Phenotypic Characterization of a Female Sterile ... - Wiley Online Library

Journal of Integrative Plant Biology 2006, 48 (3): 307−314

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Phenotypic Characterization of a Female Sterile Mutant in Rice Shuang-Cheng Li1, 2*, Li Yang1*, Qi-Ming Deng1, 2, Shi-Quan Wang1, 2, Fa-Qiang Wu1 and Ping Li1, 2** (1. Rice Research Institute, Sichuan Agricultural University, Wenjiang, Sichuan 611130, China; 2. Key Laboratory of Crop Genetic Resources and Improvement, Ministry of Education, Sichuan Agricultural University, Yaan, Sichuan 625014, China)

Abstract A female sterile mutant, derived from a spontaneous mutation, was first discovered in rice (Oryza sativa L. ssp. indica) restorer line 202R. With normal flowering, the mutant exhibits an extremely low seed-setting rate. When the mutant is crossed as a pollen donor, the seeds set normally; whereas when it is used as a pollen receiver, no seeds are obtained even with mixed pollen grains of different varieties sprinkled over the stigmas. The floret of the mutant, consisting of six stamens and one pistil, looks the same as that of the wild type in the malefemale organs, except that less than 10% of the mutant florets have three stigmas on the ovary. Although the mutant has a low seed-setting rate, its pollen fertility is approximately 87.1%, which is equal to that of the wild type. In addition, more than 90% of the mature embryo sacs of the mutant have complete inner structures. At every stage after pollination, the sperm, embryo, and endosperm are not found in the mutant embryo sac, whereas the disintegration of the egg cell that does not accomplish fertilization is visible. Through observations with a fluorescence microscope, we have found that the pollen grains germinate normally, whereas the pollen tube abnormally elongates in the style-transmitting tissue. The mutant pollen tubes display various defects in the style, such as slower elongation, conversed elongation, distorted elongation, swollen tips, or branched tips. As a result, the growth of the pollen tubes ceases in the style, and, therefore, the pollen tubes cannot reach the embryo sac and the process of double fertilization is blocked. Based on these observations, we conclude that this mutant, designated as fs-202R, is a novel type of female sterile mutation in rice, which causes the arrest of the elongation of the pollen tube. Key words: elongation; embryo sac; female sterility; mutant; pollen tube; rice. Li SC, Yang L, Deng QM, Wang SQ, Wu FQ, Li P (2006). Phenotypic characterization of a female sterile mutant in rice. J Integrat Plant Biol 48(3), 307−314.

Female sterility is found widely in plants such as Pinus tabulaeformis Carr., soybean, Zea maize L., and rice (Yokoo et al. 1984; Ling et al. 1991; Ilarslan et al. 1997, 1999, 2003; Pamer et al. 2000; Cheng et al. 2002) and this phenomenon is mainly caused by the abnormal development of female organs, including the development of the ovule, the formation of the embryo sac, and the

Received 17 Jun. 2005

Accepted 27 Dec. 2005

Supported by the Hi-Tech Research and Development (863) Program of China (2003AA212030) and the Program for Changjiang Scholars and Innovative Research Team in University (IRT0453). * These authors contributed equally to the work. ** Author for correspondence. Tel: +86 (0)28 8272 2497; Fax: +86 (0)28 8272 6875; E-mail: .

growth of the embryo. According to the angiosperm’s embryo genesiology, female sterility can be classified into three types. The first type is one in which there are no female organs or only incomplete female organs. In this type of sterility, the flower has no differentiation of the female organs or its carpels change into male organs. Ling et al. (1991) considered these plants as monandrous plants. The second type of female sterility is one in which the female organs lack a normal embryo sac owing to blocked development of the ovule or the megasporocyte. Abnormal meiosis of the megasporocyte or abnormal mitosis of the functional megaspore is the key factor responsible for this type of sterility (Kazimierska et al. 1995; Ouyang et al. 1996; Rim et al. 1998). The third type of female sterility is one in which there is a normal mature embryo sac but abnormal development of the embryo after pollination (Bouharmont et al. 1985; Kazimierska et al.

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1995). Over the past two decades, considerable advances have been achieved in our understanding of female sterility in plants. Many loci for a male-sterile, female-sterile mutant, a female partially sterile mutant, and an ovule lethal mutant of soybean have been mapped on the molecular linkage group (Palmer et al. 2000, Kato et al. 2003a, 2003b, 2004). Some embryo sac fertility loci relevant to hybrid breakdown and spikelet infertility of intersubspecific hybrids rice have also been mapped (Kubo et al. 2005; Song et al. 2005). Furthermore, several genes important for ovule development have been identified in Arabidopsis. Lang et al. (1994) indicated that sin1 affected female fertility and interacted with its recessive modifier mod1 in Arabidopsis. Further investigation found that the female-specific infertility of sin− plants was due to abnormal ovule integument development and aberrant differentiation of the megagametophyte in a subset of ovules and that sin1 was a gene that was required for ovule development and the initiation of flowering in Arabidopsis (Ray et al. 1996). The BELL1 gene, encoding a homeodomain protein (Reiser et al. 1995), controls the expression of the floral organ identity gene AG within the ovule and thereby controls morphogenesis of ovule integuments (Modrusan et al. 1994; Ray et al. 1994). The SUPERMAN gene, another cadastral gene that restricts the spatial expression pattern of the floral organ identity gene AP3 (Sakai et al. 1995), is important in ovule integument development (Gaiser et al. 1995). The organ identity gene AP2 is also known to control ovule morphogenesis (Modrusan et al. 1994). De Martinis et al. (1999) proved that silencing gene expression of the ethylene-forming enzyme resulted in the arrest of ovule development in transgenic tobacco plants. Observations revealed that, in the transgenic plants, megasporogenesis did not occur and ovules did not reach maturity. After pollination, pollen tubes could reach the ovary, but could not penetrate the micropyle and missed the embryo sac, possibly because of the absence of the appropriate guiding system. Greef et al. (1997) developed an effective system to control female sterility in plants comprised of a foreign DNA incorporated into the nuclear genome of the cell. The foreign DNA compromises a female organ-specific expressed gene encoding a protein or polypeptide that can kill or significantly disturb the metabolism, functioning or development of cells. Wu and Liu (1995) also developed a premature female sterile line in wheat and succeeded in maintaining and restoring the female sterile characteristic for breeding procedures.

Most reports on female sterility in rice are focused on mutants derived from the progenies of cross between indica and japonica rice. In addition, almost all these mutants belong to the structural infertility group, in which infertility is caused by abnormal development of the embryo sacs (Oka et al. 1957; Oka and Doida 1962; Ouyang et al. 1996). However, in the present study, we report a female sterile material originating from a spontaneous mutation and characterize it as a novel type of female sterile mutant in rice through anatomical, cytological, and fluorescence microscopic observations.

Results Mutant phenotype and cross-test The morphology of the mutant was the same as that of wide-type rice 202R, except that the mutant exhibited a lower seed-setting rate. The seed-setting rate of 202R by selfing is above 90% (Figure 1A), whereas that of the mutant did not exceed 5% (averaging 2.9% and ranging from 0.4% to 4.7%; Figure 1B). When the mutant was crossed as male parents, the seed-setting rate was normal (averaging 36.7% and ranging from 19.2% to 67.8%), which was close to that of the control cross in which 202R was used as a pollen donor (Figure 1D,E; Table 1). However, when the mutant was crossed as female parents, we could not obtain a single seed. Even after mixed pollen grains of different varieties were sprinkled over the stigmas, the seed-setting rate of this cross was zero (Table 1; Figure 1C). In contrast, seeds set normally when the wild-type rice 202R was used as a pollen receiver (Table 1). So, we deduced that the mutant, designated as fs202R, was a female-specific sterile mutant and that the mutation did not affect male gametophyte function. Anatomical observations and pollen fertility No obvious differences were found between the wild-type florets and most of the mutant florets. An individual mutant floret had six stamens and one pistil, with each pistil consisting of one ovary and two folded feather-like stigmas, as seen in 202R. The shape and position of the pistils were both normal. However, approximately 10% of the mutant florets had one more stigma compared with the wild type, which was obviously smaller than the other

Table 1. Seed-setting rate of the cross-test Materials

Seed-setting rate (%) 202R

527R

9311

D62B

G46A

fs-202R ( )

25.7 ± 2.6

19.2 ± 3.0

41.3 ± 3.9

24.6 ± 2.5

67.8 ± 4.2

202R( )

28.3 ± 4.2

20.8 ± 1.9

39.8 ± 5.2

22.1 ± 4.2

71.2 ± 4.6

0

0

0

0

0

26.7 ± 4.0

19.5 ± 2.7

22.8 ± 5.0

20.3 ± 2.7

22.3 ± 3.9

fs-202R ( ) 202R( )

, crossed as male parents;

, crossed as female parents.

Mixed pollen grains

Female Sterility of Blocked Pollen Tube Growth 309

Figure 1. Seed setting by selfing and hybridization. (A) Selfing of 202R, seeding normally. (B) Selfing of fs-202R, sterility. (C) Fs-202R crossed as female parents ( ) × (9311+527R+202R+D62B) crossed as male parents ( ), no seeds obtained. (D) D62B × fs-202R , seeding normally. (E) G46A × fs-202R , high seed-setting rate.

normal stigmas (Figure 2A,B). It seemed that there was no obvious connection between sterility and the redundant stigma, because more than 90% of the florets looked the same as those of the wild type, although they were sterile eventually. Thus, it may be reasonable to consider that the redundant stigma was only a physiological aspect as a result of the mutation. Investigations showed that pollen grains of the mutant were normal in morphology and staining gradation compared with those of the wild type (Figure 2C,D), and both shared a fertile ratio of approximately 87.1%. Therefore, we concluded that it was the functional loss of the female apparatus, rather than that of its male counterparts, that actually led to the sterility of the mutant. Cytological observations More than 50 mature embryo sacs of the mutant were examined under an optical microscope. We found that almost all the mature embryo sacs were of the typical polygonum type and more than 90% of the embryo sacs had complete inner components. An embryo sac of the mutant usually consisted of one egg cell, two synergid cells, one central cell with two polar nuclei, and a group of antipodal cells, just as those seen in the typical embryo sac of rice. In addition, all the apparatus in the mature embryo sac showed

apparent polarity and were in the appropriate positions (Figure 3A−C). Meanwhile, we also found that when the embryo sac in the mutant reached maturation, the pollen grains also had a high staining rate, which suggested that the developmental process of the male and female organs was synchronous. Further observations of the development of the embryo in the mutant revealed that the egg cell could not complete fertilization with the sperm cell and that the embryo and the endosperm were undetectable, although disintegration of the egg cell that did not accomplish the fertilization was noted (Figure 3D,E). In contrast, fertilization, as well as the development of the embryo and the endosperm, was observed in the wild-type material 202R (Figure 3F−H). All these results prove that the mature embryo sac of the mutant is normal, but it is abortive finally, which strongly suggests that the sterility of the mutant may occur during the course of pollination. Fluorescence microscopic observations Five minutes after flowering, many pollen grains were attached to the stigmas and began to germinate; 10 min after flowering, the pollen tube had entered the stigmas. At these two stages, no significant divergences were detected between the mutant and

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Figure 2. Anatomical observations and pollen fertility. (A) A floret of fs-202R, with the arrow indicating the redundant stigma. (B) A normal floret of 202R. (C, D) Pollen fertility of fs-202R (C) and 202R (D).

202R (Figure 4A,B). Twenty minutes after flowering, the speed of the pollen tube’s elongation between the mutant and 202R diverged, with the speed of the former being apparently slower than that of the latter (Figure 4C,D,G). The pollen tubes in 202R reached the ovary, the basal ovule and the micropyle 30, 45, and 60 min after flowering, respectively (Figure 4H,K,L), whereas those in the mutant were abnormally blocked in the style-transmitting tissue during the same period with the following main problems observed: (i) a swollen tip of the pollen tube, like an air bladder (Figure 4E,O); (ii) a bent tip of the pollen tube resulting in conversed elongation (Figure 4F,I); (iii) an agglomerated tip of the pollen tube and distorted elongation (Figure 4M,N); and (iv) a branched tip of the pollen tube (Figure 4F,J). As a result, no pollen tubes were found entering the embryo sac in the mutant, even 48 h after pollination (Figure 4P,Q). However, pollen tubes were still visible in the ovary of 202R even 6 h after flowering (Figure 4R). Thus, we concluded that the mutant’s female sterility characteristic was a consequence of the disruption of the double fertilization caused by the arrest of the elongation of the pollen tube.

Discussion The morphosis of a typical female sterile plant is usually characterized by one of the following forms: no pistils, a shriveled ovary without stigmas, or normal stigmas and an ovary with aberrant inner apparatus. Apparent alterations to the female apparatus have been found in all known female sterile mutants of rice (Bouhamont et al. 1985; Kazimierska et al. 1995). As a result, it was generally believed that female sterile plants inevitably had distorted female apparatus. However, in the present study, we identified female sterile rice material originating from a spontaneous mutation that did not belong to any of the three typical phenotypes of female sterility in plants. Through anatomical and cytological observations, we found that the female organs of the

mutant investigated in the present study were, on the whole, the same as those of 202R in appearance and that the number and position of the inner structures of the mutant embryo sac were normal. Thus, it is necessary to define this novel type of plant female sterility as “no structural deficiency” sterility. Further examination revealed that the cause for the mutant’s sterility lay in the arrest of pollen tube elongation in the styletransmitting tissue owing to many types of abnormal changes. Although several pollen tube directional growth-defective mutants have been reported for Arabidopsis in previous studies (see below), the mutant fs-202R reported on herein is different from all of them. The pollen tubes in the mutants pop2 and pop3 did not elongate towards the micropyle, but elongated in random orientations within the ovary (Wilhelmi and Preuss 1996). Studies on another two mutants, namely the mutant generated from a reciprocal chromosomal translocation and the maa mutant in Arabidopsis, proved that the abnormal elongation of the pollen tube was due to the absence of a normal female gametophyte (Ray et al. 1997; Shimizu and Okada 2000). In the feronia mutant, the pollen tube continued to grow even it entered the receptive synergid of the female gametophyte, and failed to rupture and release sperm cells (Norbert et al. 2003; Rotman et al. 2003). Jiang et al. (2005) reported that, in the vgd1 mutant, the pollen was able to germinate, but the growth of pollen tubes through the transmitting tract of the style was retarded. Although the vgd1 pollen tubes grown in vitro were misshapen, they were morphologically normal when grown on stigmatic tissue. Pollen tubes in most of these mutants could reach the ovule and the abnormalities in pollen tube growth were mainly shown as a deficiency in orientation guidance rather than a cessation of elongation in the transmitting tissue. If the process of pollination can be divided into four stages, namely germination on the stigma, elongation in the transmitting tissue, entrance into the micropyle, and fertilization with the eggs, we believe that the mutant reported in the present study is a novel type of female sterility that occurs during the second


Figure 3. Observations of the embryo sac and embryo development. (A− − C) Mature embryo sac of fs-202R, in which the inner apparatus were normal. (D) Mature embryo sac of fs-202R, 48 h after flowering, with no pro-embryo and primary endosperm; only the egg cell was seen. (E) Mature embryo sac of fs-202R, 72 h after flowering, with no embryo and endosperm, but disintegration of the egg cell evident. (F− − H) Embryo sac of 202R, showing fertilization (F), the embryo development, and the endosperm development (G, H). a, antipodal cell; e, egg cell; em, embryo; endo, endosperm; pn, polar nucleus; s, synergid; sp, sperm.

stage of the navigation of the pollen tube to the embryo sac. Further investigations into the genetic and molecular bases of the sterility in this mutant will benefit a deeper understanding of the pollination of flowering plants. In flowering plants, the process of pollination involves a series of complex cellular interactions between the male and female apparatus. Many factors, such as extracellular matrix (Baldwin et al, 1992; Lee et al. 1994; Wu et al. 1995; Cheung et al. 1996; Malhó et al. 1998; Fowler et al. 1999; Lord et al. 2000; Bosch et al. 2001; Li et al. 2001; Wheeler et al. 2001; and Joly et al. 2002), hormones (De Martinis et al. 1999; Zhang et al. 2003), and Ca2+ (Yu et al. 1999a,1999b) have been reported to be involved in the growth and guidance of the pollen tube. In these reports, a lack of or quantitative change in these factors had a considerable effect on

the elongation of the pollen tube. So, we may deduce that the cause of female sterility in fs-202R is probably due to an interaction disruption between the male and female organs rather than a structural deficiency of the female organs. Further studies are currently underway in order to gain a closer insight into the female sterile characteristic of this mutant.

Materials and methods Plant materials and cross-test The fs-202R mutant was discovered in the indica rice restorer line 202R. Other rice lines (202R, G46A, 527R, 9311, and D62B)

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Figure 4. Fluorescence microscopic observation of pollen germination and pollen tube growth. (A, B) Ten minutes after flowering, the pollen tubes entered the stigmas in fs-202R (A) and 202R (B). (C, D) Twenty minutes after flowering, differences in the speed of elongation of the pollen tube between the mutant (C) and 202R (D) started to become evident. (E, F, I, J, M, N, O) Various abnormalities in the elongation of the pollen tube in fs-202R from 30 min to 6 h after flowering. (E, O) A swollen tip of the pollen tube, like an air bladder. (F, I) A bent tip of the pollen tube resulting in conversed elongation. (F, J) A branched tip of the pollen tube. (M, N) An agglomerated tip of the pollen tube and distorted elongation. (G, H, K, L) The pollen tube in 202R elongated normally in the style (G), reaching the ovary (H), the basal ovule (K), and the micropyle (L) 20, 30, 45, and 60 min after flowering, respectively. (P, Q) No pollen tubes were visible in the ovary of fs-202R during period from 60 min (P) to 48 h (Q) after flowering. (R) Pollen tubes were visible in the ovary of 202R even 6 h after flowering.


were crossed with the mutant as male parents or female parents. To eliminate technical errors, 202R was used as a control parent. In each cross, controlled pollinations were performed on two replications consisting of approximately 200 florets. All materials were supplied by the Rice Research Institute of Sichuan Agricultural University. Anatomical observations Floral structures of the mutant plant and 202R were observed under a microscope before flowering. More than 50 florets were investigated and some images were recorded photographically (China Lucky Film, Xiamen, China).

Research Institute, Sichuan Agricultural University (SAU), for constructive advice and help with anatomical and cytological observations. The authors also thank Dr. Hai Long of Triticeae Research Institute of SAU, and Professor Ying-Ze Niu of Crop’s Breeding Department of SAU, for their helpful comments on the manuscript.

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Cytological observations The ovaries of the mutant and 202R were collected and fixed with a solution of 5% formaldehyde, 5% acetic acid, and 45% ethanol (FAA) at various stages before or after pollination. Twenty-four hours later, ovaries were washed in low concentration ethanol (10%) and stained in 10% hematoxylin solution for 24−48 h. Samples were then dehydrated in a graded series of ethanol solution from 30% to 100%, incubated in dimethylbenzene twice, and dipped in the paraffin for 1 week. Sections were cut into pieces for observation of the embryo sac and the embryo development under an optical microscope. Images were recorded photographically (China Lucky Film, Xiamen, China).

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