Proteomics Analysis of Photo-Thermo-Sensitive Male Sterility Wheat Line BNS during Its Thermosensitive Period

Photo-thermo sensitive male sterile (PTMS) line is one of the important materials in utilizing heterosis in crops. Wheat line BNS (Bainong sterility) is an important nuclear-controlling PTMS line and suitable for growing and seed production in Huang Huai wheat zone in China. It has genetic stability with male sterility when sowing in autumn and male fertility when sowing in spring. Their thermosensitive periods were between stamen and pistil differentiation stage and anther connective stage and they could be regarded key periods for fertility conversion in BNS. To determine the molecular mechanisms of fertility conversion at thermosensitive period, we investigated characters of seed setting, anther and pollen grain of fertile and sterile BNS plants and compared young spike proteome patterns at their thermosensitive periods between the two BNS plants. The results showed that sterile plants had lower seed setting rate and pollen number, small pollen grain and lower pollen vitality than fertile plants. Out of protein spots reproducibly detected and analyzed on two dimensional electrophoresis gels, 76 spots showed significant changes in at least one BNS plant and 36 spots were identified by MALDI-TOF MS. The results showed that proteins involved in multiple biochemical pathways were differentially expressed at thermosensitive period between the two plants, including energy metabolism, stress response, signal transduction and regulation, protein process, amino acid and fatty acid metabolism, nucleic acid metabolism etc. Some of these proteins are reported to be involved in the abortion of anther or pollen grains in MS plants, such as energy metabolism and anti oxidative stress, and some were found to be novel proteins involved in the fertility conversion, such as phytohormones regulation. These results indicated that proteins related with anther or pollen development had expressed differently between the two BNS plants before anther development and phytohormones and signal transduction might be involved in the regulation of fertility conversion at thermosensitive period. Our studies have provided new insight to reveal the molecular mechanisms of fertility conversion at thermosensitive period in PTMS wheat.


Introduction
Male sterility (MS) has been wildly found for the production of hybrid seed in crop plants.Several CMS (cytoplasmic MS) systems have been reported in many crops and have been extensively utilized for hybrid seed production, such as in rich (Li & Yuan, 2000;Virmani, 2003) and rapeseed (Fan et al., 1986;Röbbelen, 1991).The utilization of heterosis in common wheat was made possible by the establishment of the nuclear-cytoplasm interactive male sterility in the three-line system and by the use of chemical hybridizing agents (CHA) (Zhao et al., 1999).However, wheat heterosis has not been utilized extensively in worldwide.The three-line system has a narrow origin and low degree of restoration, and the use of CHA is very limited for its cost and potential residue problems.Photo-thermo sensitive male sterile (PGMS) lines are utilized as an alternative way of exploiting heterosis in wheat.The PGMS system is advantageous for its broad restoring ability, easy maintenance and multiplication and it is considered to be more efficient than CMS system in hybrid wheat production (Jordaan, 1996;Virmani & Ilyas-Ahmed, 2001).BNS (Bainong sterility) line is a new type of ecological male sterile wheat line with PGMS character.BNS was obtained from BNY-S, a temperature sensitive nature mutant from wheat va.Bainong, and it was improved by continuous backcrossing with a wheat va.Lankao (Zhou et al., 2010).Both Bainong and Lankao were released for cultivation in HuangHuai wheat zone, which is the main wheat growing area and the wheat production in this area accounts for more than 60% of wheat production in China (Liu et al., 2011;Zhou et al., 2007).The fertility of BNS was easy to be restored by a few normal wheat varieties in this area (Li et al., 2009).BNS might be a potential, important PGMS line for wheat heterosis utilization in this area.
BNS is a genic PTMS line and its fertility and sterility are stable in different growing years.BNS has the characteristics of high sterility when it was sowed in early or medium autumn and fertility when sowed in later autumn or spring (Li et al., 2009).
As a PTMS line, the temperature played a leading role in its fertility alteration.Our previous studies proved that the temperature sensitive period was between stamen and pistil differentiation stage and anther connective stage, and this period could be regarded as critical for its fertility conversion (Wang et al., 2011).Based on continuous growing years' results, it could be concluded that the complete sterility could be obtained when air temperature goes below 11.4 o C at this fertility conversion period, and hybrid seed could be produced by crossing with restoration lines.BNS showed complete fertility when air temperature is more than 15.9 o C at the fertility conversion period, and fertile seed could be produced by self crossing (Wang et al., 2011).In addition, our research also showed that the male fertility in BNS was controlled by two major genes plus polygene, and a certain amount of cytoplasmic effects was preliminarily found.The two major genes have more effects on the fertility, and their additive effects are much more than their dominant effects (Zhang et al., 2013).
Up to now, many researches on mechanisms of male sterility have focus on the developments of anther or pollen grain.In order to understand the molecular mechanism underlying the key period of fertility conversion in male sterility, we investigated the proteome pattern differences of young spikes at fertility conversion period between fertile plants and sterile plants of BNS.This research will be helpful for elucidation of proteins regulation in conversion of fertility and sterility, and for utilization of this PTMS line in wheat heterosis.

Experimental Materials and Cultivation
PTMS winter wheat line BNS was used in this study.Materials were planted at the farm of Agronomy College of Shandong Agricultural University, in Taian City (36°11′N, 117°8′E and 135 m a.s.l), in Eastern China, during 2011-2012 growing season.Sowings occurred at two different dates, autumn (October 5, 2011) and spring (March 10, 2012).Seeds were planted in 3 replicates with plot of 2.0 m × 7 m (14 m 2 ) in this study.Two hundred seedlings were planted per square meter at equal spacing.All cultural practices, such as irrigation, fertilization, and the controls of weed, insect and disease were standard and uniform according to the local farmer cultivation practices.

Investigation of Seed Setting, and Characters of Anther and Pollen Grains
Seed setting rates: three spikes (one main and two side tillers per plant) on 10 randomly selected plants in each replication of the two sowing date plants were bagged with butter paper bags before flowering to observe seed setting in bagged spikes.Percent seed setting was calculated as two formulae given below: Seed set (%) = (Total number of kernels on spikelet base)/(Number of spikelets per spike × 2) × 100 (Chinese seed setting formula) (Song et al., 2005).
Characters of anther and pollen grains: 30 anthers of each replicate of the two sowing date plants were analyzed, of which always 10 were taken from the bottom third part, 10 from the middle third part and 10 from the top third part of the spike.Anthers just turning yellow, approximately one day before dehiscence, were removed from the floret with a pincet and measured under a binocular stereomicroscope (16×) with an ocular micrometer.The length and the width of the anther were determined.Pollen grain number pre anther was determined according to Hansen' methods (Hansen & Andersen, 1998).Brifly, the anther was deposited in a vial with 0.5 ml 0.5% aceto carmine, all pollen grains were loosened from the anther by crushing and stirring the anther.The concentration of pollens could be determined using haemocytometer method and the real number of pollen grains per anther could be calculated.At the same time, the diameter of pollen grains were determined under microscopy with an ocular micrometer.The viability of pollen grains was examined under the microscope.Anthers were taken from each spikelet in 5 spikes and squashed with a glass rod on a glass-slide to disperse pollen grains and stained with iodine-potassium iodine solution (2% I-KI).Only those being round, normal sized and dark blue stained were considered fertile (Barnabás & Kovács, 1992).

Protein Sample Extraction for Proteomics
Proteomics analysis for wheat line BNS during its thermosensitive period were conducted State Key Laboratory of Crop Biology, Shandong Agricultural University.For proteomic analysis, young spikes at the thermosensitive period (between the stage of differentiations of pistil and stamen and the stage of anther connective) were sampled according to the observation under stereomicroscope.Proteins were extracted using a two step trichloroacetic acid/acetone protein extraction protocol with minor modification (Cho et al., 2006).Briefly, a total of 0.3 g of young spikes were ground in liquid nitrogen with mortar and pestle (pre-cooled) into a fine powder.The powder was precipitated with ice-cold acetone containing 10% (w/v) trichloroacetic acid and 0.07% (v/v) 2-mercaptoethanol (acetone-TCA-2-ME) for 2 h at 20 o C, and then centrifuged at 40,000 g for 25 min at 4 o C. The pellets were washed with ice-cold acetone containing 0.07% (v/v) 2-ME, for 6 h at 20 o C and centrifuged again at 4 o C. Then pellets were lyophilized to powder and were stored at 80 o C until further use.Six biological replicates were extracted independently for fertile and sterile plants respectively.

Two-Dimensional Electrophoresis
Isoelectric focusing (IEF) was carried out on immobilized pH gradient (IPG) 24 cm pH 3-10 L strips (Bio-Rad).The running condition was as follows: 500 V for 1 h, followed by 1000 V for 1 h, and finally 8000 V for 14 h.The focused strips were equilibrated for 15 min in 10 ml equilibration solution containing 7 M urea, 30% (w/v) glycerol, 2.5% (w/v) SDS, 1% (w/v) DTT, and 50 mM Tris-HCl buffer, pH 8.8.Separation of proteins in the second dimension was performed by SDS-PAGE in a vertical slab of acrylamide (12% total monomer, with 2.6% cross linker) using a Dodeca Cell (Bio-Rad).For preparative gels 1.8 mg protein were loaded.The protein spots in analytical and preparative gels were visualized by colloidal CBB G-250.

In-Gel Digestion and MALDI-TOF MS Analysis
The excised protein spots were washed with ultrapure water twice at room temperature, and destained with 100 ml of 100 mM NH 4 HCO 3 /acetonitrile (50:50, v/v) for 1 h.Gel fragments were dehydrated with 50 ml of acetonitrile for 10 min and dried at room temperature.10 ml of 20 ng/ml trypsin (Promega) was added to each dried gel fragment and incubated for 45 min at 4 o C. 10 ml NH 4 HCO 3 (50 mM) was added and fragments were incubated at 37 o C overnight (about 16 h).After digestion of proteins, peptides were desalted with C18 ZipTips (Millipore Corp., Bedford, MA, USA), then spotted on MALDI plates (Bruker Daltonics, Germany) in 50% acetonitrile and 0.1% trifluoroacetic acid (TFA).Finally, peptides were co-crystallized with saturated a-cyano-4-hydroxycinnamic acid (CHCA) prepared in 50% (v/v) acetonitrile containing 1% TFA.To obtain peptide mass fingerprint (PMF) of protein spots, peptide masses were measured using autoflex MALDI-TOF MS (Bruker Daltonics, Billerica, MA, USA).External calibration was performed with a peptide calibration standard (Bruker Daltonics,Part No.: 206 195).All obtained PMFs were analyzed with the protein search engine MASCOT (Matrix Science, U.K.) against NCBI's database.Search parameters were set as follows: peptide tolerance (0.2 Da), NCBInr database, Green plants (taxonomy), carbamidomethylation of cysteine (fixed modification), and methionine oxidation (variable modification).Molecular function of proteins was annotated using the database at http://www.uniprot.org/uniprot(Wen et al., 2010).

Statistical Analysis
SPSS Version 13.0 (Lead Technologies, Chicago, IL, USA) was used to statistically analyze the results of characters of seed setting, anther and pollen grains, the intensity of protein spots by Student's t-test.Percentage of seed setting was subject to arcsine transformation prior to statistical analysis.Significance was determined at the p < 0.05 level.

Characters of Seed Setting, Anther and Pollen from Two Sowing Date Plants
The investigation results of seed setting, characters of anther and pollen from sterile and fertile plants of BNS were shown in Table 1.The seed setting rates of BNS line sowed at autumn and spring were 1.25% and 84.62% (as international calculation method), or 2.96% and 118.67% (as Chinese calculation method), respectively (P < , and they were identified with high confidence by MALDI-TOF MS.The genomic sequence data analysis of all the differentially expressed proteins identified only 38 proteins from wheat (Table 2).Among these identified proteins, 22 proteins were up-regulated in fertile plants, while 14 were up-regulated in sterile plants.Homologs of some of these proteins were identified in other plant species, and the identification of wheat proteins was low due to the incomplete status of the wheat genome database.According to the functional features described in annotation and literature data, the identified proteins were categorized into several types, including carbohydrate and energy metabolism, stress responses, signaling and regulation, antioxidant and defense pathways, protein synthesis or DNA replication process, amino acid and fatty acid metabolism, nucleic acid metabolism and other unclassified proteins.Detailed information can be found in Table 2.

Discussion
We analyzed the differences of fertility characters between of autumn sowing and spring sowing plants of PTMS winter line BNS.The results indicated that BNS was highly sterile when planted in early autumn in Taian and its seed setting rate recovered to more than 80%, and self-pollinated to normal levels when sowing in spring.This indicated that it could be used as both maintainer and sterile lines in a two-line hybrid system.The present study showed that the sterility of BNS line sowed in autumn might be due to the reduction of pollen number per anther or the decrease of pollen viability.This is consistent with previous studies in other species.In peach, a mutant with male sterility had smaller anthers and lesser number of pollen grains than fertile parental variety (Sreevalli et al., 2003).In eggplant, both the number of pollen per anther and pollen viability in the BC 5 plants with male sterility were lower than those in their fertile nucleus donor (Shiro & Yoshida, 2002).However, the reduction of pollen number per anther and the decrease of pollen viability are influenced by different mechanisms.The pollen number might be with the development of meiosis of microspore mother cell, or the daparture of pollen grains from tapetum and the realeasement from anther, and the pollen viability might be related with normal development of pollen grains after meiosis of microspore mother cell.Lee et al. (2008) reported that no pollen grain is typically visible during anthesis in a male sterile radish, but a small number of pollen grains could be observed stuck together in the dehiscing anthers in another male sterile radish.Gothandam et al. (2007) showed that pollen viability and grain production decreased significantly when treated at a low temperature in a rice MS line, and the male sterility is due to functional loss of the tapetum which is the most sensitive to low-temperature stress.
The present research showed that, the external appearances had no obvious change at their theomosensitive periods between sterile and fertile plants of BNS, but the expression of a variety of proteins were different significantly between them as shown by proteomics (Table 2, Figure 2).Vijayalakshmi and Bangarusamy (2007) have reported that high temperature induced male sterility in a thermosensitive genie male sterile rice line; and comparied with fertile plants, the male sterile plants were associated with accumulation of total phenolics and proline and reductions of soluble proteins at the critical stage of thermosensitivity.Our research indicated that there might be multiple biochemical mechanisms involved in the fertility conversion at thermosensitive period.Among these proteins, proteins related with energy metabolism, antioxidant stress and hormone regulation could be particularly prominent.
Proteins related with energy metabolism and regulation included 4 proteins.ATP synthase subunit d (mitochondrial-like) (spot 7) was up-regulated in sterile plants, and mitochondrial ATP synthase precursor (spot F18), V-type proton ATPase subunit B1 (spot F19) and ubiquinol-cytochrome c reductase complex protein (spot F13) were up regulated in fertile plants.
ATP synthase is an important enzyme that provides energy for the cell to use through the synthesis of adenosine triphosphate (ATP).ATPases are a class of enzymes that catalyze the decomposition of ATP into ADP and a free phosphate ion.This dephosphorylation reaction releases energy and this process is widely used in all known forms of life.Mitochondrion, the site of both the tricarboxylic acid cycle and oxidative phosphorylation pathway, plays a crucial role in energy and carbon metabolism in eukaryotic cells (Hatefi, 1985).Up to now, 12 mitochondrion DNA regions associated with CMS have been identified, and most of them are involved in the genes encoding F 0 F 1 -ATPase subunits (Hanson, 2004).In higher plants, the demand for ATP is highly increased during pollen development (Sabar et al., 2003), and decreased mitochondrial ATP synthesis may be a causal factor in disruption of pollen or microspore development (Yang et al., 2009).These previous researchers have reported that reduction of the proteins associated with energy production and lesser ATP equivalents detected in CMS anther and that indicated that the low level of energy production played an important role in inducing CMS.
Oue research indicated that the differences of sterile and fertile plants on energy production has taken place as early as thermosensitive period in BNS, and there is no visible anther in the young spike at this period.The ubiquinol cytochrome c reductase is a key subunit of the cytochrome bc1 complex (complex III) of the mitochondrial respiratory chain (Vedel et al., 1999).The complex shows characteristics associated with a Q-cycle mechanism of redox-driven proton translocation, including two pathways for reduction of b cytochromes by quinols and oxidant-induced reduction of b cytochromes in the presence of antimycin (Berry et al., 1991).But to our knowledge no proof shows that it is related with MS in plants.
Superoxide dismutase precusor (SOD) (spot 22) and cytosolic ascorbate Peroxidase (spots 10, 12) were found up regulated in sterile plants.Superoxide dismutases are enzymes that catalyze the dismutation of superoxide (O 2-) into oxygen and hydrogen peroxide.This is an important antioxidant defense in nearly all cells exposed to oxygen.Superoxide dismutases up regulated in sterile lines might be related with the elimination of activated oxygens in male sterile plants.In rice, compared with the maintainer line, fertile line Yuetai B, a significant decrease of SOD activity was detected in the CMS line, Yuetai A, which resulted in an increase in the reactive oxygen species (ROS) content (Wang et al., 2013).Ascorbate peroxidases (APX) are enzymes that detoxify peroxides such as hydrogen peroxide using ascorbate as a substrate.Programmed cell death during microgenesis in a Honglian CMS line of rice is correlated with oxidative stress in mitochondria.This pollen disruption was correlated with excess production of reactive oxygen species and down-regulation of the activity of superoxide dismutase (SOD) and ascorbate peroxidase (APX) in mitochondria (Li et al., 2004).
The cupin proteinbelongs to cupin superfamily, which comprises both enzymatic and non-enzymatic members, although it notably contains the non-enzymatic seed storage proteins (Dunwell et al., 2001).In seed, the cupin protein mainly competes with ABA in the activated a-subunit of GTP-binding proteins in the regulation of its germination (Santner & Estelle, 2009;Lapik & Kaufman, 2003).Once ABA interacts with GTP-binding protein receptors, other proteins related to the signal cascade are needed for signal transduction to take place.Wrab17 is a dehydrin-like protein which was shown to be highly homologous (84% amino acid identity) to ES2A, a barley GA3 (gibberellic acid)-responsive protein (Pandey et al., 2009).ABA-RESPONSIVE1 (ABR1), which is highly induced by infection with avirulent Xanthomonas campestris and it is also highly induced by treatment with ABA.The pepper ABR1, which localizes to the nucleus, negatively regulates ABA signaling in an SA-dependent manner to resist pathogen attack (Tsuda et al., 2000).Spot F2 had the highest homology to indole-3-glycerol phosphate lyase (IGL2).IGL2 is involved in auxin-mediated signal transduction.The interaction of auxin with its cellular receptors for IGL2 triggers a cascade of events resulting in response including the modification of cell wall components such as lipids, and in altering the orientation of cell wall polysaccharides (Choi & Hwang, 2011).Annexin D4 (spot F9) plays important roles in osmotic stress and ABA signaling in a Ca 2+ -dependent manner (Macdonald, 1997).
The previous research has proved that almost all the hormones are related with male fertility in plants.Flower tissue-specific auxin reduction is the primary cause of high temperature injury, which leads to the abortion of pollen development in barley and Arabidopsis (Lee et al., 2004).The tapetum-specific expression of a mutated ethylene receptor gene is a potential strategy for inducing male sterility in transgenic tobacco plants (Sakata, 2010).In B. napus ABA content in flower buds was higher in fertile plants than that in male sterile plants, and the expression of KIN1, a ABA reactive protein, also reduced in sterile flower (Zhu et al., 2010).Gibberellin-induced gene expression associated with cytoplasmic male sterility in sunflower (Duca et al., 2008).
Our present results indicated that complex plant hormone regulations might exist during the thermosensitive period.Plant hormones might have important roles in the fertility conversion.However, as for what specific hormones mainly involved and what action modes of hormone in fertility conversion are needed to be further studied.
In addition, there are many other proteins expressed differently between the two lines at thermosensitive period in BNS (Figure 2; Table 2).The proteins up-regulated in sterile line included that s-adenosylmethionine synthetase 1, Oxidase (IM1), triticin precursor, NBS-LRR type RGA, rice homologue of Tat binding protein, nitrate reductase apoenzyme, KNOX class homeodomain protein and hypothetical protein.The proteins up regulated in fertile plants included starch synthase isoform IV, putative peptidyl-prolyl cis-trans isomerase family protein, DNA ligase, histone H3, TPA (acidic ribosomal protein P2a-3), chaperonin isoform 1, ADP-ribosylation factor, retrotransposon protein, chitinase 3, pyruvate decarboxylase, isopentenyl pyrophosphate isomerase, cysteine proteinase, chilling inducible protein, RicMT.These proteins might have some roles in the fertility conversion, and they are also worthy of further research.
In conclusion, sterile plants of BNS had lower seed setting rate, lower pollen number, small pollen grain and lower pollen vitality than fertile plants.Proteins involved in multiple pathways were differentially expressed in response to fertility differences at thermosensitive period.Proteins related with pollen abortion had expressed differently at thermosentive period and plant hormones might be involved in the regulation of fertility conversion at this period.

Table 2 .
Up regulated proteins displayed and identified on gels after 2-DE followed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis in sterile and fertile plants of BNS