Genetic Analysis of Streaked and Abnormal Floret Mutant st-fon

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1 Rice Science, 2013, 20(4): Copyright 2013, China National Rice Research Institute Published by Elsevier BV. All rights reserved DOI: /S (13) Genetic Analysis of Streaked and Abnormal Floret Mutant st-fon CHEN De-xi 1, 2, LI Ting 2, QU Guang-lin 2, HUANG Wen-juan 2, HE Zhong-quan 1, LI Shi-gui 2 ( 1 Institute of Plant Protection, Sichuan Academy of Agricultural Sciences, Chengdu , China; 2 Rice Research Institute, Sichuan Agricultural University, Chengdu , China) Abstract: A double mutant with streaked leaf and abnormal floret was found and temporarily named streaked leaf and floral organ number mutant (st-fon). For this mutant, besides white streak appeared on culm, leaves and panicles, the number of floral organs increased and florets cracked. The extreme phenotype was that several small florets grew from one floret or branch rachis in small florets extended and developed into panicles. By using transmission electron microscope to observe the ultrastructure of white histocytes of leaves at the seedling stage, the white tissues which showed abnormal plastids, lamellas and thylakoids could not develop into normal chloroplast, and the development of chloroplast was blocked at the early growth stage of plastid. Scanning electron microscope and paraffin section were also used to observe the development of floral organs, and the results indicated that the development of floral meristem was out of order and unlimited, whereas in the twisty leaves, vascular bundle sheath cells grew excessively, or some bubbly cells increased. Genetic analyses carried out by means of cross and backcross with four normal-leaf-color materials revealed that the mutant is of cytoplasm inheritance. Key words: rice; streaked leaf; floral organ; floret; mutant; chloroplast; anatomical structure; plasmatic inheritance Chlorophyll deficient mutants are ideal materials for basic research on photosynthesis, chlorophyll biosynthesis, structure, function, heredity, differentiation and growth of chloroplast, as well as organelle transplantation, cell fusion and gene expression and regulation in higher plants. Flower development is the most apparent feature of flowering plants at the reproductive growth stage, and floral organ formation is one of the important genetic characteristics of flowering plants. The process of the morphological development of flowers provides a suitable system for the study on the molecular mechanism of gene regulation. Based on the molecular biology research on floral organ mutants of the model plant Arabidopsis thaliana and snapdragon, the genetic control mechanism of flower organ development of dicotyledonous plants was basically elucidated. Rice floral development directly affects rice quality and yield, but it differs significantly from dicotyledonous plants in the development features of flowers and floral meristem (Whipple et al, 2007). The studies on the development of flower organs in monocotyledonous Received: 17 September 2012; Accepted: 22 December 2012 Corresponding author: LI Shi-gui (lishigui_sc@263.net) plants are far from perfect, lagging behind that of the dicotyledonous plants. At present, people have established a large number of rice mutants, and found a lot of leaf mutants and flower mutants. Mutants of streaked leaf in rice mainly include wp1 and wp2 (Sanchez and Khush, 1994), wp(t) (Li et al, 2003), virescent-1 (v1) (Kusumi et al, 1997), virescent-2 (v2) (Sugimoto et al, 2004, 2007), Oswm (Li et al, 2007) and st(t) (Sang et al, 2010). wp(t) is a white-panicle mutant. The midribs of a small number of leaves are white, and the gene has been mapped on chromosome 1. v1 and v2 are thermo-sensitive leaf color mutants. V1 gene controls the transformation from proplastid to functional chloroplast, and V2 gene suppresses chloroplast differentiation by disturbing the chloroplast translating mechanism at the early stage of chloroplast development. Both Oswm and Oswm2 show white midribs. Oswm has no effect on other characters, while leaves close to white midribs in Oswm2 become yellowish, and the agronomic traits of Oswm2 get worse. They are located on chromosomes 4 and 7, respectively (Hu et al, 2008). st(t) is a white stripe-leaf mutant identified from the descendants of an indica restorer line Jinhui 10 which is bred by mutagenesis

2 268 and has good agronomic traits. The characters of st(t) are controlled by a pair of recessive nuclear genes, and were mapped on chromosome 6 (Sang et al, 2010). Reports are rare about the mutants that their flowers and leaves mutate in company, including those without midrib in leaves and mutants whose pistils develop abnormally, e.g. dl-1, dl-2, dl-sup1, dl-sup2 (Nagasawa et al, 2003), DLR (Wang et al, 2004), dl-sup6 (Li et al, 2011a) and dl(t) (Luo et al, 2001). DL is a member of plant transcription factor, YABBY, which regulates the specification of carpel identity and promotes cell proliferation of the central region to control the formation of leaf midribs (Nagasawa et al, 1996). For the mutant with flowers and leaves mutated in company found in the study, during the whole growth period, streaks appeared on culm, leaves and panicles, and the total content of chlorophyll and the net photosynthetic rate were much lower than those of the wild type, the number of floral organs increased and the primordium of florets divided unequally (Chen et al, 2006). When the mutants were cultivated in Hainan, China, there were some more extreme phenotypes. The phenotypes of the floral organs are similar to those of mutants fon1 (Suzaki et al, 2004), fon2 (Suzaki et al, 2006), fon3 (Jiang et al, 2005), fon4 (Chu et al, 2006; Chu and Zhang, 2007) and fon5 (Zhang et al, 2008). However, there is no homologous transition from lodicules to organs that are similar to the palea/lemma structures. Therefore, it is necessary to further identify details and do genetic analysis in order to clarify the mutation mechanism. Rice materials MATERIALS AND METHODS The rice (Oryza sativa L.) mutant, st-fon, was from the mutant strains of immature embryos of rice variety Shuhui 527 produced by gene gun transforming, which did not contain foreign genes and the mutant characters were stable. Experiment method Phenotype and anatomical structure observation Morphology characters of the mutants were carefully observed during the whole growth period, and the numbers of stamens, pistils, paleae, lemmas and lodicules of a great number of florets randomly selected were calculated. At the 2-leaf stage, the second leaf was taken and treated according to He et al (2001). After double fixation by glutaraldehyde and osmic acid, the Rice Science, Vol. 20, No. 4, 2013 materials were dehydrated gradually, and sliced after replacement and embedding, then dyed with uranyl acetate and lead citrate liquid, and observed with an H600 transmission electron microscope (Hitachi, Japan). Young spikelets were selected at different growth stages, fixed with glutaraldehyde, and dehydrated with ethanol alcohol in gradient, and then transferred into an HCP-2 type critical point drying machine (Hitachi, Japan). After being dried, the materials were coated under vacuum and observed by HITACHS-450 SEM (Hitachi, Japan). Paraffin section Florets, leaves and culm of the mutants were taken and fixed with FAA liquid (50% ethyl alcohol 89 ml, glacial acetic acid 6 ml and formalin 5 ml) and then stored. The paraffin section process was according to Zhang et al (2007). Agronomic trait survey Ten mutants and the wild types in the field were randomly selected. Their agronomic traits, such as plant height, effective panicle number per plant, grain number per panicle, seed-setting rate and 1000-grain weight, were recorded and the average values were used. Genetic analysis The mutants and wild-types, 9311, Zhenshan B and new dwarf source were performed with reciprocal cross, respectively. The mutants/wild types, wild types/mutants backcrossed with the wild-types and mutants, respectively. Some mutants/wild types and wild types/mutants self-crossed. Finally, the seeds were harvested and the growth of seedlings was computed. RESULTS Morphological properties of mutants Across the growth period of the mutants, some leaves were all albinos, as shown in Fig. 1. At the reproductive growth stage, the florets grew abnormally. About 700 florets from 50 plants were randomly analyzed, and the results indicated that the number of floral organs of the mutants increased in various degrees (Table 1), including pistils (1 2), stamens (6 12), lodicules (2 6), more paleae and lemmas, and newly increased palea/lemma-like lodicules (2 5) (Fig. 2-A to -C). Only a small number of florets were lack of paleae, stamens or ovaries. Inflorescences or pedicels appeared on paleae/lemmas, lodicules or ovaries of the florets, where new florets grew. Some florets cracked, inside

3 CHEN De-xi, et al. Characterization and Genetic Analysis of Streaked and Abnormal Floret Mutant st-fon 269 where new inflorescences grew, and the paleae/lemmas of some florets contained unknown conglobation. About 40% of those florets cracked and could not close. Histological analysis of mutant leaves and florets To investigate the variation at tissue level, leaf blades, leaf sheaths and florets of mutants were fixed, dehydrated and dyed. After that, paraffin section was conducted. The results indicated that the color of the streaked section in the mutant leaves was very light, whereas normal leaves were deep when they were dyed with hematoxylin (Fig. 3-A). There were many Fig. 1. Phenotypes of st-fon mutant at different growth stages. A, Phenotypes of the wild type at the tillering stage; B, C, D and E, Phenotypes of the st-fon mutant at the seedling, tillering, before and after heading stages, respectively; F and G, Seeds of the mutant. Fig. 2. Floral morphology of mutant. A, Floral morphology of the mutant and the wild type; B, Floral phenotype of the mutant in extreme cases; C, A separated floret from the mutant.

4 270 Rice Science, Vol. 20, No. 4, 2013 Table 1. Average number of floret organs in wild type and mutant. Material No. of plants Lemma Palea Lodicule Palea/lemma-like lodicule Stamen Carpel Other State of floret Wild type Closed Mutant % conglobation 40% crack Wild type, n = 10; Mutant, n = 50. alveolar cells with big size at the distorted leaf sections. The forked leaves had no main veins, but had secondary veins (Fig. 3-B to -F). Leaves produced some neoplasms and leaf blades or leaf sheaths were distorted in the mutant, mainly because of the abnormal growth of bundle sheath cells (Fig. 3-A and -G). A normal rice floret had a pair of glumes, a palea/lemma, a pair of lodicules close to lemma, six stamens and one pistil (Fig. 4-A), whereas the floret of the mutant had many paleae/lemmas and ovaries or new inflorescences (Fig. 4-B to -H). Moreover, the floret contained differentiated stamens, pistils and ovaries, as well as undifferentiated inflorescences. Comparison of leaf cell structure between wild type and mutant under electronic microscope Observation on mutant leaves showed that the abnormal phenotype of chloroplast occurred at the tillering stage (Chen et al, 2006), so leaves of the mutant at the 2-leaf stage were observed. The cells in a totally albino leaf contained very little stuff, and the chloroplasts did not develop (Fig. 5-A), which might be a sign of death. Plastids existed in the white tissues of streaked leaves, including proplastids with double-membrance which contained many messy foam-like structures but without lamellar structures (Fig. 5-B), and plastids with an initial lamella of concentric structure but without starch granule (Fig. 5-C). In the transition region, fully developed chloroplast cells were clearly visible, but the arrangement of granum thylakoids in chloroplasts was out of order, and the cells were relatively large (Fig. 5-D and -E). Close to the large cells, there were some very small cells (Fig. 5-F) and these small cells usually only had a swollen chloroplast that occupied more than 2/3 of the entire volume of the cell. The adjacent cells contained undeveloped chloroplasts, and the stuff in the cells was very little (Fig. 5-D to -G). However, the electronic density of cells in green areas was normal. The chloroplast grana were stacked neatly, and contained starch granules (Fig. 5-H). The observation indicated that the development of chloroplasts was blocked at the early stage of plasmid development, and Fig. 3. Leaf histological analysis of mutant. A, Slightly dyed part in leaf of the mutant; B, The misshapen leaf vein of the mutant (arrow mark); C, Enlarged bulliform cells of contorted mutant leaf (arrow mark); D, Split leaf sector of mutant leaf; E, Siliciffied mutant leaf; F, The appendage of mutant leaf (arrow mark); G, The vascular bundle sheath cell growing excessively in leaf sheath of mutant leaf (arrow mark).

5 CHEN De-xi, et al. Characterization and Genetic Analysis of Streaked and Abnormal Floret Mutant st-fon 271 Fig. 4. Spikelet histological analysis in st-fon mutant. A, The cross section of a wild type spikelet contains one lemma, one palea in whorl 1, two lodicules in whorl 2 interior to the lemma, six stamens in whorl 3 and a carpel in whorl 4; B to E, The cross sections of spikelet in the st-fon mutant; B, The number of floral organs in st-fon increased centripetally; C, Indefinite inflorescence of a spikelet in the st-fon mutant; D and E, Spikelet consisting of multi-floret and inflorescence; F, The longitudinal section of mutant floret; G, Multiple paleae and lemmas of spikelet in the st-fon mutant; H, Additional inflorescences in the mutant (arrow mark). Fig. 5. Cell structures of mutant leaf at seedling stage (two leaves and one heart). A, Cell structures of white sectors of a mutant leaf, 4 000; B, Vesicle of plastid in cells from white sectors of a mutant leaf, 8 000; C, Concentric circular plastid in cells of white sectors of a mutant leaf, ; D, A lot of vacuole-like structures and osmiophilic droplets in cells of white sectors of a mutant leaf, ; E, The cells including big cells (arrow mark) of transition sector in a mutant leaf, 4 000; F, The cells including small cell of transition sector in a mutant leaf (arrow mark), ; G, Disordered arrangement among thylakoids of chloroplasts of transition sector in a mutant leaf, ; H, Structure of chloroplast of green sector in a mutant leaf, was associated with the assembly of the thylakoids. Early development stage of mutant florets The generating process of rice floret primordium comprises several stages: after the formation of protective glume primordium, lemma primordia are formed 180 degree opposite to the position of the second protective glume primordium, and palea primordia are formed 180 degree opposite to the lemma primordia; then, two lodicule primordia are formed on both sides of the palea, and finally equal concentric wheels formed by six stamen primordia are around the pistil primordia (Fig. 6-A). In mutants, palea/lemma primordia were formed normally during the differentiated period, and later mutants had different characteristics from the wild type in the

6 272 Rice Science, Vol. 20, No. 4, 2013 development of floret primordia. The primordia of florets were larger than normal ones, and unequal division emerged, and extra palea/lemma primordia were formed in mutants. Big lodicule primordia emerged between glume primordia and palea or lemma and developed into glume-like lodicule, and the palea and lemma of the further developed florets were still not folded (Fig. 6-B to -E). Stamen primordia developed out of synchronization, which led to different sizes of stamens. Some developed into more than one stamen, and non-concentric wheels were arranged around the pistils. Carpel primordium was divided into two carpel primordia, which later developed into two ovaries (Fig. 6-F). Floral meristems of mutants were bigger than those of the wild type, and their shapes were irregular (Fig. 6-G, -H and -I); the differentiation of floral meristems was limitness, and the floral meristems could grow unlimitedly. The pedicel of a floret was derived from the floral meristem, but not converted from floral organs (Fig. 6-J, -K and -L). Investigation of agronomic traits According to Table 2, affected by leaf mutation and photosynthesis, the plant height of the mutant was shorter and the length of flag leaf of the mutant was longer than that of the wild type. The length of panicle, effective panicle number per plant and 1000-grain weight were similar. However, seed-setting rate and grain number per panicle of the mutant were obviously lower than those of the wild type. Genetic analysis of mutants In the four combinations in which the mutants were female parents, there were streaked plants and albino plants in F 1 generations, and albino plants died when they grew to a certain stage (2 3 leaves). However, F 1 seedlings of the three reciprocal combinations were all green plants. It was obvious that the leaf color of F 1 seedling was different between crosses and reciprocal crosses, which illustrated that the leaf color mutations Fig. 6. Scanning electron microscope of morphogenesis in mutant floral organ. A, A wild type floret including six stamens normally arranged around the pistil, 700; B, After the lemma and palea primordium had established, central floral primordium unequally split, 500; C, After the lemma, palea and lodicule primordium had established, additional lemma/palea-like lodicules originated from aside of lemma, 450; D, Stamen primordium was formed and abnormally arranged around the pistil with irregular shape, 540; E, The carpel primordium with irregular shape, 500; F, The multiple stamen primordium developed, and the pistil primordium unequally split, 450; G, Multiple additional lemma/palea-like lodicules developed, and central floral primordium prolonged and increased, 500; H, Multifid central primordium, 550; I, After the stamen primordium had established, two cylindrical central floral primordium formed, 500; J, The abnormal protrusion in floral meristem, 600; K, Cylindrical central primordium in the pistil primordium position, 330; L, After the lemma, palea, lodicule, additional lemma/palea-like lodicules and lemma primordium had established, central primordium became narrowed cylindrical primordium, 400. le, Lemma; pa, Palea; apa, Additional palea; ll, Lodicule-like organ; lo, Lodicule; st, Stamen; ca, Carpel; fm, Floral meristem.

7 CHEN De-xi, et al. Characterization and Genetic Analysis of Streaked and Abnormal Floret Mutant st-fon 273 Table 2. Agronomic traits of mutant and wild type. Agronomic trait Mutant Wild type Difference Plant height (cm) ± 2.5** ± Length of flag leaf (cm) 48.5 ± 5.3** 39.0 ± Width of flag leaf (cm) 1.6 ± ± Length of panicle (cm) 27.1 ± ± No. of effective panicles per plant 15.0 ± ± Grain number per panicle ± 8.9** ± Seed-setting rate (%) 23.6 ± 8.4** 84.2 ± grain weight (g) 33.8 ± ± ** indicate significant differences at P < 0.01 between the mutant and wild type. Data are presented as mean SD. The positive value of difference means increase and the negative value means decrease compared with wild type. were cytoplasmic mutations (Table 3). Among F 2 groups, four cross combinations showed no normal green plant, and all of them consisted of streaked plants and albino plants. On the contrary, among three reciprocal cross combinations in which mutants were male parents, F 2 showed no streaked plant, and all of them were composed of green plants. The streaked status of the mutant did not accord with the classical Mendelian ratio. Separation in leaf color of F 2 populations further proved that mutations were plasmatic mutations controlled by female parents, and floral organ mutations and leaf mutations were controlled by the same mutant genes (Table 4). Therefore, the mutant discovered in this experiment is indeed a mutant controlled by cytogene. DISCUSSION Leaf color mutation is a mutant character with high mutation frequencies in higher plants and can be identified easily. In rice, mutant types of leaf color are rich. In addition to rice chromosome 12, a total of more than 80 genes causing leaf color mutation have been found in other chromosomes, and the mutations are mostly albino leaf mutations and yellow mutations. The streaked rice mutants are less reported, for which the main reason may be that the mutant trait is easily influenced by environments, and can not be stably inherited. The rice mutant with streaked character during the whole growth period is few. The st-fon mutants in the study showed the streaked character of culm and leaves during the whole growth period. The histological observation revealed abnormal development of cytosome in the streaked regions. According to Whatley (1983), the development of plastids in leaf needs to go through five stages: bioplastid, amyloplast, thylakoid, granum plastid and mature chloroplast. In the present experiment, the results showed that the plastid development could go to the fourth stage, granum plastid. Although concentric-circle-shaped initial lamella or cup-shaped plastid could be formed, granalamella was not discovered. Prolamellar bodies and concentric circle lamella are developmental features of plastid under dim or dark conditions. For this mutant, the presence of these structures is apparently not because of insufficient light, but probably because of insufficient ability of making use of light energy. The abnormality of plastid structure can be caused by the mutation of plastogene, or by both nuclear gene mutation and plastogene mutation (Rodermel, 2002). Many examples on leaf color mutation controlled by cytogene in plants, such as geranium and purple jasmine, had been reported. The mutation trait only controlled by cytogenes is extremely rare, since Table 3. Segregation of color in F 1, F 2 from negative and positive crossing of mutants with other normal plants. Combination Property of F 1 No. of normal plants No. of streaked plants and white plants Mutant/9311 Streaked plants and albino plants Mutant/Zhenshan B Streaked plants and albino plants Mutant/New dwarf source Streaked plants and albino plants Mutant/Wild type Streaked plants and albino plants /Mutant All green plants Zhenshan B/Mutant All green plants Wild type/mutant All green plants F 2 Table 4. Leaf color changes in BCF 1 generation from F 1 backcrossed with wild type or mutant. Combination Property Floret status (9311/Mutant) F 1 //Mutant All green plants Normal (Mutant/Wild type) F 1 //Wild type Streaked plants and albino plants Abnormal (Mutant/Wild type) F 1 //Mutant Streaked plants and albino plants Abnormal (Wild type/mutant) F 1 //Wild type All green plants Normal (Wild type/mutant) F 1 //Mutant All green plants Normal

8 274 cytoplasmic mutations are caused by organelle DNA mutations. Mitochondria and chloroplasts are principle carriers of non-chromosomal DNA. There are many copies (genomes) of DNA in each organelle, and mutation generally affects only one of them. In addition, the mutant organelle or individual mutant is often lack of competence, and cytoplasmic mutations are rarely found, therefore, there are rare reports about rice cytoplasmic mutants. Qian et al (1996) reported the white and green cytoplasmic mutants found in F 4 hybrid seedlings from the cross of Xiushui 11 and Chunjiang 03. The st-fon mutant showed different results between crosses and reciprocal crosses, and the streaked phenotypes of the offspring are always the same as those of the female parents, without separation. In this study, for the st-fon, besides streak appeared on leaves, floral organs were abnormal, and the early development of florets was significantly different from that of wild types. The number of paleae, lemmas, lodicules, stamens and pistils increased in some florets. It was found that there was a gene ij, controlling white streaks, located on chromosome 7 in maize (Han et al, 1992). Homozygosis ijij seedlings showed white and green streaked leaves, but the development of floral organs was not affected (Han et al, 1992). The mutant wslwp with white streaked leaves and white panicles showed no abnormal floral organs (Jin et al, 2011). Development of rice floral organs is a complex biological process, in which a number of genes are involved, and the functional mutation of any genes may cause an abnormal formation of floral organs (Li et al, 2011b). It was previously reported that the double mutants of leaves and floral organs in rice were controlled by nuclear genes, and the corresponding characters were caused by the missing of some genes in A, B and C, such as dl-1, dl-2, dl-sup1, dl-sup2 and dl(t), which are located at dl(loop) seat on the short arm of chromosome 3. The mutant in the study showed enhanced expression of A, B and C group genes at different extents, which was similar to fon mutants. The difference is that streak appeared on leaves of st-fon mutant, and lodicules experience no homeosis transformations to palea/lemma-like organs. Currently, FON1 gene was located on chromosome 11, and this gene encodes a LRR receptor binding protein, homologous with Arabidopsis CLV receptor protein (Suzaki et al, 2004). FON4 was also located on chromosome 11, and encodes a CLAVATA3 (CLV)-like secretory protein. Li et al (2007) found a mutant similar to fon in rice as well. fon(t) was located on chromosome 6. clv1, clv2 Rice Science, Vol. 20, No. 4, 2013 and clv3 mutants in Arabidopsis affect the development of culm meristems and floral meristems, resulting in the growth of culm meristems and floral meristems, and consequently the number of floral organs increases, causing more rounds and more organs each round (Clark et al, 1993, 1995; Kayes and Clark, 1998). OsLRK1 is a gene similar to CLV in rice, leading to an increase in the number of floral organs after RNA interferences (Kim et al, 2000). Rice lsh1 mutants are controlled by SEP genes called OsMADS1, showing leaf-like paleae/lemmas, and creating new flowers in the same florets (Jeon et al, 2000). Mutant osmads34 shows larger number of inflorescences and florets (Gao et al, 2010). In this study, some floral meristems of mutant st-fon showed no lodicule, stamen or pistil differentiations after they differentiated paleae and lemmas. 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