an A nucleotide exhibit ordinary splicing, resulting in the spreading-stub phenotype, whereas alleles with G show abnormal splicing that represses gene expression and causes erect stem phenotypes (Yu et al. 2007, Komori et al. 2009). Jiang et al. (2012) reported that this type of SNP was found ubiquitously among a wide range of cultivated rice, as well as wild rice species. Ku et al. (2011) demonstrated a putative TAC1 orthlog in maize that also regulated the tiller angle in this species.

This spreading-stub phenotype has been regarded as a negative trait to be avoided in rice breeding, particularly for

japonica-type cultivars (Yamamoto et al. 1997, Yan and Hwa 2008). However, this phenotype might facilitate light harvesting of and light penetration into the plant canopy, resulting in improved photosynthetic efficiency of the canopy and lodging resistance (Jiang et al. 2012, Kato 2014). In addition, ventilation within the canopy could be improved in spreading-stub phenotype, also resulting in improved tolerance to pests (Jiang et al. 2012, Kato 2014). Therefore, the spreading-stub phenotype could be considered as a novel plant architecture providing higher performance in rice breeding.

Fig. 1. Tiller angle (θ1), branching angle (θ2) and bending

angle (θ3) of the primary tiller from the stem base of rice plants.

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Kato (2014) observed the histology of the spreading stub at the base of the stems at the tillering stage of rice. A rice tiller differentiates from the tiller bud at several non-elongating inter-nodes of the mother stem, and initiates its outgrowth. This tiller, also consists of several inter-nodes, and shows upward bending once near its mother stem (Fig. 1). Kato (2014) concluded that

larger tiller angle (θ1 in Fig. 1) in spreading-stub plants was

attributed mostly to smaller bending angle (θ3), rather than larger

branching angle (θ2) (Note θ1 =θ2 θ3). This smaller bending angle

in spreading-stub plants might be attributed to weaker negative gravitropism of the stems.

Okamura et al. (2013a, 2015) reported that a mutant derived from the insertion of the rice retrotransposon, Tos17, into the OsAGPL1 locus, apparently showed a spreading-stub phenotype from the tillering stage. The knock-down of OsAGPL1 by the Tos17 insertion caused a reduction in starch granules in pulvinus cells of the leaf sheathes, because OsAGPL1 regulates starch metabolism in source organs. Because the starch granules in pulvinus cells was suggested to be a sensor of gravitropism (Hashiguchi et al. 2013, Wu et al. 2013), Okamura et al. (2013a, 2015) concluded that the spreading stub appeared in insertional mutants was attributed to weakened negative gravitropism of their stems. Okamura et al. (2013b) confirmed the decreased negative gravitropism in the insertional mutants, but they examined only a single main stem at the young seedling stage of their materials. The objective of the present study, therefore, was to conﬁ rm the weakened negative gravitropism in rice spreading-stub cultivars at tillering stage when tillers are actually spreading. Starch contents of the base of stems were also examined in the cultivars used.

Materials and Methods

Rice cultivars Nakateshinsenbon (japonica-type), Milyang 23 (indica-type) and Nanjing 11 (indica-type) were used. These japonica-type and indica-type cultivars showed typical erect and spreading-stub phenotypes, respectively.

To confirm the alleles of these three cultivars at the TAC1 locus, the genomic region including the SNP site for the spreading-stub phenotype was ampliﬁ ed by PCR using forward primer 5 -CTATCTGCTTAACGCTCCACATTA-3 and reverse primer 5 -GAGACAGGGATTGAGTGGATTAGTA-3 , and template DNA isolated from young leaves using the CTAB method (Doyle and Doyle 1987). PCR was conducted using KOD FX Neo Polymerase Kit (TOYOBO Co. Ltd., Osaka, Japan). Amplified fragments were resolved by 2% agarose gel electrophoresis, stained with ethidium bromide, and isolated with a MiniElute Gel Extraction Kit (QIAGEN K.K., Tokyo, Japan). The obtained fragments were sequenced directly in both directions using a Dye Termination Cycle Sequencing Kit

(Beckman Coulter Inc., Brea, CA) with forward primer 5 -TAGTCGAACTGGGAAGAATTGCTG-3 and reverse primer 5 - A C A A C A A A A G G A A G A G G A C T G A A C - 3 , a n d a multicapillary electrophoresis system (CEQ-2000XL, Beckman Coulter Inc.). All procedures were performed in accordance with the respective manufactures instructions.

In 2014, seeds of the three cultivars were sown in a nursery box on 14 May, grown in a greenhouse, and transplanted into pots with one plant per pot on 11 June. On 2 July, when the plants already had several tillers, the pots were slanted at approximately 45° from the perpendicular (slanting treatment) (Fig. 2A). At this stage, the stems of the rice plants were roughly placed within a plane, which is defined as the tiller plane . Thus, in the slanting treatment, the tiller plane was slanted at approximately 45° from the perpendicular plane. At 14 days after the start of treatment (14 DAT), the pots were restored to their initial upright angle, termed restoring treatment, and kept until 28 DAT (14 days after the restoring treatment) (Fig. 2B). The tilting angle , which was the angle between the tiller plane and perpendicular plane was measured for each plant at every 7 days from 0 DAT to 28 DAT, using photographs (Fig. 2D). If the tillers rose upward because of negative gravitropism, this angle should be positive. Control plots that did not undergo slanting treatment were also prepared. Spreading angle , which is the angle between the two outer-most tillers (Fig. 2C), was measured for the plants in the control plot at the same time points as the tilting angle for pots receiving the slanting treatment. In every cultivar, four pots each were assigned to the slanting treatment and control as four replicates.

Fig. 2. Slanting treatment (A) and restoring treatment (B) against pot-planted rice plants. Spreading angle (C) and tilting angle (D) of a plant experienced after slanting and restoring treatments.

In 2016, seeds of the three cultivars were again sown in a nursery box on 17 May and transplanted into a paddy ﬁ eld of the

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Faculty of Biology-Oriented Science and Technology, Kindai University, Kinokawa, Wakayama, Japan, on 8 June, with a separation of 15 cm × 30 cm and a single plant per hill. Fertilizers were applied at transplanting at the rate of

N:P2O5:K2O = 6:6:6 g m-2. Six culms (two culms from three

plants each) per cultivar were sampled one month after transplanting by correcting separately the upper part of the leaf sheathes and the lower part of the leaf sheathes (about 10 mm in length) including the pulvinus cells. Immediately, the samples were dried in an oven at 80°C for three days, and then homogenized with a mixer mill (M 301, Retsch Technology GmbH, Haan, Germany). Starch contents of these tissues were determined enzymatically using a Starch Assay Kit, GO/A (STA-20, Sigma-Aldrich Japan KK, Tokyo, Japan), in accordance with the manufactures instruction, after removing alcohol-soluble sugars using three 20-min extractions with hot 80% ethanol. Three replicates were performed, in which two culms per plant were combined, because of the small amounts of sampled tissue. Starch contents (% dry weight) in the lower and upper parts of the leaf sheathes, and the ratio of the former to the latter were obtained.

An analysis of variance in one-way classification was performed for spreading angle and tilting angle at every DAT, and for three traits of starch content. Three cultivars were compared for their means using the least square difference at P<0.05, when mean of squares for cultivars was signiﬁ cant. Sum of squares was divided into that of comparison between erect-type and spreading-erect-type (df=1), and residual sum of squares after subtracting that of the comparison (i.e. within two spreading-types) (df=1), and subjected to F-test.

Results and Discussion

Sequence analysis of the SNP site in the TAC1 locus clearly conﬁ rmed that A and G bases existed at this site in the two spreading-type cultivars (Milyang 23 and Nanjing 11) and the erect-type cultivar (Nakateshinsenbon), respectively. No other polymorphisms were found within the region examined.

Nanjing 11, a spreading-type cultivar, appeared to have a higher spreading angle than the other two cultivars already at 0 DAT (Fig. 3A). The other spreading-type cultivar, Milyang 23, showed a similar angle to Nakatesinsenbon, an erect-type cultivar, at 0 DAT, and increased from 0 DAT to 7 DAT to a level similar to Nanjing 11. All three cultivars examined kept their respective angles from 7 DAT to 14 DAT, and the angles then decreased slightly with further growth. The spreading-type cultivars showed signiﬁ cantly (P<0.01) higher spreading angles than the erect-type cultivar throughout the period examined except for 0 DAT (Table 1, Fig. 3A).

Table 1. Analysis of variance [F -value (P -value)] for the diﬀ erence between erect-type and spreading-type rice cultivars of spreading and tilting angles at diﬀ erent days after slanting treatment during the tillering stage (From 14 days after the treatment, recovering treatment was started)

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Fig. 3. Changes in the spreading angle (A) and tilting angle (B) of an erect-type cultivar and two spreading-type cultivars during slanting treatment (from 0 day to 14 days after slanting treatment, DAT), and recovering treatment (from 14 DAT to 28 DAT).

Means ± SE (n=4). Means with the same letter or no letters were not signifi cantly diff erent (P>0.05) at the respective DAT using a least signifi cant diff erence test.

As shown in Fig. 3B, all three cultivars increased their tilting angles considerably from 0 DAT to 7 DAT, and then mostly kept these levels to 14 DAT. After the start of restoring treatment, all three cultivars again decreased their angles, and by 28 DAT the angles had returned nearly to the level at 0 DAT. This means that both spreading-type and erect-type cultivars at tillering stage responded immediately to the slanting and restoring treatments, indicating that they clearly expressed negative gravitropism in present to the two treatments. This result was in contrast to the ﬁ ndings of Okamura et al. (2013b) in which gravitropism was observed in the young control seedlings with a single stem but not in a mutant with reduced starch content (Okamura et al. 2013a).

The degree of responses to the treatments, on the other hand, depended on cultivars with different spreading angles: the erect-type cultivar, Nakateshinsenbon, showed the highest tilting angle during the slanting treatment and the lowest during the restoring treatment, compared with the spreading-type cultivars, Milyang

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23 and Nanjing 11. Significant differences (P<0.05) were observed between erect-type and spreading-type cultivars at 14 DAT and 28 DAT (Table 1, Fig. 3B). This result clearly indicated that the spreading-type cultivars showed weaker negative gravitropism than the erect-type cultivar at the tillering stage.

Starch contents in the lower part of the leaf sheath in the three cultivars showed no clear difference between the erect-type and spreading-type cultivars: erect-type Nakatesinsenbon did not differ significantly (P>0.05) from the two spreading-types, Milyang 23 and Nanjing 11, although Nanjing 11 showed signifi cantly higher content than Milyang 23 (Fig. 4A, Table 2). On the other hand, Nanjing 11 had higher starch content in the upper part of leaf sheath than the two other cultivars, although these differences were not signifi cant (P>0.05) (Fig. 4B, Table 2). Because the lower part of leaf sheath in the present experiment not only included the pulvinus cells but also ordinary leaf sheath tissues, the higher starch content of this part in Nanjing 11 might be attributed to a higher content in leaf sheath tissues other than pulvinus cells. The ratio of the starch contents in the lower part to the upper part of the leaf sheaths was calculated (Fig. 4C). The ratios of the spreading-type cultivars were clearly and signifi cantly (P<0.01) lower than the ratio of the erect-type cultivar (Table 2).

Table 2. Analysis of variance [F-value (P-value)] for the starch contents (% dry weight) of the lower part (LP) and the upper part (UP) of leaf sheaths, and the ratio of LP to UP (The LP includes pulvinus cells)

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Fig. 4. Starch contents of the lower part (A) and the upper part (B) of leaf sheaths, and the ratio of the lower to upper (C) of the leaf sheaths in an erect-type cultivar and two spreading-type cultivars at tillering stage.

Naka, Nakateshinsenbon (erect-type); M 23, Milyang 23 (spreading-type); N 11, Nanjing 11 (spreading-type). Means ± SE (n=3). Means with the same letter or no letters were not significantly different (P >0.05) using a least significant diﬀ erence test.

The present experiment evaluated two spreading-type rice cultivars for their degrees of negative gravitropism in response

to artiﬁ cial treatments to induce responses in gravitoropism. The spreading-stub phenotype of these cultivars was conﬁ rmed to be caused by the SNP in the fourth intron of TAC1 (Yu et al. 2007). The results suggested that the degrees of negative gravitropism of the two spreading-type rice cultivars were lower than that of the erect-type cultivar in the tillering stage when the tillers showed spreading characteristics. This shows a good agreement with Okamura et al. (2013b), in which no or weaker negative gravitropism was observed in the seedling stage of the loss-of-function mutant for OsAGPL1 showing a spreading-stub phenotype in adult stages. Okamura et al. (2013a) and Okamura et al. (2015) also concluded that the decrease in starch granules in pulvinus cells of the mutant resulted in weakened negative gravitropism of stems. The shortage of starch in pulvinus cells was also implied, altough indirectly, in the present experiment using ordinary spreading-type cultivars, as in the mutant. Moreover, the present result corresponded with the result of Kato (2014), in which larger tiller angle in spreading-stub plants was attributed to a smaller bending angle of the tiller base, probably resulting from weaker negative gravitropism. In conclusion, there are strong evidences in rice for the relationship between the expression of spreading-stub phenotype and weakened negative gravitropism probably because of the shortage of sensory materials (starch granules). No investigation has been made for the behavior of auxins or other physiological substances relating to gravitropism. In addition, only three cultivars differing in spreading-stub were evaluated in the present experiment. Further studies on this subject could provide a base for the utilization of this spreading-stub phenotype in the breeding of plants with a novel architecture.

Acknowledgements

We sincerely thank to D. Morino, for his technical assistance.

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