Seed-Specific Expression of OsDWF4, a Rate-Limiting Gene Involved

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Agricultural and Environmental Chemistry

Seed-specific expression of OsDWF4, a rate-limiting gene involved in BR biosynthesis, improves both grain yield and quality in rice Qian-Feng Li, Jia-Wen Yu, Jun Lu, Hong-Yuan Fei, Ming Luo, BuWei Cao, Li-Chun Huang, Changquan Zhang, and Qiaoquan Liu J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 03 Apr 2018 Downloaded from http://pubs.acs.org on April 3, 2018

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Journal of Agricultural and Food Chemistry

Article

Seed-specific expression of OsDWF4, a rate-limiting gene involved in BR biosynthesis, improves both grain yield and quality in rice

Qian-Feng Li†‡, Jia-Wen Yu†, Jun Lu†, Hong-Yuan Fei†, Ming Luo§, Bu-Wei Cao †ÈLi-Chun Huang†, Chang-Quan Zhang†‡, Qiao-Quan Liu†‡*

†Key Laboratory of Crop Genetics and Physiology of Jiangsu Province/Key Laboratory of Plant Functional Genomics of the Ministry of Education, College of Agriculture, Yangzhou University, Yangzhou 225009, China ‡Co-Innovation Center for Modern Production Technology of Grain Crops of Jiangsu Province/Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education, Yangzhou University, Yangzhou 225009, China §Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China.

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ABSTRACT: Brassinosteroids (BRs) are essential plant-specific steroidal hormones that regulate diverse growth and developmental processes in plants. We evaluated the effects of OsDWF4, a gene that encodes a rate-limiting enzyme in BR biosynthesis, on both rice yield and quality when driven by the Gt1 or Ubi promoter, which correspond to seed-specific or constitutive expression, respectively. Generally, transgenic plants expressing OsDWF4 showed increased grain yield with more tillers and longer and heavier seeds. Moreover, the starch physicochemical properties of the transgenic rice were also improved. Interestingly, OsDWF4 was found to exert different effects on either rice yield or quality when driven by the different promoters. The overall performance of the pGt1::OsDWF4 lines was better than that of the pUbi::OsDWF4 lines. Our data not only demonstrate the effects of OsDWF4 overexpression on both rice yield and quality, but also suggest that a seed-specific promoter is a good choice in BR-mediated rice breeding programs.

KEYWORDS: Oryza sativa L.; brassinosteroids; OsDWF4 gene; seed-specific promoter; grain yield; grain quality


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INTRODUCTION Brassinosteroids (BRs) are a group of plant steroidal hormones that are structurally similar to animal steroids. BR is ubiquitous in the plant kingdom, and is well-known mainly for its roles in regulating a broad spectrum of plant developmental and physiological events.1-3 BR-deficient or -insensitive mutants display various growth defects, including dwarfism, dark green leaves, male sterility, delayed flowering, and senescence.4-5 Recently, the combined application of genetic, molecular, biochemical, and proteomic approaches have identified almost all major components in the BR signaling pathway in Arabidopsis, from the cytomembrane localized BR receptor BR INSENSITIVE 1 (BRI1) to the downstream nuclear transcription factors BRASSINAZOLE RESISTANT1 (BZR1) and bri1-EMSSUPPRESSOR 1 (BES1), which makes the BR signaling pathway one of the most well-understood plant hormone signal transduction pathways.5 Moreover, BR biosynthesis is also well characterized, although it occurs in an intricate network pathway.6,7 Briefly, the process of BR biosynthesis basically involves the conversion of campesterol into campestanol, followed by the formation of castasterone, and eventually leads to the formation of brassinolide (BL) from castesterone.8 Previous studies have shown that several key genes are involved in BR biosynthesis,

such

as

de-etiolated-2

(DET2),

dwarf4

(DWF4),

and

constitutive

photomorphogenesis and dwarfism (CPD). BR biosynthesis is mainly regulated by modulation of the expression of BR biosynthetic genes in a feedback regulation mode, which relies on an intact BR signaling pathway and the BZR1 transcription factor.9-12 In addition to the feedback regulation module, expression of BR biosynthetic genes is also regulated by developmental programs and tissue-specific expression patterns.13



Recent progress in our understanding of BR biosynthesis and signaling facilitates its future application in crop breeding. In fact, mutants defective in either gibberellin (GA) biosynthesis or signaling, another critical growth promoting phytohormone, generated semi-dwarf but high-yielding crop varieties that facilitated the Green Revolution.14,15 Similar to GA, BR is also involved in the control of various key agronomic traits in rice, including plant height, leaf angle, and seed size, among others.16-19 Furthermore, BR directly interacts with GA in both hormone biosynthesis and signaling levels through a well established molecular crosstalk framework.20-23 Therefore, BR is considered to be an ideal biotechnological target for crop breeding, and it is important to evaluate the effects of key components of BR on crops.24 Among the cloned BR biosynthesis genes, DWF4 is most well characterized, and shows potential applications in crop breeding. For example, overexpression of DWF4 in tomato, Arabidopsis, tobacco, and Brassica napus has been shown to increase plant height, vegetative growth, and seed yield.25-28 Most importantly, modulation of OsDWF4 expression in rice affects seed size, seed weight, and plant architecture.26,29-31 Indeed, both overproduction and a mild deficiency of BR caused by modulating OsDWF4 expression can enhance grain yield under certain growth conditions. For example, the overexpression of OsDWF4 in stems, leaves, and roots of rice plants produced more and heavier seeds, thus enhancing per-plant grain yield.31 In contrast, a slight reduction in OsDWF4 expression resulted in lower per-plant grain yield but increased per-plot grain yield due to the erect leaf plant phenotype, which enabled higher density planting of rice.30,32 All of the above results suggest that manipulation of BR biosynthesis can improve crop yield, and that OsDWF4 is indeed an ideal genetic target for rice breeding.


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Although starch comprises approximately 90% of the total rice endosperm weight, it is not only the major contributor to grain weight, but also determines rice grain qualities. While previous studies have indicated that BR can promote the transport of sucrose and other sugars to the endosperm and embryo, they mainly focused on evaluating its effect on rice yield.31,33 Whether modulation of BR biosynthesis could also affect starch biosynthesis and rice quality is still unclear. Therefore, transgenic rice lines overexpressing OsDWF4 could be used not only to evaluate the effects of BR biosynthesis on rice yield, but also to help explore how BR affects starch biosynthesis and rice grain qualities. Unlike some other phytohormones, such as auxin and cytokinin, BR does not undergo long distance transport and only functions locally where it is synthesized.34 Thus, the regulation of expression levels and the spatiotemporal expression patterns of BR biosynthesis genes are essential for proper plant growth and development. It is generally accepted that promoters are key cis-elements that control target gene expression. However, no systematic study has been reported that examines how different promoters affect rice yield and quality by modulating BR biosynthesis. At present, a number of promoters are widely used in plant biotechnology, such as the cauliﬂower mosaic virus (CaMV) 35S promoter and the maize ubiquitin (Ubi) gene promoter. In this study, the Ubi and glutelin 1 (Gt1) promoters, which are commonly used in rice biotechnology and correspond to constitutive and seed-specific expression patterns, respectively,35,36 were selected to drive the overexpression of OsDWF4. Detailed comparisons between pGt1::OsDWF4 and pUbi::OsDWF4 were then performed. Our results indicate that overexpression of OsDWF4, especially when under control of the seed-specific Gt1 promoter, can positively affect both rice yield and grain quality, thus highlighting the



importance of selecting suitable gene promoters in BR-involved rice breeding programs.

MATERIALS AND METHODS Plant Materials, Growth Conditions and Agronomic Trait Investigations. Tissues and developing seeds of the japonica rice cultivar ‘Nipponbare’, were used for expression analysis of the OsDWF4 gene. ‘Yandao 8’ (Y8), an elite japonica rice cultivar grown in China, was used for transformation with the OsDWF4 gene. ‘Wuxiangjing 9’ (WX9) was used for transformation with the beta-glucuronidase (GUS) reporter gene fused with Gt1 and Ubi promoters, respectively. All rice lines, including ‘Nipponbare’, Y8, WX9, and their respective transgenics, were planted in the same experimental field at Yangzhou University (Yangzhou, Jiangsu province, China) under identical climatic conditions. Three replicate plots were used for the experiments, and the plots were arranged in a randomized block pattern, with 6 rows per plot and 10 plants per row. The main agronomic traits were recorded after seed maturity. The mature seeds were then harvested from the main panicle in the middle of each plot and air-dried for subsequent grain morphology and rice quality analyses. Data for each sample represents the mean of the three plots. Transgene Constructs and Rice Transformation. The rice GT1 promoter and the maize Ubi promoter were cloned separately into the binary vector pCAMBIA1300 to generate the vectors p696G and p696U, respectively, which could be used to drive the endosperm-specific (p696G) and constitutive (p696U) expression of target genes. In the present study, OsDWF4, a key gene that regulates BR biosynthesis, was cloned into the two vectors. Subsequently, the two constructs were transformed into Agrobacterium tumefaciens EHA105 and then


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introduced into primary calli derived from mature seeds of japonica rice cultivar Y8 via Agrobacterium-mediated transformation. The pGT1::GUS and pUbi::GUS transgenic rice lines were generated previously in our lab as described.36 GUS Activity Assay. The GUS enzyme activity assay was performed as described.36 Briefly, for histochemical analysis, various rice tissue samples were collected at 12 days after flowering (DAF) and incubated overnight at 37oC with 0.5 g L−1 of X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide). For quantitative determination of GUS activity, a fluorometric assay was conducted by grinding various tissue samples into a powder in a mortar with liquid nitrogen, and total proteins were then extracted in GUS extraction buffer. The reaction buffer containing 1 mmol L−1 4-methyl umbelliferyl-β-D-glucuronide was the used for determination of GUS activity. Hygromycin Resistance Assay. To identify and isolate homozygous transgenic lines, a hygromycin resistance assay was performed. In brief, fresh leaf blades from T2 generation plants of the pGt1::OsDWF4 and pUbi::OsDWF4 transgenic lines were collected, cut into segments ~2 cm long, and then incubated in ddH2O containing 0.5 mg/L 6-BA (6-benzylaminopurine) and 50 mg/L hygromycin. The leaves were then incubated in a growth chamber (26°C, 12-h light/12-h dark cycles) for three days before collecting data. RNA Isolation and Quantitative Real-Time PCR Assays. Total RNA was extracted from various rice tissues or organs using the RNeasy Plant Mini Kit (Qiagen). The extracted RNA was pre-treated with DNase I (Qiagen), and first-strand cDNA was reverse transcribed from 2 µg total RNA by using the SuperScript First-Strand Synthesis System (Invitrogen). qRT-PCR was then performed using the SYBR Premix Ex TaqTM II system (TaKaRa) and the CFX96



TouchTM real-time PCR detection system (Bio-Rad). The Ubiquitin Conjugase (UBC) gene was used as the internal control for normalization of gene expression. The primer sequences used to amplify genes in qRT-PCR assays are given in Table S1. Lamina Joint Bending Assay. The lamina joint assay using excised leaf segments was performed as described previously with small modifications.37 Synchronous seedlings were selected two days after germination and grown in the dark for seven days at 28oC. The entire segments, containing 1 cm of the second leaf blade and leaf sheath, were floated on MilliQ water for 24 h and then incubated in 2.5 mM maleic acid potassium solution for 48 h at 28oC in the dark. The angle between the lamina and the sheath was then measured. Flour Preparation and Physicochemical Analyses. Air-dried mature seeds were first dehusked with a rice huller (model SY88-TH, Korea) and then polished with a grain polisher (Kett, Tokyo, Japan). Milled rice samples were sealed in bags and stored at 4°C until analysis. Polished rice samples were ground into flour in a mill (FOSS 1093 Cyclotec Sample Mill, Sweden) equipped with a 0.15 mm screen. The rice apparent amylose content (AAC) was then determined using the iodine colorimetric method.38 Gel consistency (GC) and Rapid Visco Analyzer (RVA) profiles were performed as previously described.39 A Differential Scanning Calorimeter (DSC 200 F3, Netzsch Instruments NA LLC; Burlington, MA) was used to evaluate starch gelatinization and retrogradation temperatures as described.40 All tests were performed in triplicate. Gel Permeation Chromatography (GPC) Analysis. Samples of rice flour (5 mg) were debranched with isoamylase (EC3.2.1.68, E-ISAMY, Megazyme) and the relative molecular weight distribution of the debranched starch was determined by GPC with a PLGPC 220


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system (Polymer Laboratories Varian, Inc., Amherst, MA, USA) as described previously.41 Generally, three columns (PL110-6100, -6300, and -6525) with a differential refractive index detector (DRI) were included in the PL-GPC 220 system. The GPC data were transformed through integral equations based on dextrans of known molecular weights and then used to draw the molecular weight distribution curves. By using dextran standards, the GPC data are reported as dextran-equivalent molecular weight, denoted as MW. Statistical Analysis. For sample characterization in this study, at least two replicate measurements were performed unless otherwise specified. All data represent the means of biological repeats from three plots with standard deviation (SD). For experiments with single pair-wise comparisons, Student’s t test was used to determine the level of significance. For experiments with multiple comparisons, the data were analyzed by one-way analysis of variance (ANOVA) with Bonferroni’s correction.

RESULTS Characterization of the Expression Pattern of OsDWF4 in Wild-Type Plants and Under Control of Two Commonly-Used Promoters. Quantitative real-time PCR (qRT-PCR) was performed to examine the expression of OsDWF4 in different rice tissues. The result showed that highest levels of OsDWF4-specific mRNA were in rice leaves, followed by young differentiated panicles, while the differences were small in the other tested tissues (Figure 1A). Next, we further examined OsDWF4 expression in the developing seeds to determine its temporal expression pattern. The result showed that OsDWF4 expression was stable at the early stage (0 to 10 DAF) and then increased at the late stage (15 to 20 DAF) of



seed development, although there was a decline around 15 DAF (Figure 1B). To investigate potential future applications of OsDWF4 in rice breeding. Promoter sequences from the rice Gt1 and maize Ubi genes were selected for this study, representing seed-specific and constitutive expression patterns, respectively. First, both the Gt1 and Ubi promoters were fused with the GUS reporter gene and introduced into rice via agrobacterium-mediated transformation. Tissues from the resulting pGt1::GUS and pUbi::GUS transgenic lines and wild-type rice plants were then stained for histochemical analysis (Figure 2A). The results showed that an intense blue coloration due to the presence of GUS was only detected in the cross-sections of developing seeds for the pGt1::GUS transgenic line, while only very weak or no blue color was observed in the other tissues examined. In the pUbi::GUS transgenic line, strong blue coloration was detected in all test tissues. Furthermore, fluorometric measurements were performed to quantify the exact levels of GUS activity in the same tissues. In general, the quantitative data were consistent with the histochemical staining results (Figure 2B). The quantitative results showed that GUS activity in the developing seeds of the pGt1::GUS transgenic line, including the embryo and endosperm, was considerably higher than in the wild-type control. However, it is worth noting that although the observed GUS activity was also weak in the leaf sheaths and stems of the pGt1::GUS transgenic line, it was significantly higher than that in the wild-type control. Correspondingly, all test tissues of the pUbi::GUS transgenic rice line exhibited very high GUS activities, consistent with the constitutive expression pattern of the reporter gene. Therefore, we found that the Gt1 and Ubi promoters were able to direct dominant expression of the target gene in seeds and constitutive expression in all tissues of rice, respectively,


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which is suitable for our research. Generation and Analysis of OsDWF4-Expressing Transgenic Rice Lines. First, the full-length CDS of OsDWF4 was amplified and cloned into the binary vectors p696G and p696U (Figure 3A and Figure S1A). The p696G and p696U vectors are modifications of the pCAMBIA1300 vector based on insertion of the Gt1 and Ubi promoters to give seed-specific and constitutive expression, respectively. The binary vectors pGt1::OsDWF4 and pUbi::OsDWF4 were constructed and introduced into Y8, an elite japonica rice cultivar from China, via agrobacterium-mediated transformation. We were able to generate 28 pGt1::OsDWF4 and 18 pUbi::OsDWF4 independent transgenic lines. The homozygous transgenic lines were then screened by using both the hygromycin resistance assay and PCR genotyping (Figure S1B and S1C). In the T2 generation, we selected a total of 15 and 8 homozygous transgenic lines for pGt1::OsDWF4 and pUbi::OsDWF4, respectively. To select several representative transgenic lines for further analysis, OsDWF4 expression was examined in the mature seeds of a number of pGt1::OsDWF4 and pUbi::OsDWF4 transgenic lines. Generally, the transcript abundance for endogenous OsDWF4 was only ~1% that of the Ubiquitin-conjugating enzyme E2 (UBC) reference gene in the wild-type control. However, the expression of OsDWF4 in almost all the tested transgenic rice lines driven by the Gt1 or Ubi promoters, was significantly higher than in the control (Figure 3B). Thus, we selected three representative transgenic lines (designated Lines 1 to 3), with similar OsDWF4 expression levels, for both pGt1::OsDWF4 and pUbi::OsDWF4. The relative levels of OsDWF4-specific mRNA were then carefully compared in different tissues of the transgenic plants (Figure 3C). The results showed that the highest level of OsDWF4 expression was in



the seeds of pGt1::OsDWF4 transgenic plants, and was almost 200-fold higher than in seeds of the wild-type control. The expression levels of OsDWF4 in the other tissues of pGt1::OsDWF4 transgenic rice, although much lower than that in the seeds, were still 2- to 9-fold higher than in the control. Also, the expression of OsDWF4 in all tested tissues of the pUbi::OsDWF4 transgenic lines was at least 100-fold higher than in the control, with the highest levels detected in the leaves. Therefore, we successfully overexpressed OsDWF4 in the selected representative transgenic rice lines, and the expression showed distinct spatial patterns, depending on the promoter used. Field Performance of OsDWF4 Transgenic Rice Lines Driven by Different Promoters. Agronomic traits of the selected transgenic lines were examined. Generally, the two different types of transgenic lines exhibited opposite changes in plant height. In comparison with the wild-type control, the pGt1::OsDWF4 transgenic rice plants showed a slight increase in plant height, while the pUbi::OsDWF4 plants were slightly shorter in height (Figure 4A). Further analysis of the internode lengths showed that except for the first internode, the other three internodes of the pGt1::OsDWF4 transgenic plants were longer than those in the wild-type, especially the fourth internode, which was almost twice the length of the control. In the pUbi::OsDWF4 transgenic plants, except for the fourth internode, the lengths of the other internodes, as well as the panicle, were shorter than those of the wild-type (Figure 4B). The quantitative data is shown in Figure 4C. Therefore, the length of the fourth internode is mainly responsible for the differences in plant heights. In addition, we observed that the leaf angles in pGt1::OsDWF4 transgenic plants were slightly larger, and those in pUbi::OsDWF4 plants were much larger, than leaf angles in the wild-type control Y8 (Figure 4A). To further


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confirm the higher leaf angle phenotype, we performed the lamina joint inclination assay, which is one of the most sensitive tests of the BR response in rice.42 The result showed that overexpression of OsDWF4 did indeed enhance bending of the lamina joint, which effect is consistent with BL-treated samples. Moreover, the degree of lamina joint bending in pUbi::OsDWF4 was larger than in pGt1::OsDWF4 plants (Figure 4D and E), which was consistent with the observed plant morphologies (Figure 4A). We then analyzed other agronomic traits of rice, including the length and width of the flag leaves, tiller numbers, grain numbers, seed setting rate, 1000-grain weight, and grain yield per plant (Figure 5A-G). The results showed that OsDWF4 overexpression, when driven by either promoter, resulted in significant reductions in the length and width of flag leaves, but increased tiller number, 1000-grain weight, and per-plant grain yield (Figure 5A-C, F and G). It is noteworthy that grain yield per plant was significantly increased in all the OsDWF4 transgenic rice lines, and was highest in the pGt1::OsDWF4 lines, confirming the potential of OsDWF4 overexpression to enhance grain yield, and demonstrating the importance of selecting a suitable promoter. There were no significant differences observed for grain number and setting rate in the pUbi::OsDWF4 and pGt1::OsDWF4 transgenic lines compared to the wild-type control (Figure 5C and D). Overexpression of OsDWF4 Affects Rice Grain Morphology and Appearance Quality. The results of our grain shape analysis showed that OsDWF4 overexpression slightly increased grain length, but had no effect on grain width (Figure 6A, B, E and F). After the grains were dehusked, the size of the brown rice grains was measured, which showed that the length of the OsDWF4 transgenic rice grains was significantly increased compared to the



control, while the width was unchanged (Figure 6C, D, G and H). The brown rice grains were then further polished and the appearance characteristics of the grains were evaluated by using a rice quality detector (SC-E, Wanshen, China). The data showed that the degree of chalkiness of the OsDWF4 transgenic rice grains was increased to a certain extant, although the difference was not statistically significant due to the large variation. But the chalky rice grain percentage of the OsDWF4 overexpression lines was significantly increased, and was especially serious in the pUbi::OsDWF4 transgenic lines. The results of the seed morphology assay demonstrated that although both seed-specific and constitutive overexpression of OsDWF4 can significantly increase brown rice length, the change in chalky rice grain percentage was much smaller in the pGt1::OsDWF4 lines, suggesting that seed-specific enhancement of BR biosynthesis is an effective way to maintain better rice grain appearance. Overexpression of OsDWF4 Modifies the Expression of BR and GA Metabolism Genes. Since OsDWF4 is one of the key genes involved in BR biosynthesis, the OsDWF4 overexpression lines exhibited typical phenotypes of plants with higher BR levels, including increased lamina joint bending and longer seeds. Previous studies have reported the interaction between BR and GA biosynthesis, and have demonstrated that BR regulates cell elongation by modulating GA metabolism in rice.23 Therefore, qRT-PCR assays were performed to monitor the expression of BR and GA metabolism genes in developing seeds of the OsDWF4 overexpression lines. First, we examined the relative transcription levels of ebisu dwarf (D2) and dwarf 11 (D11), two key genes that encode rate-limiting enzymes in BR biosynthesis. The data showed that the expression of D2 decreased significantly in both the pGt1::OsDWF4 and pUbi::OsDWF4 transgenic lines, while expression of D11 showed a


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significant increase compared to the control (Figure 7A), implying a complex regulation of BR biosynthesis in vivo. Several typical GA metabolism genes that are involved in either GA biosynthesis or deactivation were then selected for expression assays (Figure 7B). Generally, the expression of all four selected GA metabolism genes showed little change in the pGt1::OsDWF4 transgenic lines. However, expression of the GA biosynthesis gene gibberellin 20-oxidase 2 (GA20ox-2) decreased significantly in the pUbi::OsDWF4 transgenic lines, while expression of GA2-oxidase-1 (GA2ox-1) and GA2ox-3, two genes responsible for GA deactivation, showed a smaller (although significant) decrease in the pUbi::OsDWF4 lines. Thus, we hypothesized that OsDWF4 overexpression modulates transcription of both BR and GA metabolism genes, and subsequently affects rice agronomic traits, although the effects are dependent on the OsDWF4 expression pattern. Characterization of Grain Quality and Starch Fine Structure in the Transgenic Rice Lines Overexpressing OsDWF4. Previous studies have mainly focused on potential applications of the BR pathway in improving rice yield. Whether changes in BR biosynthesis or signaling will affect rice qualities, especially those pertaining to cooking and eating, which determine rice market values and consumer acceptance, are still mostly unknown. Therefore, we analyzed the AAC, GC, DSC, and RVA properties of rice flours to assess the effects of OsDWF4 overexpression on rice quality. The results showed that there was no difference in the AAC (Figure 8A), but the GC values were slightly lower in both of the transgenic rice lines (Figure 8B). Moreover, the DSC result showed that the transgenic flour samples had similar gelatinization curves to the control Y8, suggesting that OsDWF4 overexpression had little effect on the thermal properties of rice flours, with only enthalpy of gelatinization (∆H)



declining in all transgenic samples (Figure 8C, D; Table S2). The RVA assay, which shows the pasting properties of rice flour and reflects its apparent viscosity changes during heating and cooling, can be used to predict the texture of cooked rice. Generally, rice flours from both the pGt1::OsDWF4 and pUbi::OsDWF4 transgenic lines shared similar changes in apparent viscosity when compared with the wild-type control flour. In more detail, the viscosity of rice flour from the transgenic lines was enhanced due to the appreciably elevated Peak Viscosity (PKV) (Figure 8E, F), corresponding to potentially improved rice eating and cooking qualities. To further evaluate the effect of OsDWF4 overexpression on starch fine structure, the starch was completely debranched and separated by GPC. The GPC results gave three well-resolved fractions of rice starch (Figure 8G and H), which represent, from left to right, low molecular-weight molecules of amylopectin, higher molecular-weight molecules of amylopectin, and the amylose fraction, respectively. The fine structures of starch from the pGt1::OsDWF4 lines seemed different from the Y8 control, with more low-molecular-weight molecules of amylopectin accumulating in the starch from the pGt1::OsDWF4 transgenic lines (Figure 8G). However, the phenomenon was not observed in starch from the pUbi::OsDWF4 transgenic lines (Figure 8H), implying that distinct expression patterns of OsDWF4 could have different effects on rice eating and cooking qualities. To further explore how OsDWF4 overexpression caused changes in the starch characteristics, we examined the expression of a number of key genes involved in starch biosynthesis. We found that the expression of most of the selected genes, including ADP-glucose pyrophosphorylase small subunit 2b (AGPS2b), Granule-bound starch synthase I (GBSSI), Starch synthase I (SSSI), and Starch synthase III-2 (SSSIII-2), was increased in the pGt1::OsDWF4 transgenic lines


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(Figure 9A). In the pUbi::OsDWF4 plants, the expression of AGPS2b, GBSSI, SSSIII-2, and Starch branching enzyme IIb (SBEIIb) was increased, while expression of Starch branching enzyme I (SBEI) was decreased (Figure 9B). Thus, transcriptional regulation of starch biosynthesis genes by BR could contribute to the observed changes in starch physicochemical properties.

DISCUSSION Although exogenous application of synthetic BR could noticably enhance yield in various plant species, the actual yields were variable, and depended upon the application mode, plant growth stage, and environmental conditions. In addition, the cost for synthesizing BR is high.24 All of these limitations discouraged the application of BR to agriculture. Nevertheless, modulating the endogenous BR pathways by directly manipulating BR biosynthesis or signaling genes could enhance crop yield in an economical and uniform way.43 Moreover, it should be recalled that the success of the Green Revolution was due to the production of crops that are deficient in or insensitive to GA, another growth-promoting phytohormone. Therefore, genetic modulation of BR pathways should be a novel and promising approach to improve crop yield. A number of studies have been carried out to study the effects of BR-related genes on crop yield improvement. In rice, OsDWF4 is one of the most important genes, and it has been extensively studied. Previous studies demonstrated that reducing the level of OsDWF4 expression could enhance rice yield under conditions of high plant density, while increasing its expression could improve per-plant grain yield.30-32 However, other than its potential application in increasing grain yield in rice, it is still unclear whether changes in



OsDWF4 gene expression will affect rice quality, another critical point emphasized in modern rice breeding. Moreover, although it is well accepted that BR regulates a diverse group of important agronomic traits in rice, at present we still can not generate an ideal rice variety with all the BR-controlled traits improved. Therefore, selection of suitable promoters, the critical cis-element in transgenic studies, is a feasible way to optimize the overall effects of BR. OsDWF4 could be expressed at an appropriate level and pattern, thus maintaining the delicate balance of endogenous BR and to ensure superior trait performance. In the present study, the Ubi and Gt1 gene promoters, two commonly used promoters in transgenic rice research,35 were carefully characterized and compared. These particular promoters were selected to improve the endogenous BR content either in the whole plant or mainly in the seeds. Generally, overexpression of OsDWF4 led to consistent changes in a number of agronomic traits except plant height, which increased in the pGt1::OsDWF4 and declined in the pUbi::OsDWF4 transgenic plants. The leaf angles, tiller numbers, brown rice length, 1000-grain weight, chalky rice grain percentage, and grain yield per-plant were all increased, but the length and width of the flag leaf decreased in both transgenic lines. Although most of the agronomic changes were consistent, the distinct spatial expression patterns determined by the Gt1 and Ubi promoters still resulted in some variations in the agronomic traits. For example, the leaf angles and the percentage of chalky rice grains are much larger in the pUbi::OsDWF4 transgenic plants, but seed length is higher in the pGt1::OsDWF4 transgenic plants. Detailed comparisons between the pGt1::OsDWF4 and pUbi::OsDWF4 transgenic rice lines are summarized in Table S3. If we consider the key agronomic traits, including plant height, tiller number, leaf angle,


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seed size, and grain yield, that are involved in rice yield, the overall performance of the pGt1::OsDWF4 transgenic lines was better than for the pUbi::OsDWF4 lines. There are two possible reasons for this: the first is that ectopic expression of a target gene driven by a constitutive promoter often leads to unexpected phenotypes due to the negative effects of accumulated

molecules

on

certain

cellular

processes

or

energy

consumption.44

Condition-dependent or tissue-specific promoters that show high levels of specificity can overcome this disadvantage and direct optimal expression of introduced genes without wasting cellular and metabolic resources.45,46 Therefore, plants in which the expression of OsDWF4 was driven by the seed-specific Gt1 promoter displayed better agronomic performance in general than did the pUbi::OsDWF4 lines. A second possible reason for the difference in performance is that the effect of BR on cell elongation and plant growth depend on concentration. Generally, low concentrations of BR induces, while high concentrations of BR suppresses, the cell elongation process.47 In addition to the seeds, more BR would be expected to accumulate in most other tissues of pUbi::OsDWF4 compared to pGt1::OsDWF4 plants, which could cause the reduced plant height observed in the pUbi::OsDWF4 plants. It is noteworthy that although the Gt1 gene is considered to be expressed in a seed-specific manner, its promoter can also drive target gene expression in some other organs or tissues of rice, including the leaf, leaf sheath, and stems, albeit at lower levels than in the seeds (Figure 2B and 3C). Furthermore, when considering the high sensitivity of BR, it was not unexpected that the leaf angle and plant height in the pGt1::OsDWF4 transgenic plants were also slightly increased. The worldwide demand for high quality rice keeps increasing, especially in terms of the



eating and cooking qualities (ECQs) that determine its acceptance by consumers and also its economic value in export markets. Therefore, improving rice grain quality is a major focus of modern breeding programs.48 Interestingly, we found that the quality of OsDWF4 transgenic rice, especially the ECQ, was improved to a certain extent. In fact, a number of studies have mentioned the regulatory roles of BR in starch metabolism, which determines rice quality. Starch is the major component of rice endosperm; thus the composition and physicochemical properties of the starch will affect rice quality. Schluter et al. (2002) showed that starch content was reduced in the leaves of BR deficient Arabidopsis plants.49 Also, Oh et al. (2011) showed that overexpression of wild-type or modified BRI1 both significantly increased starch content in Arabidopsis leaves.33 In cucumber, the exogenous application of BR was shown to promote the accumulation of sucrose and starch in the leaves.50 In rice, knockdown of D11 or OsBZR1 expression causes reduced seed size and weight, as well as reduced starch accumulation.51 Although these results indicate that modulation of BR biosynthesis or signaling can indeed affect starch accumulation, no systemic analysis has been performed to evaluate the effect of BR on rice quality, which is another important consideration for rice breeders and consumers at present. In general, rice grain quality includes appearance, milling quality, cooking and eating quality, and nutritional quality. In the present study, although the overexpression of OsDWF4 did not change grain AC, it did affect the pasting property of the rice starch, as evaluated by the RVA test. In general, several RVA parameters changed in response to OsDWF4 overexpression, such as increased PKV, Hot Paste Viscosity (HPV), Cool Paste Viscosity (CPV), and Breakdown (BDV) and decreased Setback Value (SBV), implying that the ECQ of the transgenic rice was improved. In addition, GC and DSC also


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showed some minor changes between the transgenic lines and the wild-type control. Although consistent improvements were observed in most rice quality parameters for both groups of OsDWF4 transgenic lines, there were some variations. For example, there are less chalky rice grains in the pGt1::OsDWF4 transgenics. Chalkiness is a key parameter that could influence both rice processing quality and appearance quality. During grain processing, chalky grains crack easily and thus the head rice yield (HRY) is reduced.52 Also, consumers prefer to choose high quality rice with less chalkiness.53 Therefore, chalky rice will lead to a remarkable financial loss to both rice farmers and traders. Some factors, including high temperature and grain shape, will affect the formation of chalky grain.54, 55 Noteworthy, if the rice grain becomes slender, that is with a large length-to-width ratio, the chalkiness degree of rice will decrease.56, 57 Therefore, it should be an effective way to overcome the chalkiness drawback of OsDWF4 transgenic rice by modulating its grain shape. In fact, the seed length of pGt1::OsDWF4 grain is a little longer than that of the pUbi::OsDWF4 lines. Furthermore, variation in the chain length distribution of debranched starch was only detected in the endosperm of pGt1::OsDWF4 rice, which might be one possible reason for the differences in the physicochemical properties of pGt1::OsDWF4 vs. pUbi::OsDWF4 transgenic rice. In conclusion, we have demonstrated that overexpression of OsDWF4 has generally positive effects on both rice yield and ECQs. In addition, our study highlighted the fact that it is better to use a seed-specific promoter than a constitutive promoter to drive OsDWF4 expression. Therefore, genetic modulation of BR biosynthesis by choosing the appropriate promoter(s) will be valuable in the breeding of elite rice varieties with significant improvements in both grain yield and quality.



AUTHOR INFORMATION Corresponding Author *(Q.Q. Liu) College of Agriculture, Yangzhou University, Yangzhou 225009, China. Phone: +86 514 8797 9242. Email: [email protected]. ORCID Qiao-Quan Liu: 0000-0001-5543-5798 Notes The authors declare no competing financial interest.

ABBREVIATIONS USED AAC, apparent amylose content; AGPS2b, ADP-glucose pyrophosphorylase small subunit 2b; ANOVA, analysis of variance; BDV, Breakdown; BES1, bri1-EMSSUPPRESSOR 1; BRI1, BR INSENSITIVE 1; BZR1, BRASSINAZOLE RESISTANT1; CaMV, cauliﬂower mosaic virus; CPD, constitutive photomorphogenesis and dwarfism; CPV, Cool Paste Viscosity; D2, ebisu dwarf; D11, dwarf 11; DET2, de-etiolated-2; DSC, differential scanning calorimeter; DWF4, dwarf4; ECQs, eating and cooking qualities; GA, gibberellin; GA2ox-1, GA2-oxidase-1; GA20ox-2, gibberellin 20-oxidase 2; GBSSI, granule-bound starch synthase I; GC, gel consistency; GPC, Gel permeation chromatography; Gt1, glutelin 1; GUS, beta-glucuronidase; HPT, hygromycin resistance gene; HPV, Hot Paste Viscosity; PaT, paste temper; PeT, peak time; PKV, Peak Viscosity; RVA, Rapid Visco Analyzer; SBEI, Starch branching enzyme I; SBEIIb, Starch branching enzyme IIb; SBV, Setback Value; SD, standard deviation; SSSI, Starch synthase I; SSSIII-2, Starch synthase III-2; Tc, conclusion


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temperature; tNos, Nopaline synthase terminator; To, onset temperature; Tp, peak temperature; UBC, Ubiquitin Conjugase; Ubi, ubiquitin; WT, wild-type; ∆H, enthalpy of gelatinization;

FUNDING This study was financially supported by the Ministry of Agriculture of China (2016ZX08009003-004), the National Natural Science Foundation of China (31601275 and 31771745), Guangdong Provincial Key Laboratory of Applied Botany Foundation (AB2016004), the China Postdoctoral Science Foundation (2014M560450), Jiangsu Provincial Department of Education of China (17KJA210001, PAPD and Qinglan Projects), the Innovative and Entrepreneurial Talent of Jiangsu province and the Top Talent Supporting Program of Yangzhou University.

SUPPORTING INFORMATION DESCRIPTION Generation of OsDWF4 transgenic rice plants and screening of homozygous lines (Figure S1). PCR primers (Table S1), parameters of the DSC of rice flours (Table S2) and a summary of the comparison between pGt1::OsDWF4 and pUbi::OsDWF4 transgenic rice lines (Table S3).

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FIGURE CAPTIONS Figure 1. Expression of OsDWF4 in various organs and developing seeds of the japonica rice cultivar ‘Nipponbare’. (A) Organ-specific expression of OsDWF4 in wild-type rice relative to the expression of UBC. (B) Expression profile of OsDWF4 in developing seeds of ‘Nipponbare’ relative to UBC from 0 to 20 days after flowering (DAF). Total RNA was isolated from young seedling shoots, young differentiated panicles (Panicles-1), panicles 4 days after heading (Panicles-2), stems, leaf blades, leaf sheaths, and developing seeds 0, 3, 5, 7, 10, 15, and 20 DAF. qRT-PCR was performed and the UBC gene was used as an internal reference for normalization of gene expression. Error bars represent the SDs and the different lower case letters indicate statistically significant differences at P

Seed-Specific Expression of OsDWF4, a Rate-Limiting Gene Involved

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