水稻蛋白贮存中伴侣蛋白的作用

∗ Corresponding author: E-mail, takaiwa@nias.affrc.go.jp ; Fax, + 81-29-838-8397 . Plant Cell Physiol. 50(8): 1532–1543 (2009) doi:10.1093/pcp/pcp098, available online at www.pcp.oxfordjournals.org © The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org Seed storage proteins are specifi cally and highly synthesized during seed maturation and are deposited into protein bodies (PBs) via the endoplasmic reticulum (ER) lumen. The accumulation process is mediated by ER chaperones such as luminal binding protein (BiP) and protein disulfi de isomerase (PDI). To examine the role of ER chaperones and the relationship between ER chaperones and levels of accumulation of seed storage proteins, we generated transgenic rice plants in which the rice BiP and PDI genes were overexpressed in an endosperm-specifi c manner under the control of the rice seed storage protein glutelin promoter. The seed phenotype of the PDI-overexpressing transformant was almost identical to that of the wild type, whereas overexpression of BiP resulted in transgenic rice seed that displayed an opaque phenotype with fl oury and shrunken features. In the BiP-overexpressing line, the levels of accumulation of seed storage proteins and starch contents were signifi cantly lower compared with the wild type. Interestingly, overproduction of BiP in the endosperm of the transformant not only altered the morphological structure of ER-derived PB-I, but also generated unusual new PB-like structures composed of a high electron density matrix containing glutelin and BiP and a low electron density matrix containing prolamins. Notably, polysomes were attached around the aberrant PB-like structures, indicating that this aberrant structure is an ER-derived PB-I derivative. These results suggested that the PB-like structure may be formed in the ER lumen, resulting in inhibition of translation, folding and transport of seed proteins. Keywords: BiP • Chaperone proteins • ER stress • PB • PDI • Quality control • Storage proteins . Abbreviations : BiP , binding protein ; BSA , bovine serum albumin ; CBB , Coomassie Brilliant Blue ; DAF , days after fl owering ; ER , endoplasmic reticulum ; GluB-1 , rice glutelin B-1 ; PB-I/II , protein body type I/II ; PBS , phosphate-buffered saline ; PDI , protein disulfi de isomerase ; PSV , protein storage vacuole ; RT–PCR , reverse transcription–PCR ; SEM , scanning electron microscopy ; TEM , transmission electron microscopy ; UPR , unfolded protein response. Introduction Transgenic plants are being used as bioreactors for the pro- duction of pharmaceutical proteins and industrial enzymes. These high value products have been synthesized in leaves ( Abranches et al. 2005 ), cell cultures ( Hellwig et al. 2004 ) and storage organs, such as seeds and tubers ( Fischer et al. 2004 , Stoger et al. 2005 , Takaiwa et al. 2007 ). When transgenic plants are used as a production platform as an alternative to conventional fermentation systems, the important issue is to enhance yields in the plants. To achieve this, it is necessary to optimize several factors required for high levels of expres- sion ( Streatfi eld 2007 , Takaiwa 2007 ). For instance, selection of a strong promoter whose expression is tissue specifi c in plants may be more advantageous than selection of a consti- tutive promoter. Furthermore, addition of the 5 ′ - and 3 ′ - untranslated regions to the transgene is necessary to stabilize Overexpression of BiP has Inhibitory Effects on the Accumulation of Seed Storage Proteins in Endosperm Cells of Rice Hiroshi Yasuda 1 , 2 , Sakiko Hirose 1 , 3 , Taiji Kawakatsu 1 , Yuhya Wakasa 1 and Fumio Takaiwa 1 , ∗ 1 Transgenic Crop Research and Development Center, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki, 305-8602 Japan 2 Research Team for Crop Cold Tolerance, National Agricultural Research Center for Hokkaido region, Hitsujigaoka 1, Toyohira-ku, Sapporo, Hokkaido, 062-8555 Japan 3 Rice Biotechnology Research Team, National Institute of Crop Sciences, Kannondai 2-1-18, Tsukuba, Ibaraki, 305-8518 Japan 1532 Plant Cell Physiol. 50(8): 1532–1543 (2009) doi:10.1093/pcp/pcp098 © The Author 2009. Regular Paper at China Academ y of Agricultural Sciences on M arch 20, 2010 http://pcp.oxfordjournals.org D ow nloaded from the mRNA, and codon optimization for the target tissue is a critical factor for boosting expression ( Green 1993 , Richter et al. 2000 ). Subcellular localization also has a signifi cant infl uence on the yield of recombinant products ( Yasuda et al. 2006 ). When the recombinant proteins are expressed as secre- tory proteins by adding a signal peptide at the N-terminus to transport them into the lumen of the endoplasmic reticulum (ER), the proteins accumulate to higher levels than proteins lacking a signal peptide, even if the transcripts are similarly abundant ( Takagi et al. 2005 ). The addition of the KDEL tet- rapeptide as an ER retention signal at the C-terminus of recombinant proteins is also effective for obtaining higher levels of accumulation than recombinant proteins lacking a KDEL ( Wandelt et al. 1992 , Stoger et al. 2000 ). These results indicated that entry of recombinant proteins into the secre- tory pathway and retention by the ER permits higher levels of accumulation, and the ER serves as a storage compart- ment for recombinant proteins in specifi c cells. The ER has many functions, such as entry into the secre- tory pathway, folding, assembly, glycosylation and transport of nascent proteins, sequestration of calcium, and lipid syn- thesis and storage. One of the most important functions of the ER is quality control of nascent proteins ( Galili et al. 1998 ), which is accomplished by ER chaperone proteins such as luminal binding protein (BiP) and protein disulfi de isomerase (PDI) in the ER lumen. The gene encoding BiP has been isolated from maize, rice, Arabidopsis and pumpkin. Several studies have demonstrated that BiPs are involved in the synthesis of high levels of storage proteins and stress responses ( Koizumi 1996 , Hatano et al. 1997 , Muench et al. 1997 ). The ER chaperones are implicated in not only assisting in the folding and assembling of nascent proteins but also in post-translational regulation. Therefore, when foreign pro- tein genes were highly expressed as secretory proteins in transgenic plants, synthesis of ER-resident chaperone pro- teins increased to assist with the folding and assembly of for- eign proteins ( Nuttall et al. 2002 ). We have also recently observed that BiP and PDI levels were enhanced about 4- and 3-fold, respectively, in ER-derived protein bodies (PBs) in transgenic rice seed by high accumulation (40–60 µg grain –1 ) of an artifi cial 7Crp peptide composed of seven T-cell epitopes derived from cedar pollen allergens ( Takaiwa et al. 2009 ). Therefore, given that the direct participation of chaperone proteins BiP and PDI in the active folding of pro- teins in the ER can be one of the key factors for determining production levels of foreign proteins, alleviation of ER stress by overexpression of these chaperones in the targeted tissue may lead to yield enhancement of foreign products. Practi- cally, improvement in the folding and secretion effi ciency by overexpressing chaperone proteins and resulting increases in accumulation levels of foreign proteins were previously reported in tobacco ( Leborgne-Castel et al. 1999 ), yeast ( Smith et al. 2004 , Zhang et al. 2006 ), insect cells ( Kato et al. 2005 ) and mammalian cells (Chung et al. 2003). Thus, levels of accumulation of recombinant proteins in rice seed are expected to be enhanced by artifi cially manipulating ER chaperone levels. In preparation for this purpose, we fi rst generated transgenic rice overexpressing BiP or PDI under the control of a seed storage protein promoter to examine the relationship between chaperones and levels of accumu- lation of endogenous seed storage proteins in rice, and whether production of seed storage proteins in endosperm cells could be enhanced by controlling ER chaperone levels. Unexpectedly, levels of the seed proteins were not enhanced in either transformant. Although the kernel phenotype of the PDI-overexpressing transformant was nearly identical to that of the wild type, the kernels of the BiP-overexpressing transformant exhibited fl oury and shrunken features. In addition, synthesis of seed storage proteins and starch was also severely suppressed in BiP-overexpressing transformants compared with the wild type. Thus, extreme overexpression of BiP had a considerable inhibitory effect on seed develop- ment and deposition of storage proteins into PBs. Results Production of transgenic rice plants overexpressing PDI and BiP chaperones In order to elucidate the role of ER chaperones in rice endosperm, transgenic rice plants were generated in which rice BiP and PDI cDNAs were overexpressed under the con- trol of the endosperm-specifi c glutelin GluB-1 promoter ( Fig. 1 ). For the BiP- and PDI-overexpressing constructs, 30 and 35 independent transformants were produced, respec- tively. The highest expression line of both constructs was screened by immunoblot analysis using anti-BiP and anti- PDI antibodies on blots of total proteins extracted from mature seeds of individual transformants (data not shown). To obtain homozygous lines, transformants were advanced at least two generations (T 3 ) by self-crossing; inheritance of phenotype and gene transfer were confi rmed in the progeny. The highest BiP-overexpressing line exhibited a semi-dwarf phenotype and the fertility was slightly reduced. The semi-dwarf phenotype and the low fertility in the BiP- overexpressing transformants might result from growth retardation of the seedling at the germination and seedling stages due to an insuffi cient supply of nutrients or an inhibi- tory effect on growth by leaky expression of BiP in leaf and stem. The latter possibility was excluded by analysis of reverse transcription–PCR (RT–PCR) using total RNAs from various tissues, because the exogenous BiP was specifi cally expressed in maturing seed and was not expressed in other tissues under the control of the glutelin GluB-1 promoter (Supplementary Fig. S1). Furthermore, it was shown that 1533 Overproduction of BiP and PDI in rice seed Plant Cell Physiol. 50(8): 1532–1543 (2009) doi:10.1093/pcp/pcp098 © The Author 2009. at China Academ y of Agricultural Sciences on M arch 20, 2010 http://pcp.oxfordjournals.org D ow nloaded from the inhibitory effect on plant height was observed in other transgenic lines exhibiting the severe kernel phenotype (data not shown). These results indicate that the semi-dwarf phe- notype is not caused by non-specifi c weak expression of exogenous BiP in vegetative tissue, but by a poor level of nutrient stock in rice grains required for seedling growth because of abnormal seed. In contrast, growth of the highest PDI-overexpressing line was almost identical to that of the wild type. Fig. 2 shows the overall phenotype ( Fig. 2A–C ) and transverse section ( Fig. 2D–F ) of the kernels of the BiP- and PDI-overexpressing transformant. The kernel of the BiP-overexpressing line was opaque in appearance with fl oury and shrunken features ( Fig. 2B, E ). In contrast, the kernel of the PDI-overexpressing transformant was hardly different in morphological struc- ture from that of the wild type ( Fig. 2C, F ). Starch and total protein contents in the kernels of the BiP-overexpressing transformant Since the kernels of the BiP-overexpressing transformant exhibited fl oury and shrunken features, we investigated a few properties of mature seed of the transformant. The dry weight of 100 grains of the transformant was reduced to about half of that of the wild type ( Fig. 3A ). Starch and pro- tein contents per grain of the transformants were also reduced to approximately 40 and 50 % of those of the wild type, respectively ( Fig. 3B, C ). Observations of starch granules in the kernel of the BiP-overexpressing transformant Since the starch content was signifi cantly lower in the kernel of the BiP-overexpressing transformant ( Fig. 3B ), we observed starch granules in the kernels of the transformant using scanning electron microscopy (SEM). In the wild type, the sectioned endosperm appeared to be tightly packed ( Fig. 4A ). This tight structure was refl ected in the structure of isolated starch granules that were polygonal, of similar size with sharp edges ( Fig. 4C ). In contrast, the endosperm in the BiP-overexpressing transformant appeared to be a loosely packed and fragile structure ( Fig. 4B ), and the iso- lated starch granules had rounded edges ( Fig. 4D ). Further- more, the sizes of starch granules in the BiP-overexpressing transformant were different from each other ( Fig. 4B, D ). These morphological changes in the starch granules may be one of the reasons for the fl oury and shrunken features of the BiP-overexpressing transformant. Effect of overexpression of ER chaperones on accumulation of seed storage proteins To investigate whether there are any effects on accumula- tion of seed proteins by overexpression of BiP and PDI ER chaperones, total seed proteins extracted from mature seed were subjected to SDS–PAGE or immunoblot analysis ( Fig. 5 ). In mature kernels of the highest BiP-overexpressing trans- genic rice, a marked increase in BiP level was detected as a distinctly visible band by Coomassie brilliant blue (CBB) staining on SDS–PAGE ( Fig. 5A ). Additional bands with lower molecular weights (approximately 25 kDa and smaller) result- ing from degradation of the 75 kDa intact BiP were detected by immunoblot analysis using anti-BiP serum ( Fig. 5B , BiP, lane 2). The BiP level in the transgenic line was 14.7-fold higher than that of the wild type. As shown in Fig. 5 , expres- sion of seed storage proteins in the developing seed signifi - cantly decreased as a whole, except for the glutelin GluC precursor ( Fig. 5B ). This result suggests that the effect on folding and assembly of glutelins by high amounts of BiP may be a bottleneck in traffi cking or deposition of storage proteins into PB-IIs, resulting in enhancement of glutelin pGPTV-HPT vector Hind III pAg7 LB EcoRI RB 1.4k GluB-1-P Nco I Nos-TBiP PDI1.4k GluB-1-P Nos-T SacI HPT 35S-P Fig. 1 Diagram of transgene constructs used in this experiment. LB, left border; pAg7, gene 7 terminator; HPT, hygromycin phosphotransferase; 35S-P, caulifl ower mosaic virus 35S promoter; 1.4 k GluB-1-P, 1.4 kb glutelin GluB-1 promoter; Nos-T, nopaline synthase terminator; RB, right border. 1534 H. Yasuda et al. Plant Cell Physiol. 50(8): 1532–1543 (2009) doi:10.1093/pcp/pcp098 © The Author 2009. at China Academ y of Agricultural Sciences on M arch 20, 2010 http://pcp.oxfordjournals.org D ow nloaded from precursors in the ER and a decrease in mature glutelin acidic and basic subunits ( Fig. 5A , lane 2). Furthermore, accumula- tion of the 26 kDa globulin, 13 kDa cysteine-poor prolamin and 16 kDa prolamin was also severely suppressed in the transformant ( Fig. 5B , lane 2). On the other hand, overex- pression of BiP had little effect on expression of the other ER chaperone, PDI ( Fig. 5B , PDI, lane 2). In mature seeds from the highest PDI-overexpressing transgenic rice plant, the amount of PDI was 4.7-fold higher than that of the wild type ( Fig. 5B , PDI, lane 3). It is interest- ing to note that overexpression of rice PDI had little effect on the overall expression pattern of seed proteins including BiP ( Fig. 5A, B , lane 3) except for a slight decrease in the 13 kDa cysteine-poor prolamin and a slight increase in the 10 and 16 kDa prolamins. These results indicate that overpro- duction of PDI has little detrimental effect on seed development. Electron microscopy of maturing subaleurone cells in the BiP-overexpressing transformants Since the accumulation of seed storage protein per grain decreased in the BiP-overexpressing transformant ( Figs. 3, 5 ), we decided to observe intracellular structures of the matur- ing subaleurone cells in the transformant [15–20 days after fl owering (DAF)] using transmission electron microscopy 2500 2000 1500 1000 500 0 D ry w ei gh t (m g) /10 0 g rai ns 25 20 15 10 5 0 St ar ch (m g) /gr ain 3.0 2.5 2.0 1.5 1.0 0.5 0 Pr ot ei n (m g) /gr ain A B C Fig. 3 Comparison of dry weight (A), starch content (B) and total protein content (C) of mature kernels from the wild type (yellow box) and the BiP-overexpressing transformant (violet box). A B C D E F Fig. 2 Observation of surface (A–C) and transverse sections (D–F) of the wild type (A and D) and BiP- (B and E) and PDI-overexpressing transformants (C and F). Mature kernels from the transformants and wild type were harvested and hand-sectioned with a razor blade for the transverse sections. Bars = 1 mm. 1535 Overproduction of BiP and PDI in rice seed Plant Cell Physiol. 50(8): 1532–1543 (2009) doi:10.1093/pcp/pcp098 © The Author 2009. at China Academ y of Agricultural Sciences on M arch 20, 2010 http://pcp.oxfordjournals.org D ow nloaded from A B B C D Fig. 4 Morphology of hand-sectioned endosperm surface and starch granules of the wild type (A and C) and the BiP-overexpressing transformant (B and D). (A) and (B) SEM of hand-sectioned endosperm surface. (C) and (D) SEM of starch granules. Bars: 200 µm in (A) and (B), 10 µm in (C) and (D). A 1 2 3 1 2 3 1 2 3M 150 75 50 37 25 20 15 BiP glutelins acidic subunit glutelins basic subunit 13kD prolamins globulin glutelins precursor B BiP PDI GluA 26kD globulin 16kD prolamin GluB 13kD Cys-rich prolamin 10kD prolamin precursor acidic subunit GluC 13kD Cys-poor prolamin precursor acidic subunit 75kD 25kD precursor 60kD (kD) acidic subunit Fig. 5 Comparison of seed storage proteins and ER chaperone proteins (BiP and PDI) in mature kernels of the wild type (lane 1) and BiP- (lane 2) and PDI- (lane 3) overexpressing transformants. Protein extracts were separated by SDS–PAGE and stained with CBB (A) or transferred to a PVDF membrane and incubated with antibodies directed against seed storage proteins or ER chaperone proteins, respectively (B). 1536 H. Yasuda et al. Plant Cell Physiol. 50(8): 1532–1543 (2009) doi:10.1093/pcp/pcp098 © The Author 2009. at China Academ y of Agricultural Sciences on M arch 20, 2010 http://pcp.oxfordjournals.org D ow nloaded from (TEM). In the endosperm cells of the wild type, there are two types of typical PBs within the same cell; one is the ER- derived protein body called PB-I. The other is the protein storage vacuole (PSV), called PB-II, in which seed storage proteins are deposited via the secretory pathway through the Golgi bodies and/or via a direct membrane traffi cking route through PAC (precursor-accumulating) vesicles ( Tanaka et al. 1980 , Krishnan et al. 1986 , Takaiwa et al. 1999 , Takahashi et al. 2005 ). The former structure is a spherical, 1–2 µm in diameter, and electron-lucent PB containing sev- eral types of prolamins (10, 13 and 16 kDa), whereas the latter,