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Abstract The study was aimed to explore the effect and mechanism of brassinolide （BL） regulated starch metabolism in rice endosperm during seed germination. The radicle elongation of rice seeds treated with 1 μmol/L BL was inhibited during germination. The analysis on rice seeds with a GUS-fused promoter showed that BL had different regulatory effects on Wx， SBEI， and AGPS1. The effect of BL treatment on the physicochemical properties of rice starch was further investigated by scanning electron microscopy， Fourier transform infrared spectroscopy， and X-ray diffraction. The results showed that the starch treated with BL maintained better crystallinity and orderly structure， indicating that during the germination process， the degradation of starch in endosperm was slow， which might be one of the reasons for the slow radicle growth in the early stages of seed germination. This study provided important clues for further analysis of the molecular mechanisms underlying BR-regulated rice seed germination.
Key words Seed germination； Brassinolide； Starch metabolism； Rice； Starch structure； Physicochemical properties
Rice is the staple food of more than 1/2 of the worlds population， and starch is the main component of rice， accounting for 80%-90% of the dry quality of rice. Therefore， the composition， structure and physicochemical properties of starch have important effects on the function and quality of rice. Starch can be divided into transient starch and reserve starch according to its function. Transient starch exists in the photosynthetic tissues of plants and accumulates during the day and degrades at night. As an important carbon source， it provides the materials and energy required for growth and development when plants cannot perform photosynthesis. Reserve starch is mainly distributed in heterotrophic tissues， such as roots， seeds， or tubers， and can be stored for a long period of time. Sucrose， the initial substrate for its synthesis， comes from the product of photosynthesis[1]. An important role of reserve starch is to provide material and energy for the germination of seeds. According to the molecular structure， starch is divided into amylose and amylopectin. Amylose is a glucose polymer bonded with α-1，4 glycosidic bonds. It is a linear macromolecule with no or few branches. On the other hand， amylopectin is a glucose polymer which bonded with α-1，6 glucosidic branched bonds to long chains formed by α-1，4 glycosidically bonded glucose residues. The number of branched chains varies greatly， and the branch lengths are also inconsistent. The molecular weight of amylopectin is much larger than that of amylose[2]. In addition， rice seeds are also important agricultural means of production， and their dormancy and germination characteristics can directly affect rice quality and yield. For example， if the seed is dormant to a certain degree， it can avoid ear sprouting during ripening and reduce the loss of rice yield and quality. The germination of rice seeds is the first step in rice production， and good seed germination is the basis for rice seedling morphogenesis. Whether the seeds can be successfully broken and germinated after sowing， so as to quickly reach the standards of early seedlings， strong seedlings and full seedlings， will directly correlate the use value and yield of rice， and thus affect food security of China. Therefore， the study on seed dormancy and germination has been highly concerned by scholars at home and abroad. Seed germination refers to a series of ordered physiological and morphogenic processes of seeds from the beginning of water absorption to the protrusion of radicles. Seed germination is affected by a variety of internal and external factors， including seed moisture content， maturity， dormancy， temperature， light， and plant hormones[3]. Plant hormones play an important role in the regulation of seed germination. The two most important hormones are gibberellin （GA） and abscisic acid （ABA）， which antagonize each other in the control of seed germination[4]. In addition， the role of phytohormone brassinosteroid （BR） in seed germination has also been reported， including the physiological effects and preliminary regulatory mechanisms of BR in seed germination[5-7]. However， in general， the studies related to BR-regulated seed germination are rare， especially in rice， not only a monocotyledonous model plant but also an important crop. Moreover， the effects and mechanisms of BR regulated seed germination are still unclear.
The BR family contains dozens of natural brassinosteroids， of which BL is the most active one， and is therefore commonly used in research related to plant hormones. In this study， we first analyzed the effect of BL treatment on rice seed germination， and then investigated the influences of BL on the expression of some key genes for starch synthesis by using the transgenic rice with GUS-fused promoter. Moreover， the effect of BL treatment on the structure and physicochemical properties of rice starch was further investigated by scanning electron microscopy， Fourier transform infrared spectroscopy， and X-ray diffraction. The results of this study can provide important information for the subsequent in-depth study of how BR affect rice germination by regulating starch metabolism and further analyzing the molecular mechanism of BR regulated rice germination. Materials and Methods
Rice materials and growth conditions
In this test， mature rice seeds were used as the materials to carry out research on seed germination， including Japonica rice Nipponbare， Japonica rice 9983， and its transgenic rice with modified genes of proAGPS1-GUS， proWx-GUS， and proSBE1-GUS. The test materials were planted in the test base of the Agricultural College of Yangzhou University according to the wide-narrow row spacing. The plant spacing was （25.2 cm + 16.2 cm） × 12.8 cm， and each plot had 2 rows with 10 plants per row. During the whole growth period， the test plots were irrigated to the aquifers with precision management， strict control of diseases and insect pests， and seeds were harvested at maturity for analysis of subsequent germination tests.
Rice seed germination test
Coats were manually removed from the seeds of Nipponbare， 9983 and the trangenetic rice with GUS-fused promoter. Then， the seeds were sterilized with 70% ethanol twice， washed with ultrapure water twice， and then placed on circular dishes. Each circular dish contained 1μmol/L of BL or its solvent DMSO control solution， which was then placed in a dark environment at 28 ℃ with 70% relative humidity for germination test. Samples were taken every 12 h until reaching 72 h， with photos taken.
Determination of GUS activity
At 72 h after germination of the transgenic rice seeds with GUS-infused promoter， materials were collected for quantitative GUS fluorescence measurement， using the method of Jefferson et al.[8]. The total protein content was measured according to the method of Bradford[9].
Rice starch extraction
After 36 h of Nipponbare seed germination， materials were collected for starch extraction and subsequent physicochemical analysis of starch. In order to reduce the damage to the starch structure during the alkaline extraction process， the extraction of rice starch in this test was performed using the method of wet grinding combined with neutral protease， which made some improvement based on the method of Zhu et al.[10]. The specific procedures went as follows： 10.0 g of polished rice was taken and added with an appropriate volume of alkaline water （pH of 8.0 -8.5）. After soaking at room temperature overnight and decanting the supernatant， 0.001 mol/L NaOH solution of 1 times of the solid volume was added， and after fully mixing using tissue homogenizer， 0.001 mol/L NaOH solution of 1 times of the solid volume was added again. And then 1 mon/L NaOH was used to adjust pH value to 9.5. Then， the homogenate was added with 0.5 g of alkaline protease， followed by magnetic stirring for 18 h. The digested homogenate was sieved by the 200-mesh sieve， and centrifuged at 3 600 g for 20 min. After suspending the sediment using deionized water， the homogenate was centrifuged again at 3 600 g for 20 min. The above cleaning process was repeated three times to remove the residual ions in the starch. Finally， the starch was washed for 3 times with 95% ethanol， and after drying at 40 ℃， it was placed in a plastic bag a refrigerator at 4 ℃ for later use. Observation of microstructure of starch granules
Starch morphology was observed using a Philips XL30ESEM environmental scanning electron microscope. First， the samples were pretreated first， that is， the starch granule samples were dispersed on the sample plate with anhydrous ethanol， then treated with an IB-5 ion sputter （Eiko Co.， Japan） for 30 min， and plated with a layer of Pt gold ions. Afterwards， the environmental scanning electron microscope was used to take pictures of the samples under different magnifications.
X-ray diffraction analysis of starch
X-ray diffraction （XRD） is one of the most useful techniques for studying the geometric properties of three-dimensional structures of crystals and the properties of molecules themselves. The X-ray diffraction pattern of the test materials was obtained using a polycrystalline X-ray diffractometer （D8-ADVANCE of Borke AXS Co.， Germany）. Starch samples were equilibrated at room temperature for 24 h before analysis. The diffractometer operating parameters were 40mA and 45kV， and the scanning range of diffraction angle （2θ） was 4-40°， 0.1° every 0.2 s. The crystal peaks and the total diffraction area were obtained by the mechanical polar planimeter method， and the crystallinity was expressed as the percentage of crystal area to the total area.
Fourier transform infrared spectroscopy （ATR-FTIR） analysis of starch
The starch powder was placed in a closed container containing saturated aqueous solution of sodium chloride to absorb water for 7 d. The sampled were then added to the OMNI sampler and the starch samples were subjected to total reflection spectral scanning using an ATR holder. The scanning wave number range was 4 000-800 cm-1 with a resolution of 4 cm-1. Using deionized water as the blank， DTGS detector was used， and the scanning totaled 64 times. The accessory software to FTIR was used to analyze the spectrum. First， the spectrum with the wave number of 1 200-800 cm-1 was selected. After adjusting the baseline， half-peak breath of 19 cm-1 and enhancement factor of 1.9 to deconvolute the selected spectrum to obtain the deconvolution spectrum. Origin 6.0 was used to plot the spectrum， and the heights （i.e.， peak intensities） from the peaks at 1 047 cm， 1 022 cm， and 995 cm-1 to baseline were measured， thereby calculating the peak intensity ratios of 1 045 cm-1/1 022 cm-1 and 1 022 cm-1/995 cm-1.
Results and Analysis
Effect of BL treatment on rice germination Seed germination is accompanied by continuous water absorption by seeds， and the seeds that break dormancy change in fresh quality， germination rate， and embryonic germ. The germination of seeds can be divided into three stages. The first stage （the first 20 h） is the stage of rapid swelling and water absorption， and there is no obvious change on the phenotype； the second stage （20-50 h） is the stage of metabolic reactivation， when various metabolic processes are activated accompanied by the appearance and elongation of coleoptiles； the third stage （after 50 h） is another stage of rapid water absorption for growth， accompanied by the appearance of radicles[11]. In order to test the effect of applying BL on the germination of rice seeds during germination， Nipponbare seeds were treated with 1 μmol/L of BL solution and the corresponding DMSO control solution. From 0-72 h， germination was photographed every 12 h， and the prominent radicles and coleoptiles could be clearly observed at 72 h and their lengths were further measured. As shown in Fig. 1， there was no significant change in the seeds between the two treatments after 24 h imbibition. After 36 h， the embryos of the seeds swelled and the coleoptiles began to appear； afterwards， the coleoptiles further extended， and the radicles appeared at 72 h. Overall， the length of radicles treated with DMSO control solution was significantly longer than that of the BL treatment group. The lengths of germs and radicles of seeds germinated for 72 h were measured. The results showed that there was no significant difference in the length of radicles between BL treatment and control， but the length of radicles treated with DMSO was significantly longer than that of BL treatment， presumably because the concentration of 1 μmol/L of BL was relatively high for the radicles of seed， which could inhibit the elongation of radicles. It has reported that high concentration of BL could inhibit the elongation of roots in dicotyledonous plant Arabidopsis thaliana. Tanaka et al. found that 1 μmol/L of BL promoted the hypocotyl elongation of A. thaliana seedlings， but significantly inhibited root elongation， and the root length was only about 30% of the control group[12]. This study had similar results， indicating that roots were more sensitive to BL treatment.
Agricultural Biotechnology2018
Effect of BL treatment on the expression of several key genes involved in starch synthesis
In order to identify the target proteins regulated by BR in embryos of germinating rice seeds， so as to analyze the molecular mechanism of BR regulation on seed germination， Nipponbare seeds treated with BR synthesized inhibitor BRZ and the seeds of BR-insensitive mutant d61 were used as research materials. iTRAQ proteomic method was used to identify BR-regulated target proteins in the embryos of germinated seeds[13]. The results showed that the target proteins under the regulation of BR pathway included 3 key enzymes involved in starch metabolism， namely Wx， SBE1， and AGPS2， and the expression of the 3 enzymes reduced significantly in rice seeds with defective BR synthesis and damaged BR signaling pathways （Fig. 2-A）. Previously， we created a batch of transgenic rice varieties with GUS-fused promoter for starch synthesis， which included Wx， SBE1， and AGPS1， the isozyme of AGPS2. Therefore， these rice materials were used to further investigate how BL treatment affected the expression of these genes in the endosperm. Quantitative analysis of GUS showed that the expression quantity of Wx was highest in the endosperm of germinated seeds， while the expression of SBE1 was the lowest. The comparison of BL treatment with the control group showed that BL significantly inhibited the expression of Wx， only about 50% of the control treatment； while BL had no obvious effect on the expression of SBE1， but BL could obviously induce the expression of AGPS1 （Fig. 2-B）. The comparison between the results of this study and the results of previous proteomics showed that inhibition of BR synthesis or signal transduction could consistently reduce the expression of these 3 enzymes in embryos， whereas in the endosperm， the regulation of BL treatment on the transcriptional levels of the 3 genes were different from the previous research. The expression of AGPS1 increased significantly， while that of Wx decreased remarkably， and the expression of SBE1 showed no significant change. It was speculated that since the detected tissues of expression were different （endosperm and embryo）， the modes of expression were also different （transcription level and translation level）. The results indicated that BR could specifically regulate rice starch metabolism at different expression levels and in different tissues during seed germination， while its specific molecular mechanism remained to be further studied. Effect of BL treatment on the physicochemical properties of rice starch
The above results confirmed that BR could affect seed germination by regulating the expression of genes related to starch metabolism. Further tests were made to analyze the effects of BL treatment on rice starch metabolism from the physicochemical properties of starch. The test materials were the germinated Nipponbare seeds which were in the second stage of germination （after 36 h） and treated with BL and control solution respectively. The starch in the endosperm was extracted by an improved starch extraction method and observed by environmental scanning electron microscopy （SEM） to determine the effect of BL treatment on the morphological structure of starch granules in the initial stage of seed germination. In general， the surface of nearly 1/2 starch granules of BL-treated rice seeds was still relatively smooth， while the surface of the rest starch granules was initially degraded， forming obvious pores； whereas in the control group， the proportion of pores from the degraded starch granules was significantly higher than that of the BL-treated group （Fig. 3）， which at least implied that BL treatment could affect the degradation rate of starch granules in rice endosperm at the early stage of seed germination.
To investigate whether BL treatment affected the fine structure of rice starch， the crystallinity and ordered structure of starch in the 2 groups of materials were further analyzed. Starch granule structure includes crystalline region and amorphous region. The crystallization region is mainly composed of amylopectin molecules with a double helix structure， which is structurally stable and is not easily destroyed by external forces. The amorphous region is composed of amylose molecules with loose structure， which is easy to be destroyed by external forces and chemical components[14]. At present， XRD technology is a relatively mature method to determine the crystal type and calculate the crystallinity. It is easy to operate， and the result is reliable. The XRD results were as shown in Fig.4， and the crystallinity data of the tested samples showed that the crystallinity of the BL treated samples was significantly higher than that of the control group （Table 1）.
ATR-FTIR was used to analyze the attenuated total reflection of the starch short-range ordered structure. The deconvolution of the original data by setting the enhancement factor was shown in Fig. 5. Studies have shown that the peak intensity ratios of 1 045 cm-1/1 022 cm-1 and 1 022 cm-1/995 cm-1 in the spectrum are regarded as indicators of the ordered structure of starch， and the larger the ratio， the higher the degree of order. The calculated results were shown in Table 1. The results showed that the peak intensity ratio of 1 022 cm-1/995 cm-1 of the BL treated samples was significantly higher than that of the control samples. The above results indicated that the starch in BL treated samples maintained better crystallinity and more ordered structure， implying that the degradation of starch in endosperm during germination was slow， which may be one of the reasons for the slower growth of seed radicles in the early stages of seed germination. Discussion
Studying the regulation mechanism of seed germination and increasing the seed germination rate and germination potential in production have always been the focus of plant seed research. The process of seed germination is influenced and regulated by a variety of intrinsic and external factors， including plant hormones. BR is an important steroid plant hormone involved in the regulation of many processes of plant growth and development. At present， remarkable progresses have been made in the studies of BR synthesis and degradation， physiological effects， functions in environmental stress， and signal transduction pathways. However， there are few studies on the mechanism of BR-regulated plant seed germination and seedling morphogenesis， and most of the molecular mechanisms are still unknown.
In addition， starch is the most important component of important rice seeds， and its accumulation， composition and structure have remarkable influence on the yield and quality of rice. It has been found that BR is involved in the starch metabolism of plants. In A. thaliana， the starch content of BR deficient plants， namely CPD-antisense and cbb1 mutant plants， were significantly reduced[15]. Oh et al. found that overexpression of the wild-type and modified （Y831F） BR receptor gene BRI1 in the BR-insensitive mutant bri1-5 increased the starch content in leaves[16]. In addition to A. thaliana， related studies in other plants have also demonstrated that BR can affect the starch metabolism of plants. In cucumber， exogenous spraying of BR can significantly increase the content of sucrose and starch in cucumber leaves[17]. In rice， spraying BR in the early heading stage and during heading stage reduces the starch content in rice leaf sheaths and stems， while the starch content in seeds increases significantly[18]. Wu et al. created transgenic rice plants expressing genes encoding sterol C-22 hydroxylases of maize， rice or A. thaliana driven by a promoter that was active in only the stems， leaves and roots， thus enhancing BR levels of rice. They found that the content of glucose in rice flag leaves increased and the process of converting glucose into starch in rice seeds was enhanced[19]. The results of these tests show that BR can indeed affect the starch metabolism of plants and can promote the accumulation of starch in plant leaves and seeds. In addition to the starch synthesis and accumulation involved in seed development， starch metabolism during seed germination is also an important part， but little is known yet how BR regulates the seed germination by affecting starch metabolism. In this study， BR was used to treat the germinated seeds. On the one hand， the regulation of BR on the expression of key genes in starch metabolism was studied， which clarifies that BR has different regulation modes for Wx and SBE1 in the embryos and endosperms of rice seeds， further demonstrating that the phytohormone-regulated plant growth and development is tissue-specific， and its potential regulatory mechanism may be different. On the other hand， the effects of BR treatment on the degradation rate and crystallinity degree of rice starch were studied by scanning electron microscope （SEM） and XRD spectroscopy analysis， indicating that BR plays an important role in the early stage of seed germination and has a notable effect on the starch degradation process. The results of this study are consistent with those of Han et al.， whose proteomics study showed that the phosphorylation levels of BSK1， BSU1 and BIN2 in the BR signal transduction pathway have significantly increased in the early stage of rice seed germination， suggesting that the BR signaling pathway is strengthened at the early stage of rice seed germination[20]. The results of this study provide important information for the regulation network of starch metabolism by BR， and also lay the foundation for the further analysis of the underlying molecular mechanisms.
References
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[2] JAMES MG， DENYER K， MYERS AM. Starch synthesis in the cereal endosperm[J]. Current Opinion in Plant Biology， 2003， 6（3）： 215-222.
[3] FINICH-SAVAGE WE， LEUBNER-METZGER G. Seed dormancy and the control of germination[J]. The New Phytologist， 2006， 171（3）： 501-523.
[4] SHU K， LIU XD， XIE Q， et al. Two faces of one seed： hormonal regulation of dormancy and germination[J]. Molecular Plant， 2016， 9（1）： 34-45.
[5] STEBER CM， MCCOURT P. A role for brassinosteroids in germination in Arabidopsis[J]. Plant Physiology， 2001， 125（2）： 763-769.
[6] CHEN JG， PANDEY S， HUANG JR， et al. GCR1 can act independently of heterotrimeric G–protein in response to brassinosteroids and gibberellins in Arabidopsis seed germination[J]. Plant Physiology， 2004， 135（2）： 907-915.
[7] XI W， YU H. MOTHER OF FT AND TFL1 regulates seed germination and fertility relevant to the brassinosteroid signaling pathway[J]. Plant Signaling & Behavior， 2010， 5（10）：1315-1317.
[8] JEFFERSON RA， KAVANAGH TA， BEVAN MW. GUS fusions： beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants[J]. EMBO Journal， 1987， 6（13）： 3901-3907. [9] BRADFOR MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry， 1976， 72（1/2）： 248-254.
[10] ZHU LJ， LIU QQ， SANG YJ， et al. Underlying reasons for waxy rice flours having different pasting properties[J]. Food Chemistry， 2010， 120（1）：94-100.
[11] HE DL， YANG PF. Proteomics of rice seed germination[J]. Frontiers in Plant Science， 2013， 4（S1）：246.
[12] TANAKA K， NAKAMURA Y， ASAMI T， et al. Physiological roles of brassinosteroids in early growth of Arabidopsis： brassinosteroids have a synergistic relationship with gibberellins as well as auxin in light-grown hypocotyls elongation[J]. Journal of Plant Growth Regulation， 2003， 22（3）： 259-271.
[13] LI QF， XIONG M， XU P， et al. Dissection of brassinosteroid-regulated proteins in rice embryos during germination by quantitative proteomics[J]. Scientific Reports， 2016， 6：34583.
[14] ZHANG CQ， ZHOU LH， ZHU ZB， et al. Characterization of grain quality and starch fine structure of two japonica rice （Oryzasativa） cultivars with good sensory properties at different temperatures during the filling stage[J]. Journal of Agricultural and Food Chemistry， 2016， 64（20）： 4048-4057.
[15] SCHRODER F， LISSO J， OBATA T， et al. Consequences of induced brassinosteroid deficiency in Arabidopsis leaves[J]. BMC Plant Biology， 2014， 14（1）： 1-14.
[16] OH MH， SUN JD， OH DH， et al. Enhancing Arabidopsis leaf growth by engineering the BRASSINOSTEROID INSENSITIVE1 receptor kinase[J]. Plant Physiology， 2011， 157（1）： 120-131.
[17] YU JQ， HUANG LF， HU WH， et al. A role for brassinosteroids in the regulation of photosynthesis in Cucumis sativus[J]. Journal of Experimental Botany， 2004， 55（399）： 1135-1143.
[18] FUJII S， SAKA H. Distribution of assimilates to each organ in rice plants exposed to a low temperature at the ripening stage， and the effect of brassinolide on the distribution[J]. Plant Production Science， 2001， 4（2）： 136-144.
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Key words Seed germination； Brassinolide； Starch metabolism； Rice； Starch structure； Physicochemical properties
Rice is the staple food of more than 1/2 of the worlds population， and starch is the main component of rice， accounting for 80%-90% of the dry quality of rice. Therefore， the composition， structure and physicochemical properties of starch have important effects on the function and quality of rice. Starch can be divided into transient starch and reserve starch according to its function. Transient starch exists in the photosynthetic tissues of plants and accumulates during the day and degrades at night. As an important carbon source， it provides the materials and energy required for growth and development when plants cannot perform photosynthesis. Reserve starch is mainly distributed in heterotrophic tissues， such as roots， seeds， or tubers， and can be stored for a long period of time. Sucrose， the initial substrate for its synthesis， comes from the product of photosynthesis[1]. An important role of reserve starch is to provide material and energy for the germination of seeds. According to the molecular structure， starch is divided into amylose and amylopectin. Amylose is a glucose polymer bonded with α-1，4 glycosidic bonds. It is a linear macromolecule with no or few branches. On the other hand， amylopectin is a glucose polymer which bonded with α-1，6 glucosidic branched bonds to long chains formed by α-1，4 glycosidically bonded glucose residues. The number of branched chains varies greatly， and the branch lengths are also inconsistent. The molecular weight of amylopectin is much larger than that of amylose[2]. In addition， rice seeds are also important agricultural means of production， and their dormancy and germination characteristics can directly affect rice quality and yield. For example， if the seed is dormant to a certain degree， it can avoid ear sprouting during ripening and reduce the loss of rice yield and quality. The germination of rice seeds is the first step in rice production， and good seed germination is the basis for rice seedling morphogenesis. Whether the seeds can be successfully broken and germinated after sowing， so as to quickly reach the standards of early seedlings， strong seedlings and full seedlings， will directly correlate the use value and yield of rice， and thus affect food security of China. Therefore， the study on seed dormancy and germination has been highly concerned by scholars at home and abroad. Seed germination refers to a series of ordered physiological and morphogenic processes of seeds from the beginning of water absorption to the protrusion of radicles. Seed germination is affected by a variety of internal and external factors， including seed moisture content， maturity， dormancy， temperature， light， and plant hormones[3]. Plant hormones play an important role in the regulation of seed germination. The two most important hormones are gibberellin （GA） and abscisic acid （ABA）， which antagonize each other in the control of seed germination[4]. In addition， the role of phytohormone brassinosteroid （BR） in seed germination has also been reported， including the physiological effects and preliminary regulatory mechanisms of BR in seed germination[5-7]. However， in general， the studies related to BR-regulated seed germination are rare， especially in rice， not only a monocotyledonous model plant but also an important crop. Moreover， the effects and mechanisms of BR regulated seed germination are still unclear.
The BR family contains dozens of natural brassinosteroids， of which BL is the most active one， and is therefore commonly used in research related to plant hormones. In this study， we first analyzed the effect of BL treatment on rice seed germination， and then investigated the influences of BL on the expression of some key genes for starch synthesis by using the transgenic rice with GUS-fused promoter. Moreover， the effect of BL treatment on the structure and physicochemical properties of rice starch was further investigated by scanning electron microscopy， Fourier transform infrared spectroscopy， and X-ray diffraction. The results of this study can provide important information for the subsequent in-depth study of how BR affect rice germination by regulating starch metabolism and further analyzing the molecular mechanism of BR regulated rice germination. Materials and Methods
Rice materials and growth conditions
In this test， mature rice seeds were used as the materials to carry out research on seed germination， including Japonica rice Nipponbare， Japonica rice 9983， and its transgenic rice with modified genes of proAGPS1-GUS， proWx-GUS， and proSBE1-GUS. The test materials were planted in the test base of the Agricultural College of Yangzhou University according to the wide-narrow row spacing. The plant spacing was （25.2 cm + 16.2 cm） × 12.8 cm， and each plot had 2 rows with 10 plants per row. During the whole growth period， the test plots were irrigated to the aquifers with precision management， strict control of diseases and insect pests， and seeds were harvested at maturity for analysis of subsequent germination tests.
Rice seed germination test
Coats were manually removed from the seeds of Nipponbare， 9983 and the trangenetic rice with GUS-fused promoter. Then， the seeds were sterilized with 70% ethanol twice， washed with ultrapure water twice， and then placed on circular dishes. Each circular dish contained 1μmol/L of BL or its solvent DMSO control solution， which was then placed in a dark environment at 28 ℃ with 70% relative humidity for germination test. Samples were taken every 12 h until reaching 72 h， with photos taken.
Determination of GUS activity
At 72 h after germination of the transgenic rice seeds with GUS-infused promoter， materials were collected for quantitative GUS fluorescence measurement， using the method of Jefferson et al.[8]. The total protein content was measured according to the method of Bradford[9].
Rice starch extraction
After 36 h of Nipponbare seed germination， materials were collected for starch extraction and subsequent physicochemical analysis of starch. In order to reduce the damage to the starch structure during the alkaline extraction process， the extraction of rice starch in this test was performed using the method of wet grinding combined with neutral protease， which made some improvement based on the method of Zhu et al.[10]. The specific procedures went as follows： 10.0 g of polished rice was taken and added with an appropriate volume of alkaline water （pH of 8.0 -8.5）. After soaking at room temperature overnight and decanting the supernatant， 0.001 mol/L NaOH solution of 1 times of the solid volume was added， and after fully mixing using tissue homogenizer， 0.001 mol/L NaOH solution of 1 times of the solid volume was added again. And then 1 mon/L NaOH was used to adjust pH value to 9.5. Then， the homogenate was added with 0.5 g of alkaline protease， followed by magnetic stirring for 18 h. The digested homogenate was sieved by the 200-mesh sieve， and centrifuged at 3 600 g for 20 min. After suspending the sediment using deionized water， the homogenate was centrifuged again at 3 600 g for 20 min. The above cleaning process was repeated three times to remove the residual ions in the starch. Finally， the starch was washed for 3 times with 95% ethanol， and after drying at 40 ℃， it was placed in a plastic bag a refrigerator at 4 ℃ for later use. Observation of microstructure of starch granules
Starch morphology was observed using a Philips XL30ESEM environmental scanning electron microscope. First， the samples were pretreated first， that is， the starch granule samples were dispersed on the sample plate with anhydrous ethanol， then treated with an IB-5 ion sputter （Eiko Co.， Japan） for 30 min， and plated with a layer of Pt gold ions. Afterwards， the environmental scanning electron microscope was used to take pictures of the samples under different magnifications.
X-ray diffraction analysis of starch
X-ray diffraction （XRD） is one of the most useful techniques for studying the geometric properties of three-dimensional structures of crystals and the properties of molecules themselves. The X-ray diffraction pattern of the test materials was obtained using a polycrystalline X-ray diffractometer （D8-ADVANCE of Borke AXS Co.， Germany）. Starch samples were equilibrated at room temperature for 24 h before analysis. The diffractometer operating parameters were 40mA and 45kV， and the scanning range of diffraction angle （2θ） was 4-40°， 0.1° every 0.2 s. The crystal peaks and the total diffraction area were obtained by the mechanical polar planimeter method， and the crystallinity was expressed as the percentage of crystal area to the total area.
Fourier transform infrared spectroscopy （ATR-FTIR） analysis of starch
The starch powder was placed in a closed container containing saturated aqueous solution of sodium chloride to absorb water for 7 d. The sampled were then added to the OMNI sampler and the starch samples were subjected to total reflection spectral scanning using an ATR holder. The scanning wave number range was 4 000-800 cm-1 with a resolution of 4 cm-1. Using deionized water as the blank， DTGS detector was used， and the scanning totaled 64 times. The accessory software to FTIR was used to analyze the spectrum. First， the spectrum with the wave number of 1 200-800 cm-1 was selected. After adjusting the baseline， half-peak breath of 19 cm-1 and enhancement factor of 1.9 to deconvolute the selected spectrum to obtain the deconvolution spectrum. Origin 6.0 was used to plot the spectrum， and the heights （i.e.， peak intensities） from the peaks at 1 047 cm， 1 022 cm， and 995 cm-1 to baseline were measured， thereby calculating the peak intensity ratios of 1 045 cm-1/1 022 cm-1 and 1 022 cm-1/995 cm-1.
Results and Analysis
Effect of BL treatment on rice germination Seed germination is accompanied by continuous water absorption by seeds， and the seeds that break dormancy change in fresh quality， germination rate， and embryonic germ. The germination of seeds can be divided into three stages. The first stage （the first 20 h） is the stage of rapid swelling and water absorption， and there is no obvious change on the phenotype； the second stage （20-50 h） is the stage of metabolic reactivation， when various metabolic processes are activated accompanied by the appearance and elongation of coleoptiles； the third stage （after 50 h） is another stage of rapid water absorption for growth， accompanied by the appearance of radicles[11]. In order to test the effect of applying BL on the germination of rice seeds during germination， Nipponbare seeds were treated with 1 μmol/L of BL solution and the corresponding DMSO control solution. From 0-72 h， germination was photographed every 12 h， and the prominent radicles and coleoptiles could be clearly observed at 72 h and their lengths were further measured. As shown in Fig. 1， there was no significant change in the seeds between the two treatments after 24 h imbibition. After 36 h， the embryos of the seeds swelled and the coleoptiles began to appear； afterwards， the coleoptiles further extended， and the radicles appeared at 72 h. Overall， the length of radicles treated with DMSO control solution was significantly longer than that of the BL treatment group. The lengths of germs and radicles of seeds germinated for 72 h were measured. The results showed that there was no significant difference in the length of radicles between BL treatment and control， but the length of radicles treated with DMSO was significantly longer than that of BL treatment， presumably because the concentration of 1 μmol/L of BL was relatively high for the radicles of seed， which could inhibit the elongation of radicles. It has reported that high concentration of BL could inhibit the elongation of roots in dicotyledonous plant Arabidopsis thaliana. Tanaka et al. found that 1 μmol/L of BL promoted the hypocotyl elongation of A. thaliana seedlings， but significantly inhibited root elongation， and the root length was only about 30% of the control group[12]. This study had similar results， indicating that roots were more sensitive to BL treatment.
Agricultural Biotechnology2018
Effect of BL treatment on the expression of several key genes involved in starch synthesis
In order to identify the target proteins regulated by BR in embryos of germinating rice seeds， so as to analyze the molecular mechanism of BR regulation on seed germination， Nipponbare seeds treated with BR synthesized inhibitor BRZ and the seeds of BR-insensitive mutant d61 were used as research materials. iTRAQ proteomic method was used to identify BR-regulated target proteins in the embryos of germinated seeds[13]. The results showed that the target proteins under the regulation of BR pathway included 3 key enzymes involved in starch metabolism， namely Wx， SBE1， and AGPS2， and the expression of the 3 enzymes reduced significantly in rice seeds with defective BR synthesis and damaged BR signaling pathways （Fig. 2-A）. Previously， we created a batch of transgenic rice varieties with GUS-fused promoter for starch synthesis， which included Wx， SBE1， and AGPS1， the isozyme of AGPS2. Therefore， these rice materials were used to further investigate how BL treatment affected the expression of these genes in the endosperm. Quantitative analysis of GUS showed that the expression quantity of Wx was highest in the endosperm of germinated seeds， while the expression of SBE1 was the lowest. The comparison of BL treatment with the control group showed that BL significantly inhibited the expression of Wx， only about 50% of the control treatment； while BL had no obvious effect on the expression of SBE1， but BL could obviously induce the expression of AGPS1 （Fig. 2-B）. The comparison between the results of this study and the results of previous proteomics showed that inhibition of BR synthesis or signal transduction could consistently reduce the expression of these 3 enzymes in embryos， whereas in the endosperm， the regulation of BL treatment on the transcriptional levels of the 3 genes were different from the previous research. The expression of AGPS1 increased significantly， while that of Wx decreased remarkably， and the expression of SBE1 showed no significant change. It was speculated that since the detected tissues of expression were different （endosperm and embryo）， the modes of expression were also different （transcription level and translation level）. The results indicated that BR could specifically regulate rice starch metabolism at different expression levels and in different tissues during seed germination， while its specific molecular mechanism remained to be further studied. Effect of BL treatment on the physicochemical properties of rice starch
The above results confirmed that BR could affect seed germination by regulating the expression of genes related to starch metabolism. Further tests were made to analyze the effects of BL treatment on rice starch metabolism from the physicochemical properties of starch. The test materials were the germinated Nipponbare seeds which were in the second stage of germination （after 36 h） and treated with BL and control solution respectively. The starch in the endosperm was extracted by an improved starch extraction method and observed by environmental scanning electron microscopy （SEM） to determine the effect of BL treatment on the morphological structure of starch granules in the initial stage of seed germination. In general， the surface of nearly 1/2 starch granules of BL-treated rice seeds was still relatively smooth， while the surface of the rest starch granules was initially degraded， forming obvious pores； whereas in the control group， the proportion of pores from the degraded starch granules was significantly higher than that of the BL-treated group （Fig. 3）， which at least implied that BL treatment could affect the degradation rate of starch granules in rice endosperm at the early stage of seed germination.
To investigate whether BL treatment affected the fine structure of rice starch， the crystallinity and ordered structure of starch in the 2 groups of materials were further analyzed. Starch granule structure includes crystalline region and amorphous region. The crystallization region is mainly composed of amylopectin molecules with a double helix structure， which is structurally stable and is not easily destroyed by external forces. The amorphous region is composed of amylose molecules with loose structure， which is easy to be destroyed by external forces and chemical components[14]. At present， XRD technology is a relatively mature method to determine the crystal type and calculate the crystallinity. It is easy to operate， and the result is reliable. The XRD results were as shown in Fig.4， and the crystallinity data of the tested samples showed that the crystallinity of the BL treated samples was significantly higher than that of the control group （Table 1）.
ATR-FTIR was used to analyze the attenuated total reflection of the starch short-range ordered structure. The deconvolution of the original data by setting the enhancement factor was shown in Fig. 5. Studies have shown that the peak intensity ratios of 1 045 cm-1/1 022 cm-1 and 1 022 cm-1/995 cm-1 in the spectrum are regarded as indicators of the ordered structure of starch， and the larger the ratio， the higher the degree of order. The calculated results were shown in Table 1. The results showed that the peak intensity ratio of 1 022 cm-1/995 cm-1 of the BL treated samples was significantly higher than that of the control samples. The above results indicated that the starch in BL treated samples maintained better crystallinity and more ordered structure， implying that the degradation of starch in endosperm during germination was slow， which may be one of the reasons for the slower growth of seed radicles in the early stages of seed germination. Discussion
Studying the regulation mechanism of seed germination and increasing the seed germination rate and germination potential in production have always been the focus of plant seed research. The process of seed germination is influenced and regulated by a variety of intrinsic and external factors， including plant hormones. BR is an important steroid plant hormone involved in the regulation of many processes of plant growth and development. At present， remarkable progresses have been made in the studies of BR synthesis and degradation， physiological effects， functions in environmental stress， and signal transduction pathways. However， there are few studies on the mechanism of BR-regulated plant seed germination and seedling morphogenesis， and most of the molecular mechanisms are still unknown.
In addition， starch is the most important component of important rice seeds， and its accumulation， composition and structure have remarkable influence on the yield and quality of rice. It has been found that BR is involved in the starch metabolism of plants. In A. thaliana， the starch content of BR deficient plants， namely CPD-antisense and cbb1 mutant plants， were significantly reduced[15]. Oh et al. found that overexpression of the wild-type and modified （Y831F） BR receptor gene BRI1 in the BR-insensitive mutant bri1-5 increased the starch content in leaves[16]. In addition to A. thaliana， related studies in other plants have also demonstrated that BR can affect the starch metabolism of plants. In cucumber， exogenous spraying of BR can significantly increase the content of sucrose and starch in cucumber leaves[17]. In rice， spraying BR in the early heading stage and during heading stage reduces the starch content in rice leaf sheaths and stems， while the starch content in seeds increases significantly[18]. Wu et al. created transgenic rice plants expressing genes encoding sterol C-22 hydroxylases of maize， rice or A. thaliana driven by a promoter that was active in only the stems， leaves and roots， thus enhancing BR levels of rice. They found that the content of glucose in rice flag leaves increased and the process of converting glucose into starch in rice seeds was enhanced[19]. The results of these tests show that BR can indeed affect the starch metabolism of plants and can promote the accumulation of starch in plant leaves and seeds. In addition to the starch synthesis and accumulation involved in seed development， starch metabolism during seed germination is also an important part， but little is known yet how BR regulates the seed germination by affecting starch metabolism. In this study， BR was used to treat the germinated seeds. On the one hand， the regulation of BR on the expression of key genes in starch metabolism was studied， which clarifies that BR has different regulation modes for Wx and SBE1 in the embryos and endosperms of rice seeds， further demonstrating that the phytohormone-regulated plant growth and development is tissue-specific， and its potential regulatory mechanism may be different. On the other hand， the effects of BR treatment on the degradation rate and crystallinity degree of rice starch were studied by scanning electron microscope （SEM） and XRD spectroscopy analysis， indicating that BR plays an important role in the early stage of seed germination and has a notable effect on the starch degradation process. The results of this study are consistent with those of Han et al.， whose proteomics study showed that the phosphorylation levels of BSK1， BSU1 and BIN2 in the BR signal transduction pathway have significantly increased in the early stage of rice seed germination， suggesting that the BR signaling pathway is strengthened at the early stage of rice seed germination[20]. The results of this study provide important information for the regulation network of starch metabolism by BR， and also lay the foundation for the further analysis of the underlying molecular mechanisms.
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