Tapetum Degeneration Retardation is Critical for Aliphatic Metabolism and Gene Regulation during Rice Pollen Development

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1 Molecular Plant Volume 1 Number 4 Pages July 2008 RESEARCH ARTICLE Tapetum Degeneration Retardation is Critical for Aliphatic Metabolism and Gene Regulation during Rice Pollen Development Da-Sheng Zhang a,2, Wan-Qi Liang a,2, Zheng Yuan a,nali a, Jing Shi a, Jue Wang a, Yu-Min Liu b, Wen-Juan Yu b and Da-Bing Zhang a,1 a Shanghai Jiao Tong University Shanghai Institutes for Biological Sciences Pennsylvania State University Joint Center for Life Sciences, School of Life Science and Biotechnology, Key Laboratory of Microbial Metabolism, Ministry of Education, Shanghai Jiao Tong University, Shanghai , China b Instrumental Analysis Center of Shanghai Jiao Tong University, Shanghai , China ABSTRACT As a complex wall system in flowering plants, the pollen outer wall mainly contains aliphatic sporopollenin; however, the mechanism for synthesizing these lipidic precursors during pollen development remains less well understood. Here, we report on the function of the rice tapetum-expressing TDR (Tapetum Degeneration Retardation) gene in aliphatic metabolism and its regulatory role during rice pollen development. The observations of transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analyses suggested that pollen wall formation was significantly altered in the tdr mutant. The contents of aliphatic compositions of anther were greatly changed in the tdr mutant revealed by GC MS (gas chromatography mass spectrometry) testing, particularly less accumulated in fatty acids, primary alcohols, alkanes and alkenes, and an abnormal increase in secondary alcohols with carbon lengths from C29 to C35 in tdr. Microarray data revealed that a group of genes putatively involved in lipid transport and metabolism were significantly altered in the tdr mutant, indicating the critical role of TDR in the formation of the pollen wall. Also, a wide range of genes (236 in total 154 up-regulated and 82 down-regulated) exhibited statistically significant expressional differences between wild-type and tdr. In addition to its function in promoting tapetum PCD, TDR possibly plays crucial regulatory roles in several basic biological processes during rice pollen development. Key words: tapetum; pollen wall; aliphatic metabolism; microarray; gene expression; rice. INTRODUCTION Rice (Oryza sativa) is one of the most important crops in the world with a significantly smaller genome size, and it has become an excellent monocot model for genetic, molecular, and genomic studies (IRGSP, 2005). In rice breeding, the fertility of pollen grains is critical for rice yield, particularly for hybrid rice production. Rice pollen is produced during anther development that is a critical process in the lifecycle of sexual plants (Scott et al., 2004; Itoh et al., 2005). Briefly, anther primordial cells generate meiocytes and four-lobed cell layers, including the epidermis, endothecium, middle layer, and tapetum. After meiosis, microspores are released from tetrads, at which the tapetum is thought to function as a nutrient source for developing microspores (Ma, 2005). The mature pollen wall is a multilayered structure with a pectocellulosic intine surrounded by a sporopollenin-based exine, which itself contains two layers the inner nexine and the outer sexine (Scott et al., 2004). Sporopollenin is one of the complex biopolymers that confer the exine high resistance to biological and environmental stresses (Meuter-Gerhards et al., 1999), and experimental evidence has proved that sporopollenin consists mainly of fatty acid derivatives and phenolic compounds (Piffanelli et al., 1998; Blackmore et al., 2007). At the earlier pollen developmental stage, the majority of exine constituents are derived from tapetum, which secretes lipidic precursors to the pollen surface, leading to the formation of sculptured exine. At the later pollen developmental stage, tapetum produces mainly lipidic components of pollen coat/tryphine, which are deposited into exine cavities (Piffanelli et al., 1998). During the past few years, there have been considerable achievements in pollen development and elucidation of the 1 To whom correspondence should be addressed. zhangdb@sjtu. edu.cn, fax , tel These authors contributed equally to this work. ª The Author Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS. doi: /mp/ssn028, Advance Access publication 23 June 2008 Received 5 February 2008; accepted 4 April 2008

2 600 Zhang et al. d TDR Controls Rice Anther Aliphatic Metabolism interaction between the sporophytic tapetum and the developing pollen wall using molecular, genetic, ultrastructural, and biochemical approaches. For instance, the MALE STERILITY 2 (MS2) protein as one putative fatty acyl reductase is predicted to be able to convert fatty acids to fatty alcohols (Aarts et al., 1997). DEFECTIVE EXINE1 (DEX1) encodes a novel membrane-associated protein, and, in the dex1 mutant, primexine deposition is delayed and significantly reduced (Paxson- Sowders et al., 1997, 2001). NO EXINE FORMATION 1 (NEF1) is one putative plastid membrane-integrated protein, and, in the nef1 mutant, sporopollenin fails to be deposited within the locule and on the locule wall (Ariizumi et al., 2004). The FACELESS POLLEN-1 (FLP1) is a putative lipid transfer protein and the flp1 pollen surface is nearly smooth and a reticulate pattern (Ariizumi et al., 2003). CalS5 encodes a callose synthase, which is responsible for the synthesis of callose deposited at the primary cell wall of meiocytes, tetrads, and microspores (Dong et al., 2005). The rice Wax-Deficient Anther1 (WDA1) gene, which is preferentially expressed in anther epidermal cells, is likely involved in the biosynthesis of very-long-chain fatty acids and in the establishment of pollen exine (Jung et al., 2006). Additionally, several transcription factors have been reported to affect pollen wall development. One is MALE STE- RILITY 1 (MS1), which is one putative nuclear protein with a PHD-finger motif, and may function by regulating the transcription of tapetal genes involved in pollen wall development (Wilson et al., 2001; Ito and Shinozaki, 2002; Vizcay-Barrena and Wilson, 2006; Ito et al., 2007; Yang et al., 2007). Dysfunctional tapetum1 (DYT1) encoding a putative bhlh transcription factor has been proved to function in pollen wall formation at the early developmental stage (Zhang et al., 2006). Previously, we reported the isolation and characterization of a rice male developmental gene named Tapetum Degeneration Retardation (TDR), which is preferentially expressed in tapetum and encodes a putative basic helixloop-helix protein. We proved that TDR controls rice tapetum development and degeneration by positively regulating tapetum programmed cell death (PCD). Moreover, in the tdr mutant, no obvious exine deposition was observed at the early young microspore stage (Li et al., 2006). In this study, we further characterized the developmental defects of the microspore exine and anther outer surface in the tdr mutant by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) observations. In agreement with these observations, we observed the changed contents of aliphatic compositions in the tdr anther by gas chromatography mass spectrometry (GC MS) testing. Also, global expressional analysis of tdr revealed that approximately 236 genes exhibited statistically significant expressional differences between wild-type and tdr during pollen development. In particular, we observed that the expression of 28 genes putatively related to aliphatic metabolism was greatly changed, implying that TDR likely acts as one key regulator of lipidic metabolism during rice pollen development. RESULTS AND DISCUSSION TDR Controls Pollen Wall Development Previously, we proved that TDR is strongly expressed in tapetum from the meiosis stage to the young microspore stage and the tdr plant seems normal in vegetative and floral development but fails to produce any viable pollen, with complete male sterility. The main morphological defect in the tdr mutant is that the tapetum seems to be abnormally expanded without degradation and the microspores collapse at the later stages. The mechanism of TDR regulating tapetum development and degradation is likely by positively triggering programmed cell death (PCD) of tapetal cells during pollen development (Li et al., 2006). To better understand the role of TDR during pollen development, we performed a more detailed analysis of tdr mutants. Consistent with previous observations, at the early tetrad stage, we observed no significant differences in the anther wall layers and microspores between the wild-type and the tdr mutant (Figure 1A and 1C). In Cosmos bipinnatus, after the completion of meiosis (at the beginning of the tetrad stage), primexine (a microfibrillar matrix consisting mainly of cellulose) starts deposition within the microspore (Blackmore and Barnes, 1985), and it is proved that primexine serves as an elaborate template patterning the deposition of sporopollenin precursors and their following polymerization (Heslop-Harrison, 1968). In wild-type and tdr, we observed that the primexine matrix or glycoclayx was about nm thick outside the microspore plasma membrane (Figure 1B and 1D). At the young microspore stage in wild-type anthers, microspores were released from tetrads, tapetal cells had deeply stained cytoplasm but no longer had large vacuoles, and the middle layer was hardly visible (Figure 1E). Also, the TEM data revealed that the microspores developed electron-dense materials, namely probaculae and protectum (Figure 1F, arrow indicated), generated within the primexine as sporopollenin deposition; the microspore plasma membrane also seemed undulating. During formation of the outermost layer of primexine, the tectum accumulates more sporopollenin. It is obvious that the conversion of primexine into exine is largely through the polymerized sporopollenin; however, the mechanism of this process is not clear (Edlund et al., 2004; Scott et al., 2004). In contrast, the tdr tapetal cells continued to expand and remained vacuolated, the middle layers were still clearly visible, and no obvious exine formation was observed in the tdr microspore only in the primexine structure outside the microspore with undulating plasma membrane (Figure 1G and 1H). From the end of the tetrad stage to the mitosis stage is the free microspore stage, in which the morphology of the exine is mainly complete, and the tapetum is very active in metabolism, with significant changes. Previously, we proved that the tapetum PCD in rice anther is initiated from the tetrad stage, then the tapetal cells degenerate, whereas the tdr tapetum is abnormally expended, with

3 Zhang et al. d TDR Controls Rice Anther Aliphatic Metabolism 601 Figure 1. Transmission Electron Microscopy Analyses of Pollen Walls and Scanning Electron Microscopy Analyses of tdr. (A) Normal developing tetrads encased by callose wall in wild-type at the tetrad stage. (B) Magnified image of microspore wall showing the primexine at the tetrad stage in wild-type. (C) The developing tetrads encased by callose wall in tdr at the tetrad stage. (D) Magnified image of microspore wall structure at the tetrad stage in the tdr mutant. (E) At the young microspore stage, wild-type tapetal cells became collapsed in conjunction with the dissolution of callose walls for release of the developing microspores. (F) The exine of microspore was established with distinct primexine and probaculae layers in wild-type pollen at the young microspore stage. (G) The tdr tapetal cells became enlarged with large vacuoles and did not degenerate and result in an abnormal microspore in shape at the young microspore stage. (H) Magnified image of the delayed formation of exine structure at the young microspore stage in the tdr mutant. (I) The wild-type developed microspores with normal pollen wall at the vacuolated pollen stage. (J) Magnified image of pollen wall structure at the vacuolated pollen stage in wild-type. Pollen exine configuration formed approximately with distinct tectum, bacula, and nexine layers. (K) The tdr mutant showed overly expanded tapetum with more irregular microspores at the vacuolated pollen stage. (L) In the tdr mutant, the persistent primexine with sexine and nexine but no sporopollenin or pollen coat deposition was observed at the vacuolated pollen stage. (M) The wild-type anther showed degenerated tapetum and vacuolated microspores at the mature pollen stage. (N) Magnified image of incrassated pollen wall structure at the mature pollen stage in the wild-type.

4 602 Zhang et al. d TDR Controls Rice Anther Aliphatic Metabolism significantly delayed degradation (Li et al., 2006). It is known that the tapetal cells have the ability to synthesize sporopollenin precursors for exine patterning (Piffanelli et al., 1998). At the vacuolated pollen stage, the wild-type formed pollen exine with nearly distinct tectum, bacula, and nexine layers, and intine (Figure 1I and 1J), whereas, in the tdr mutant, only the preliminary structure of sexine and nexine without sporopollenin or pollen coat deposition was observed (Figure 1K and 1L, arrow indicated). At the mature pollen stage, more sporopollenin accumulated on the pollen wall in wild-type (Figure 1M and 1N). On the contrary, in the tdr mutant, the extremely expanded tapetal cells occupied the majority of the locule, microspores began to collapse and the pollen wall persisted with remnant thinner layers and sporadically distributed irregular sporopollenin deposition (Figure1O and 1P, arrow indicated). It is known that sporopollenin is largely made up of a series of related polymers derived from very-long-chain fatty acids (VLCFAs) plus more modest numbers of oxygenated aromatic rings and phenylpropanoids (Piffanelli et al., 1998). Thus, we speculate that TDR possibly functions in regulating the synthesis of lipidic precursors during rice pollen wall development. Anther epidermis is covered with a layer of wax, protecting the anther from various environmental stresses, and cuticular wax components predominantly comprise long-chain aliphatic compounds derived from VLCFAs (Kunst and Samuels, 2003). Varnier et al. (2005) proposed that anther PCD is a progressive process, initially being triggered in the tapetum, and the radial extension of PCD in the anther cell layers is from the tapetum to the peripheral layers in Lilium, which suggests that the lack of PCD in the tdr tapetum could further impact on the other somatic anther cell layers (Varnier et al., 2005). To study whether TDR affects the development of anther cuticular wax, scanning electron microscopy (SEM) observation was performed. At the young microspore stage, there were no obvious differences in the anther surface between the wild-type and the tdr mutant, and their anther outer surfaces appeared smooth except for a few cells synthesizing wax precursors (Figure 1Q and 1R). At the vacuolated pollen stage, the epidermis of wild-type anthers was covered with abundant wax crystals (Figure 1S), while the tdr anther epidermis had less accumulated wax (Figure 1T). At the mature pollen stage, the epidermis of wild-type anthers formed more compact reticular structures (Figure 1U), whereas the tdr epidermis lacked a normal reticulate pattern (Figure 1V). These implied that TDR might indirectly affect anther epidermis wax formation by lipid signaling, since TDR is mainly expressed in tapetum. Lipidic Compositions are Altered in tdr The abnormalities of the pollen wall and anther surface in tdr suggested possible defects in the synthesis of lipid molecules. To test this hypothesis, we employed gas chromatography mass spectrometry (GC MS) analysis to compare the aliphatic components between the wild-type and the mutant anther at the vacuolated pollen stage. We ground up the whole anthers and submersed them in chloroform to extract lipidic compositions as previously described (Hauke and Schreiber, 1998), and the residual anther material was treated with ultrasonics in cyclohexane to re-extract the remained lipids (Wang et al., 2002; Chen et al., 2005). Then, these lipidic samples, including cuticular wax, intracellular and intercellular soluble lipids, were combined for analysis. Generally, fatty acids can be converted to primary fatty alcohols, alkanes and alkenes, and these aliphatic molecules are crucial for pollen wall and wax formation (Kolattukudy and Rogers, 1986; Schneider-Belhaddad and Kolattukudy, 2000). GC MS analysis indicated that wild-type anther had detectable alkanes, alkenes, fatty acids, and alcohols, with carbon chain lengths for alkanes and alkenes ranging from C23 to C39 and for fatty acids and alcohols from C18 to C36 (Figure 2). Compared with wild-type anther, the tdr mutant anther contained significantly changed lipidic contents (Figure 2 and Table 1). The contents of these lipids in wild-type anther were composed of lg mg 1 fatty acids, lg mg 1 fatty alcohols, lg mg 1 alkanes, and lg mg 1 alkenes. By contrast, in the tdr mutant anther, the contents of fatty acids, alkanes, and alkenes decreased to 1.787, 5.299, and lg mg 1, respectively, while the levels of fatty alcohols increased to lg mg 1. Obviously, the content of fatty acids, alkanes, and alkenes in tdr anther decreased to 29.4, 31.1, and 47.2% of wild-type, respectively. In plant, alkanes and alkenes are formed by elongation of saturated fatty acyl chains and modified by the decarbonylation pathway (Kunst and Samuels, 2003); thus, the reduction of fatty acids in tdr possibly causes the decrease in alkane and alkene formation. Generally, primary fatty alcohols are produced from VLCFAs by the acyl reduction pathway and secondary fatty alcohols formed by the decarbonylation pathway (Millar et al., 1999). In tdr, the reduction in primary (O) In the tdr mutant, the extremely expanded tapetal cells occupied the majority of the locule, and the microspores began to collapse at the mature pollen stage. (P) The tdr mutant microspores began to collapse and pollen wall disappeared with a remaining thin wall at the mature pollen stage. (Q) Wild-type anther showed a smooth epidermis at the young microspore stage. (R) At the young microspore stage, the tdr mutant anther showed a smooth epidermis. (S) At the vacuolated pollen stage, abundant wax crystals covered at the epidermises of the wild-type anthers. (T) Epidermis of the tdr mutant anthers had delayed synthesis of wax at the vacuolated pollen stage. (U) More compact reticular structures on the epidermis of the wild-type anthers at the mature pollen stage. (V) The tdr epidermis surface lacked a normal reticulate pattern at the mature pollen stage. Tds, tetrads; Msp, microspore; Pe, primexine; Pb, probaculae; Pt, protectum; Te, tectum; Ba, bacula; Ne, nexine; T, tapetum; U, ubisch body. Bars = 0.5 lm in (A) (P) and 5 lm in (Q) (V).

5 Zhang et al. d TDR Controls Rice Anther Aliphatic Metabolism 603 Figure 2. Lipids Constituents of Wild-Type and the tdr Anthers. (A) Constituents analysis of wild-type and tdr for fatty acids in anther. (B) Constituents analysis of wild-type and tdr for fatty alcohols in anther. (C) Constituents analysis of wild-type and tdr for alkanes in anther. (D) Constituents analysis of wild-type and tdr for alkenes in anther. White bars represent levels of each compound in the wild-type, and black bars represent them in the tdr mutant. Error bars indicate SD (n = 3). alcohols (only 0.41% compared with wild-type) proves that the acyl reduction pathway in the mutant is greatly blocked. On the contrary, the level of secondary alcohols in tdr was dramatically increased to 6.81-fold compared with wild-type, especially in C29, C31, and C33, implying that the secondary fatty alcohol synthetic pathway is possibly activated in tdr. To date, either biochemically or molecularly, enzymes involved in the production of secondary alcohols from alkanes remain poorly understood (Kunst and Samuels, 2003). Thus, these lipidic changes in the tdr mutant, especially in the fatty acid synthesis pathway and decarbonylation pathway, also offers one unique system to reveal the molecular mechanism for lipidic metabolism in plants. The tdr Mutant Exhibits Changed Gene Expression Pattern To further reveal the regulatory role of TDR during rice pollen development, especially its role in lipid metabolism, we employed microarray analysis using the RNAs prepared from wild-type and tdr. Since TDR is strongly expressed from the meiosis stage to the young microspore stage, and it is difficult to isolate anther at the early stage, we collected the developing spikelets containing anthers at the meiosis stage, the tetrad stage, and the young microspore stage, respectively, and extracted RNAs from these materials. The equally mixed RNAs from three different stages were used for hybridization. The hybridized Affymetrix rice genome array contained the probes corresponding to the transcripts representing two rice cultivars, with approximately japonica transcripts and Table 1. Aliphatic Composition of Anther for Wild-Type and tdr. Wild-Type (lg mg 1 ) tdr (lg mg 1 ) Fatty acids Primary alcohols Secondary alcohols Alkanes Alkenes Values shown are means 6 SD (n = 3) transcripts representing the indica cultivar, respectively. The transcripts with statistically significant differences in the p-value of less than 0.06 from three replicates were selected for further analyses (Supplemental Table 1). The genes in all three biological replicates with a greater than two-fold change in expressional levels were used for functional category analyses between wild-type and the tdr mutant. In total, 236 genes exhibited significantly different expression levels in tdr compared with wild-type, including 154 up-regulated genes and 82 down-regulated genes (Table 2). To deduce the functions of these changed genes in tdr identified from the microarray analysis, the cdna sequence from each of these 236 genes was searched in Gramene ( The TIGR Rice Genome Annotation Database and Resource ( NCBI ( and Clusters of Orthologous Groups (COG; databases. These genes

6 604 Zhang et al. d TDR Controls Rice Anther Aliphatic Metabolism Table 2. Functional Classification by COG Analysis of Genes that Were Up-Regulated or Down-Regulated at Least Two-Fold in tdr. Table 3. Up-Regulated and Down-Regulated Genes in Lipid Metabolism and Secondary Metabolites Biosynthesis in tdr. Function Up Down Total Down-regulated gene Putative function INFORMATION STORAGE AND PROCESSING 26 Translation, ribosomal structure and biogenesis RNA processing and modification Transcription Replication, recombination and repair Chromatin structure and dynamics CELLULAR PROCESSES AND SIGNALING 45 Cell cycle control, cell division, chromosome partitioning Defense mechanisms Signal transduction mechanisms Cell wall/membrane/envelope biogenesis Intracellular trafficking, secretion, and vesicular transport Posttranslational modification, protein turnover, chaperones METABOLISM 95 Energy production and conversion Carbohydrate transport and metabolism Amino acid transport and metabolism Nucleotide transport and metabolism Coenzyme transport and metabolism Lipid transport and metabolism Inorganic ion transport and metabolism Secondary metabolites biosynthesis, transport and catabolism POORLY CHARACTERIZED 70 General function prediction only Function unknown Total For detailed gene function, see Supplemental Table 1. were then functionally grouped into four categories: (1) information storage and processing, (2) cellular processes and signaling, (3) metabolism, and (4) genes with unknown functions, and could be further divided into 21 subgroups (for details, see Supplemental Table 1). The genes related to nuclear structure, cell motility, cytoskeleton, and extracellular structures were not included in these 236 genes, implying that TDR may have less of a function in regulating these pathways. The majority of the up-regulated or down-regulated genes in the tdr anthers were grouped in metabolism, transcription, cell wall/membrane/envelope biogenesis, posttranslational modification, and signal transduction. To verify the results of microarray analyses, we selected eight differentially expressed genes (Table 3) for RT PCR analysis (for RT PCR primer sequences, see Supplemental Table 4), namely Os08g43290 (Osc4), Os10g34360 (YY2), Os08g38810 (OsRAFTIN1), Os03g07250 (P450), Os03g04120 (Acyl-CoA Synthetase, OsACS), and Os05g49900 (Chalcone Synthase, OsCHS), Os02g40784 CER1, putative, expressed Os06g43044 GDSL-like Lipase/Acylhydrolase Os03g04120 Acyl-CoA synthetase Os08g43290 Osc4, protease inhibitor/seed storage/ltp family protein Os03g50960 Protease inhibitor/seed storage/ltp family protein Os05g49830 Lipase family protein Os08g38810 OsRAFTIN1a protein Os02g38290 Cytochrome P450 Os03g07140 Putative dehydrogenase domain of multifunctions Os03g07250 Cytochrome P450 Os05g49900 Chalcone and stilbene synthases 2 Os10g34360 YY2, chalcone and stilbene synthases Os10g35180 ABC-2 type transporter family protein Os11g24180 SNG2, putative, serine carboxypeptidase; serine carboxypeptidase S10 family protein Os12g13930 Beta-ketoacyl-ACP reductase Up-regulated gene Os11g31900 Os04g48750 Os10g30290 Os07g47210 Os06g47910 Os06g14630 Os03g46150 Os02g33380 Os02g02340 Os01g17170 Os10g41780 Os10g10180 Os04g58200 Putative function Acyl carrier protein/nadh-ubiquinone oxidoreductase Predicted steroid reductase Predicted lipase Predicted lipase Predicted lipase Predicted lipase Protease inhibitor/seed storage/ltp family protein Pectinesterase inhibitor domain containing protein Acyltransferase family protein Magnesium-protoporphyrin IX monomethyl ester cyclase Short-chain alcohol dehydrogenases SAM-dependent methyltransferases Short-chain alcohol dehydrogenases Os03g07140 (OsMS2, Arabidopsis MS2 homolog), and Os02g40784 (OsCER1, Arabidopsis CER1 homolog). Results of RT PCR analysis revealed that these genes were mainly expressed during pollen development (Figure 3), implying that these genes may function in rice pollen development. However, the decrease in expression of these eight genes was observed in tdr during pollen development (Figure 3). In contrast to these eight genes, compared with wild-type, no obvious altered expression of WDA1 was observed in tdr. All these were consistent with the microarray data.

7 Zhang et al. d TDR Controls Rice Anther Aliphatic Metabolism 605 To further quantitatively evaluate the conventional RT PCR result, we selected five genes, namely Os02g40784 (OsCER1), Os03g07250 (P450), YY2, Os03g07140 (OsMS2), and Os05g49900 (OsCHS), for real-time PCR analysis. Compared with wild-type, the expression levels of Os02g40784, Os03g07250, YY2, Os03g07140, and Os05g49900 at the young microspore stage were reduced to 72.6, 92.4, 97.9, 92.4, and 92.5% in tdr, respectively (Table 4). The correlation between the fold changes estimated from quantitative real-time PCR versus those from the microarray analysis was Both correlations were statistically significant below the 0.05 level, indicating the reliability of our microarray data. TDR Regulates Lipid Metabolism during Pollen Development Tapetum is metabolically active and plays a major role in pollen development by contributing to microspore release, nutrition, Figure 3. RT PCR Analysis of Lipid Tansport and Metabolism- Related Genes in Wild-Type and the tdr Mutant. ACTIN served as a control. Bands shown were RT PCR products. Ms, the microsporocyte stage; M, the meiosis stage; Y, the young microspore stage; V, the vacuolated pollen stage. Table 4. Comparison of the Changes of Five Genes using Real- Time PCR and Microarray. Changed fold in real-time PCR Os02g Os03g Os10g Os03g Os05g Correlation Changed fold in microarray pollen wall synthesis, and pollen coat deposition; consistent with this, we observed that the expression of 95 genes related to metabolism in the tdr mutant was greatly changed (40.3% in total genes) (Table 2). These genes were predicted to function in lipid metabolism and secondary metabolites biosynthesis (29.5% in total metabolism genes), carbohydrate transport and metabolism (25.2%), energy production and conversion (24.2%), and amino acid transport and metabolism (6%). Enzymatic conversion is a core event for lipidic metabolism. In tdr, the expression of 22 genes encoding putative enzymes possibly involved in lipid metabolism was altered (10 downregulated and 12 up-regulated; Table 3). In plants, de-novo biosynthesis of fatty acids occurs in plastids using two enzyme systems, namely heteromeric acetyl-co-a carboxylase (ACCase) and fatty acid synthase (FAS), which results in acyl-acp moieties that are 16 or 18 carbons long (O Hara et al., 2001). Beta-ketoacyl-ACP reductase is linked to FAS in reducing beta-ketoacyl-coa to beta-hydroxyacyl-coa (Fehling and Mukherjee, 1991). The activity of acyl-coa synthetase varies coincidentally during the period of lipid accumulation in rice seed (Ichihara et al., 2003). Consistent with the reduction of fatty acids in the tdr anther, the expression decrease in Os03g04120 (Acyl-CoA synthetase) and Os12g13930 (betaketoacyl-acp reductase) possibly involved in early fatty acid synthesis was observed. In fatty alcohols synthesis, Os03g07140 (OsMS2) (a homologous gene of Arabidopsis MS2, which regulates pollen wall development likely through converting fatty acids to primary alcohol (Aarts et al., 1997)), was also down-regulated (Figure 3). This was consistent with the reduced level of primary alcohols in the tdr anther. Plant waxes predominantly comprise alcohols, alkenes, alkanes, and esters derived from VLCFAs, ranging in chain length from C20 to C34 (Kunst and Samuels, 2003). In Arabidopsis, the CER1 gene encodes an aldehyde decarbonylase involved in the convention of long-chain aldehydes to alkanes a key step in wax biosynthesis (Aarts et al., 1995). In the tdr microarray analysis, expression of the CER1 homologous gene OsCER1 (56% with Arabidopsis CER1) was reduced and, in agreement with this, a slight reduction in alkanes was observed in the tdr anther. However, the expression of another important wax biosynthesis gene, Wda1 (56% with Arabidopsis CER1 and 63% with OsCER1), remained an undetectable change in the tdr microarray data (Figure 3), suggesting that OsCER1 and WDA1 are likely to be involved in the independent pathway of wax biosynthesis. In wda1, the epicuticular wax crystals were absent in the outer layer of the anther, while the rough epicuticular wax crystals could be observed in tdr. Several cytochrome P450 enzymes have been shown to be involved in the synthesis of cutin monomers by hydroxylating methyl groups at the x-chain terminus of fatty acids (Kandel et al., 2006), and, recently, CYP703A2 was proved as a conserved cytochrome P450 in land plants catalyzing in-chain hydroxylation of lauric acid for sporopollenin synthesis (Morant et al., 2007). Another cytochrome P450 enzyme, CYP96A15, is the mid-chain alkane hydroxylase responsible for formation of

8 606 Zhang et al. d TDR Controls Rice Anther Aliphatic Metabolism secondary alcohols and ketones in Arabidopsis stem cuticular wax (Greer et al., 2007). CYP86, CYP94, CYP96, and CYP704 were grouped together in the CYP86 clan of non A-type P450s (Nelson et al., 2004). In tdr, the expression of two P450 genes (Os02g38290 belongs to CYP 94A2 and Os03g07250 belongs to CYP86A2) was decreased, suggesting their possible role in lipid metabolism during pollen development. In addition, during pollen wall development, good correlation between the expression pattern of anther-specific CHS genes and the predicted male-specific components makes the attractive hypothesis that CHS enzymes are likely involved in the biosynthetic pathway related to exine synthesis plausible (Atanassov et al., 1998). One down-regulated gene in tdr, YY 2, which was specifically expressed in anthers at the uninucleate microspore stage (Figure 3), exhibited substantial homology to chalcone synthase (Hihara et al., 1996). Another chalcone synthase (Os05g49900) was also down-regulated in tdr. Furthermore, we also observed that the predicted GDSL-like lipases (Os10g30290, Os07g47210, Os06g47910, and Os06g14630) were up-regulated in tdr. GDSL enzymes have thioesterase, protease, arylesterase, and lysophospholipase activity and have broad hydrolytic activity toward three kinds of substrates, including acyl-coas, esters, and amino acid derivatives (Akoh et al., 2004). Lipase is also proved to be involved in the pollen stigma interaction for efficient pollination and pollen coat formation (Mayfield et al., 2001). Transference of lipidic molecules from the tapetum to the microspore surface is considered to be one essential step for pollen wall formation. In the tdr mutant, the expression of five genes putatively related to lipid transfer was found to be greatly altered, namely Osc 4 (Os08g43290), Os03g50960, Os03g46150, OsRAFTIN1, and Os10g Osc 4 was predicted to have homology with putative lipid transfer protein (LTP) in rice (Hihara et al., 1996). It is likely that the antherspecific LTPs possibly participate in the transport of fatty acids and/or other sporopollenin precursors from the tapetum to the microspore during exine deposition (Xue et al., 1994). In the microarray data, the expression of Osc4 and Os03g50960 was decreased (42-fold and 4.6-fold, respectively) in tdr, whereas another LTP gene (Os03g46150) was up-regulated in tdr (four-fold). OsRAFTIN1 has been proved to be accumulated in rice Ubisch bodies and possibly function in transporting molecules from tapetum to pollen walls via orbicules (Wang et al., 2003). In wild-type, we observed strong expression of OsRAFTIN1 by both microarray and RT PCR analyses (Figure 3); however, significant expression reduction of OsRAF- TIN1 was observed in tdr, suggesting the defects in transporting lipidic molecules from the tapetum to pollen walls via orbicules in tdr. Arabidopsis CER5 (an ATP-binding cassette sub-family member) is a key component of the export pathway for cuticular lipids, and cer5 has a strong reduction in the alkanes of surface waxes (Pighin et al., 2004; Bird et al., 2007). Os10g35180 (an ATP-binding cassette sub-family member), which is similar to CER5 with 56% identity, was downregulated in tdr. Undeveloped Tapetum1 (Udt1), which is required for the differentiation of secondary parietal cells to mature tapetal cells, is considered as a putative upstream transcription factor of TDR (Jung et al., 2005). Six genes possibly involved in lipid metabolism (Os08g43290, Os12g13930, Os03g07140, Os01g26000, Os07g41650, and Os05g49900) were found to be down-regulated in both the udt1 and tdr microarray data (Supplemental Table 3), suggesting that UDT1 and TDR possibly have similar regulatory functions during rice pollen development. Genes Implicated in Programmed Cell Death Are Changed in the tdr Mutant Tapetum degeneration is proposed to be triggered by a programmed cell death (PCD) process during later pollen development stages (Varnier et al., 2005), and, in the tdr mutant, we found that the tapetum PCD was retarded (Li et al., 2006). The predictable effectors that carry out cell death-associated processes, including proteases, nucleases, and reactive oxygen species, may be involved in animal PCD (Wu and Cheun, 2000). In agreement with this, previously, we proved that TDR has the ability in vivo to directly regulate rice Cys protease gene OsCP1 and the Osc6 gene (Li et al., 2006). However, less evidence related to the PCD effectors has been revealed in plants. In the microarray data, we observed that a group of genes possibly involved in PCD was changed in tdr. For example, Os03g59090 (one homolog of Lls1 (Lethal leaf spot1) encoding a cell death suppressor protein) was up-regulated in the tdr mutant. In maize, the Lls1 protein is required to limit the spread of cell death in mature leaves (Gray et al., 1997). In signal transduction pathway, calcium ion functions as an intracellular messenger in regulating a plethora of cellular processes from acclimative stress responses to survival and PCD (Hoeberichts and Woltering, 2003; Ng and McAinsh, 2003). Three genes Os03g21380 (putative encoding calcium-binding protein CAST), Os05g41270 (putative encoding calciumdependent protein kinase), and Os08g38600 (putative encoding copine-6, calcium-dependent membrane binding protein) were down-regulated in tdr. Another group of genes related to PCD were heat shock transcription factors (HSFs), which are thought to regulate the expression of heat shock proteins (HSPs) through binding to the promoter sequences located on the HSP genes, known as heat shock elements. HSPs have been shown to play important roles in repairing the damaged proteins and survival of cells (Miller and Mittler, 2006). In rice, HsfA4a, encoding a putative heat stress transcription factor protein, acts as an anti-apoptotic factor to defend against disease lesion mimics phenotypes (Yamanouchi et al., 2002). In the tdr mutant, three HSF genes (Os10g28340, Os09g35790, and Os04g48030) were found to be up-regulated; possibly, these genes contribute to the delayed PCD observed in the tdr taptetum. Caspases are cysteine proteases that play key roles in mediating PCD in animal systems (Cryns and Yuan, 1998). However, to date, no caspase homologs have been identified in plants.

9 Zhang et al. d TDR Controls Rice Anther Aliphatic Metabolism 607 But cell death in plants exhibits similar morphological features to caspase-mediated apoptosis in animals, suggesting that plant PCD is executed by caspase-like proteases. As substitutes, there are metacaspase and vacuolar processing enzymes (VPEs) as the possible executors of plant PCD (Woltering, 2004; Hara- Nishimura et al., 2005). In the tdr miroarray data, two cysteine protease (Os09g38920 and Os08g44270) were found to be upregulated in the tdr mutant. Recent evidence indicated that saturated VLCFAs ranging from C20:0 to C30:0 may be involved in activating ethylene biosynthesis (Qin et al., 2007), and the ethylene is a trigger in PCD (de et al., 2002; Steffens and Sauter, 2005). In addition, plant lipid transfer protein (LTP) can also initiate the apoptotic cascade at the mitochondrial level (Crimi et al., 2006). The down-regulated LTP family genes in tdr might block tapetum PCD with the abnormal features of mitochondria (Li et al., 2006). These give a strong hint that TDR possibly regulates tapetum PCD through controlling lipid molecules as an initiator. TDR Controls Basic Biological Activities during Pollen Development Among the 236 genes exhibiting statistically expressionsignificant differences between wild-type and the tdr mutant, a high proportion of the genes changed in tdr are associated with metabolism, transcription, cell wall/membrane/envelope biogenesis, posttranslational modification, and signal transduction, as indicated. In addition to the role of TDR in tapetum PCD and lipid metabolism, TDR is likely one master regulator controlling energy production and conversion, carbohydrates metabolism and others. In the microarray data, 23 upregulated genes were predicted to be involved in energy production and conversion in the tdr mutant (Supplemental Table 2), including the genes encoding chloroplast components, Photosystem I (PSI) and Photosystem II (PSII) multi-protein complexes and chlorophyll a b binding proteins. Another group of changed genes in the tdr mutant were possibly related to carbohydrate transport and metabolism, such as Os12g03860, Os03g43720, and Os02g49260, which were predicted to belong to permeases of a major facilitator superfamily related to sucrose transfer. In plants, sucrose is the major transportation form of photo-assimilated carbon as a source of carbon skeletons and energy for plant organs (sink organs) (Lemoine, 2000). Some other gene products were likely involved in tricarboxylic acid cycle pathway, such as beta-fructosidases, beta-glucosidase, fructose-1,6-bisphosphatase, glycosyl transferases, hexokinase-1, galactosyltransferase, beta-glucuronidase, etc. (Supplemental Table 2). Additionally, some down-regulated geneswererelatedtoaminoacidtransportandmetabolismintdr, such as the two genes AK and Os03g38540, probably encoding amino acid transferases. In addition, a group of tapetum-specific transcription factors were reported to play key roles in tapetum and pollen development (Ma, 2005). In the tdr mutant, 18 putative transcription factors were shown to exhibit changed expression, namely bhlh-type transcription factor (basic helix-loop-helix), HSF (heat shock transcription factor), zinc finger, and Myb family transcription factors (see Table 1). As to bhlh-type transcription factors, the TDR gene itself (Os02g02820) was shown to have approximately eight-fold changes. These suggested that TDR may play an important role in regulating gene expression through regulating other transcription factors. Conclusion In short, here, we report the unique role of the rice TDR gene controlling pollen outer wall formation by regulating the lipid metabolism. The tdr mutant developed abnormal anther walls, epidermis, and its microspore had defects in the establishment exine. Intriguing changes of lipdic compositions, particularly the decrease of fatty acids and increase of secondary alcohols, were observed in the tdr anther, suggesting one specific regulatory role of TDR in lipid metabolism during pollen development. Also, this phenotype is quite different from some valuable isolated mutants involved in lipid molecule syntheses and wax production during anther development. In agreement with this, TDR has the ability to regulate the genes related to lipid transport and metabolism. Also, loss of function of TDR caused the expression changes of a total of 236 genes (154 up-regulated and 82 down-regulated), revealing that TDR is one master regulator for tapetum PCD, and some other basic biological processes during pollen development. It will be interesting to uncover the detailed regulatory network of TDR in rice pollen development in the future. METHODS Microarray Experiment and Analysis Rice plants were grown in the paddy field of Shanghai Jiao Tong University. The spikelets from the tetrad stage to the young microspore stage of wild-type and the tdr mutant were collected for total RNA extraction, using Trizol reagent (Invitrogen). Hybridization, washing, staining, scanning, and data collection were performed by Gene Tech Biotechnology Company (Shanghai), according to the standard Affymetrix protocol. The Affymetrix rice genome array was used in the array experiment. Detailed procedures for microarray analyses were according to the standard Affymetrix protocol (Affymetrix, Santa Clara, CA, USA, manual). Each microarray chip (six in total) was scanned once with the GeneArrayTM scanner 3000 (GeneChipâSystem). The hybridization signals for all the experiments were scaled to the same target intensity of 100 to compare expression profiles between samples. The Signal Log Ratio estimated the magnitude and direction of change of a transcript when two arrays were compared. The value for gene expression was the average of the three corresponding duplicates, and changes in transcript levels greater than two-fold were used for functional category

10 608 Zhang et al. d TDR Controls Rice Anther Aliphatic Metabolism analyses. The statistical method used was as per the Statistical Algorithms Reference Guide (Affymetrix protocol). RT PCR Total RNAs were isolated using Trizol reagent (Generay Biotech (Shanghai) Co. Ltd) from rice (Oryza sativa) spikelets (at the microsporocyte stage, the meiosis stage, and the young microspore stage, respectively) and anthers at the vacuolated pollen stage. Then, RNA was extracted and reverse-transcribed as previously described (Li et al., 2006). All the primers for RT PCR are listed in Supplemental Table 4 online, with ACTIN as an internal control. Quantitative Real-Time PCR Os02g40784 (OsCER1), Os03g07250 (P450), Os10g34360 (YY2), Os05g49900 (OsCHS), and Os03g07140 (OsMS2) use the young microspore stage anther s cdna as the template. The primers for real-time PCR were the same as the primers for RT PCR. All tested primer pairs were confirmed to yield a single band upon PCR amplification as assayed via agarose gel electrophoresis and dissociation curve analysis. Quantitative real-time PCR was performed on a Rotor-Gene RG3000A detection system (Corbett RESEARCH, Australia) using SYBR Green I master mix (Generay Biotech, Co. Ltd, Shanghai). All PCR experiments were conducted using 40 cycles with 94 C for 20 s, 58 C for 20 s and 72 C for 20 s) in a reaction mixture containing 10 pmol of each primer and 3 mm magnesium chloride and a 1:10 dilution of each cdna pool (per biological replicate) as a template; all reactions were performed in triplicate, with ACTIN as the reference gene for all comparisons. Fluorescence signal was detected at 72 C and then set as the threshold. The cycle numbers of fluorescence curves reaching the threshold set was the Ct values. The mean Ct value of each gene was calculated and used for fold change calculations using the method described by Roter-Gene version 6.0 (Build 38) software. Transmission Electron Microscopic and Scanning Electron Microscopy Analyses Anthers for TEM and flowers for SEM of the wild-type and the tdr mutant at various stages were fixed and observed as previously described (Li et al., 2006). Analysis of Lipids Components Lipids components were analyzed as described previously (Jung et al., 2006). Briefly, 4 mg freeze-dried anthers was ground up and extracted with 700 ll of chloroform for 1 min and the chloroform extract was transferred to a new vial. Then, the remaining anther materials were subsequently submersed in 1 ml cyclohexane for 2 h with ultrasonic treatment at 70 C (Wang et al., 2002). Then, these lipids samples were combined for subsequent testing. Also, the combined lipidic samples were spiked with 5 lg methylnonadecanoate (Sigma) as an internal standard. The solvent was evaporated under a nitrogen stream, and compounds containing free hydroxyl and carboxyl groups were converted to their trimethylsilyl ethers and esters with 40 ll of bis-(n,n-trimethylsilyl)-tri-fluoroacetamide (Sigma) in 160 ll of pyridine for 40 min at 56 C before gas chromatography mass spectrometry (GC MS) analysis. SUPPLEMENTARY DATA Supplementary Data are available at oxfordjournals.org. FUNDING This work was supported by funds from the National Key Basic Research Developments Program of the Ministry of Science and Technology, P.R. China (2007CB108700), National 863 High-Tech Project (2006AA10A102), National Natural Science Foundation of China ( and ), and Shanghai Leading Academic Discipline Project (B205). ACKNOWLEDGMENTS We thank Z.-J. Luo and M.-J. Chen for providing the tdr mutant, X.-X. Li for his valuable suggestion on the microarray data analyses, T. Zhou for microarray data analyses, and X.-Y. Gao and J.-Q. Li for SEM and TEM observation. 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