- Email: [email protected]
Thermo-sensitive sterility and self-sterility underlie the partial seed abortion phenotype of Litchi chinensis
Scientia Horticulturae 247 (2019) 156–164
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Thermo-sensitive sterility and self-sterility underlie the partial seed abortion phenotype of Litchi chinensis
T
Dan-Rong Xiea, Xiao-Sha Maa, Mohammad Zillur Rahmanb, Ming-Chao Yanga, Xu-Ming Huanga, ⁎ Jian-Guo Lia, Hui-Cong Wanga,c, a
Guangdong Litchi Engineering Research Center, College of Horticulture, South China Agricultural University, Guangzhou, China Bangladesh Agricultural Research Institute (BARI), Gazipur, Bangladesh c School of Advanced Agriculture and Bioengineering, Yangtze Normal University, Foling, Chongqing, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Seed development Thermo-sensitive sterile Self-sterile Litchi chinensis Sonn Epigenetic regulation
The seed abortion ratio is one of the most important fruit quality factors in Litchi chinensis. However, unstable seed abortion ratio in some cultivars severely affects its market value. Here, seed development of litchi cultivar ‘Guiwei’ was surveyed in plants from different orchards for five consecutive production years, applying pollen from different sources and involving exposure to different temperatures. The seed weight ranged from < 0.3 g to > 3.0 g and the seed abortion ratio ranged from 12% to 93%. We conclude that ‘Guiwei’ seed development is thermo-sensitive base on the results that the seed abortion ratio was strongly positive correlated to the average minimum temperature under field conditions and that a significantly higher seed abortion ratio was seen at 22/ 26 °C than at 18/22 °C under growth chambers. The pollen source experiment indicated the self-sterillity of ‘Guiwei’ with self-pollination resulted in > 92% aborted seeds, compared with 40%–80% in outcrossed fruits. Both thermo-sensitive and selfed sterile fruit contributed to the variable seed development phenotype, with temperature having a greater effect than the pollen source. Based on the non-Mendelian control of seed development according to the results of a reciprocal cross, we propose that the partial seed abortion phenotype seen in ‘Guiwei’ is epigenetically regulated.
1. Introduction A plant that is able to produce a fruit without seeds, traces of aborted seeds, or a much-reduced number of seeds is considered to be ‘seedless’ (Varoquaux et al., 2000). Two main mechanisms are responsible for the formation of seedless fruits: 1) parthenocarpy, where fruits develop in the absence of fertilization; and 2) stenospermocarpy, where fruits contain partially formed seeds that have aborted after fertilization. Seedlessness is an especially desirable trait for fleshy fruit crops, and for centuries both breeders and researchers have attempted to develop seedless fruits for easier consumption. Seed development is determined by the coordinated growth of the integument, the endosperm, and the embryo (Sun et al., 2010), and various factors have been reported to control seed development in Arabidopsis thaliana, as well as in field and fruit crops. In some cases, seed development depends on the genotype of the maternal tissues, while in others it is dependent on the genotype of the filial tissues. The development of the seed coat (maternal integument) has been reported to significantly influence the final seed size (Adamski et al., 2009; Fang ⁎
et al., 2012). The absence of an outer integument, due to a deletion of the INO (inner no outer) locus, results in the spontaneous seedless phenotype of Annona squamosa (sugar apple) (Lora et al., 2011). In addition, several regulatory pathways, including the HAIKU (IKU), ubiquitin-proteasome, G-protein, and mitogen-activated protein kinase signaling pathways, as well as various phytohormones and transcription factors are known to affect seed size by influencing the endosperm and/ or maternal tissue growth (Li and Li, 2016). Compared to A. thaliana and field crops, factors that control seed development in fruit crops are less well studied. Litchi (Litchi chinensis Sonn.), a widely cultivated subtropical evergreen fruit crop, produces fruit with an edible aril enclosing a single seed, surrounded by a pericarp. According to the type of seed development, litchi can be classified into three categories: seedless, abortive (shriveled) and normal (Wang et al., 2017). Litchi with small sterile seeds are considered to be particularly economically important as they have a larger edible part and a better taste. Cultivar ‘Wuheli’ is the only commercial litchi cultivar that produces seedless fruit. This feature is due to embryo sac sterility and therefore an absence of fertilization (parthenocarpy) (Feng et al.,
Corresponding author at: College of Horticulture, South China Agricultural University, Guangzhou, 510642, China. E-mail address: [email protected] (H.-C. Wang).
https://doi.org/10.1016/j.scienta.2018.11.083 Received 16 September 2018; Received in revised form 27 November 2018; Accepted 28 November 2018 Available online 17 December 2018 0304-4238/ © 2018 Elsevier B.V. All rights reserved.
Scientia Horticulturae 247 (2019) 156–164
D.-R. Xie et al.
reached the pollen diameter. Twenty inflorescences from each tree were randomly selected for each pollen source. Each tree was treated as one replicate and three trees with a similar blooming date were selected for each female donor cultivar (three replicates). Opened flowers and flowers of uncertain sex were removed from each selected inflorescence in advance, leaving only approximately 20 unopened female flowers, which were bagged with paper bags before pollination. Female flowers were hand-pollinated with pollen from different pollen sources in the morning, when the bifurcated stigma opened into a V shape. After pollination, the bag was returned until the stigma was brown at around three days after pollination.
2010). The shriveled seeds in other litchi cultivars are caused by stenospermocarpy, resulting from embryo abortion after fertilization (Huang, 2005), and they are more popular in the litchi industry due to a relatively higher productivity and a higher flesh recovery rate. In a previous study, we demonstrated that lower levels and activity of the cell wall invertase (CWIN) protein are associated with aberrant seed development of ‘Nuomici’, a stenospermic litchi cultivar bearing 95% fruit with shriveled seeds (Zhang et al., 2018). ‘Guiwei’, however, is a cultivar bearing fruit with seed sizes that vary between seasons, orchards, and even trees in the same orchard. Although the partial seed abortion phenotype of ‘Guiwei’ is well known, little information is available about the factors that regulate its seed size. Here we describe the novel phenomenon of temperature sensitive sterility combined with self-sterility in ‘Guiwei’, which we identified by correlating air temperature during key seed developmental stages for five consecutive years in plants from different orchards. We found a strong correlation between seed development and minimum temperature, and verified this in a temperature controlled trial with potted trees. Seed development is influenced by the genotype of filial tissues, and pollen sources have indeed been shown to have a direct effect on seed development in litchi cv. ‘73-S-20′ (Chu et al., 2015). We therefore also evaluated the effects of the pollen source on ‘Guiwei’ seed development and found that self-pollination tended to result in fruit with aborted seeds, while cross-pollination tended to produce large-seeded fruit. The possible mechanism that determines temperature sensitive sterility and self-sterility is discussed.
2.3. Pollen in-situ germination Pollen in-situ germination and pollen tube growth in the style and ovary were observed as described by Martin (1959) with slight modifications. ‘Guiwei’ pistils were pollinated with ‘Guiwei’ pollen (selfpollination) at female flower blooming and pistils were picked 2 h, 6 h, 8 h, 16 h, 1d, 2d, 3d, and 4d after pollination. The sampled pistils were immediately fixed in Carnoy’s fixative solution (ethyl alcohol: glacial acetic acid = 3:1) for 12 h. The fixed pistils were cut along the dorsal suture after gradient rehydration and softened in 4% NaOH for 6 h. The softened pistils were then stained with 0.1% aniline blue and pressed to a suitable thickness before observation under a fluorescence microscope (Zeiss Imager D2) and imaging with a Zeiss AxioCam HRc camera. 2.4. Semi-thin sectioning
2. Materials and methods The preparation and light microscopic observation of plastic embedded sections was performed as previously described (Pugh et al., 2010) with modifications. ‘Guiwei’ and ‘Huaizhi’ female flowers were self-pollinated and ovaries were sampled at 6, 10 and 15 days after pollination (DAP). Ovules were separated and fixed overnight at 4 °C in a paraformaldehyde (3%) and glutaraldehyde (2%) solution. After rinsing in a 0.1 M phosphate buffer (pH 7.4), ovules were dehydrated in increasing concentrations of ethanol and acetone. The material was then embedded in Epon 812 epoxy resin (Sigma 45359) overnight at 70 °C. Sections (2 μm) were cut with a Leica RM2235 microtome equipped with a glass knife and stained with methylene-azure I and aniline red. The sections were observed under an Olympus DP70 light microscope equipped with a DP 12 digital camera.
2.1. Plant materials Litchi cv. ‘Guiwei’ fruit were picked at the stage of commercial maturity from different production areas to study the variation in seed development between production locations from 2014 to 2018. The longitude, latitude and official meteorological data of the sampling orchards were recorded. Ten trees were randomly selected at each production location. Thrity to Fifty fruits were randomly collected from each tree, weighed and the seed abortion ratio was investigated. Ten replicates were used for seed weight and seed abortion ratio assays. For the temperature controlled experiment, 5-year-old air-layered ‘Guiwei’ trees were grown in 30 L pots containing loam, mushroom cinder and coconut chaff (v:v:v, 3:1:1). Uniformly potted trees with an average of 1 m height and about 30 terminal shoots were selected for the experiment. Two weeks after pollination with ‘Xuehuaizi’ pollen, sixteen trees were equally divided into two groups, which were transferred to growth chambers maintained at night/day temperatures of 22/26 °C and 18/22 °C and a 12-h photoperiod. Two weeks later the potted trees were transferred to an open field. The fruit were harvested at maturity and seed weight and seed development were recorded. A single plotted tree served as one replicate. Eight replicates were uased in this experiment. Pollination treatments were carried out with a 30-year-old commercial litchi orchard in Shenzhen in the Guangdong province of China. The trees were cultivated according to standard horticultural practices and methods for disease and insect control. Pollen samples were collected from the four cultivars ‘Guiwei’,’ Xuehuaizi’, ‘Heiye’ and ‘Nuomici’ in an experimental orchard at the South China Agricultural University at male flower blooming time.
2.5. Statistical analysis Data were subjected to one-way analysis of variance (ANOVA). Differences at P < 0.05 were considered statistically significant and means were compared using a Duncan’s multiple range test at 5% level, with IBM SPSS statistics version 22.0 (IBM Corp., Armonk, NY). Pearson correlation coefficients between seed abortion ratio or seed weight and minimum temperature were calculated and subjected to two-tailed tests to determine significance at P < 0.05. 3. Results 3.1. The partial abortion phenotype in ‘Guiwei’ cultivar Litchi fruits normally contain one chestnut-brown to dark-brown, ovoid to oblong seed. In some cultivars, such as ‘Nuomici’ and ‘Lühebao’, a high proportion of seeds may be abortive (Huang, 2005) with a small and shriveled phenotype known as ‘chicken tongues’. However, the ‘Guiwei’ cultivar is a typical partial seed abortion cultivar, bearing fruit with different seed sizes (Fig. 1). Based on the development of the liquid endosperm, ‘Guiwei’ seed development can be divided into two types. Type I refers to fruit where liquid endosperm development fails at a very early stage. In litchis, the harmonious development of the fruit as a whole results from correlative influences
2.2. Pollen germination test and pollination treatments All pollen samples were collected one year before the pollination experiments and stored in a container with silica gel at −80 °C. Pollen germination was observed under a microscope at the end of a 6-hour incubation on 1% agarose and 10% sucrose medium. The pollen grains were considered to have germinated when the pollen tube length 157
Scientia Horticulturae 247 (2019) 156–164
D.-R. Xie et al.
Fig. 1. Different types of fruit and seeds in the litchi cultivar ‘Guiwei’. A. Longitudinal sections of fruit with seed at different stages of development. B. Seed weight and images of different seed sizes at maturity.
(Table S1). We recorded the orchard maximum and minimum temperature and calculated the average values from female bloom to 45 days after anthesis when the normally developed embryo (mainly cotyledon) fills the seed coat. In the Shenzhen orchard, late spring chilling occurred in 2015 and 2018, while a relatively consistent late spring temperature was observed in the other three years (Fig. S1). As a consequence of the different air temperatures and flowering times in the different years, the average maximum and minimum temperatures varied considerably, especially the minimum temperature. Subsequently, the correlation between minimum temperature, seed abortion ratio and seed weight were evaluated among different production years in the same orchards, among trees with different blooming date in the same orchard, or among different orchards in the same production year (Fig. 3). A strong positive correlation between the minimum temperature and seed abortion ratio, and a significant negative correlation between minimum temperature and seed weight, were observed in five production years or three blooming dates in the same orchard. Although the correlation coefficient was not as high as the value in the same orchard, there was a significant positive correlation between the minimum temperature and the seed abortion ratio in different orchards. Seed shriveling was artificially induced in the plump seed litchi cultivar ‘Huaizhi using maleic hydrazide. The optimum treatment time was 15 to 25 days after female bloom, when the liquid endosperm volume increased sharply (Huang, 2005). We analyzed the temperature data from an orchard with different female blooming dates, and thus different abortion ratios, and deduced that chilling two weeks after bloom may have an important effect in seed development (Fig. 4A). Potted ‘Guiwei’ trees were moved to growth chambers with 18/22 °C or 22/26 °C night/day temperature two weeks after bloom, where they were remained for an additional two weeks. A total of 40% of fruit from trees grown at 18/22 °C showed seed abortion, with an average seed weight of approximately 1.5 g, while 80% of fruit from trees grown at 22/26 °C showed seed abortion, with an average seed weight of approximately 0.35 g (Fig. 4B, C).
operating from tissue to tissue. The lifespan of the liquid endosperm was positive correlated the development of seed coat (Huang and Qiu, 1987). A cavity can be observed between the young ovule and pericarp due to the poorly developed liquid endosperm and thus stunted seed coat (Fig. 1A). Generally, type I seeds are less than 0.3 g at maturity. Since normal liquid development favors the growth of a seed coat, a fully developed seed coat indicates a long lifespan of the liquid endosperm. Type II refers to fruit with a longer liquid endosperm lifespan, reflecting a well-developed seed coat. However, the embryo of type II fruits may degenerate at different time of seed development or develop normally, resulting in different seed weights. The seed weight of type II fruits usually ranges from ∼0.3 g to 3.0 g (Fig. 1B). 3.2. Variation in seed development between production orchards and years To identify the factors influencing ‘Guiwei’ seed abortion, we investigated seed development in fruit from orchards at different geographical positions over five consecutive years. The seed development data from six orchards in 2016 and 2018 are listed in Fig. 2. The average seed size and abortion ratio varied considerably among orchards and production years in the same orchard, with seed weight ranging from 0.58 g to 2.09 g and seed abortion ratio from 28.2% to 90.4%. In 2016, orchards at higher latitudes (Zengcheng and Chaozhou) tended to produce fruits with heavier seeds, while in 2018 the orchard (Zhanjiang) at the lowest latitude showed the lowest abortion ratio and thus the highest average seed weight. The seed weight distribution charts show an obviously skewed distribution in all orchards tested. Although there was notable variation among orchards and production years, the majority of seeds in most of the orchards weighed less than 0.6 g and had a shriveled phenotype. 3.3. Seed development is related to air temperature Temperature is an important ecological factor affecting plant development, and since, in the same orchard, earlier blooming panicles tend to bear fruit with bigger seeds, we targeted temperature as a potential key environmental factor affecting ‘Guiwei’ seed development 158
Scientia Horticulturae 247 (2019) 156–164
D.-R. Xie et al.
Fig. 2. The distribution and average seed weight (left coordinate) and average abortion ratio (right coordinate) of ‘Guiwei’ fruit from orchards at different geographical locations in 2016 and 2018.
3.4. Effects of pollen sources on ‘Guiwei’ seed development
3.5. Microscopic analysis of pollen tube in situ growth and ovule development in self-pollinated ‘Guiwei’ seeds
Xiang et al. (2001) reported that the rate of stenopermocarpy in ‘Guiwei’ varied depending on the pollen source, which may also be a reason for the variation in seed size in ‘Guiwei’. In the current study, we performed out hand-pollination trials (Table 1). The pollen germination rate ranged from 11.6% to 62.1%, with ‘Nuomici’ showing the highest value, followed by ‘Guiwei’ and ‘Huaizhi’, while ‘Xuehuaizi’ was the lowest. The pollen germination rate did not affect seed development, but the pollen genotype had significant effects on seed weight and abortion ratio when ‘Guiwei’ was used as the maternal plant. Self-pollination resulted in the highest seed abortion (> 92%) and thus the lowest seed weight in both the 2015 and 2016 trials. In contrast, crosspollination and open pollination resulted in less seed abortion and thus larger seeded fruits, with a greater degree in 2015 than in 2016. In 2015, the lowest percentage of seed abortion and largest seeds was seen in ‘Guiwei’ pollinated with ‘Huaizhi’, followed by fruits receiving pollen from ‘Nuomici’ and ‘Xuehuaizi’. In 2016, however, no significant difference in the seed abortion ratio and seed weight was observed in the three cross-pollination experiments. When ‘Huaizhi’ was used as the maternal plant receiving pollen from ‘Guiwei’ and from ‘Huaizhi’, the seed abortion ratio and seed weight did not vary with pollen source.
We observed that under normal field conditions, self-pollination resulted in a high seed abortion ratio (> 92%), while cross-pollination produced less seed abortive fruit (40% to 80%) (Table 1). Self-incompatibility, where the interaction of matching S-determinants from the pistil and pollen allow “self” recognition, resulting in growth arrest and subsequent programmed cell death of incompatible pollen, is usually the reason for self-sterility (Wu et al., 2011). Here, we observed the pollen tube growth in the styles of self-pollinated ‘Guiwei’. As shown in Fig. 5, the pollen grains germinated normally on the stigma at 2 h after pollination (HAP), and pollen tube tips arrived at the basal ovules and the micropyles at 6 HAP. At about 48 HAP, a pollen tube growing through the micropyle and reaching the embryo sac was clearly observed. To investigate the seed abortion process in more detail, we examined cross-sections of the ovules using light microscopy and compared liquid endosperm and embryo development in the self-sterile ‘Guiwei’ and the self-fertile ‘Huaizhi’ (Fig. 6 A–F). At 6 DAP, a smaller inner ovule cavity and fewer endosperm nuclei were observed in the ‘Guiwei’ ovules than in those of ‘Huaizhi’. With the development of the embryo sac, the endosperm nucleus and embryo were clearly observed in the ovule of ‘Guiwei’ at 10 DAP, and at 15 DAP, an enlarged embryo with four cells was seen in the ovule of ‘Huaizhi’, while the ‘Guiwei’ 159
Scientia Horticulturae 247 (2019) 156–164
D.-R. Xie et al.
Fig. 3. Average seed weight and abortion ratio and the correlation with average minimum temperature during key seed development stages in fruit from different production years, different blooming dates and different orchards. The vertical bars represent the standard error of ten replicates (n = 10). Correlation coefficient r with ‘*’ indicates significant correlation at P < 0.05.
4.2. Thermo-sensitive ovule sterility
embryo remained at the pro-embryo stage. These results suggested that the self-sterility of ‘Guiwei’ was not related to self-incompatibility, but rather with a delay in endosperm development and degeneration of the embryo.
In litchi, stenospermocarpy can occur after unfavorable weather (Stern et al., 1996). Here, temperature was targeted as a potential key factor influencing seed abortion among panicles with different blooming dates (Table S1). In plants, pollen formation and fertility are known to be highly sensitive to temperature (Peng et al., 2004; Zhou et al., 2012; Sakata et al., 2014). In this study, the results from experiments conducted under field conditions showed a highly positive correlation between the seed abortion ratio and the average minimum temperature during crucial seed development stages. In addition, in growth chamber experiments a significantly higher seed abortion rate (smaller seeds) was seen in plants growing at 22/26 °C than at 18/22 °C, indicating that ‘Guiwei’ seed development is thermo-sensitive (Fig. 3–4). Seed development (seed weight and seed abortion ratio) was influenced by a change in the temperature regime and an ovule sterility-fertility transition temperature seemingly ranged from 20 to 22 °C, with higher temperatures increasing the incidence of seed abortion. This result will be of great help to the zoning of ‘Guiwei’. To produce ‘Guiwei’ fruit with high seed abortion rate, growers should select planting area with low chance of later spring chilling and an average minimum temperature during the key seed development stage above 22 °C. Heat stress is major environmental factor that limits the growth and productivity of various crop species (Barnabás et al., 2008; Sita et al., 2017). During lentil (Lens culinaris) seed filling, seed size and yield were shown to be strongly affected when the plants were subjected to heat stress (> 32 °C) (Sehgal et al., 2018). Litchi has adapted to the warm subtropics and the crop yield is best in climates with a hot, humid summer. It makes sence that the seed abortion rate is higher at higher temperature, but a post-anthesis air temperature of 22–26 °C does not represent a heat stress temperature for litchi.
4. Discussion 4.1. The partial seed abortion phenotype Different plant species, and even varieties in the same species, produce seeds of widely varying sizes. Seed size is crucial for plant fitness and seed yield (Moles et al., 2005), and is also a major factor in the quality of many fleshy fruit crops. In general, seed size is predominantly controlled by the genotype of maternal and filial tissues (the endosperm and embryo), although seed growth is environmentally influenced (Li and Li, 2016). Apart from the parthenocarpically seedless litchi, there are three types of litchi cultivars based on seed size: bigseeded (∼2.0 g), small-seeded (∼1.2 g), and seed-abortive (∼0.6 g) (Zhang et al., 2018). Seed size is a characteristic of a specific cultivar and usually remains relatively stable between production seasons and areas. Here, we describe an unusual litchi cultivar, ‘Guiwei’, that has a complex seed development phenotype. ‘Guiwei’ is one of the most commercially important litchi cultivars and produces big-seeded, smallseeded, and seed-abortive fruits with seed weights ranging from < 0.3 g to > 3.0 g (Fig. 1). While seed weight may be a quantitative character, a clearly skewed seed weight distribution was observed in all the orchards tested (Fig. 2). Although there was variation between orchards and production years, the majority of fruits in most of the orchards were seed-abortive, with a seed weight less than 0.6 g. Previous studies have termed this cultivar a partial abortion cultivar (Lü et al., 1985), but the cause of the partial seed abortion has not been characterized. 160
Scientia Horticulturae 247 (2019) 156–164
D.-R. Xie et al.
Fig. 4. The role of air temperature in determining ‘Guiwei’ seed development. A. Temperature data from an orchard in Zhanjiang and the different days after anthesis when the late spring chilling appeared. Arrows indicate blooming date. Brackets indicate the days after anthesis (DAA) when late spring chilling presents. B. The seed weight and abortion ratio of fruits from potted ‘Guiwei’ trees grown in growth chambers at 18/22 °C (night/day temperature) or 22/26 °C. * represent significant difference at P < 0.05 (t-test, n = 8). C. Image of seeds grown at 18/22 °C and 22/26 °C.
fertility when the temperature is ∼21-23 °C (Zhou et al., 2012). To date, nine types of TGMS rice lines have been isolated and studies revealed that mutations within a 21-nt small RNA, osa-smR5864w, an RNase ZS1, and receptor-like kinases can cause TGMS traits (Yu et al., 2017). In contrast, the mechanism underlying thermo-sensitive ovule sterility in ‘Guiwei’ remains unknown.
In wheat (Triticum aestivum), higher post-anthesis temperatures were reported to increase the rate but reduce the duration of seed filling, and longer seed filling duration usually results in larger seeds (Nasehzadeh and Ellis, 2017). In litchi, 28 days after anthesis represents a transition point between the cell division stage and the filling stage of seed development, and seed abortion occurs mainly during the cell division stage (Zhang et al., 2018). In the present study, higher temperatures were shown to result in a failure in liquid endosperm production and embryo development (seed abortion) and thus mainly affected the cell division stage. These results suggested that the temperature determined the cell fate of filial tissues but not the seed filling of ‘Guiwei’. A similar effect of temperature on cell development was seen in thermo-sensitive genic male sterile (TGMS) rice (Oryza sativa) lines. Rice line PA64S exhibits male sterility at temperatures higher than 23.5 °C during anther development, but it converts to male
4.3. Self-sterility is independent of self-incompatibility in ‘Guiwei’ We observed that warm temperatures caused a sequential failure in liquid endosperm and embryo development, resulting in partial or complete seed abortion. However, in different orchards the determination coefficient (R2) between the seed abortion ratio or seed weight and minimum temperature was much smaller than the values in the same orchards (Fig. 3), suggesting that other factors may affect ‘Guiwei’
Table 1 Pollen germination rate of different litchi cultivars and seed development of ‘Guiwei’ and ‘Huaizhi’ in response to different pollen sources. Cross
‘Guiwei’ בGuiwei’ ‘Guiwei’ בXuehuaizi’ ‘Guiwei’ בHuaizhi’ ‘Guiwei’ בNuomici’ ‘Guiwei’ × Free ‘Huaizhi’ בGuiwei’ ‘Huaizhi’ בHuaizhi’
Pollen germination rate (%)
25.4 ± 1.6 b 11.6 ± 1.0 d 15.8 ± 0.7 c 62.1 ± 2.0 a ———— 25.4 ± 1.6* 15.8 ± 0.7
2015
2016
Seed weight (g)
Abortion rate (%)
0.36 1.38 1.88 1.41 1.16 2.17 2.06
93.3 ± 3.3 65.3 ± 2.8 39.2 ± 7.1 54.2 ± 7.1 63.9 ± 7.1 2.3 ± 0.67 3.1 ± 0.19
± ± ± ± ± ± ±
0.08 0.07 0.33 0.14 0.07 0.04 0.07
c ab a ab b
a b c bc b
Seed weight (g)
Abortion rate (%)
0.33 0.74 0.76 1.01 0.98 2.01 1.81
92.6 ± 3.4 76.8 ± 6.7 76.7 ± 7.3 65.3 ± 9.8 76.6 ± 6.5 5.7 ± 0.89 4.5 ± 0.56
± ± ± ± ± ± ±
0.05 0.15 0.14 0.29 0.10 0.06 0.09
b a a a a
a a b b b
Note: Different letters after the values when the ‘Guiwei’ cultivar was used as the maternal parent indicate significant differences at P < 0.05 among pollen sources according to Duncan’s Multiple New Range Test (n = 3). * represent significant differences at P < 0.05 (t-test, n = 3) between ‘Huaizhi’ self-pollination and crosses with ‘Guiwei’ pollen. 161
Scientia Horticulturae 247 (2019) 156–164
D.-R. Xie et al.
Fig. 5. In situ germination and pollen tube growth of ‘Guiwei’ pollen. A. Pollen germinating on the stigma 2 h after pollination. B. Pollen tubes growing across the style 6 h after pollination. C. A pollen tube growing through the micropyle and reaching the embryo sac 2 days after pollination. The yellow arrows indicate the pollen and the red arrows indicate the pollen tubes. Scale bars = 100 μm.
in selfed fruits (Chu et al., 2015). The influence of pollen genotype on the seed characteristics of litchi suggested a strong xenia effect, which has previously been demonstrated for different fruit species (Sabir, 2015), although the actual mechanism remains unclear. The high ratio of shriveled seeds in self-pollinated ‘Guiwei’ fruit is indicative of self-sterility (Table 1). Normal pollen tube growth in the styles and developed endosperm and embryo indicated self-compatibility in ‘Guiwei’ (Fig. 5–6). These results suggested that the self-sterilty of ‘Guiwei’ was independed from self-incompatibility. Similar to ‘Nuomici’ (Zhang et al., 2018), sequential liquid endosperm and embryo development failure was observed in self-pollinated ovules of ‘Guiwei’ (Fig. 6). In outcrossed ‘Guiwei’ fruits, 61.8% to 23.2% seeds had well-developed embryos and the proportion was higher in 2015 than in 2016 probably due to lower minimum temperature in 2015 than in 2016 (Fig. S1). The percentages of abortive seed decreased and seed
seed development. Although litchi appears to be self-compatible, crosspollination was found to have a significant effect on fruit and seed weight (Stern et al., 1993). Most litchi plantations grow more than one cultivar, providing a possibility for outcrossing. Using isozyme analysis to study the parentage, the average hybrid production rate due to crosspollination in litchi orchards having two cultivars was shown to range from 17% to 87% among cultivars and orchards (Stern et al., 1993). In the present study, ‘Guiwei’ was pollinated with pollen from four different cultivars and seed development was characterized. In two consecutive years, self-pollinated fruit had the lowest seed weight and a significantly higher abortive seed ratio (> 92%) than did outcrossed fruit (Table 1). We conclude that pollen source was an important factor in ‘Guiwei’ seed development, consistent with earlier results from the litchi cultivar ‘73-S-20′ outcrossed with ‘Haak Yip’ pollen, which produced 81.2% seeds containing a living embryo, compared with 33.3%
Fig. 6. Endosperm and embryo development in self-pollinated ‘Guiwei’ and ‘Huaizhi’. A–C. The embryo sac of ‘Guiwei’ litchi at 6, 10 and 15 days after pollination (DAP). D–F. The embryo sac of ‘Huaizhi’ litchi at 6, 10 and 15 DAP. The red arrows indicate the endosperm nuclei. The black arrows indicate the embryos. en, endosperm; ii, inner integument; oi, outer integument. Scale bars = 50 μm. 162
Scientia Horticulturae 247 (2019) 156–164
D.-R. Xie et al.
sizes, including stenospermic cultivars. In the present study, a strong correlation between seed development of litchi cv. ‘Guiwei’ with minimum temperature was observed. And furthermore, the role of temperature in seed development was verified using a temperature control trial with potted trees. In addition, the effects of pollen sources on the seed development were also evaluated. The results clearly suggested that both thermo-sensitivity and selfed sterility contributed to the variable seed development phenotype of ‘Guiwei’ litchi, with temperature having a greater effect than the pollen source. We propose that the partial seed abortion of ‘Guiwei’ is epigenetically regulated base on non-Mendelian seed development phenotype, the seed weight distribution and thermo-sensitive sterile phenotype.
weight increased by outcrossing comparing with selfed, suggesting that outcross is one of the caused for low seed abortion rate of ‘Guiwei’. Thus, isolated planting of ‘Guiwei’ is of great important to produce fruits with high seed abortion rate. 4.4. ‘Guiwei’ seed abortion may be associated with epigenetic control Similar to the stable shriveled seed ‘Nuomici’ phenotype or normal seed ‘Huaizhi’ phenotype, partial seed abortion is an inherent characteristic of ‘Guiwei’. The strong xenia effect in ‘Guiwei’ suggests that the seed phenotypes depend on the genotype of the filial tissues (endosperm and embryo) but not on the genotype of the maternal ovule. ‘Guiwei’ pollinated with ‘Huaizhi’ pollen produced 39.2% and 76.7% seed-abortive fruits in 2015 and 2016, respectively, but the seed development of reciprocal crosses was unaltered (Table 1). These results suggest that the genetic control of ‘Guiwei’ seed development does not conform to Mendelian inheritance. Non-Mendelian control of seed development have been previously demonstrated in maize (Zea mays) and A. thaliana (Berger and Chaudhury, 2009). Seed abortion in litchi is marked by reduced proliferation of the endosperm and then reduced embryo development (Zhang et al., 2018). During the seed abortion of selfed ‘Guiwei’, a smaller central cell and fewer endosperm nuclei were observed at 6 days after anthesis, and stunted embryo development was observed around 15 days after anthesis (Fig. 6). In dicotyledons, the cellularized endosperm acts as a nourishing tissue for embryo development, and failed endosperm development will ultimately cause embryo developmental arrest (LafonPlacette and Köhler, 2014). The liquid endosperm developmental status in ‘Guiwei’ correlated well with the occurrence of seed abortion (Fig. 1), and we conclude that retarded liquid endosperm growth is the main cause of seed abortion in ‘Guiwei’. Epigenetic regulation plays an important role in regulating seed development in A. thaliana (Sun et al., 2010), and a large group of polycomb (PcG) proteins including FERTILIZATION INDEPENDENT SEED 2 (FIS2), FERTILIZATION-INDEPENDENT ENDOSPERM (FIE/ FIS3), MEDEA (MEA/FIS1), MULTICOPY SUPRESSOR OF IRA (MSI1), and SWINGER (SWN), are known to be involved in endosperm development (Pien and Grossniklaus, 2007). However, in contrast to the progress in understanding the epigenetic regulation of endosperm development in A. thaliana, key events that control self-sterility in litchi remain to be characterized. The ‘Guiwei’ cultivar also showed a thermo-sterile phenotype. It is not known whether thermo-sterility in ‘Guiwei’ involves the same seed abortion mechanism as self-sterility, but the influence of temperature on seed development in ‘Guiwei’ seems to be more important than the pollen source. The seed abortion ratio of early blooming panicles in the Zhanjian orchard was less than 12%, and the seed abortion ratio of outcrossed ‘Guiwei’ grown at 22/26 °C (80%) was significantly higher than at 18/22 °C (40%) (Fig. 3–4). Plants have evolved sophisticated epigenetic regulatory systems to adjust growth and development in accordance with ambient temperature (Liu and He, 2014). A substitution of C-to-G in noncoding RNA p/tms12-1 was reported to produce a mutant small RNA, osa-smR5864 m, which was shown to be associated with the male sterility transition of PA64S rice (Zhou et al., 2012). Hu et al. (2015) also suggested that DNA methylation or RNA-dependent DNA methylation was involved in the regulation of photoperiod- and thermo-sensitive male sterility PA64S rice. Taken together, the non-Mendelian control of seed development in artificial pollination experiments and the seed fertility transition under different temperature regimes suggests that ‘Guiwei’ seed abortion may be associated with epigenetic regulation.
Author details XDR performed the most of the experiments and MXA and RMZ carried out seed development analysis under the supervision of WHC. YMC, HXM and LJG performed data analysis and interpreted the results. WHC designed the experiment, discussed the data and drafted the manuscript. All authors have read and approved the final manuscript. Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Acknowledgements This study was supported by the China Litchi and Longan Industry Technology Research System (project No. CARS-33-11), the Innovation Team Project of the Department of Education of Guangdong Province (2016KCXTD 011) and the Guangzhou science and technology project (201804020063). The authors would like to thank Dr. Fuchu Hu (Key laboratory of tropical fruit tree biology of Hainan Province), Yongzan Wei (South Subtropical Crops Research Institute) and Junsheng Zhao (Fruit Bureau of Maoming, Guangdong Privince) for their help in sampling. We thank PlantScribe (www.plantscribe.com) for editing this manuscript. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.scienta.2018.11.083. References Adamski, N.M., Anastasiou, E., Eriksson, S., O’Neill, C.M., Lenhard, M., 2009. Local maternal control of seed size by KLUH/CYP78A5-dependent growth signalling. Proc. Natl. Acad. Sci. U. S. A. 106, 20115–20120. Barnabás, B., Jäger, K., Fehér, A., 2008. The effect of drought and heat stress on reproductive processes in cereals. Plant. Cell. Environ. 31, 11–38. Berger, F., Chaudhury, A., 2009. Parental memories shape seeds. Trends Plant. Sci. 14, 550–556. Chu, Y.C., Lin, T.S., Chang, J.C., 2015. Pollen effects on fruit set, seed weight, and shriveling of’ 73-S-20’ litchi- with special reference to artificial induction of parthenocarpy. HortSci 50, 369–373. Fang, W., Wang, Z., Cui, R., Li, J., Li, Y., 2012. Maternal control of seed size by EOD3/ CYP78A6 in Arabidopsis thaliana. Plant. J. 70, 929–939. Feng, W., Zhang, L., Li, S.G., Chen, Y.Y., Luo, S.R., 2010. Comparison on embryonic development process of three cultivars of Litchi chinensis. Subtrop. Plant. Res. Comm. 31, 736–739 Chinese with English abstract. Hu, J., Chen, X., Zhang, H., Ding, Z., 2015. Genome-wide analysis of DNA methylation in photoperiod- and thermo-sensitive male sterile rice Peiai 64S. BMC Genomics 16, 102. Huang, H., 2005. Fruit set, development and maturation. In: Menzel, C.M., Waite, G.K. (Eds.), Litchi and longan: Botany, Production, and Use. CABI Publishing Press, London, UK p115-140. Huang, H.B., Qiu, Y.X., 1987. Growth correlations and assimilate partitioning in the arillate fruit of Litchi chinensis Sonn. Aust. J. Plant. Physiol. 14, 181–188. Lafon-Placette, C., Köhler, C., 2014. Embryo and endosperm, partners in seed
5. Conclusions Seed development is of great importance in litchi fruit yield and quality and diverse cultivars have been generated with different seed 163
Scientia Horticulturae 247 (2019) 156–164
D.-R. Xie et al.
Nayyar, H., 2018. Influence of drought and heat stress, applied independently or in combination during seed development, on qualitative and quantitative aspects of seeds of lentil (Lens culinaris Medikus) genotypes, differing in drought sensitivity. Plant. Cell. Environ. 1–14. Sita, K., Sehgal, A., Kumar, J., Kumar, S., Singh, S., Siddique, K.H.M., Nayyar, H., 2017. Identification of high-temperature tolerant lentil (Lens culinaris Medik.) genotypes through leaf and pollen traits. Front. Plant. Sci. 8, 744. Stern, R.A., Gazit, S., El-Batsri, R., Degani, C., 1993. Pollen parent effect on outcrossing rate, yield, and fruit characteristics of ‘Floridian’ and ‘Mauritius’ lychee. J. Amer. Soc. Hort. Sci. 118, 109–114. Stern, R.A., Eisenstein, D., Voet, H., Gazit, S., 1996. Anatomical structure of two-day-old litchi ovules in relation to fruit set and yield. J. Hortic. Sci. 71, 661–671. Sun, X., Shantharaj, D., Kang, X., Ni, M., 2010. Transcriptional and hormonal signaling control of Arabidopsis seed development. Curr. Opin. Plant. Biol. 13, 611–620. Varoquaux, F., Blanvillain, R., Delseny, M., Gallois, P., 2000. Less is better: New approaches for seedless fruit production. Trends Biotechnol. 18, 233–242. Wang, H.C., Lai, B., Huang, X.M., 2017. Litchi fruit set, development, and maturation. In: Kumar, M., Kumar, V., Prasad, A.V. (Eds.), In: The Lychee Biotechnology. Springer, Singapore P1-30. Wu, J., Wang, S., Gu, Y., Zhang, S., Publicover, S.J., Franklintong, V.E., 2011. Self-incompatibility in papaver rhoeas activates nonspecific cation conductance permeable to Ca2+ and K+. Plant. Physiol. 155, 963–973. Xiang, X., Ou, L.X., Qiu, Y.P., Yuan, P.Y., Chen, J.Z., 2001. Enbryo abortion and pollen parent effects in ‘Nuomici’ and ‘Guiwei’ litchi. Acta Hort. 558, 257–260. Yu, J., Han, J., Kim, Y.J., Song, M., Yang, Z., He, Y., Fu, R., Luo, Z., Hu, J., Liang, W., Zhang, D., 2017. Two rice receptor-like kinases maintian male fertility under changing tenperatures. Proc. Natl. Acad. Sci. U. S. A. 114, 12327–12332. Zhang, J.Q., Wu, Z.C., Hu, F.C., Liu, L., Huang, X.M., Zhao, J.T., Wang, H.C., 2018. Abberant seed development in Litchi chinensis is associated with the impaired expression of cell wall invertase genes. Hort. Res. 5, 39. Zhou, H., Liu, Q., Li, J., Jiang, D., Zhou, L., Wu, P., Lu, S., Li, F., Zhu, L., Liu, Z., Chen, L., Liu, Y.G., Zhuang, C., 2012. Photoperiod- and thermo-sensitive genic male sterility in rice are caused by a point mutation in a novel noncoding RNA that produces a small RNA. Cell. Res. 22, 649–660.
development. Curr. Opin. Plant. Biol. 17, 64–69. Li, N., Li, Y., 2016. Signaling pathways of seed size control in plants. Curr. Opin. Plant. Biol. 33, 23–32. Liu, J.Z., He, Z.H., 2014. Epigenetic regulation of heat stress response in plants. Chin. Sci. Bull. 59, 631–639 Chinese with English abstract. Lora, J., Hormaza, J.I., Herrero, M., Gasser, C.S., 2011. Seedless fruits and the disruption of a conserved genetic pathway in angiosperm ovule development. Proc. Natl. Acad. Sci. U. S. A. 108, 5461–5465. Lü, L.X., Chen, J.L., Chen, X.J., 1985. An observation on the process of embryo development in litchi. Subtrop. Plant. Res. Comm. 1, 1–5 Chinese with English abstract. Martin, F.W., 1959. Staining and observing pollen tubes in the style by means of fluorescence. Stain Technol. 34 (3), 125–128. Moles, A.T., Ackerly, D.D., Webb, C.O., Tweddle, J.C., Dickie, J.B., Pitman, A.J., Westoby, M., 2005. Factors that shape seed mass evolution. Proc. Natl. Acad. Sci. U. S. A. 102, 10540–10544. Nasehzadeh, M., Ellis, R.H., 2017. Wheat seed weight and quality differ temporally in sensitivity to warm or cool conditions during seed development and maturation. Ann. Bot. 120, 479–493. Peng, S., Huang, J., Sheehy, J.E., Laza, R.C., Visperas, R.M., Zhong, X., Centeno, G.S., Khush, G.S., Cassman, K.G., 2004. Rice yields decline with higher night temperature from global warming. Proc. Natl. Acad. Sci. U. S. A. 101, 9971–9975. Pien, S., Grossniklaus, U., 2007. Polycomb group and trithorax group proteins in Arabidopsis. Biochim. Biophys. Acta 1769, 375–382. Pugh, D.A., Offler, C.E., Talbot, M.J., Ruan, Y., 2010. Evidence for the role of transfer cells in the evolutionary increase in seed and fiber biomass yield in cotton. Mol. Phys. 3, 1075–1086. Sabir, A., 2015. Xenia and metaxenia in grapes: differences in berry and seed characteristics of maternal grape cv. ‘Narince’ (Vitis vinifera L.) As influenced by different pollen sources. Plant Biol. 17, 567–573. Sakata, T., Oda, S., Tsunaga, Y., Shomura, H., Kawagishi-Kobayashi, M., Aya, K., Saeki, K., Endo, T., Nagano, K., Kojima, M., Sakakibara, H., Watanabe, M., Matsuoka, M., 2014. Reduction of gibberellin by low temperature disrupts pollen development in rice. Plant. Physiol. 164, 2011–2019. Sehgal, A., Sita, K., Bhandari, K., Kumar, S., Kumar, J., Prasad, P.V.V., Siddique, K.H.M.,
164
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Thermo-sensitive sterility and self-sterility underlie the partial seed abortion phenotype of Litchi chinensis
T
Dan-Rong Xiea, Xiao-Sha Maa, Mohammad Zillur Rahmanb, Ming-Chao Yanga, Xu-Ming Huanga, ⁎ Jian-Guo Lia, Hui-Cong Wanga,c, a
Guangdong Litchi Engineering Research Center, College of Horticulture, South China Agricultural University, Guangzhou, China Bangladesh Agricultural Research Institute (BARI), Gazipur, Bangladesh c School of Advanced Agriculture and Bioengineering, Yangtze Normal University, Foling, Chongqing, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Seed development Thermo-sensitive sterile Self-sterile Litchi chinensis Sonn Epigenetic regulation
The seed abortion ratio is one of the most important fruit quality factors in Litchi chinensis. However, unstable seed abortion ratio in some cultivars severely affects its market value. Here, seed development of litchi cultivar ‘Guiwei’ was surveyed in plants from different orchards for five consecutive production years, applying pollen from different sources and involving exposure to different temperatures. The seed weight ranged from < 0.3 g to > 3.0 g and the seed abortion ratio ranged from 12% to 93%. We conclude that ‘Guiwei’ seed development is thermo-sensitive base on the results that the seed abortion ratio was strongly positive correlated to the average minimum temperature under field conditions and that a significantly higher seed abortion ratio was seen at 22/ 26 °C than at 18/22 °C under growth chambers. The pollen source experiment indicated the self-sterillity of ‘Guiwei’ with self-pollination resulted in > 92% aborted seeds, compared with 40%–80% in outcrossed fruits. Both thermo-sensitive and selfed sterile fruit contributed to the variable seed development phenotype, with temperature having a greater effect than the pollen source. Based on the non-Mendelian control of seed development according to the results of a reciprocal cross, we propose that the partial seed abortion phenotype seen in ‘Guiwei’ is epigenetically regulated.
1. Introduction A plant that is able to produce a fruit without seeds, traces of aborted seeds, or a much-reduced number of seeds is considered to be ‘seedless’ (Varoquaux et al., 2000). Two main mechanisms are responsible for the formation of seedless fruits: 1) parthenocarpy, where fruits develop in the absence of fertilization; and 2) stenospermocarpy, where fruits contain partially formed seeds that have aborted after fertilization. Seedlessness is an especially desirable trait for fleshy fruit crops, and for centuries both breeders and researchers have attempted to develop seedless fruits for easier consumption. Seed development is determined by the coordinated growth of the integument, the endosperm, and the embryo (Sun et al., 2010), and various factors have been reported to control seed development in Arabidopsis thaliana, as well as in field and fruit crops. In some cases, seed development depends on the genotype of the maternal tissues, while in others it is dependent on the genotype of the filial tissues. The development of the seed coat (maternal integument) has been reported to significantly influence the final seed size (Adamski et al., 2009; Fang ⁎
et al., 2012). The absence of an outer integument, due to a deletion of the INO (inner no outer) locus, results in the spontaneous seedless phenotype of Annona squamosa (sugar apple) (Lora et al., 2011). In addition, several regulatory pathways, including the HAIKU (IKU), ubiquitin-proteasome, G-protein, and mitogen-activated protein kinase signaling pathways, as well as various phytohormones and transcription factors are known to affect seed size by influencing the endosperm and/ or maternal tissue growth (Li and Li, 2016). Compared to A. thaliana and field crops, factors that control seed development in fruit crops are less well studied. Litchi (Litchi chinensis Sonn.), a widely cultivated subtropical evergreen fruit crop, produces fruit with an edible aril enclosing a single seed, surrounded by a pericarp. According to the type of seed development, litchi can be classified into three categories: seedless, abortive (shriveled) and normal (Wang et al., 2017). Litchi with small sterile seeds are considered to be particularly economically important as they have a larger edible part and a better taste. Cultivar ‘Wuheli’ is the only commercial litchi cultivar that produces seedless fruit. This feature is due to embryo sac sterility and therefore an absence of fertilization (parthenocarpy) (Feng et al.,
Corresponding author at: College of Horticulture, South China Agricultural University, Guangzhou, 510642, China. E-mail address: [email protected] (H.-C. Wang).
https://doi.org/10.1016/j.scienta.2018.11.083 Received 16 September 2018; Received in revised form 27 November 2018; Accepted 28 November 2018 Available online 17 December 2018 0304-4238/ © 2018 Elsevier B.V. All rights reserved.
Scientia Horticulturae 247 (2019) 156–164
D.-R. Xie et al.
reached the pollen diameter. Twenty inflorescences from each tree were randomly selected for each pollen source. Each tree was treated as one replicate and three trees with a similar blooming date were selected for each female donor cultivar (three replicates). Opened flowers and flowers of uncertain sex were removed from each selected inflorescence in advance, leaving only approximately 20 unopened female flowers, which were bagged with paper bags before pollination. Female flowers were hand-pollinated with pollen from different pollen sources in the morning, when the bifurcated stigma opened into a V shape. After pollination, the bag was returned until the stigma was brown at around three days after pollination.
2010). The shriveled seeds in other litchi cultivars are caused by stenospermocarpy, resulting from embryo abortion after fertilization (Huang, 2005), and they are more popular in the litchi industry due to a relatively higher productivity and a higher flesh recovery rate. In a previous study, we demonstrated that lower levels and activity of the cell wall invertase (CWIN) protein are associated with aberrant seed development of ‘Nuomici’, a stenospermic litchi cultivar bearing 95% fruit with shriveled seeds (Zhang et al., 2018). ‘Guiwei’, however, is a cultivar bearing fruit with seed sizes that vary between seasons, orchards, and even trees in the same orchard. Although the partial seed abortion phenotype of ‘Guiwei’ is well known, little information is available about the factors that regulate its seed size. Here we describe the novel phenomenon of temperature sensitive sterility combined with self-sterility in ‘Guiwei’, which we identified by correlating air temperature during key seed developmental stages for five consecutive years in plants from different orchards. We found a strong correlation between seed development and minimum temperature, and verified this in a temperature controlled trial with potted trees. Seed development is influenced by the genotype of filial tissues, and pollen sources have indeed been shown to have a direct effect on seed development in litchi cv. ‘73-S-20′ (Chu et al., 2015). We therefore also evaluated the effects of the pollen source on ‘Guiwei’ seed development and found that self-pollination tended to result in fruit with aborted seeds, while cross-pollination tended to produce large-seeded fruit. The possible mechanism that determines temperature sensitive sterility and self-sterility is discussed.
2.3. Pollen in-situ germination Pollen in-situ germination and pollen tube growth in the style and ovary were observed as described by Martin (1959) with slight modifications. ‘Guiwei’ pistils were pollinated with ‘Guiwei’ pollen (selfpollination) at female flower blooming and pistils were picked 2 h, 6 h, 8 h, 16 h, 1d, 2d, 3d, and 4d after pollination. The sampled pistils were immediately fixed in Carnoy’s fixative solution (ethyl alcohol: glacial acetic acid = 3:1) for 12 h. The fixed pistils were cut along the dorsal suture after gradient rehydration and softened in 4% NaOH for 6 h. The softened pistils were then stained with 0.1% aniline blue and pressed to a suitable thickness before observation under a fluorescence microscope (Zeiss Imager D2) and imaging with a Zeiss AxioCam HRc camera. 2.4. Semi-thin sectioning
2. Materials and methods The preparation and light microscopic observation of plastic embedded sections was performed as previously described (Pugh et al., 2010) with modifications. ‘Guiwei’ and ‘Huaizhi’ female flowers were self-pollinated and ovaries were sampled at 6, 10 and 15 days after pollination (DAP). Ovules were separated and fixed overnight at 4 °C in a paraformaldehyde (3%) and glutaraldehyde (2%) solution. After rinsing in a 0.1 M phosphate buffer (pH 7.4), ovules were dehydrated in increasing concentrations of ethanol and acetone. The material was then embedded in Epon 812 epoxy resin (Sigma 45359) overnight at 70 °C. Sections (2 μm) were cut with a Leica RM2235 microtome equipped with a glass knife and stained with methylene-azure I and aniline red. The sections were observed under an Olympus DP70 light microscope equipped with a DP 12 digital camera.
2.1. Plant materials Litchi cv. ‘Guiwei’ fruit were picked at the stage of commercial maturity from different production areas to study the variation in seed development between production locations from 2014 to 2018. The longitude, latitude and official meteorological data of the sampling orchards were recorded. Ten trees were randomly selected at each production location. Thrity to Fifty fruits were randomly collected from each tree, weighed and the seed abortion ratio was investigated. Ten replicates were used for seed weight and seed abortion ratio assays. For the temperature controlled experiment, 5-year-old air-layered ‘Guiwei’ trees were grown in 30 L pots containing loam, mushroom cinder and coconut chaff (v:v:v, 3:1:1). Uniformly potted trees with an average of 1 m height and about 30 terminal shoots were selected for the experiment. Two weeks after pollination with ‘Xuehuaizi’ pollen, sixteen trees were equally divided into two groups, which were transferred to growth chambers maintained at night/day temperatures of 22/26 °C and 18/22 °C and a 12-h photoperiod. Two weeks later the potted trees were transferred to an open field. The fruit were harvested at maturity and seed weight and seed development were recorded. A single plotted tree served as one replicate. Eight replicates were uased in this experiment. Pollination treatments were carried out with a 30-year-old commercial litchi orchard in Shenzhen in the Guangdong province of China. The trees were cultivated according to standard horticultural practices and methods for disease and insect control. Pollen samples were collected from the four cultivars ‘Guiwei’,’ Xuehuaizi’, ‘Heiye’ and ‘Nuomici’ in an experimental orchard at the South China Agricultural University at male flower blooming time.
2.5. Statistical analysis Data were subjected to one-way analysis of variance (ANOVA). Differences at P < 0.05 were considered statistically significant and means were compared using a Duncan’s multiple range test at 5% level, with IBM SPSS statistics version 22.0 (IBM Corp., Armonk, NY). Pearson correlation coefficients between seed abortion ratio or seed weight and minimum temperature were calculated and subjected to two-tailed tests to determine significance at P < 0.05. 3. Results 3.1. The partial abortion phenotype in ‘Guiwei’ cultivar Litchi fruits normally contain one chestnut-brown to dark-brown, ovoid to oblong seed. In some cultivars, such as ‘Nuomici’ and ‘Lühebao’, a high proportion of seeds may be abortive (Huang, 2005) with a small and shriveled phenotype known as ‘chicken tongues’. However, the ‘Guiwei’ cultivar is a typical partial seed abortion cultivar, bearing fruit with different seed sizes (Fig. 1). Based on the development of the liquid endosperm, ‘Guiwei’ seed development can be divided into two types. Type I refers to fruit where liquid endosperm development fails at a very early stage. In litchis, the harmonious development of the fruit as a whole results from correlative influences
2.2. Pollen germination test and pollination treatments All pollen samples were collected one year before the pollination experiments and stored in a container with silica gel at −80 °C. Pollen germination was observed under a microscope at the end of a 6-hour incubation on 1% agarose and 10% sucrose medium. The pollen grains were considered to have germinated when the pollen tube length 157
Scientia Horticulturae 247 (2019) 156–164
D.-R. Xie et al.
Fig. 1. Different types of fruit and seeds in the litchi cultivar ‘Guiwei’. A. Longitudinal sections of fruit with seed at different stages of development. B. Seed weight and images of different seed sizes at maturity.
(Table S1). We recorded the orchard maximum and minimum temperature and calculated the average values from female bloom to 45 days after anthesis when the normally developed embryo (mainly cotyledon) fills the seed coat. In the Shenzhen orchard, late spring chilling occurred in 2015 and 2018, while a relatively consistent late spring temperature was observed in the other three years (Fig. S1). As a consequence of the different air temperatures and flowering times in the different years, the average maximum and minimum temperatures varied considerably, especially the minimum temperature. Subsequently, the correlation between minimum temperature, seed abortion ratio and seed weight were evaluated among different production years in the same orchards, among trees with different blooming date in the same orchard, or among different orchards in the same production year (Fig. 3). A strong positive correlation between the minimum temperature and seed abortion ratio, and a significant negative correlation between minimum temperature and seed weight, were observed in five production years or three blooming dates in the same orchard. Although the correlation coefficient was not as high as the value in the same orchard, there was a significant positive correlation between the minimum temperature and the seed abortion ratio in different orchards. Seed shriveling was artificially induced in the plump seed litchi cultivar ‘Huaizhi using maleic hydrazide. The optimum treatment time was 15 to 25 days after female bloom, when the liquid endosperm volume increased sharply (Huang, 2005). We analyzed the temperature data from an orchard with different female blooming dates, and thus different abortion ratios, and deduced that chilling two weeks after bloom may have an important effect in seed development (Fig. 4A). Potted ‘Guiwei’ trees were moved to growth chambers with 18/22 °C or 22/26 °C night/day temperature two weeks after bloom, where they were remained for an additional two weeks. A total of 40% of fruit from trees grown at 18/22 °C showed seed abortion, with an average seed weight of approximately 1.5 g, while 80% of fruit from trees grown at 22/26 °C showed seed abortion, with an average seed weight of approximately 0.35 g (Fig. 4B, C).
operating from tissue to tissue. The lifespan of the liquid endosperm was positive correlated the development of seed coat (Huang and Qiu, 1987). A cavity can be observed between the young ovule and pericarp due to the poorly developed liquid endosperm and thus stunted seed coat (Fig. 1A). Generally, type I seeds are less than 0.3 g at maturity. Since normal liquid development favors the growth of a seed coat, a fully developed seed coat indicates a long lifespan of the liquid endosperm. Type II refers to fruit with a longer liquid endosperm lifespan, reflecting a well-developed seed coat. However, the embryo of type II fruits may degenerate at different time of seed development or develop normally, resulting in different seed weights. The seed weight of type II fruits usually ranges from ∼0.3 g to 3.0 g (Fig. 1B). 3.2. Variation in seed development between production orchards and years To identify the factors influencing ‘Guiwei’ seed abortion, we investigated seed development in fruit from orchards at different geographical positions over five consecutive years. The seed development data from six orchards in 2016 and 2018 are listed in Fig. 2. The average seed size and abortion ratio varied considerably among orchards and production years in the same orchard, with seed weight ranging from 0.58 g to 2.09 g and seed abortion ratio from 28.2% to 90.4%. In 2016, orchards at higher latitudes (Zengcheng and Chaozhou) tended to produce fruits with heavier seeds, while in 2018 the orchard (Zhanjiang) at the lowest latitude showed the lowest abortion ratio and thus the highest average seed weight. The seed weight distribution charts show an obviously skewed distribution in all orchards tested. Although there was notable variation among orchards and production years, the majority of seeds in most of the orchards weighed less than 0.6 g and had a shriveled phenotype. 3.3. Seed development is related to air temperature Temperature is an important ecological factor affecting plant development, and since, in the same orchard, earlier blooming panicles tend to bear fruit with bigger seeds, we targeted temperature as a potential key environmental factor affecting ‘Guiwei’ seed development 158
Scientia Horticulturae 247 (2019) 156–164
D.-R. Xie et al.
Fig. 2. The distribution and average seed weight (left coordinate) and average abortion ratio (right coordinate) of ‘Guiwei’ fruit from orchards at different geographical locations in 2016 and 2018.
3.4. Effects of pollen sources on ‘Guiwei’ seed development
3.5. Microscopic analysis of pollen tube in situ growth and ovule development in self-pollinated ‘Guiwei’ seeds
Xiang et al. (2001) reported that the rate of stenopermocarpy in ‘Guiwei’ varied depending on the pollen source, which may also be a reason for the variation in seed size in ‘Guiwei’. In the current study, we performed out hand-pollination trials (Table 1). The pollen germination rate ranged from 11.6% to 62.1%, with ‘Nuomici’ showing the highest value, followed by ‘Guiwei’ and ‘Huaizhi’, while ‘Xuehuaizi’ was the lowest. The pollen germination rate did not affect seed development, but the pollen genotype had significant effects on seed weight and abortion ratio when ‘Guiwei’ was used as the maternal plant. Self-pollination resulted in the highest seed abortion (> 92%) and thus the lowest seed weight in both the 2015 and 2016 trials. In contrast, crosspollination and open pollination resulted in less seed abortion and thus larger seeded fruits, with a greater degree in 2015 than in 2016. In 2015, the lowest percentage of seed abortion and largest seeds was seen in ‘Guiwei’ pollinated with ‘Huaizhi’, followed by fruits receiving pollen from ‘Nuomici’ and ‘Xuehuaizi’. In 2016, however, no significant difference in the seed abortion ratio and seed weight was observed in the three cross-pollination experiments. When ‘Huaizhi’ was used as the maternal plant receiving pollen from ‘Guiwei’ and from ‘Huaizhi’, the seed abortion ratio and seed weight did not vary with pollen source.
We observed that under normal field conditions, self-pollination resulted in a high seed abortion ratio (> 92%), while cross-pollination produced less seed abortive fruit (40% to 80%) (Table 1). Self-incompatibility, where the interaction of matching S-determinants from the pistil and pollen allow “self” recognition, resulting in growth arrest and subsequent programmed cell death of incompatible pollen, is usually the reason for self-sterility (Wu et al., 2011). Here, we observed the pollen tube growth in the styles of self-pollinated ‘Guiwei’. As shown in Fig. 5, the pollen grains germinated normally on the stigma at 2 h after pollination (HAP), and pollen tube tips arrived at the basal ovules and the micropyles at 6 HAP. At about 48 HAP, a pollen tube growing through the micropyle and reaching the embryo sac was clearly observed. To investigate the seed abortion process in more detail, we examined cross-sections of the ovules using light microscopy and compared liquid endosperm and embryo development in the self-sterile ‘Guiwei’ and the self-fertile ‘Huaizhi’ (Fig. 6 A–F). At 6 DAP, a smaller inner ovule cavity and fewer endosperm nuclei were observed in the ‘Guiwei’ ovules than in those of ‘Huaizhi’. With the development of the embryo sac, the endosperm nucleus and embryo were clearly observed in the ovule of ‘Guiwei’ at 10 DAP, and at 15 DAP, an enlarged embryo with four cells was seen in the ovule of ‘Huaizhi’, while the ‘Guiwei’ 159
Scientia Horticulturae 247 (2019) 156–164
D.-R. Xie et al.
Fig. 3. Average seed weight and abortion ratio and the correlation with average minimum temperature during key seed development stages in fruit from different production years, different blooming dates and different orchards. The vertical bars represent the standard error of ten replicates (n = 10). Correlation coefficient r with ‘*’ indicates significant correlation at P < 0.05.
4.2. Thermo-sensitive ovule sterility
embryo remained at the pro-embryo stage. These results suggested that the self-sterility of ‘Guiwei’ was not related to self-incompatibility, but rather with a delay in endosperm development and degeneration of the embryo.
In litchi, stenospermocarpy can occur after unfavorable weather (Stern et al., 1996). Here, temperature was targeted as a potential key factor influencing seed abortion among panicles with different blooming dates (Table S1). In plants, pollen formation and fertility are known to be highly sensitive to temperature (Peng et al., 2004; Zhou et al., 2012; Sakata et al., 2014). In this study, the results from experiments conducted under field conditions showed a highly positive correlation between the seed abortion ratio and the average minimum temperature during crucial seed development stages. In addition, in growth chamber experiments a significantly higher seed abortion rate (smaller seeds) was seen in plants growing at 22/26 °C than at 18/22 °C, indicating that ‘Guiwei’ seed development is thermo-sensitive (Fig. 3–4). Seed development (seed weight and seed abortion ratio) was influenced by a change in the temperature regime and an ovule sterility-fertility transition temperature seemingly ranged from 20 to 22 °C, with higher temperatures increasing the incidence of seed abortion. This result will be of great help to the zoning of ‘Guiwei’. To produce ‘Guiwei’ fruit with high seed abortion rate, growers should select planting area with low chance of later spring chilling and an average minimum temperature during the key seed development stage above 22 °C. Heat stress is major environmental factor that limits the growth and productivity of various crop species (Barnabás et al., 2008; Sita et al., 2017). During lentil (Lens culinaris) seed filling, seed size and yield were shown to be strongly affected when the plants were subjected to heat stress (> 32 °C) (Sehgal et al., 2018). Litchi has adapted to the warm subtropics and the crop yield is best in climates with a hot, humid summer. It makes sence that the seed abortion rate is higher at higher temperature, but a post-anthesis air temperature of 22–26 °C does not represent a heat stress temperature for litchi.
4. Discussion 4.1. The partial seed abortion phenotype Different plant species, and even varieties in the same species, produce seeds of widely varying sizes. Seed size is crucial for plant fitness and seed yield (Moles et al., 2005), and is also a major factor in the quality of many fleshy fruit crops. In general, seed size is predominantly controlled by the genotype of maternal and filial tissues (the endosperm and embryo), although seed growth is environmentally influenced (Li and Li, 2016). Apart from the parthenocarpically seedless litchi, there are three types of litchi cultivars based on seed size: bigseeded (∼2.0 g), small-seeded (∼1.2 g), and seed-abortive (∼0.6 g) (Zhang et al., 2018). Seed size is a characteristic of a specific cultivar and usually remains relatively stable between production seasons and areas. Here, we describe an unusual litchi cultivar, ‘Guiwei’, that has a complex seed development phenotype. ‘Guiwei’ is one of the most commercially important litchi cultivars and produces big-seeded, smallseeded, and seed-abortive fruits with seed weights ranging from < 0.3 g to > 3.0 g (Fig. 1). While seed weight may be a quantitative character, a clearly skewed seed weight distribution was observed in all the orchards tested (Fig. 2). Although there was variation between orchards and production years, the majority of fruits in most of the orchards were seed-abortive, with a seed weight less than 0.6 g. Previous studies have termed this cultivar a partial abortion cultivar (Lü et al., 1985), but the cause of the partial seed abortion has not been characterized. 160
Scientia Horticulturae 247 (2019) 156–164
D.-R. Xie et al.
Fig. 4. The role of air temperature in determining ‘Guiwei’ seed development. A. Temperature data from an orchard in Zhanjiang and the different days after anthesis when the late spring chilling appeared. Arrows indicate blooming date. Brackets indicate the days after anthesis (DAA) when late spring chilling presents. B. The seed weight and abortion ratio of fruits from potted ‘Guiwei’ trees grown in growth chambers at 18/22 °C (night/day temperature) or 22/26 °C. * represent significant difference at P < 0.05 (t-test, n = 8). C. Image of seeds grown at 18/22 °C and 22/26 °C.
fertility when the temperature is ∼21-23 °C (Zhou et al., 2012). To date, nine types of TGMS rice lines have been isolated and studies revealed that mutations within a 21-nt small RNA, osa-smR5864w, an RNase ZS1, and receptor-like kinases can cause TGMS traits (Yu et al., 2017). In contrast, the mechanism underlying thermo-sensitive ovule sterility in ‘Guiwei’ remains unknown.
In wheat (Triticum aestivum), higher post-anthesis temperatures were reported to increase the rate but reduce the duration of seed filling, and longer seed filling duration usually results in larger seeds (Nasehzadeh and Ellis, 2017). In litchi, 28 days after anthesis represents a transition point between the cell division stage and the filling stage of seed development, and seed abortion occurs mainly during the cell division stage (Zhang et al., 2018). In the present study, higher temperatures were shown to result in a failure in liquid endosperm production and embryo development (seed abortion) and thus mainly affected the cell division stage. These results suggested that the temperature determined the cell fate of filial tissues but not the seed filling of ‘Guiwei’. A similar effect of temperature on cell development was seen in thermo-sensitive genic male sterile (TGMS) rice (Oryza sativa) lines. Rice line PA64S exhibits male sterility at temperatures higher than 23.5 °C during anther development, but it converts to male
4.3. Self-sterility is independent of self-incompatibility in ‘Guiwei’ We observed that warm temperatures caused a sequential failure in liquid endosperm and embryo development, resulting in partial or complete seed abortion. However, in different orchards the determination coefficient (R2) between the seed abortion ratio or seed weight and minimum temperature was much smaller than the values in the same orchards (Fig. 3), suggesting that other factors may affect ‘Guiwei’
Table 1 Pollen germination rate of different litchi cultivars and seed development of ‘Guiwei’ and ‘Huaizhi’ in response to different pollen sources. Cross
‘Guiwei’ בGuiwei’ ‘Guiwei’ בXuehuaizi’ ‘Guiwei’ בHuaizhi’ ‘Guiwei’ בNuomici’ ‘Guiwei’ × Free ‘Huaizhi’ בGuiwei’ ‘Huaizhi’ בHuaizhi’
Pollen germination rate (%)
25.4 ± 1.6 b 11.6 ± 1.0 d 15.8 ± 0.7 c 62.1 ± 2.0 a ———— 25.4 ± 1.6* 15.8 ± 0.7
2015
2016
Seed weight (g)
Abortion rate (%)
0.36 1.38 1.88 1.41 1.16 2.17 2.06
93.3 ± 3.3 65.3 ± 2.8 39.2 ± 7.1 54.2 ± 7.1 63.9 ± 7.1 2.3 ± 0.67 3.1 ± 0.19
± ± ± ± ± ± ±
0.08 0.07 0.33 0.14 0.07 0.04 0.07
c ab a ab b
a b c bc b
Seed weight (g)
Abortion rate (%)
0.33 0.74 0.76 1.01 0.98 2.01 1.81
92.6 ± 3.4 76.8 ± 6.7 76.7 ± 7.3 65.3 ± 9.8 76.6 ± 6.5 5.7 ± 0.89 4.5 ± 0.56
± ± ± ± ± ± ±
0.05 0.15 0.14 0.29 0.10 0.06 0.09
b a a a a
a a b b b
Note: Different letters after the values when the ‘Guiwei’ cultivar was used as the maternal parent indicate significant differences at P < 0.05 among pollen sources according to Duncan’s Multiple New Range Test (n = 3). * represent significant differences at P < 0.05 (t-test, n = 3) between ‘Huaizhi’ self-pollination and crosses with ‘Guiwei’ pollen. 161
Scientia Horticulturae 247 (2019) 156–164
D.-R. Xie et al.
Fig. 5. In situ germination and pollen tube growth of ‘Guiwei’ pollen. A. Pollen germinating on the stigma 2 h after pollination. B. Pollen tubes growing across the style 6 h after pollination. C. A pollen tube growing through the micropyle and reaching the embryo sac 2 days after pollination. The yellow arrows indicate the pollen and the red arrows indicate the pollen tubes. Scale bars = 100 μm.
in selfed fruits (Chu et al., 2015). The influence of pollen genotype on the seed characteristics of litchi suggested a strong xenia effect, which has previously been demonstrated for different fruit species (Sabir, 2015), although the actual mechanism remains unclear. The high ratio of shriveled seeds in self-pollinated ‘Guiwei’ fruit is indicative of self-sterility (Table 1). Normal pollen tube growth in the styles and developed endosperm and embryo indicated self-compatibility in ‘Guiwei’ (Fig. 5–6). These results suggested that the self-sterilty of ‘Guiwei’ was independed from self-incompatibility. Similar to ‘Nuomici’ (Zhang et al., 2018), sequential liquid endosperm and embryo development failure was observed in self-pollinated ovules of ‘Guiwei’ (Fig. 6). In outcrossed ‘Guiwei’ fruits, 61.8% to 23.2% seeds had well-developed embryos and the proportion was higher in 2015 than in 2016 probably due to lower minimum temperature in 2015 than in 2016 (Fig. S1). The percentages of abortive seed decreased and seed
seed development. Although litchi appears to be self-compatible, crosspollination was found to have a significant effect on fruit and seed weight (Stern et al., 1993). Most litchi plantations grow more than one cultivar, providing a possibility for outcrossing. Using isozyme analysis to study the parentage, the average hybrid production rate due to crosspollination in litchi orchards having two cultivars was shown to range from 17% to 87% among cultivars and orchards (Stern et al., 1993). In the present study, ‘Guiwei’ was pollinated with pollen from four different cultivars and seed development was characterized. In two consecutive years, self-pollinated fruit had the lowest seed weight and a significantly higher abortive seed ratio (> 92%) than did outcrossed fruit (Table 1). We conclude that pollen source was an important factor in ‘Guiwei’ seed development, consistent with earlier results from the litchi cultivar ‘73-S-20′ outcrossed with ‘Haak Yip’ pollen, which produced 81.2% seeds containing a living embryo, compared with 33.3%
Fig. 6. Endosperm and embryo development in self-pollinated ‘Guiwei’ and ‘Huaizhi’. A–C. The embryo sac of ‘Guiwei’ litchi at 6, 10 and 15 days after pollination (DAP). D–F. The embryo sac of ‘Huaizhi’ litchi at 6, 10 and 15 DAP. The red arrows indicate the endosperm nuclei. The black arrows indicate the embryos. en, endosperm; ii, inner integument; oi, outer integument. Scale bars = 50 μm. 162
Scientia Horticulturae 247 (2019) 156–164
D.-R. Xie et al.
sizes, including stenospermic cultivars. In the present study, a strong correlation between seed development of litchi cv. ‘Guiwei’ with minimum temperature was observed. And furthermore, the role of temperature in seed development was verified using a temperature control trial with potted trees. In addition, the effects of pollen sources on the seed development were also evaluated. The results clearly suggested that both thermo-sensitivity and selfed sterility contributed to the variable seed development phenotype of ‘Guiwei’ litchi, with temperature having a greater effect than the pollen source. We propose that the partial seed abortion of ‘Guiwei’ is epigenetically regulated base on non-Mendelian seed development phenotype, the seed weight distribution and thermo-sensitive sterile phenotype.
weight increased by outcrossing comparing with selfed, suggesting that outcross is one of the caused for low seed abortion rate of ‘Guiwei’. Thus, isolated planting of ‘Guiwei’ is of great important to produce fruits with high seed abortion rate. 4.4. ‘Guiwei’ seed abortion may be associated with epigenetic control Similar to the stable shriveled seed ‘Nuomici’ phenotype or normal seed ‘Huaizhi’ phenotype, partial seed abortion is an inherent characteristic of ‘Guiwei’. The strong xenia effect in ‘Guiwei’ suggests that the seed phenotypes depend on the genotype of the filial tissues (endosperm and embryo) but not on the genotype of the maternal ovule. ‘Guiwei’ pollinated with ‘Huaizhi’ pollen produced 39.2% and 76.7% seed-abortive fruits in 2015 and 2016, respectively, but the seed development of reciprocal crosses was unaltered (Table 1). These results suggest that the genetic control of ‘Guiwei’ seed development does not conform to Mendelian inheritance. Non-Mendelian control of seed development have been previously demonstrated in maize (Zea mays) and A. thaliana (Berger and Chaudhury, 2009). Seed abortion in litchi is marked by reduced proliferation of the endosperm and then reduced embryo development (Zhang et al., 2018). During the seed abortion of selfed ‘Guiwei’, a smaller central cell and fewer endosperm nuclei were observed at 6 days after anthesis, and stunted embryo development was observed around 15 days after anthesis (Fig. 6). In dicotyledons, the cellularized endosperm acts as a nourishing tissue for embryo development, and failed endosperm development will ultimately cause embryo developmental arrest (LafonPlacette and Köhler, 2014). The liquid endosperm developmental status in ‘Guiwei’ correlated well with the occurrence of seed abortion (Fig. 1), and we conclude that retarded liquid endosperm growth is the main cause of seed abortion in ‘Guiwei’. Epigenetic regulation plays an important role in regulating seed development in A. thaliana (Sun et al., 2010), and a large group of polycomb (PcG) proteins including FERTILIZATION INDEPENDENT SEED 2 (FIS2), FERTILIZATION-INDEPENDENT ENDOSPERM (FIE/ FIS3), MEDEA (MEA/FIS1), MULTICOPY SUPRESSOR OF IRA (MSI1), and SWINGER (SWN), are known to be involved in endosperm development (Pien and Grossniklaus, 2007). However, in contrast to the progress in understanding the epigenetic regulation of endosperm development in A. thaliana, key events that control self-sterility in litchi remain to be characterized. The ‘Guiwei’ cultivar also showed a thermo-sterile phenotype. It is not known whether thermo-sterility in ‘Guiwei’ involves the same seed abortion mechanism as self-sterility, but the influence of temperature on seed development in ‘Guiwei’ seems to be more important than the pollen source. The seed abortion ratio of early blooming panicles in the Zhanjian orchard was less than 12%, and the seed abortion ratio of outcrossed ‘Guiwei’ grown at 22/26 °C (80%) was significantly higher than at 18/22 °C (40%) (Fig. 3–4). Plants have evolved sophisticated epigenetic regulatory systems to adjust growth and development in accordance with ambient temperature (Liu and He, 2014). A substitution of C-to-G in noncoding RNA p/tms12-1 was reported to produce a mutant small RNA, osa-smR5864 m, which was shown to be associated with the male sterility transition of PA64S rice (Zhou et al., 2012). Hu et al. (2015) also suggested that DNA methylation or RNA-dependent DNA methylation was involved in the regulation of photoperiod- and thermo-sensitive male sterility PA64S rice. Taken together, the non-Mendelian control of seed development in artificial pollination experiments and the seed fertility transition under different temperature regimes suggests that ‘Guiwei’ seed abortion may be associated with epigenetic regulation.
Author details XDR performed the most of the experiments and MXA and RMZ carried out seed development analysis under the supervision of WHC. YMC, HXM and LJG performed data analysis and interpreted the results. WHC designed the experiment, discussed the data and drafted the manuscript. All authors have read and approved the final manuscript. Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Acknowledgements This study was supported by the China Litchi and Longan Industry Technology Research System (project No. CARS-33-11), the Innovation Team Project of the Department of Education of Guangdong Province (2016KCXTD 011) and the Guangzhou science and technology project (201804020063). The authors would like to thank Dr. Fuchu Hu (Key laboratory of tropical fruit tree biology of Hainan Province), Yongzan Wei (South Subtropical Crops Research Institute) and Junsheng Zhao (Fruit Bureau of Maoming, Guangdong Privince) for their help in sampling. We thank PlantScribe (www.plantscribe.com) for editing this manuscript. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.scienta.2018.11.083. References Adamski, N.M., Anastasiou, E., Eriksson, S., O’Neill, C.M., Lenhard, M., 2009. Local maternal control of seed size by KLUH/CYP78A5-dependent growth signalling. Proc. Natl. Acad. Sci. U. S. A. 106, 20115–20120. Barnabás, B., Jäger, K., Fehér, A., 2008. The effect of drought and heat stress on reproductive processes in cereals. Plant. Cell. Environ. 31, 11–38. Berger, F., Chaudhury, A., 2009. Parental memories shape seeds. Trends Plant. Sci. 14, 550–556. Chu, Y.C., Lin, T.S., Chang, J.C., 2015. Pollen effects on fruit set, seed weight, and shriveling of’ 73-S-20’ litchi- with special reference to artificial induction of parthenocarpy. HortSci 50, 369–373. Fang, W., Wang, Z., Cui, R., Li, J., Li, Y., 2012. Maternal control of seed size by EOD3/ CYP78A6 in Arabidopsis thaliana. Plant. J. 70, 929–939. Feng, W., Zhang, L., Li, S.G., Chen, Y.Y., Luo, S.R., 2010. Comparison on embryonic development process of three cultivars of Litchi chinensis. Subtrop. Plant. Res. Comm. 31, 736–739 Chinese with English abstract. Hu, J., Chen, X., Zhang, H., Ding, Z., 2015. Genome-wide analysis of DNA methylation in photoperiod- and thermo-sensitive male sterile rice Peiai 64S. BMC Genomics 16, 102. Huang, H., 2005. Fruit set, development and maturation. In: Menzel, C.M., Waite, G.K. (Eds.), Litchi and longan: Botany, Production, and Use. CABI Publishing Press, London, UK p115-140. Huang, H.B., Qiu, Y.X., 1987. Growth correlations and assimilate partitioning in the arillate fruit of Litchi chinensis Sonn. Aust. J. Plant. Physiol. 14, 181–188. Lafon-Placette, C., Köhler, C., 2014. Embryo and endosperm, partners in seed
5. Conclusions Seed development is of great importance in litchi fruit yield and quality and diverse cultivars have been generated with different seed 163
Scientia Horticulturae 247 (2019) 156–164
D.-R. Xie et al.
Nayyar, H., 2018. Influence of drought and heat stress, applied independently or in combination during seed development, on qualitative and quantitative aspects of seeds of lentil (Lens culinaris Medikus) genotypes, differing in drought sensitivity. Plant. Cell. Environ. 1–14. Sita, K., Sehgal, A., Kumar, J., Kumar, S., Singh, S., Siddique, K.H.M., Nayyar, H., 2017. Identification of high-temperature tolerant lentil (Lens culinaris Medik.) genotypes through leaf and pollen traits. Front. Plant. Sci. 8, 744. Stern, R.A., Gazit, S., El-Batsri, R., Degani, C., 1993. Pollen parent effect on outcrossing rate, yield, and fruit characteristics of ‘Floridian’ and ‘Mauritius’ lychee. J. Amer. Soc. Hort. Sci. 118, 109–114. Stern, R.A., Eisenstein, D., Voet, H., Gazit, S., 1996. Anatomical structure of two-day-old litchi ovules in relation to fruit set and yield. J. Hortic. Sci. 71, 661–671. Sun, X., Shantharaj, D., Kang, X., Ni, M., 2010. Transcriptional and hormonal signaling control of Arabidopsis seed development. Curr. Opin. Plant. Biol. 13, 611–620. Varoquaux, F., Blanvillain, R., Delseny, M., Gallois, P., 2000. Less is better: New approaches for seedless fruit production. Trends Biotechnol. 18, 233–242. Wang, H.C., Lai, B., Huang, X.M., 2017. Litchi fruit set, development, and maturation. In: Kumar, M., Kumar, V., Prasad, A.V. (Eds.), In: The Lychee Biotechnology. Springer, Singapore P1-30. Wu, J., Wang, S., Gu, Y., Zhang, S., Publicover, S.J., Franklintong, V.E., 2011. Self-incompatibility in papaver rhoeas activates nonspecific cation conductance permeable to Ca2+ and K+. Plant. Physiol. 155, 963–973. Xiang, X., Ou, L.X., Qiu, Y.P., Yuan, P.Y., Chen, J.Z., 2001. Enbryo abortion and pollen parent effects in ‘Nuomici’ and ‘Guiwei’ litchi. Acta Hort. 558, 257–260. Yu, J., Han, J., Kim, Y.J., Song, M., Yang, Z., He, Y., Fu, R., Luo, Z., Hu, J., Liang, W., Zhang, D., 2017. Two rice receptor-like kinases maintian male fertility under changing tenperatures. Proc. Natl. Acad. Sci. U. S. A. 114, 12327–12332. Zhang, J.Q., Wu, Z.C., Hu, F.C., Liu, L., Huang, X.M., Zhao, J.T., Wang, H.C., 2018. Abberant seed development in Litchi chinensis is associated with the impaired expression of cell wall invertase genes. Hort. Res. 5, 39. Zhou, H., Liu, Q., Li, J., Jiang, D., Zhou, L., Wu, P., Lu, S., Li, F., Zhu, L., Liu, Z., Chen, L., Liu, Y.G., Zhuang, C., 2012. Photoperiod- and thermo-sensitive genic male sterility in rice are caused by a point mutation in a novel noncoding RNA that produces a small RNA. Cell. Res. 22, 649–660.
development. Curr. Opin. Plant. Biol. 17, 64–69. Li, N., Li, Y., 2016. Signaling pathways of seed size control in plants. Curr. Opin. Plant. Biol. 33, 23–32. Liu, J.Z., He, Z.H., 2014. Epigenetic regulation of heat stress response in plants. Chin. Sci. Bull. 59, 631–639 Chinese with English abstract. Lora, J., Hormaza, J.I., Herrero, M., Gasser, C.S., 2011. Seedless fruits and the disruption of a conserved genetic pathway in angiosperm ovule development. Proc. Natl. Acad. Sci. U. S. A. 108, 5461–5465. Lü, L.X., Chen, J.L., Chen, X.J., 1985. An observation on the process of embryo development in litchi. Subtrop. Plant. Res. Comm. 1, 1–5 Chinese with English abstract. Martin, F.W., 1959. Staining and observing pollen tubes in the style by means of fluorescence. Stain Technol. 34 (3), 125–128. Moles, A.T., Ackerly, D.D., Webb, C.O., Tweddle, J.C., Dickie, J.B., Pitman, A.J., Westoby, M., 2005. Factors that shape seed mass evolution. Proc. Natl. Acad. Sci. U. S. A. 102, 10540–10544. Nasehzadeh, M., Ellis, R.H., 2017. Wheat seed weight and quality differ temporally in sensitivity to warm or cool conditions during seed development and maturation. Ann. Bot. 120, 479–493. Peng, S., Huang, J., Sheehy, J.E., Laza, R.C., Visperas, R.M., Zhong, X., Centeno, G.S., Khush, G.S., Cassman, K.G., 2004. Rice yields decline with higher night temperature from global warming. Proc. Natl. Acad. Sci. U. S. A. 101, 9971–9975. Pien, S., Grossniklaus, U., 2007. Polycomb group and trithorax group proteins in Arabidopsis. Biochim. Biophys. Acta 1769, 375–382. Pugh, D.A., Offler, C.E., Talbot, M.J., Ruan, Y., 2010. Evidence for the role of transfer cells in the evolutionary increase in seed and fiber biomass yield in cotton. Mol. Phys. 3, 1075–1086. Sabir, A., 2015. Xenia and metaxenia in grapes: differences in berry and seed characteristics of maternal grape cv. ‘Narince’ (Vitis vinifera L.) As influenced by different pollen sources. Plant Biol. 17, 567–573. Sakata, T., Oda, S., Tsunaga, Y., Shomura, H., Kawagishi-Kobayashi, M., Aya, K., Saeki, K., Endo, T., Nagano, K., Kojima, M., Sakakibara, H., Watanabe, M., Matsuoka, M., 2014. Reduction of gibberellin by low temperature disrupts pollen development in rice. Plant. Physiol. 164, 2011–2019. Sehgal, A., Sita, K., Bhandari, K., Kumar, S., Kumar, J., Prasad, P.V.V., Siddique, K.H.M.,
164